INTERACTIONS BETWEEN ENDEMIC BORDETELLA SPECIES AND HOST IMMUNITY

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1 The Pennsylvania State University The Graduate School College of Agricultural Sciences INTERACTIONS BETWEEN ENDEMIC BORDETELLA SPECIES AND HOST IMMUNITY A Thesis in Pathobiology by Daniel Nathan Wolfe 2007 Daniel N. Wolfe Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy December 2007

2 The thesis of Daniel N. Wolfe was reviewed and approved* by the following: Eric T. Harvill Associate Professor of Microbiology and Infectious Diseases Thesis Adviser Chair of Committee Reka Z. Albert Associate Professor of Physics Avery August Associate Professor of Immunology Biao He Associate Professor of Virology Mary J. Kennett Professor of Veterinary and Biomedical Sciences Vivek Kapur Head of the Department of Veterinary and Biomedical Sciences *Signatures are on file in the Graduate School. ii

3 ABSTRACT Bordetella pertussis and Bordetella parapertussis are bacterial pathogens that cause whooping cough, a disease that is re-emerging in vaccinated populations. The diseases caused by these bacteria are clinically indistinguishable, but pertussis toxin, which is only expressed by B. pertussis, has a variety of effects on the host immune response and hinders the clearance of B. pertussis from the respiratory tract. This led us to investigate the host factors that overcome the effects of pertussis toxin. In a mouse model of infection, Tumor Necrosis Factor-α was required to limit leukocyte accumulation and survive during infection by B. pertussis, but not a pertussis toxin-deficient strain of B. pertussis. Interferon-γ combatted the effects of pertussis toxin by contributing to the recruitment of leukocytes and the antibody-mediated clearance of B. pertussis. Interestingly, B. parapertussis does not express pertussis toxin but causes the same disease in the same host. We sought to elucidate the host factors that protect against B. parapertussis infection and how this bacterium may evade host immunity. Antibodies, T cells, and neutrophils were crucial to the elimination of B. parapertussis from the respiratory tract. However, B. parapertussis did not stimulate an early Toll-like receptor-4-mediated recruitment of leukocytes to the site of infection, resulting in the evasion of rapid clearance. Interferon-γ overcame the lack of Toll-like receptor-4 stimulation by contributing to the inflammatory response, but Interferon-γ production was inhibited by Interleukin-10, facilitating the persistence of the infection. Ultimately, both B. pertussis and B. parapertussis infections induce protective immunity. Since ecological theory predicts that two closely related immunizing pathogens can not coexist in the same host population, we assessed the cross reactivity of immunity to these pathogens. Immunity induced by B. parapertussis protected against both species while immunity induced by B. pertussis only protected against B. pertussis. O antigen enabled B. iii

4 parapertussis to avoid cross immunity and may be a key protective antigen. These studies contribute to the understanding of interactions between the bordetellae and host immunity and will guide any future vaccine design against B. parapertussis. iv

5 TABLE OF CONTENTS List of Figures.ix List of Abbreviations..xiii Acknowledgements.xv Chapter 1. INTRODUCTION.1 The bordetellae 2 B. pertussis and B. parapertussis; disease and prevalence..2 Current vaccine strategies 5 Evolution of the classical bordetellae..6 Differences between B. pertussis and B. parapertussis...7 Immunity to the bordetellae.8 Preview 9 References..10 Chapter 2. DELAYED ROLE OF TUMOR NECROSIS FACTOR-α IN OVERCOMING THE EFFECTS OF PERTUSSIS TOXIN..20 Abstract...21 Introduction.21 Materials and Methods 22 Results.25 Discussion 37 References 40 v

6 Chapter 3. INTERFERON-γ PRODUCTION BY T CELLS OVERCOMES THE INHIBITORY EFFECTS OF PERTUSSIS TOXIN ON BACTERIAL CLEARANCE...48 Abstract 49 Introduction..49 Materials and Methods.51 Results..53 Discussion 57 References 60 Chapter 4. CLEARANCE OF BORDETELLA PARAPERTUSSIS FROM THE LOWER RESPIRATORY TRACT REQUIRES HUMORAL AND CELLULAR IMMUNITY.65 Abstract...66 Introduction..66 Materials and Methods.68 Results..70 Discussion.77 References.80 Chapter 5. A LACK OF TOLL-LIKE RECEPTOR-4 STIMULATION ENABLES BORDETELLA PARAPERTUSSIS TO AVOID RAPID ANTIBODY-MEDIATED CLEARANCE...87 Abstract...88 Introduction...88 Materials and Methods..90 vi

7 Results...92 Discussion 101 References 103 Chapter 6. INDUCTION OF INTERLEUKIN-10 BY BORDETELLA PARAPERTUSSIS LIMITS THE INTERFERON-γ RESPONSE AND BACTERIAL CLEARANCE Abstract Introduction Materials and Methods..113 Results Discussion.122 References.125 Chapter 7. O ANTIGEN ENBABLES BORDETELLA PARAPERTUSSIS TO AVOID BORDETELLA PERTUSSIS-INDUCED IMMUNITY. 132 Abstract Introduction 133 Materials and Methods Results 137 Discussion..149 References..152 Chapter 8. SUMMARY AND SIGNIFICANCE Impact of Ptx on host immunity and disease. 162 Evasion of protective immunity by Bordetella parapertussis vii

8 Public health impact References..171 viii

9 LIST OF FIGURES Figure 2.1: Survival curve of wild type versus TNF-α -/- mice infected with B. pertussis.26 Figure 2.2: Production of TNF-α in response to B. pertussis.26 Figure 2.3: Quantification of bacteria and leukocytes in the lungs of wild type and TNF-α -/- mice..27 Figure 2.4: Effect of depleting TNF-α on B. pertussis numbers in the lungs.29 Figure 2.5: Cytokine responses to B. pertussis in the murine lungs...30 Figure 2.6: Measurement of airway resistance by whole body plethysmography..31 Figure 2.7: Survival curve of B. pertussis-infected TNF-α -/- mice depleted of CD4 + T cells, CD8 + T cells, or neutrophils.32 Figure 2.8 : Effects of depleting CD4 + T cells or neutrophils on bacterial numbers, leukocyte accumulation, and airway resistance 34 Figure 2.9 : Survival of TNF-α -/- mice infected with a Ptx-deficient strain of B. pertussis 36 Figure 2.10 : Quantification of bacterial numbers and leukocytes in the lungs of wild type and TNF-α-deficient mice infected with B. pertussisδptx Figure 3.1 : Effect of T cells on the clearance of B. pertussis from the lungs.53 Figure 3.2 : Role of T cell subsets in the antibody-mediated clearance of B. pertussis..54 Figure 3.3 : IFN-γ production by splenocytes from B. pertussis-infected mice.. 56 Figure 3.4 : Role of IFN-γ in the antibody-mediated clearance of B. pertussis.. 56 Figure 3.5 : Effect of IFN-γ on leukocyte accumulation in the lungs during B. pertussis infection Figure 3.6 : Effect of IFN-γ on myeloperoxidase activity in response to B. pertussis...58 ix

10 Figure 4.1 : Role of adaptive immune response in protecting against B. parapertussis infection..71 Figure 4.2 : Mice deficient in antibody production are unable to clear B. parapertussis from the lower respiratory tract..72 Figure 4.3 : Mucosal antibodies are not crucial to clearance of B. parapertussis from the lower respiratory tract 73 Figure 4.4 : T cells are required for the function of serum antibodies in clearing B. parapertussis from the lower respiratory tract 74 Figure 4.5 : Complement is required for the function of serum antibodies against B. parapertussis 75 Figure 4.6 : Neutrophils are required for the function of serum antibodies against B. parapertussis 76 Figure 5.1 : Numbers of B. parapertussis and leukocytes in the lungs over time..93 Figure 5.2 : Leukocyte numbers in the lungs of wild type versus TLR4-deficient mice...94 Figure 5.3 : Effect of B. parapertussis on TLR4 signaling induced by other bacteria in vitro..95 Figure 5.4 : Effect of B. parapertussis on TLR4 signaling induced by other bacteria in vivo..96 Figure 5.5 : Effect of B. bronchiseptica on B. parapertussis growth in vivo...97 Figure 5.6 : Effect of B. bronchiseptica on antibody-mediated clearance of B. parapertussis..99 Figure 5.7 : Effect of TLR4 on the rapid clearance of B. parapertussis x

11 Figure 6.1 : Reduction of B. parapertussis numbers in the lungs correlates with increased CD4 + T cell numbers. 115 Figure 6.2 : T cells produce IFN-γ which contributes to bacterial clearance Figure 6.3 : IFN-γ contributes to leukocyte accumulation in response to B. parapertussis infection Figure 6.4 : IL-10 production in response to B. parapertussis..121 Figure 6.5 : Effect of IL-10 on the IFN-γ response to B. parapertussis Figure 6.6 : Effect of IL-10 on clearance of B. parapertussis from the lungs Figure 7.1 : B. pertussis colonization of naive or immunized mice Figure 7.2 : B. parapertussis colonization of naive or immunized mice Figure 7.3 : Cross-protection between B. pertussis and B. parapertussis upon challenge 70 days post-inoculation Figure 7.4 : IFN-γ production by splenocytes from naïve or B. pertussis- or B. parapertussisimmune hosts Figure 7.5 : Leukocyte accumulation in the lungs of mice upon challenge with B. pertussis or B. parapertussis 143 Figure 7.6 : Recognition of B. pertussis and B. parapertussis by antibodies from convalescent phase serum..144 Figure 7.7 : Effect of passive transfer of immune serum to B. pertussis-immune mice on B. parapertussis numbers.145 Figure 7.8 : Antibody recognition of live B. pertussis, B. parapertussis, and O antigen-deficient B. parapertussis by immune serum.146 xi

12 Figure 7.9 : Ability of the O antigen-deficient strain of B. parapertussis to colonize B. pertussisimmune hosts Figure 7.10 : Protection conferred against B. parapertussis by O antigen-deficient B. parapertussis Figure 8.1: Schematic of clearance of B. pertussis from the respiratory tract 163 Figure 8.2: Schematic of clearance of B. parapertussis from the respiratory tract.167 Figure 8.3: Cross reactivity of immune responses to B. parapertussis and B. pertussis.169 xii

13 LIST OF ABBREVIATIONS BAL: bronchoalveolar lavage CFU : colony forming unit CR3: complement receptor 3 ELISA: enzyme linked immunosorbent assay g: gravity IACUC: institutional animal care and use committee IFN: interferon Ig: immunoglobulin IL: interleukin I.P. : intraperitoneal IS: insertion sequence KC: keratinocyte chemoattractant LOS: lipooligosaccharide LPS: lipopolysaccharide MHC: major histocompatability complex MIP: macrophage inflammatory protein PBS: phosphate buffered saline PBS-T : phosphate buffered saline with tween PCR: polymerase chain reaction Penh: enhanced respiratory pause index PFU : plaque forming unit Ptx: pertussis toxin xiii

14 TGF : tissue growth factor TLR : toll-like receptor TNF : tumor necrosis factor xiv

15 ACKNOWLEDGEMENTS Parts of this thesis have been published in peer-reviewed research journals. Chapter 2: Delayed Role of Tumore Necrosis Factor-α in Overcoming the Effects of Pertussis Toxin was published in the Journal of Infectious Diseases; Volume 196, Issue 8, pages Figures and were published in that article. Chapter 4: Clearance of Bordetella parapertussis from the Lower Respiratory Tract Requires Humoral and Cellular Immunity was published in Infection and Immunity; Volume 73, Issue 10, pages Figures were published in that article. Chapter 7: O antigen Enables Bordetella parapertussis to Avoid Bordetella pertussis-induced Immunity has been published in Infection and Immunity; Volume 75, Issue 10, pages Figures , 7.4, and were published in this article. All appropriate permissions have been obtained to reproduce any figures or text for these articles. I would like to thank the following people for scientific discussions regarding my thesis work: Dr. Eric Harvill as well as past and current members of his laboratory, my committee (Dr. Avery August, Dr. Mary Kennett, Dr. Biao He, and Dr. Reka Albert), and Penn State s Center for Infectious Disease Dynamics. Furthermore, I would like to thank the following people for the donation of materials and/or the use of equipment: Dr. Pamela Hankey, Dr. Sandeep Prabhu, Dr. Robert Paulson, Dr. Avery August, Dr. Drussila Burns, Dr. Rick Wetsel, and Dr. Innocent Mbawuike. I would like to thank the staff of Penn State s Flow Cytometry facility (Elaine Kunze, Susan Magargee, and Nicole Bern) for technical assistance. Finally, I would like to thank my family and friends, especially my wife Katie, for their support during my thesis research. xv

16 Chapter 1: Introduction 1

17 The bordetellae: The bordetellae (Bordetella pertussis, B. parapertussis hu, B. parapertussis ov, B. bronchiseptica, B. avium, B. hinzii, B. holmesii, B. petrii, and B. trematum) are a group of gram negative bacteria, two of which (B. pertussis and B. parapertussis hu ) cause whooping cough in humans (1). There are approximately 50 million cases of whooping cough resulting in 300,000 deaths worldwide annually (2). Although typically considered a childhood disease, infections by these pathogens and the associated diseases can occur at any age. Since the following research does not involve B. parapertussis ov, the ovine-adapted strain of B. parapertussis, the humanadapted strain, B. parapertussis hu, will simply be referred to as B. parapertussis. B. pertussis and B. parapertussis; disease and prevalence: B. pertussis, B. parapertussis, and B. bronchiseptica are known as the classical bordetellae and have been studied extensively due to their impact on human and animal health. B. bronchiseptica infects a wide range of mammals and infections by this pathogen can result in anything from asymptomatic infections to severe respiratory disease (1). B. pertussis and B. parapertussis are both etiologic agents of whooping cough. Classical whooping cough consists of a paroxysmal cough that can last for months and lead to apnea, hypoxia, vomiting, and, in severe cases, coma and death (1), but many infections result in mild or subclinical disease. B. parapertussis disease has been shown to be milder and shorter in duration than that caused by B. pertussis in some studies, but reports have varied regarding this topic (3-5). Despite the potential differences in the duration of symptoms, the diseases caused by B. parapertussis and B. pertussis are clinically indistinguishable and both pathogens are capable of causing substantial morbidity and mortality. 2

18 The similarities between diseases caused by B. pertussis and B. parapertussis (3-4) make diagnosing one versus the other very difficult. Bacterial culture is still the gold standard for diagnosing infections by B. pertussis and B. parapertussis. However, these bacteria are quite fastidious and several factors (a delay in specimen collection, poor specimen collection/transportation, laboratory inexperience/contamination, expenses, etc.) can hinder the ability to culture them (1). Polymerase chain reaction (PCR) of Bordetella insertion sequence (IS) elements has made the rapid diagnosis of B. pertussis and B. parapertussis infections possible. IS481 is present in B. pertussis and B. holmesii while IS1001 is present in B. parapertussis and B. holmesii (6-8). Thus, the combination of PCR for IS481 and IS1001 can differentiate between these species. Unfortunately, problems similar to those with bacterial culture are encountered involving the collection and processing of samples. Serology has also been used to detect infections by B. pertussis and B. parapertussis. In proactive clinical studies, if serum samples are collected before and after symptom onset, physicians can monitor for increases in antibody titers against filamentous hemagglutinin with (for B. pertussis) or without (for B. parapertussis) increases in antibody titers against Ptx. However, two major problems arise when using serological assays to detect Bordetella infection; (i) serum samples prior to and post-infection are required to detect a rise in antibody titers and (ii) many laboratories only use anti-ptx antibodies as a diagnostic tool, ignoring potential B. parapertussis infections. With all of that being said, differential diagnosis of B. pertussis versus B. parapertussis does not facilitate the treatment of disease (1), thus, is rarely performed in a clinical setting. Whooping cough is currently classified as a re-emerging infectious disease by the Center for Disease Control, but B. parapertussis disease is not reportable to this agency (B. pertussis disease is reportable). The lack of reporting of B. parapertussis infections, combined with the 3

19 difficulty and rarity of differential diagnoses between B. pertussis and B. parapertussis, has prevented researchers from gaining a clear picture of the relative prevalence of these pathogens. B. parapertussis has been shown to cause anywhere from 1% to greater than 95% of whooping cough cases depending on the population, though most estimates are between 4% and 40% (reviewed in 9). Seroprevalence data has indicated that up to 60% of a human population contained B. parapertussis-specific antibodies in their serum indicating a recent B. parapertussis infection (10). Over 90% of this population was seropositive for B. pertussis (11), suggesting that B. pertussis may be more prevalent than B. parapertussis. However, the percentage of seropositive individuals depends on both the prevalence of a pathogen and the rate of decay of detectable antibody responses to that pathogen. Immunity to B. pertussis lasts for approximately 5-10 years (12), but it is not known how long immunity to B. parapertussis lasts. Adding to the difficulty of determining the relative prevalences of B. pertussis and B. parapertussis, many cases of whooping cough caused by either species go undiagnosed for a variety of reasons. For example, many cases in vaccinated individuals do not present with the classical whoop associated with the disease (13-14), making diagnosis of the disease difficult. In fact, a recent estimate suggested that although the annual incidence of notified B. pertussis cases is only 0.01% in the Netherlands, the actual incidence of infection may be as high as 6.6% (15). This would mean that as few as 1 in 600 B. pertussis infections are reported as cases of whooping cough. The rate of reporting of B. parapertussis infections would likely be lower than that of B. pertussis for the following reasons: (i) if B. parapertussis disease is milder than that caused by B. pertussis, B. parapertussis-infected individuals may be less likely to visit a physician, (ii) even if B. parapertussis is diagnosed, it is not reportable to the Center for Disease Control, and (iii) many physicians may assume cases of whooping cough to be caused by B. 4

20 pertussis since it is more well-known as the causative agent of whooping cough. Without a concentrated effort to examine the prevalence of B. parapertussis in human populations, the role of this pathogen in the resurgence of whooping cough will remain unclear. Current vaccine strategies: Despite the fact that both B. pertussis and B. parapertussis cause whooping cough, current vaccine strategies only contain B. pertussis antigens. The original vaccines that were licensed which are still used in many developing countries consisted of whole, inactivated B. pertussis cells. Upon the introduction of these vaccines, the incidence of whooping cough as a whole greatly declined (16). Furthermore, Lautrop et.al. suggested that these vaccines reduced the prevalence of both B. pertussis and B. parapertussis (17). However, more recent studies have demonstrated that B. pertussis vaccines have little, if any, efficacy against B. parapertussis infection or disease (18-22). Most developed countries switched to acellular vaccines during the 1990s due to the reactogenicity of whole-cell vaccines. Acellular vaccines consist of some combination of B. pertussis Ptx, pertactin, filamentous hemagglutinin, fimbriae 2, and fimbriae 3. Experimental studies in a mouse model have shown that the immune responses induced by acellular vaccines are effective against B. pertussis but are unable to limit colonization by B. parapertussis (18-19). Clinical studies support these results in that B. pertussis vaccination does not appear to prevent disease caused by B. parapertussis (20-22). Both experimental and clinical studies have indicated that the acellular vaccines are even less effective against B. parapertussis than the whole cell vaccines (18-22). In fact, acellular vaccines may even exacerbate B. parapertussis disease (18) and prevalence of this pathogen may have increased despite, or even because of, the introduction of these vaccines (20). 5

21 Importantly, whooping cough has been increasing in incidence over the past 20 years in countries that have maintained excellent vaccine coverage (23-27), and it is unclear what the relative roles of B. pertussis and B. parapertussis are in this re-emergence. The modification of vaccine strategies to efficiently protect against both B. pertussis and B. parapertussis would reduce the incidence of B. parapertussis disease and, consequently, whooping cough as a whole. Vaccines containing whole, heat-inactivated B. parapertussis have been used in the past to curb epidemics by this pathogen (28), but it is unclear what is involved in protective immunity against B. parapertussis. Evolution of the classical bordetellae: B. bronchiseptica, B. pertussis, and B. parapertussis are very closely related; B. pertussis and B. parapertussis appear to have evolved independently from B. bronchiseptica-like progenitors and have undergone a large-scale loss of genes during their evolution (29). Despite the fact that B. pertussis and B. parapertussis are adapted to humans and B. bronchiseptica infects a wide range of mammals, each human-adapted pathogen is still more closely related to B. bronchiseptica than they are to each other (29). Genomic analyses have suggested that the most recent common ancestor of B. pertussis and B. parapertussis existed some time between million years ago. The most recent common ancestor for B. pertussis and B. bronchiseptica existed between million years ago. Interestingly, the most recent common ancestor for B. parapertussis and B. bronchiseptica existed as recently as million years ago (29). Furthermore, strains of B. parapertussis isolated from anywhere in the world over the past 50 years are clonally identical, suggesting that this species emerged as a pathogen very recently (30-31). Together, these data suggest that B. parapertussis evolved to infect human populations some time after B. pertussis did. Since the mean age of first B. 6

22 pertussis infection for unvaccinated children is less than 5 years (1) and this pathogen induces protective immunity, the population that B. parapertussis adapted to likely had some degree of immunity to B. pertussis (32). Differences between B. pertussis and B. parapertussis: Although B. pertussis and B. parapertussis are more closely related to B. bronchiseptica than they are to each other, they still, in large part, utilize the same sets of virulence factors. However, there are two major exceptions; pertussis toxin (Ptx) and O antigen. Ptx is unique to B. pertussis. The genes for this toxin are intact in B. bronchiseptica and B. parapertussis, but there are mutations in the promoters that prevent its expression by these species (33-35). Ptx is an AB 5 type toxin that inactivates a subset of heterotrimeric G-proteins, which are common to the signaling pathways of many chemokine receptors (36). This toxin impacts the immune response by preventing the early migration of leukocytes to the lungs (37-38). Ptx affects the adaptive immune response by stimulating Interferon (IFN)-γ production independently of major histocompatability complex (MHC) interactions, driving the differentiation of Th1 T cells, and inhibiting the generation of Bordetella-specific antibodies (39-41). Some have suggested that Ptx is the key virulence determinant of B. pertussis (42), but given that B. parapertussis does not express Ptx and still causes whooping cough, this toxin may not be a key contributor to virulence. Alternatively, Ptx may be an important virulence determinant for B. pertussis, but B. parapertussis utilizes a parallel mechanism to make up for the the loss of the effects of this toxin. O antigen is expressed by B. bronchiseptica strains but was lost by B. pertussis during its evolution from a B. bronchiseptica-like progenitor (43). Interestingly, B. parapertussis has maintained the expression of O antigen (44), suggesting that it is important to the success of B. 7

23 parapertussis, but not B. pertussis, in human populations. Several functions have been described for O antigen including the evasion of complement-mediated killing, phagocytosis, and antibody binding (45-48). This virulence factor could be a key virulence factor of B. parapertussis, possibly making the effects of Ptx dispensible. Besides the differential expression of Ptx and O antigen by B. pertussis and B. parapertussis, these pathogens largely express the same sets of virulence factors. It could be expected that an immune response induced by B. pertussis antigens would confer protection against B. parapertussis because of how closely related these bacteria are. However, there is a degree of antigenic variation in the virulence factors that are shared by B. pertussis and B. parapertussis including filamentous hemagglutinin and pertactin (49). The core lipopolysaccharide (LPS) structures of these pathogens also differ substantially. B. pertussis LPS [actually lipooligosaccharide (LOS)] separates into two major bands on an SDS-PAGE gel; band A and band B, but B. parapertussis LPS consists of no band A and a truncated band B structure (50). These differences could, at least in part, explain why various B. pertussis vaccines are ineffective against B. parapertussis. Immunity to the bordetellae: Previous studies have shed light on what is involved in a protective immune response against B. bronchiseptica and B. pertussis, but much less is known about what is involved in protective immunity against B. parapertussis. Antibodies are protective against B. bronchiseptica (51). Toll-like receptor 4 (TLR4), neutrophils, Fcγ receptors, and complement are required for the antibody-mediated clearance of this pathogen from the lungs (52). In contrast, Immunoglobulin (Ig)A mediates protection against B. bronchiseptica in the upper respiratory tract (53). 8

24 Both antibodies and T cells are protective against B. pertussis (51, 54). Specifically, antibodies raised against virulence factors such as filamentous hemagglutinin, fimbriae, pertactin, and pertussis toxin (Ptx) correlate with protection against disease in clinical studies (1, 55). Interestingly, Ptx inhibits the antibody-mediated clearance of B. pertussis during the first week of infection, but its inhibitory effect is ultimately overcome and antibodies are able to clear the infection (37). These antibodies require the presence of neutrophils and Fcγ receptors in order to protect against B. pertussis (37). Prior to these studies, there was very little known regarding the protective immune response against B. parapertussis. It was known that adaptive immunity was crucial to protection, since SCID-beige mice succumbed to B. parapertussis infection (56), and B cells were required for clearance of this pathogen from the murine respiratory tract (51). Other than that, the vast majority of immunological studies regarding the bordetellae have focused on B. bronchiseptica or B. pertussis. Preview: The research described here involves the study of interactions between endemic Bordetella species and host immunity in a mouse model of infection. Since many have suggested that Ptx is a key contributor to virulence for B. pertussis, and our lab has shown that this toxin inhibits antibody-mediated clearance of B. pertussis (37), the first sections of this thesis will focus on the host factors that overcome the effects of Ptx. B. parapertussis does not express Ptx but behaves similarly to B. pertussis in murine hosts (as well as in human hosts). Thus, this pathogen likely utilizes different strategies in order to optimize the infection process. The next section of this thesis will focus on (i) how B. parapertussis is cleared from the host respiratory tract and (ii) how B. parapertussis avoids the host immune response to persist in its 9

25 host. Finally, Watanabe et.al. suggested that B. pertussis and B. parapertussis infections induce protective cross-immunity (57), but if this were true, it would be expected that one of these species would be displaced from human populations via immune-mediated competition. Furthermore, current evolutionary and immunological theories indicate that B. parapertussis likely invaded a population in which some degree of immunity to B. pertussis would have been present. In order to do so, the ability to evade B. pertussis-induced immunity would have been crucial to the success of B. parapertussis. The penultimate section of this thesis will focus on (i) whether or not infections by these pathogens induce effective cross-immunity and (ii) how B. parapertussis may evade B. pertussis-induced immunity. References: 1. Mattoo S, and J. Cherry (2005) Molecular pathogenesis, epidemiology, and clinical manifestations of respiratory infections due to Bordetella pertussis and other Bordetella subspecies. Clin Microbiol Rev 18(2): Crowcroft, N. S., C. Stein, P. Duclos, and M. Birmingham (2003) How best to estimate the global burden of pertussis? Lancet Infect Dis 3(7): Mastrantonio, P., P. Stefanelli, M. Giulano, Y. Herrera Rojas, M. Ciofi degli Atti, A. Anemona, and A.E. Tozzi (1998) Bordetella parapertussis infection in children: epidemiology, clinical symptoms, and molecular characteristics of isolates. J Clin Microbiol 36(4):

26 4. Bergfors, E., B. Trollfors, J. Taranger, T. Lagergard, V. Sundh, and G. Zackrisson (1999) Parapertussis and pertussis: differences and similarities in incidence, clinical course, and antibody responses. Int J Infect Dis 3(3): Heininger, U., K. Stehr, S. Schmitt-Grohe, C. Lorenz, R. Rost, P.D. Christenson, M. Uberall, and J.D. Cherry (1994) Clinical characteristics of illness caused by Bordetella parapertussis compared with illness caused by Bordetella pertussis. Pediatr Infect Dis J 13(4): Poddar, S. K. (2003) Detection and discrimination of B. pertussis and B. holmesii by realtime PCR targeting IS481 using a beacon probe and probe-target melting analysis. Mol Cell Probes 17(2): Reischl, U., N. Lehn, G.N. Sanden, and M.J. Loeffelholz (2001) Real-time PCR assay targeting IS481 of Bordetella pertussis and molecular basis for detecting Bordetella holmesii. J Clin Microbiol 39(5): Templeton, K. E., S.A. Scheltinga, A. van der Zee, B.M. Diederen, A.M. van Kruijssen, H. Goosens, E. Kuijper, and E.C. Claas (2003) Evaluation of real-time PCR for detection of and discrimination between Bordetella pertussis, Bordetella parapertussis, and Bordetella holmesii for clinical diagnosis. J Clin Microbiol 41(9): Watanabe, M., M. Nagai (2004) Whooping cough due to Bordetella parapertussis: an unresolved problem. Expert Rev Anti Infect Ther 2(3):

27 10. Maixnerova, M. (2003) The 2001 serological survey in the Czech Republic-- parapertussis. Cent Eur J Public Health 11(Suppl): S Maixnerova, M. (2003) The 2001 serological survey in the Czech Republic pertussis. Cent Eur J Public Health 11(Suppl):S Wendelboe, A.M., A. Van Rie, S. Salmaso, and J.A. Englund (2005) Duration of immunity against pertussis after natural infection or vaccination. Pediatr Infect Dis J 24(5Suppl):S Heininger, U., K. Klich, K. Stehr, and J.D. Cherry (1997) Clinical finding in Bordetella pertussis infections: results of a prospective multicenter surveillance study. Pediatrics 100:e Schlapfer, G., J.D. Cherry, U. Heininger, M. Uberall, S. Schmitt-Grohe, S. Laussucq, M. Just, and K. Stehr (1995) Polymerase chain reaction identification of Bordetella pertussis infections in vaccines and family members in a pertussis vaccine efficacy trial in Germany. Pediatr Infect Dis J 14: de Melker, H.E., F.G. Versteegh, J.F. Schellekens, P.F. Teunis, and M. Kretzschmar (2006) The incidence of Bordetella pertussis infections estimated in the population from a combination of serological surveys. J Infect 53(2): Cherry, J. D. (1984) The epidemiology of pertussis and pertussis immunization in the United Kingdom and the United States: a comparative study. Curr Probl Pediatr 14(2):

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35 Chapter 2: Delayed Role of Tumor Necrosis Factor-α in Overcoming the Effects of Pertussis Toxin 20

36 Abstract: Bordetella pertussis causes whooping cough, an endemic respiratory disease that is increasing in prevalence despite vaccination efforts. While host immunity is modulated by virulence factors of this pathogen, it is unclear what host factors are required to overcome the effects of these virulence factors. Here, we investigated an apparent relationship between the effects of Ptx and Tumor Necrosis Factor (TNF)-α. B. pertussis grew efficiently and caused moderate pathology in wild type mice, whereas TNF-α -/- mice had higher numbers of bacteria and leukocytes in the lungs, experienced more airway resistance, and succumbed to the infection. The depletion of CD4+ T cells or neutrophils from TNF-α -/- mice limited airway resistance and delayed mortality, indicating that these cell types were contributing to the pathogenesis of the infection. Interestingly, an isogenic B. pertussis strain lacking Ptx did not induce these effects in TNF-α -/- mice and behaved similarly in wild type and TNF-α-deficient hosts. Together, these results indicate that TNF-α is essential for the host to overcome the effects of Ptx, allowing both control of B. pertussis numbers and regulation of the inflammation induced by infection. Introduction: TNF-α is a classical pro-inflammatory cytokine, inducing the production of chemokines by a variety of cell types (1-2). Endothelial cells and circulating leukocytes exhibit an upregulation of adhesion molecules upon stimulation by this cytokine (3-4) facilitating the extravasation of leukocytes from the bloodstream. TNF-α also activates these cells by inducing efficient respiratory bursts, degranulation, and bactericidal activity (5-6). This cytokine is required for host control of a wide range of respiratory pathogens including Pneumocytsis, Respiratory Syncytial Virus, Mycobacterium, Listeria, Chlamydia, Pseudomonas, and Klebsiella (7-14). 21

37 Our lab has previously shown that TNF-α induced by Toll-like receptor (TLR)4 signaling is crucial to the early response to the animal pathogen, B. bronchiseptica (15). TNF-α-deficient mice are unable to limit B. bronchiseptica numbers and pathology, leading to death within approximately 72 hours (15). TLR4 signaling also contributes to the early TNF-α response to B. pertussis but it is not essential to survival upon infection. However, a deficiency in TLR4 results in higher numbers of B. pertussis in the respiratory tract after one week, suggesting that TNF-α may be important to protection against B. pertussis later in infection (16-17). Here, we showed that TNF-α was essential for protection against an infection of the murine respiratory tract by B. pertussis. This cytokine appeared to modulate the immune response by limiting the accumulation of leukocytes in response to B. pertussis infections. Blocking neutrophil accumulation reduced airway resistance and extended the lifespan of these mice, suggesting that mortality involves neutrophil-induced damage to the lungs. TNF-α was not required to survive an infection by, or to limit the accumulation of leukocytes in response to, a mutant of B. pertussis lacking Ptx (B. pertussisδptx). Thus, TNF-α is essential only when Ptx is expressed, and appears to modulate the neutrophil response to B. pertussis. Materials and Methods: Bacterial Strains and Growth. B. pertussis strain 536 is a streptomycin-resistant derivative of Tohama I (18). BPH101 (B. pertussis Ptx) is a Ptx mutant of 536 and was a kind gift from Dr. Drusilla Burns (FDA, Rockville, Maryland, USA) (19). Both were maintained on Bordet- Gengou agar (Difco) containing 10 % defibrinated sheep blood (Hema Resources) and 20 μg/ml streptomycin. Liquid culture bacteria were grown at 37 C overnight on a roller drum to mid-log phase in Stainer-Scholte broth. 22

38 Animal experiments. C57BL/6 and TNF-α -/- mice were obtained from Jackson laboratories (Bar Harbor, Maine, USA) and bred in our Bordetella-free, specific pathogen-free breeding rooms at The Pennsylvania State University. 4-6 week old mice were lightly sedated with 5% isofluorane (Abbott laboratories) in oxygen and inoculated by pipetting 50 μl of phosphate buffered saline (PBS) containing approximately 5 x 10 5 colony forming units (CFU) of B. pertussis or B. pertussisδptx onto the tip of the external nares as previously described (18). For co-inoculation with the control or TNF-α-expressing adenovirus, 5 x 10 8 plaque forming units (PFU) of the adenovirus in 50µL of PBS (20) was pipetted onto the external nares immediately after inoculation with B. pertussis. For survival curves, once the progression of disease was clear and death was imminent, moribund animals were euthanized to prevent unnecessary suffering. Protocols were approved by the university Institutional Animal Care and Use Committee (IACUC) and all animals were handled in accordance with institutional guidelines. Bacterial quantification. Animals were sacrificed on day 0, 3, 7, 10, or 14 days postinoculation. Lungs, trachea, nasal cavity, heart, kidneys, liver, and spleen were homogenized in 1 ml of PBS and plated onto Bordet-Gengou agar containing 20 μg/ml streptomycin at appropriate dilutions to quantify CFU. Colonies were counted after incubating for 4 days at 37 o C. Lung leukocyte and cytokine quantifications. Lungs were perfused with sterile PBS, harvested, and placed in 5 ml of Dulbecco s modified Eagle medium (HyClone, Logan, Utah, USA) supplemented with 10% fetal bovine serum. Lungs and media were then pressed through Cellector tissue sieves (Bellco Glass, Inc., NJ) for homogenization. Lung homogenate was laid over Histopaque 1119 (Sigma Aldrich, MO) and centrifuged for 30 minutes at 3000 rpm at room temperature. The leukocyte layer was collected and the total number of cells was determined by 23

39 counting at 40x magnification on a hemocytometer. Numbers of individual cell types were quantified by spinning the leukocyte layer onto a glass slide, staining the isolated cells with a modified Giemsa stain (Fisher Scientific Company), and determining the percentages of lymphocytes, macrophages, and neutrophils by cell morphology. For the quantification of cytokines and chemokines, lung homogenates were examined by enzyme linked immunosorbent assays (ELISAs) [for TNF-α, Interferon (IFN)-γ, Interleukin (IL-10), IL-4, Tissue growth factor (TGF)-β, Macrophage inflammatory protein (MIP)-1α, and keratinocyte chemoattractant (KC)], which were run according the suppliers protocols (R & D Systems, Inc., Minneapolis, MN, USA). Splenocyte restimulations. Splenocytes were isolated by homogenizing spleens, spinning at 400 x gravity (g) for 5 min at 4 C, lysing the red blood cells, and washing the cells with Dulbecco's modified Eagle cell culture medium. 2 x 10 6 cells were re-suspended in Dulbecco's modified Eagle medium supplemented with 10% fetal calf serum (HyClone), 1 mm sodium pyruvate (HyClone), 100 µg/ml penicillin and streptomycin (HyClone), and 0.005% betamercaptoethanol and placed into wells of a 96-well plate. Splenocytes were stimulated with media or 10 7 heat-killed B. pertussis. After three days, supernatant was collected and analyzed for TNF-α production by ELISA. Airway resistance. Airway resistance was evaluated by whole-body plethysmography (21). Unrestrained mice were placed into a whole-body plethysmography chamber and allowed to move freely. Airway resistance was estimated by the Enhanced Respiratory Pause (Penh) index, a measurement of the phase shift of the thoracic displacement and air flow through the nares. Penh = (Te/RT -1) x PEF/PIF where Te is expiratory time, RT is relaxation time, PEF is peak expiratory flow, and PIF is peak inspiratory flow (21). 24

40 Depletions via neutralizing antibodies. Neutrophils were depleted by intraperitoneal (I.P.) injections of 1 mg of the antibody from the hybridoma RB6-8C5 (22), 24 hours prior to and 7 days post-infection. CD4 + and CD8+ T cells were depleted by I.P. injections of 1 mg of the antibody from the hybridomas GK1.5 (23-24) and YTS168 4 (25) respectively, at days 0 and 7. Mice were depleted of TNF-α by I.P. injection of 1 mg of the antibody from the hybridoma MP6-XT3 (26) at specified time points. Statistical Analysis. The mean +/- standard deviation (error bars) was determined for each group for CFU, cytokines, leukocyte numbers, or Penh values. Two-tailed, unpaired Student s T-tests were used to determine statistical significance between groups. All experiments were performed at least two times with similar results. Results: TNF-α is essential for survival following infection by B. pertussis. To test whether the production of TNFα is important to protection against B. pertussis, wild type and TNF-α -/- mice were inoculated with 5 x 10 5 CFU in 50 μl of PBS. Wild type mice inoculated with B. pertussis showed no signs of distress for at least 105 days post-inoculation (27). However, TNF-α -/- mice began to show signs of disease around two weeks postinoculation including ruffled fur, hunched posture, and unresponsiveness, and succumbed to the infection soon after morbidity became apparent (mean survival time ~ 16.4 days) (Fig. 2.1). To determine whether this defect could be compensated for by restoring TNF-α expression in the lungs, TNF-α -/- mice were co-inoculated with B. pertussis and 5 x 10 8 PFU of an adenovirus vector expressing TNF-α or a control adenovirus. This dose of the TNF-α-expressing adenovirus produces a peak of approximately 900 pg of TNF-α around day 4 post-inoculation 25

41 (28) and prevented death in all of the TNF-α -/- mice while 75% of the mice coinoculated with the control adenovirus succumbed to the infection (Fig. 2.1). Since TNF-α appeared to be protective, its production in response to B. pertussis was assessed by monitoring splenocytes from B. pertussis-infected mice for cytokine production. Splenocytes from infected mice produced low levels of TNF-α upon exposure to media alone (Fig. 2.2). Splenocytes from mice that had been Figure 2.1: Survival curve of wild type versus TNF-α-/- mice infected with B. pertussis. Groups of eight C57BL/6 and 12 TNF-α -/- mice were inoculated with 5 x 10 5 CFU of B. pertussis in 50 μl of PBS. TNF-α -/- mice were untreated or co-infected with 5 x 10 8 PFU of control adenovirus or TNF-αexpressing adenovirus. Survival is represented as the percent living on the indicated day postinoculation. J Infect Dis 196(8): infected for three days did not produce significantly more TNF-α upon stimulation by B. pertussis, but stimulation did result in increased TNF-α production by splenocytes from mice that were infected for 7 (900 pg) and 14 (300 pg) days (Fig. 2.2). These data indicate that TNF-α is produced in response to B. pertussis, peaking after one week of infection, and is protective against this pathogen. The fact that TNF-α was not Figure 2.2: Production of TNF-α in response to B. pertussis. Splenocytes were collected from B. pertussis-infected mice at day 3, 7, or 14 postinoculation and exposed to media or heat-killed B. pertussis (Bp) for 3 days. Levels of TNF-α in the supernatant were quantified by ELISA and are expressed as the mean +/- standard deviation. Asterisks denote P-values < J Infect Dis 196(8):

42 Figure 2.3: Quantification of bacteria and leukocytes in the lungs of wild type and TNF-α - /- mice. 20 C57BL/6 and 20 TNF-α -/- mice were inoculated with B. pertussis and groups of four of each mouse strain were sacrificed on days 0, 3, 7, 10, and 14 post-inoculation. (A) Numbers of bacteria are expressed as Log 10 mean. Lymphocytes (B), macrophages (C), and neutrophils (D) were also quantified in the lungs of these mice. Cell counts are represented as the mean. Error bars represent the standard deviations and dashed lines represent the lower limit of detection. One asterisk denotes P-values < 0.05 and two asterisks denote P-values < J Infect Dis 196(8): significantly induced until day 7 suggests that some adaptive immune cell population, most likely T cells, is producing this cytokine. Reducing B. pertussis numbers and limiting leukocyte accumulation in the murine lung require TNF-α. To determine if TNF-α is important to limiting bacterial loads in the respiratory tract or systemic spread of the infection, bacterial numbers were quantified in the lungs, trachea, nasal cavity, spleen, liver, heart, kidneys, and blood of wild type and TNF-α -/- mice. Mice were inoculated with B. pertussis and sacrificed on days 0, 3, 7, 10, or 14. No differences in bacterial numbers were observed between C57BL/6 and TNF-α -/- mice up to 7 days post-inoculation. By day 10, B. pertussis numbers began to decline in wild type mice and by day 14, B. pertussisspecific antibodies were detectable (data not shown) and bacterial numbers were reduced to 27

43 approximately 10 4 CFU in the lungs. However, B. pertussis remained at high numbers in the lungs of TNF-α -/- mice (~ 5 x 10 6 CFU) at day 14 post-inoculation (Fig. 2.3A). Similar trends were observed in the tracheae and nasal cavities (data not shown). Additionally, depleting TNF-α from wild type mice led to an inability to reduce bacterial numbers in the lungs (Fig. 2.4). Bacteria were not detectable in the spleen, liver, heart, kidneys, or blood of wild type or TNF-α -/- mice, indicating that B. pertussis had not spread systemically by day 14 postinoculation (data not shown). The failure of TNF-α -/- mice to reduce numbers of B. Figure 2.4: Effect of depleting TNF-α on B. pertussis numbers in the lungs. Groups of 4 C57BL/6 mice were inoculated with B. pertussis and depleted of TNF-α 14 and 21 days later. Mice were sacrificed on day 14, 21, or 28 postinoculation to quantify bacterial numbers in the lungs. Bacterial numbers are expressed as the Log 10 mean +/- standard deviation (error bars). The dashed line represents the lower limit of detection. Asterisks denote P-values < pertussis provides a possible explanation for death in these animals. However, B. pertussis reaches approximately 10 7 CFU in the lungs of other immunodeficient mouse strains and persists at high levels for at least 105 days without causing symptomatic disease (27, 29). Thus, we speculated that mortality associated with B. pertussis in the absence of TNF-α was not directly due to high bacterial numbers. An alternative explanation for the lethality of B. pertussis infections in TNF-α -/- mice may be that TNF-α regulates the inflammatory response to this bacterium. Therefore, numbers and types of leukocytes in the lungs of wild type and TNF-α-deficient mice were quantified. 28

44 There were no significant differences between wild type and TNF-α -/- mice in the numbers of lymphocytes in the lungs throughout the infection (Fig. 2.3B). No significant differences were observed between wild type mice and those lacking TNF-α in the accumulation of macrophages or neutrophils in the lungs up to 7 days post-inoculation (Fig. 2.3C-D). However on day 14, macrophage and neutrophil numbers were low in the lungs of wild type mice (3 x 10 5 and 1 x 10 5 respectively) while the numbers in the lungs increased dramatically in TNF-α -/- mice (3 x 10 6 and 5 x 10 6 respectively) (Fig. 2.3C-D). Despite the differences in leukocyte numbers, many classical pro- and anti-inflammatory cytokines and chemokines (IFN-γ, IL-4, IL-10, IL-1β, MIP- 1α, MIP-2, and KC) were produced at similar levels in wild type and TNF-α -/- mice (Fig. 2.5). Although, TNF-α may play an important role in the activation of leukocytes in response to B. pertussis infection, as measured by the production of reactive oxygen species or nitric oxide. Together, these data suggest that there may be a defect in the resolution of inflammation, rather than an increase in the recruitment of leukocytes. 29

45 Figure 2.5: Cytokine responses to B. pertussis in the murine lungs. 20 C57BL/6 and TNF-α -/- mice were inoculated with B. pertussis and groups of 4 were sacrificed on days 0, 3, 7, 10, or 14 post-inoculation. Lungs were excised and homogenized and levels of (A) IFN-γ, (B) MIP-1α, (C) IL-4, (D) MIP-2, (E) IL-10, (F) KC, and (G) IL-1β were quantified. Cytokine levels are expressed as the mean pg/ml of lung homogenate or the mean absorbance +/- standard deviation (error bars). TNF-α -/- mice experience increased airway resistance in response to infection by B. pertussis. It was observed that B. pertussis-infected TNF-α -/- mice appeared to be having difficulty breathing after about two weeks post-inoculation. Whole body plethysmography was performed 30

46 as a measurement of airway constriction. C57BL/6 and TNF-α -/- mice were analyzed by unrestrained plethysmography prior to infection and on days 8 and/or 13 post-inoculation. On day 13 postinoculation, B. pertussis-infected wild type Figure 2.6: Measurement of airway constriction by mice showed similar levels of airway whole body plethysmography. Groups of 3 C57BL/6 and TNF-α -/- mice were inoculated with B. constriction to those of uninfected mice; pertussis. Penh values were determined by unrestrained plethysmography on the indicated day post-inoculation. Penh values are denoted for each Penh values were 0.61 and 0.68 individual mouse with the horizontal bars representing the means. Asterisks denote P-values respectively (Fig. 2.6). TNF-α -/- mice < 0.01 when compared to wild type mice infected for 13 days. J Infect Dis 196(8): infected with B. pertussis for 8 days experienced Penh values that were apparently higher (1.012) than uninfected mice, but this difference was not statistically significant. On day 13 postinoculation, these mice experienced high levels of airway constriction (Penh value of ~ 3.1) compared to infected wild type mice (Fig. 2.6). Thus, B. pertussis-induced lethality observed in the absence of TNF-α is associated with increased airway constriction. Depleting neutrophils or CD4+ T cells extends the lifespan of B. pertussis-infected TNF-α -/- mice. Since lethality correlated with elevated numbers of leukocytes in the lungs of B. pertussis-infected TNF-α -/- mice, we tested the effects of depleting certain leukocyte populations on survival. In TNF-α -/- mice, a small rise in lymphocyte infiltration at day 10 was followed by high numbers of neutrophils in the lungs on day 14 post-inoculation (Fig. 2.3B,D). To examine the contributions of these cell types to the lethality of B. pertussis in the absence of TNF-α, we 31

47 depleted CD4+ T cells, CD8+ T cells, or neutrophils prior to B. pertussis infection. Mean survival times for untreated and CD8+ T cell-depleted TNF-α -/- mice were similar (95% confidence intervals of and days respectively). However, CD4+ T cell-depleted and neutrophil-depleted TNF-α -/- mice infected with B. pertussis survived significantly longer (95% confidence intervals of days and days respectively) (Fig. 2.7). Thus, CD4+ T cells and neutrophils appear to Figure 2.7: Survival curve of B. pertussis-infected TNF-α -/- mice depleted of CD4+ T cells, CD8+ T cells, or neutrophils. Groups of eight C57BL/6 and eight TNF-α -/- mice were inoculated with B. pertussis. CD8+ T cells, CD4+ T cells, or neutrophils were depleted at days 0, 7, and 14 post-inoculation. As controls, groups of eight C57BL6 and TNF-α -/- mice were left untreated. Survival curves are presented as the percent living on the indicated day postinoculation. Asterisks denote P-values < 0.01 when compared to the mean survival time of untreated B. pertussis-infected TNF-α -/- mice. J Infect Dis 196(8): contribute to the mortality of B. pertussis infected TNF-α -/- mice. Limiting neutrophil accumulation in the lungs does not affect bacterial colonization but does decrease airway constriction. Due to the positive effects of depleting CD4+ T cells or neutrophils from B. pertussisinfected mice, the effects of these cells on bacterial colonization were assessed. C57BL/6 and TNF-α -/- mice were inoculated with B. pertussis and left untreated or depleted of CD4+ T cells or neutrophils. Untreated wild type mice harbored approximately 10 5 CFU in the lungs on day 14 post-inoculation, but mice depleted of CD4+ T cells or neutrophils harbored approximately and 10 7 CFU respectively (Fig. 2.8A). On day 14 post-inoculation, CFU were similar in 32

48 untreated, CD4+ T cell-depleted, and neutrophil-depleted TNF-α -/- mice (~10 7 CFU) (Fig. 2.8A), indicating that these cells had no measurable effect on bacterial numbers in the absence of TNFα. These data suggest that CD4+ T cells and neutrophils are unable to aid the clearance of B. pertussis in the absence of TNF-α and that the extended lifespan of CD4+ T cell- or neutrophildepleted TNF-α -/- mice infected with B. pertussis was not due to reduced bacterial numbers. 33

49 Figure 2.8: Effects of depleting CD4+ T cells or neutrophils on bacterial numbers, leukocyte accumulation, and airway constriction. Groups of 12 C57BL/6 and TNF-α -/- mice were inoculated with B. pertussis and lungs were excised for the quantification of (A) bacteria, (B) neutrophils, (C) macrophages, and (D) lymphocytes on day 14 post-inoculation. One group was left untreated, one was depleted of CD4+ T cells, and one was depleted of neutrophils. CFU are represented as the Log 10 mean +/- standard deviation (error bars) with the dashed line representing the lower limit of detection and cell counts are represented as the mean +/- standard deviation (error bars). (E) Groups of 3 TNF-α -/- mice were inoculated with B. pertussis and were left untreated, depleted of CD4+ T cells, or depleted of neutrophils. Penh values were determined by whole body plethysmography at 13 days post-inoculation. Penh values are denoted for each individual mouse with the horizontal bars representing the means. One asterisk denotes P-values < 0.05 and two asterisks denote P-values < J Infect Dis 196(8): To investigate the contribution of neutrophils and CD4+ T cells to the inflammatory response of B. pertussis-infected TNF-α -/- mice, the effects of cell depletions on the leukocyte populations in the lungs and airway resistance were analyzed. While high numbers of neutrophils were observed in untreated B. pertussis-infected TNF-α -/- mice (~5 x 10 6 ), depletion of neutrophils resulted in very low numbers of these cells (<1 x 10 5 cells) on day 14 34

50 but did not significantly affect the recruitment of macrophages or lymphocytes (Fig. 2.8B-D). Depletion of CD4+ T cells decreased the numbers of lymphocytes in the lungs by day 14 but also significantly lowered the numbers of neutrophils (~1 x 10 5 cells) (Fig. 2.8B-D). Additionally, depleting CD4+ T cells or neutrophils resulted in large decreases in airway constriction, from Penh values of ~2.7 to 1.1 and 0.95 respectively (Fig. 2.8E). These data suggest that increased airway constriction and mortality may be due to elevated numbers of neutrophils. Ptx is necessary for B. pertussis-induced mortality of TNF-α -/- mice. Despite the high numbers of neutrophils in the lungs of B. pertussis-infected TNF-α -/- mice (Fig. 2.3D), these cells were ineffective at reducing bacterial numbers (Fig. 2.3A). Since Ptx is known to block the function of neutrophils (30-31), we tested the effect of Ptx on the outcome of B. pertussis infection in TNF-α -/- mice. C57BL/6 and TNF-α -/- mice were inoculated with an isogenic strain of B. pertussis lacking Ptx (B. pertussisδptx) and monitored for survival. While wild type B. pertussis kills TNF-α -/- mice, these mice do not succumb to infections by B. pertussisδptx (Fig. 2.9) suggesting that TNF-α is required only when Ptx is expressed. In support of this, B. parapertussis, which causes the same disease as B. pertussis but does not express Ptx, does not cause lethal disease in TNF-α -/- mice and is controlled in these mice similar to wild type mice (data not shown). 35

51 Elevated numbers of B. pertussis and leukocytes are dependent on Ptx. Since infections of TNF-α -/- mice by B. pertussisδptx were not lethal, the ability of these mice to control bacterial numbers and inflammation in response to this strain was examined. Although Ptx inhibits early chemotaxis, it also stimulates a potent Th1-type immune response potentially leading to increased inflammation later in infection (32-33). C57BL/6 and TNF-α -/- mice were inoculated with B. pertussisδptx and Figure 2.9: Survival of TNF-α -/- mice infected with a Ptx-deficient strain of B. pertussis. Groups of 8 C57BL/6 and TNF-α -/- mice were inoculated with B. pertussis or B. pertussisδptx and monitored for survival. Survival curves are presented as the percent living on the indicated day post-inoculation. J Infect Dis 196(8): sacrificed on day 7 or 14 to determine if the accumulation of neutrophils in the absence of TNFα was dependent on Ptx. C57BL/6 and TNF-α -/- mice were both able to reduce B. pertussisδptx numbers to approximately CFU in the lungs by day 14 post-inoculation (Fig. 2.10A). Additionally, there were no significant differences in leukocyte numbers between wild type and TNF-α -/- mice infected with B. pertussisδptx (2.10B-D). Thus, TNF-α is important only when Ptx is expressed and appears to overcome the effects of Ptx, limiting B. pertussis colonization and preventing excessive accumulation of leukocytes in the lungs. 36

52 Figure 2.10: Quantification of bacterial numbers and leukocytes in the lungs of wild type and TNF-α-deficient mice infected with B. pertussisδptx. Groups of 8 C57BL/6 and TNF-α -/- mice were inoculated with B. pertussisδptx. Groups of 4 mice were sacrificed on day 7 or 14 post- and (D) inoculation for the quantification of (A) bacterial numbers, (B) leukocytes, (C) macrophages, neutrophils in the lungs. CFU are represented as the Log10 mean +/- standard deviation (error bars) with the dashed line representing the lower limit of detection and cell counts are represented as the mean +/- standard deviation (error bars). J Infect Dis 196(8): Discussion: The current study identifies defective TNF-α responses as a potential risk factor for B. pertussis disease. TNF-α -/- mice are unable to reduce B. pertussis numbers, limit leukocyte accumulation in the lungs, or survive the infection. Since TNF-α had no measurable effect during the first week of infection and TNF-α responses peaked after one week, it is likely that the later production of this cytokine is crucial to protection. The lifespan of B. pertussis-infected TNF-α -/- mice was extended by the inhibition of cellular influx to the lungs by depletions of CD4+ T cells or neutrophils, both of which limited neutrophil accumulation and airway constriction without affecting bacterial numbers. B. pertussisδptx numbers were reduced in the respiratory tract without TNF-α, and this cytokine was not essential to regulating the 37

53 inflammatory response to this strain. Thus, TNF-α is required only when Ptx is expressed, and appears to overcome some of the Ptx-mediated effects of B. pertussis infection. The role of TNF-α in counteracting the effects of Ptx was examined in part because of the apparent Ptx-dependent influx of leukocytes after the first few days of infection (34-35). Ptx also affects the function of neutrophils by inhibiting phospholipase C stimulation, lysosomal enzyme secretion, and the rise of intracellular calcium levels upon chemokine stimulation (30-31). Therefore, it is likely that Ptx decreases the ability of neutrophils to phagocytose and kill B. pertussis. Activation by TNF-α may be required to overcome inhibitory effects of Ptx on neutrophils, perhaps by inducing signaling molecules that act on Ptx-insensitive receptors and activate leukocytes for more efficient phagocytosis. B. pertussis infections in the absence of TNF-α appear to become fatal because of increased airway constriction which correlates with large numbers of leukocytes in the lungs. Despite the large number of neutrophils in the lungs of B. pertussis-infected TNF-α -/- mice after 14 days post-inoculation (Fig. 2.3D), these cells are ineffective at reducing bacterial numbers (Fig. 2.3A). Diminished TNF-α correlates with impaired phagocytosis in several models (13,36-37), and this cytokine has been shown to facilitate the phagocytosis of B. pertussis in vitro (38). Further more, it has been suggested that upon phagocytosis of bacteria or bacterial products, altered transcriptional regulation and/or proapoptotic effects of reactive oxygen species cause apoptosis of tissue neutrophils (39-41). These apoptotic neutrophils are subsequently phagocytosed by macrophages (42), resolving the inflammatory response. Inefficient phagocytosis of B. pertussis by TNF-α-deficient neutrophils, combined with the large number of these cells recruited to the lungs, could result in the observed cellular accumulation and airway resistance. It remains unclear if macrophages play an important role in protection against B. 38

54 pertussis, but in this model, TNF-α may be negatively regulating inflammation by facilitating the phagocytosis of B. pertussis by, and consequently the apoptosis of, neutrophils. The extensive inflammation of B. pertussis-infected lungs in the absence of TNF-α could also be more directly mediated by Ptx. This toxin significantly impacts the T cell response to B. pertussis infection by activating CD4+ T cells independent of MHC-T cell receptor interactions (32) and skewing them towards a Th1 phenotype and IFN-γ production (32-33). While Ptx inhibits the early migration of neutrophils to the lungs (30-31), the stimulation of proinflammatory Th1 responses may result in leukocyte accumulation at later time points. Ineffective phagocytosis, apoptosis, and subsequent anti-inflammatory signals may occur in the absence of TNF-α. This is consistent with the fact that the depletion of CD4+ T cells, as well as neutrophils, was protective in B. pertussis-infected TNF-α-deficient mice. Thus, TNF-α limits the accumulation of leukocytes in response to B. pertussis infection which may be mediated in part by the effects of Ptx. This study highlights an interesting relationship between TNF-α and a specific bacterial virulence factor, Ptx. Importantly, patients that are infected with B. pertussis often suffer from secondary infections by other pathogens. Other respiratory pathogens, such as Pseudomonas aeruginosa, inhibit TNF-α signaling (43-44), which could enhance the severity of B. pertussis infections. The observations that eliminating B. pertussis and resolving the inflammation that is induced by this pathogen depend on TNF-α add to our understanding of the pathogenesis and resolution of B. pertussis infections and may contribute to intervention strategies. 39

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63 Chapter 3: Interferon-γ Production by T Cells Overcomes the Inhibitory Effects of Pertussis Toxin on Bacterial Clearance 48

64 Abstract: Ptx is a virulence factor that is uniquely expressed by Bordetella pertussis, one of the causative agents of whooping cough. This toxin prevents B. pertussis-specific antibodies from eliminating this pathogen from the respiratory tract during the first week of infection by inhibiting the migration of leukocytes to the site of infection. However, the inhibition of antibody-mediated clearance by Ptx is ultimately overcome and B. pertussis is cleared from its host. Here, we used a mouse model of infection to address the host factor(s) that are required to overcome the effects of Ptx on bacterial clearance. In response to B. pertussis, T cells produced large amounts of IFN-γ. Both T cells and IFN-γ were crucial to the function of antibodies against B. pertussis during the second week of infection; more specifically, CD4+ αβ T cells were required for the antibody-mediated elimination of this pathogen. Since Ptx delays bacterial clearance by preventing the migration of leukocytes to the lungs, we hypothesized that IFN-γ is important to the recruitment of these cells. In support of this, IFN-γ contributed to the accumulation of leukocytes in the lungs of B. pertussis-infected mice. Together, these data suggest that T cells and IFN-γ overcome the inhibitory effect of Ptx on leukocyte migration, enabling the elimination of B. pertussis from the respiratory tract. Introduction: Current pertussis vaccines induce a robust serum antibody response (1), but the T cell response is dependent on the type of vaccine being used. Whole cell vaccines induce a balanced Th1/Th2 response, but acellular vaccines, which are now predominantly used in developed countries, induce a Th2-skewed response (2-4). Despite the breadth of research that has been dedicated to whooping cough, the relative importance of antibodies versus T cells in protection against B. pertussis remains somewhat controversial (5). 49

65 We and others have shown that B cell-deficient mice are unable to clear B. pertussis from the respiratory tract (6-8), suggesting that antibodies are important to bacterial clearance. Likewise, T cells are essential to protective immunity against B. pertussis (7). However, a passive transfer of serum antibodies from either vaccinated or convalescent animals did not confer protection against B. pertussis for at least 7 days post-inoculation, leading multiple groups to conclude that serum antibodies are not important to bacterial clearance (6-7). We have recently observed that serum antibodies clear B. pertussis from the murine respiratory tract between 7 and 14 days post-inoculation via a mechanism that involves Fcγ receptors and neutrophils (9). Ptx of B. pertussis delays antibody-mediated clearance by preventing the recruitment of neutrophils to the lungs (9). While studies have shed light on how B. pertussis avoids antibody-mediated clearance, it is not clear how the host immune response ultimately overcomes the inhibition of bacterial clearance by Ptx. Interestingly, the time period when adoptively transferred serum antibodies begin to affect bacterial numbers correlates with the time when T cell responses to B. pertussis become detectable (10). IFN-γ is produced in large amounts by T cells and prevents lethal infection by this pathogen (11-12). This study investigates the role of T cells and IFN-γ in the antibody-mediated clearance of B. pertussis from the murine respiratory tract. Our data show that CD4+ αβ T cells are essential to the clearance of B. pertussis, even when convalescent phase serum is passively transferred. Upon investigating the role of T cells in the clearance of this pathogen, it was found that these cells produced large amounts of IFN-γ, the timing of which correlated with the reduction of bacterial numbers. IFN-γ was required to overcome the inhibitory effects of Ptx on the migration of leukocytes to the lungs and the antibody-mediated clearance of B. pertussis. 50

66 Together, these data indicate that vaccine strategies aimed at inducing a Th1 type immune response may be more effective against B. pertussis. Materials and Methods: Bacterial Strains and Growth. B. pertussis strain 536 is a streptomycin-resistant derivative of Tohama I (13). Bacteria were maintained on Bordet-Gengou agar (Difco) containing 10 % defibrinated sheep blood (Hema Resources) and 20 μg/ml streptomycin. Liquid culture bacteria were grown at 37 C overnight on a roller drum to mid-log phase in Stainer-Scholte broth. Animal experiments. C57BL/6, RAG2 -/-, μmt, TCRαδ -/-, TCRα -/-, TCRδ -/-, and IFN-γ -/- mice were obtained from Jackson laboratories (Bar Harbor, Maine, USA) and bred in our Bordetella- The Pennsylvania State University. 4-6 week old free, specific pathogen-free breeding rooms at mice were lightly sedated with 5% isofluorane (Abbott laboratories) in oxygen and inoculated by pipetting 50 μl of PBS containing approximately 5 x 10 5 CFU of B. pertussis or B. pertussisδptx onto the tip of the external nares as previously described (13). All protocols were approved by the university IACUC and all animals were handled in accordance with institutional guidelines. Bacteria and leukocyte quantification. Animals were sacrificed on day 0, 3, 7, 14 or 28 post- in 1 ml of PBS and plated onto Bordet-Gengou agar inoculation. Lungs were homogenized containing 20 μg/ml streptomycin at appropriate dilutions to quantify CFU. Plates were incubated at 37 o C for four days prior to counting. To quantify leukocytes in the bronchoalveolar lavage (BAL) fluid, lungs were inflated with PBS + 2% fetal bovine serum twice and the liquid was extracted and placed in ice. Cells were pelletted by centrifugation for 5 minutes at 4 o C, 400 x g. Red blood cells were lysed by adding 2 ml of 0.83% ammonium chloride solution to the pellet for 2 minutes at room temperature, adding 8 ml of RPMI to neutralize the reaction, pelleting the cells by spinning as above, and resuspending in 1 ml of PBS + 2% fetal bovine 51

67 serum. The total number of leukocytes was determined by counting at 40x magnification on a hemocytometer. Splenocyte restimulations. Splenocytes were isolated by homogenizing spleens, spinning at 400 x g for 5 min at 4 C, lysing the red blood cells as above, and washing the cells with Dulbecco's modified Eagle cell culture medium. 2 x 10 6 cells were re-suspended in Dulbecco's modified Eagle medium supplemented with 10% fetal calf serum (HyClone), 1 mm sodium pyruvate (HyClone), 100 µg/ml penicillin and streptomycin (HyClone), and 0.005% betamercaptoethanol and placed into wells of a 96-well plate. Splenocytes were stimulated with media or 10 7 CFU of heat-killed B. pertussis. After three days, supernatant was collected and analyzed for IFN-γ production by ELISA according to the manufacturer s protocol (R & D Systems, Inc., Minneapolis, MN, USA). Depletions via neutralizing antibodies. CD4 + and CD8+ T cells were depleted by I.P. injections of 1 mg of the antibody from the hybridomas GK1.5 (14) and YTS168 4 (15) respectively, at days 0 and 7. IFN-γ was depleted by I.P. injection of 1 mg of the antibody from the hybridoma XMG1.2 (16) Statistical Analysis. The mean +/- standard deviation (error bars) was determined for each group for CFU, leukocyte numbers, or Penh values. Two-tailed, unpaired Student s T-tests were used to determine statistical significance between groups. All experiments were performed at least two times with similar results. 52

68 Results: CD4+ αβ T cells are essential to the antibody-mediated clearance of B. pertussis. Previous studies have shown that B cells are required for the elimination of B. pertussis from the murine respiratory tract (8). Passive transfer of serum antibodies from convalescent mice is sufficient to eliminate B. pertussis from B cell-deficient mice, but these antibodies have no measurable effect on bacterial numbers until after the first week of infection (8-9), correlating with the generation of a T cell response to B. pertussis (10). This led us to speculate that T cells may mediate the function of B. pertussis-specific antibodies. C57BL/6 and TCRαδ -/- mice were inoculated with our standard dose of B. pertussis (5 x 10 5 CFU in 50 μl of PBS) and sacrificed on day 0, 3, 7, 14, or 28 postinoculation for the quantification of bacteria in the lungs. In wild type mice, bacterial numbers rose initially, peaking at ~ CFU between days 3-7 post-inoculation, but were then slowly reduced to less than 1000 CFU by day 28 (Fig. 3.1A). In contrast, bacterial numbers were not reduced in T cell-deficient mice, remaining at ~ 10 7 CFU for at least 28 days (Fig. 3.1A). To determine if T cells were necessary for B. pertussis-specific antibodies to clear the Figure 3.1. Effect of T cells on the clearance of B. pertussis from the lungs. (A) C57BL/6 and TCRαδ -/- mice were inoculated with B. pertussis and sacrificed on day 0, 3, 7, 14, or 28 post-inoculation to quantify CFU in the lungs. (B) C57BL/6 and TCRαδ -/- mice were inoculated with B. pertussis, given I.P. injections of naïve or immune serum and sacrificed 14 days later. Bacterial numbers are expressed as the Log 10 mean +/- standard deviation. Asterisks represent P-values <

69 infection, C57BL/6 and TCRαδ -/- mice were inoculated with B. pertussis and given an I.P. injection of 200 μl of naïve serum or immune serum. While the adoptive transfer of antibodies cleared the infection from the lungs of wild type mice within two weeks, antibodies had no effect in T cell-deficient mice (Fig. 3.1B) suggesting that these cells were required for the function of antibodies against B. pertussis. We further investigated specific T cell subsets that were required for antibody-mediated clearance of B. pertussis. Groups of four RAG2 -/-, μmt, TCRαδ /, TCRα /, and TCRδ / mice were inoculated with B. pertussis, given a passive transfer of convalescent serum, and sacrificed 14 days later for the enumeration of bacteria in the lungs. Like wild type mice, μmt mice were able to reduce the infection to nearly undetectable levels by day 14 postinoculation, but RAG2 -/- and TCRαδ / were unable to do so (Fig. 3.2A) indicating that T cells are required for the function of antibodies against B. pertussis. Additionally, a passive transfer of immune serum resulted in bacteria being reduced to undetectable levels in the lungs of TCRδ /, but not TCRα / mice Figure 3.2. Role of T cell subsets in the antibody-mediated clearance of B. pertussis. (A) RAG2 -/-, μmt, TCRαδ -/-, TCRα -/-, and TCRδ -/- mice were inoculated with B. pertussis. (B) C57BL/6 mice were inoculated with B. pertussis and given I.P. injections of PBS, anti-cd4, or anti- CD8 antibodies. All mice were given I.P. injections of naïve or immune serum and sacrificed 14 days post-inoculation. Bacterial numbers are represented as the Log10 mean +/- standard deviation. Asterisks represent P-values <

70 (Fig. 3.2A) indicating that αβ T cells are crucial to the function of B. pertussis-specific antibodies. C57BL/6 mice were inoculated with B. pertussis and given an I.P. injection of PBS, anti-cd8 antibodies (15), or anti-cd4 antibodies (14) on days 0 and 7 post-inoculation. All mice were also given an I.P. injection of naïve or immune serum at the time of inoculation and sacrificed 14 days later. Treatment with immune serum resulted in elimination of B. pertussis from the lungs of mice injected with PBS or anti-cd8 antibodies, but bacterial numbers were not reduced in the lungs of mice injected with anti-cd4 antibodies (Fig. 3.2B). These data indicate that CD4+ αβ T cells are essential to the antibody-mediated clearance of B. pertussis. IFN-γ production by CD4+ T cells is essential to the function of B. pertussis-specific antibodies. Previous studies have shown that Th1 type immune responses are protective against B. pertussis (3) and IFN-γ contributes to intracellular killing of B. pertussis (12). In light of these findings, we hypothesized that the primary role of T cells in the antibody-mediated clearance of B. pertussis was IFN-γ production. Groups of four C57BL/6 and TCRαδ -/- mice were infected with B. pertussis and sacrificed on day 0, 3, 7, 14, or 28 post-inoculation. Splenocytes were isolated from these mice and IFN-γ production by these cells was measured after exposure to heat-killed B. pertussis. There was a peak of IFN-γ production (~ 6000 pg) by splenocytes from -/- C57BL/6 mice, but not TCRαδ mice, that occurred on day 7 post-inoculation (Fig. 3.3), the time when antibodies begin to affect B. pertussis numbers in the respiratory tract. IFN-γ production decreased by day 14 but rebounded by day 28 post-inoculation. 55

71 We then tested the role of IFN-γ in the antibody-mediated clearance of B. pertussis. Groups of C57BL/6 and IFN-γ -/- mice were inoculated with B. pertussis, given I.P. injections of naïve or immune serum, and sacrificed 14 days later. Bacteria were reduced to nearly undetectable levels in the Figure 3.3. IFN-γ production by splenocytes from B. pertussis-infected mice. C57BL/6 and TCRαδ -/- mice were inoculated with B. pertussis and sacrificed 0, 3, 7, 14, or 28 days later. Splenocytes were exposed to media alone (unstimulated) or heat-killed B. pertussis (Bp stimulated). IFN-γ in the supernatant was quantified and expressed as the mean +/- standard deviation. Asterisks denote P-values < IFN-γ responses overcome the inhibition of leukocyte migration by Ptx. Ptx inhibits the antibody-mediated clearance of B. pertussis by preventing the migration of leukocytes to the site of infection. If IFN-γ facilitated the antibodymediated clearance of B. pertussis by counteracting this effect, it would be expected that this cytokine would contribute lungs of wild type mice treated with immune serum, but no reduction in numbers was observed in the lungs of similarly treated IFNγ -/- mice (Fig. 3.4). Thus, IFN-γ is essential to the antibody-mediated clearance of B. pertussis. to the accumulation of leukocytes in the lungs. C57BL/6 and IFN-γ -/- mice were Figure 3.4. Role of IFN-γ in the antibody-mediated clearance of B. pertussis. C57BL/6 and IFN-γ -/- mice were inoculated with B. pertussis and given I.P. injections of naïve or immune serum and sacrificed 14 days later. Bacterial numbers in the lungs are expressed as the Log 10 mean CFU +/- standard deviation. Asterisks denote P-values <

72 inoculated with B. pertussis and sacrificed one week later to quantify leukocytes in the BAL fluid. This time point was chosen because B. pertussis induces a peak of leukocyte accumulation in the lungs of wild type mice at this time (9,17). Approximately 8.5 x 10 5 leukocytes were found in the BAL fluid of wild type mice, while only about 3 x 10 5 leukocytes were Figure 3.5: Effect of IFN-γ on leukocyte accumulation in the lungs during B. pertussis infection. C57BL/6 and IFN-γ -/- mice were inoculated with B. pertussis and sacrificed 7 days later to quantify leukocytes in the BAL fluid. Leukocyte numbers are expressed as the mean +/- standard deviation. found in the BAL fluid of IFN-γ-deficient mice (Fig. 3.5). These data suggest that IFN-γ is essential to counteract the inhibitory effect of Ptx on leukocyte migration to the lungs. In addition to its role in recruiting leukocytes to the s ite of infection, IFN-γ has been implicated in the activation of leukocytes for phagocytosis. Therefore, we measured the myeloperoxidase activity in the lungs of wild type versus IFN-γ-deficient mice. The lungs of wild type mice had more myeloperoxidase activity (~ 950 U/mL) than those of IFN-γ -/- mice (~ 700 U/mL) (Fig. 3.6). However, the difference in myeloperoxidase activity may just be a reflection of the difference in leukocyte numbers in the lungs of wild type versus IFN-γ -/- mice at this time. Together, these data show that IFN-γ contributes to the recruitment of leukocytes to the lungs upon B. pertussis challenge. Discussion: T cells are vital to the ability of antibodies to mediate the elimination of B. pertussis from its host. In a murine model of infection, this role of T cells is highlighted by the rapid 57

73 clearance of B. pertussis from convalescent hosts (18) relative to the much slower clearance observed upon adoptive transfer of B. pertussis-specific antibodies to naïve mice. Specifically, CD4+ αβ T cells are required for the function of antibodies. In response to B. pertussis, these cells produce large amounts of IFN-γ, which is also crucial to antibody-mediated protection. Ptx delays Figure 3.6. Effect of IFN-γ on myeloperoxidase antibody-mediated clearance by inhibiting the activity in response to B. pertussis. C57BL/6 -/- and IFN-γ mice were inoculated with B. migration of leukocytes to the lungs (9), but pertussis and sacrificed 7 days later to quantify myeloperoxidase activity in the lungs. IFN-γ counteracts this effect, contributing to Myeloperoxidase activity is expressed as the mean +/- standard deviation. the migration of leukocytes to the lungs and the elimination of B. pertussis. Ptx inhibits the migration of leukocytes to the lungs in response to B. pertussis infection (9,17). This toxin is a potent inhibitor of a subset of G-proteins that are common to the signaling pathways of several chemokine receptors (19). Chemokine receptors can be grouped into two classes; Ptx-sensitive and Ptx-resistant (20). Chemokines acting on Ptx-resistant receptors are likely to be responsible for the migration of leukocytes into the lungs that is observed after the initial inhibition of leukocyte migration by Ptx. IFN-γ is not a direct chemotactic source for leukocytes, but this cytokine upregulates the expression of a variety of chemokines including CCL5, CXCL9, CXCL10, and CXCL11 (21-22). CXCL9, 10, and 11 bind to CXCR3 and induce chemotactic effects that are insensitive to 58

74 Ptx (23). The IFN-γ-dependent upregulation of certain chemokines may be the key to counteracting the inhibitory effect of Ptx on the chemotaxis of leukocytes. This could potentially enable more T cells to migrate to the site of infection and produce more IFN-γ and lead to the recruitment of more neutrophils, which are essential to bacterial clearance (9). In addition to its effects on the migration of leukocytes, Ptx inhibits the functions of leukocytes in a variety of ways which could hinder phagocytic capabilities. Both phospholipase C stimulation and lysosomal enzyme secretion are inhibited by Ptx (24-25). Due to its effects on a subset of G-proteins, this toxin also renders cells defective in the downstream effects of stimulation by certain chemokines. While IFN-γ certainly contributes to the recruitment of leukocytes to the site of infection (26), this cytokine may also play a vital role in activating these cells. IFN-γ stimulation results in more efficient phagocytosis by macrophages (27-29). However, its importance in activating leukocytes to phagocytose B. pertussis remains unclear (12,30). In vaccinated human populations, the IFN-γ response to B. pertussis can largely be shaped by the vaccine that is administered. While natural infection or whole cell B. pertussis vaccines induce a balanced Th1/Th2 response, the response induced by acellular vaccines is Th2- skewed (2-4). These acellular vaccines have largely replaced the whole cell vaccines in developed countries due to concerns about the reactogenicity of the whole cell preparations. However, it has been suggested that acellular vaccines are less effective than whole cell vaccines at reducing the circulation of B. pertussis (31). Designing the next generation of vaccines to induce more of a Th1-type response could help curb the ongoing resurgence of whooping cough (32-36). 59

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77 15. Rao, M., M. Bray, C.R. Alving, P. Jahrling, and G.R. Matyas (2002) Induction of immune responses in mice and monkeys to Ebola virus after immunization with liposomeencapsulated irradiated Ebola virus: protection in mice requires CD4(+) T cells. J Virol 76: Abrams, J.S., M.G. Roncarolo, H. Yssel, U. Andersson, G.J. Gleich, and J.E. Silver (1992) Strategies of anti-cytokine monoclonal antibody development: immunoassay of IL-10 and IL-5 in clinical samples. Immunol Rev 127: Carbonetti, N.H., G.V. Artamonova, C. Andreasen, and N. Bushar (2005) Pertussis toxin and adenylate cyclase toxin provide a one-two punch for establishment of Bordetella pertussis infection of the respiratory tract. Infect Immun 73(5): Wolfe, D.N., E.M. Goebel, O.N. Bjornstad, O. Restif, and E.T. Harvill (2007) O antigen Enables Bordetella parapertussis to Avoid Bordetella pertussis-induced Immunity. Infect Immun 75(10): Katada, T., M. Tamura, and M. Ui (1983) The A protomer of islet-activating protein, pertussis toxin, as an active peptide catalyzing ADP-ribosylation of a membrane protein. Arch Biochem Biophys 224: Luther, S.A. and J.G. Cyster (2001) Chemokines as regulators of T cell differentiation. Nat Immunol 2(2): Millward, J.M., M. Caruso, I.L. Campbell, J. Gauldie, and T. Owens (2007) IFN-gammainduced chemokines synergize with Ptx to promote T cell entry to the central nervous system. J Immunol 178(12):

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79 29. Biggs, B.A., M. Hewish, S. Kent, K. Hayes, and S.M. Crowe (1995) HIV-1 infection of human macrophages impairs phagocytosis and killing of Toxoplasma gondii. J Immunol 154: Mobberley-Schuman, P.S. and A.A. Weiss (2005) Influence of CR3 (CD11b/CD18) expression on phagocytosis of Bordetella pertussis by human neutrophils. Infect Immun 73(11): Mattoo, S. and J.D. Cherry (2005) Molecular pathogenesis, epidemiology, and clinical manifestations of respiratory infections due to Bordetella pertussis and other Bordetella subspecies. Clin Microbiol Rev 18(2): de Melker, H.E., J.F. Schellekens, S.E. Neppelenbroek, F.R. Mooi, H.C. Rumke, and M.A. Conyn-van Spaendonck (2000) Reemergence of pertussis in the highly vaccinated population of the Netherlands: observations on surveillance data. Emerg Infect Dis 6(4): von Konig, C. H., S. Halperin, M. Riffelmann, and N. Guiso (2002) Pertussis of adults and infants. Lancet Infect Dis 2: Skowronski, D. M., G. De Serres, D. MacDonald, W. Wu, C. Shaw, J. Macnabb, S. Champagne, D.M. Patrick, and S.A. Halperin (2002) The changing age and seasonal profile of pertussis in Canada. J Infect Dis 185: Celentano, L.P., M. Massari, D. Paramatti, S. Salmaso, A.E. Tozzi; EUVAC-NET Group (2005) Resurgence of pertussis in Europe. Pediatr Infect Dis J 24(9): Centers for Disease Control and Prevention (2002) Pertussis--United States, JAMA 287(8):

80 Chapter 4: Clearance of Bordetella parapertussis from the Lower Respiratory Tract Requires Humoral and Cellular Immunity 65

81 Abstract: Bordetella parapertussis and Bordetella pertussis are closely related species that cause whooping cough, an acute, immunizing disease. However, these pathogens are able to coexist in the same host populations, suggesting that the protective immunity induced by each may not efficiently cross protect against the other. Furthermore, vaccines consisting of B. pertussis antigens are ineffective against B. parapertussis infection and disease. Before we can address why B. pertussis vaccines are ineffective against B. parapertussis and how these pathogens may coexist, we must first determine which host immune factors are important to protection against B. parapertussis. The present study explores the mechanisms by which B. parapertussis is cleared from the lower respiratory tract. Serum antibodies are necessary for elimination of this bacterium; CD4 + T cells, complement, and neutrophils are required for serum antibody-mediated clearance. Mice lacking immunoglobulin (Ig)A had no defect in their ability to control or clear infection. Interestingly, the reduction of bacterial numbers upon an adoptive transfer of immune serum did not require Fc receptors that are required for antibody-mediated clearance of B. pertussis. Together these data support a model for the mechanism of protective immunity to B. parapertussis that is similar but distinct from that of B. pertussis. Introduction: B. pertussis has received the vast majority of attention from the scientific community, since it has been historically associated with whooping cough, but the recent evidence of increases in the prevalence of B. parapertussis (1) have spurred heightened interest. Therefore, the relevant immune functions that provide protective immunity to B. parapertussis may be of increasing importance. 66

82 B. bronchiseptica is closely related to B. pertusssis and B. parapertussis and mechanisms by which this bacterium is eliminated from the respiratory tract have been elucidated. Antibodies are essential to the clearance of this pathogen from murine lungs. IgA mediates protection in the upper respiratory tract (2), but serum antibodies are protective in the lower respiratory tract (3). Complement, neutrophils, TLR4, Complement receptor 3, and Fcγ receptors are required for the function of serum antibodies (4), suggesting the following model. B. bronchiseptica stimulates TLR4 which leads to the recruitment of neutrophils to the lungs. These neutrophils phagocytose bacteria that are coated by antibodies and complement component C3b via Fcγ receptors and complement receptor 3 respectively. B. pertussis is cleared by a similar mechanism except T cells, and not complement, are required. Here we will address the mechanisms by which B. parapertussis is eliminated from its host. While it has previously been suggested that infection by B. parapertussis or B. pertussis induces an immune response to B. parapertussis, that response has largely gone uncharacterized (5-6). To understand the mechanisms of protective immunity to B. parapertussis, we dissected the host immune functions necessary for elimination of the bacterium from the lower respiratory tract. Here we show that clearance of B. parapertussis from the lower respiratory tract requires antibodies, T cells, and neutrophils. However, unlike immunity to B. pertussis, Fcγ receptors are not essential to the reduction of bacterial numbers, while the complement cascade is required. Serum antibodies and helper T cells together are sufficient to eliminate B. parapertussis from the lower respiratory tract, while mucosal antibodies and cytotoxic T lymphocytes are not required. These data suggest that mechanisms of protective immunity to B. parapertussis and B. pertussis are not identical. 67

83 Materials and Methods: Bacterial strains and growth. The B. parapertussis strain used in this study, 12822G, is an isolate from German clinical trials with a gentamicin resistance marker inserted. It was maintained on Bordet-Gengou agar (Difco) containing 7.5% defibrinated sheep blood (Hema Resources) and appropriate antibiotics (20 µg/ml gentamicin). The original strain from German clinical trials without gentamicin resistance has been described previously (7). Liquid culture bacteria were grown at 37 C overnight on a roller drum to mid-log phase in Stainer- Scholte broth. Animal experiments. C57BL/6, RAG2 /, µmt, TCR /, C5 /, and Cd11b / mice were obtained from Jackson Laboratory (Bar Harbor, Maine). FcR2 / γ-common / mice were obtained from Taconic Laboratories (Germantown, New York). C3 / mice, backcrossed extensively onto a C57BL/6 background, have been described elsewhere and were a gift of Rick Wetsel (8). IgA mice were a kind gift from Innocent Mbawuike (9). All mice were bred in a Bordetella-free environment. Mice were slightly sedated with isofluorane (Abbott Laboratories) and inoculated by pipetting 50 µl of PBS containing approximately 5 x 10 5 CFU of B. parapertussis onto the tip of the external nares. For time course experiments, groups of three or four animals were sacrificed on day 3, 7, 14, 28, 49, 70, or 105 postinoculation. Lung and systemic colonization was quantified by homogenizing tissues in PBS, plating onto Bordet-Gengou blood agar containing 20 µg/ml gentamicin, and counting the number of colonies. Statistical analysis was performed using the Student s T-test when comparing numbers of CFU. P values less than 0.05 were considered statistically significant. Adoptive transfer experiments were conducted by injecting 200 µl of convalescent-phase serum I.P. at the time of inoculation. Convalescent-phase serum samples were collected from wild-type mice infected with B. parapertussis 28 days 68 /

84 postinoculation. For the production of survival curves, once the progression of disease was clear, moribund animals were euthanized to prevent unnecessary stress. All animals were handled in accordance with institutional guidelines. Cell depletions. Neutrophil depletion was performed by injecting 1 mg of the monoclonal antibody from the RB6-8C5 hybridoma I.P. (10-11) 24 hours prior to and 7 days after infection. CD4 + T cells were depleted by I.P. injections of 1 mg of the monoclonal antibody from the GK1.5 hybridoma at days 0 and 7 (12). Similarly, CD8 + T cells were depleted by I.P. injection of 1 mg of the monoclonal antibody from the YTS168 4 hybridoma at days 0 and 7 (13). Enzyme-linked immunosorbent assays (ELISAs). Bacteria were grown overnight to an optical density at 600 nm of 0.7, heat inactivated, diluted in carbonate buffer, and used to coat the wells on 96-well plates. Plates were stored at 4 C (wells filled with PBS containing Tween 20 [PBS-T] plus 1% bovine serum albumin) until use. A 1:50 dilution of convalescent-phase serum samples from different mouse strains was added to the first wells and serially diluted 1:2 across the plates. The plates were incubated for 2 h at 37 C in a humidified chamber and then washed three times with PBS-T. Polyvalent anti-mouse secondary antibodies were used to look at the total titer (14), and specific isotypes were determined by using the appropriate secondary antibodies (Southern Biotechnology Associates and Pharmingen). The plates were then incubated at 37 C in a humidified chamber for 1 h before they were washed four times with PBS-T. 2,2'-Azino-bis(3- ethylbenz-thiazoline-6-sulfonic acid) in a phospho-citrate buffer and hydrogen peroxide were added to the wells, which were then incubated at room temperature in the dark for 30 min. A sodium fluoride solution was added to the wells to stop the reaction, and the plates were read at an absorbance of 405 nm. 69

85 Results: An adaptive immune response is required to survive infection by B. parapertussis. In both humans and mice, B. parapertussis causes an acute infection of the lower respiratory tract. CFU in the lungs have been shown to increase rapidly more than 100-fold during the first week of infection but decline thereafter in wild-type mice (3). In SCID-beige mice, which are deficient in B, T, and natural killer cells, this decline is not observed and the infection is lethal (15), indicating that these cells are necessary to control B. parapertussis. In order to identify which aspects of the adaptive immune response are required for control and clearance, different immunodeficient strains of mice were infected with B. parapertussis. Groups of 10 C57BL/6 (wild-type), RAG2 / (B- and T-cell-deficient), µmt (B-cell-deficient), and TCR-α / (Tαß-cell-deficient) mice were inoculated with B. parapertussis. Wild-type, µmt, and TCR-α / mice survived 105 days postinoculation without any signs of distress and were then euthanized. On day 17 postinoculation, RAG2 / mice began to show symptoms of disease, such as hunched posture, decreased level of activity, and ruffled fur. These mice began dying on day 18, and all were dead by day 28 post-inoculation (Fig.4.1A). In a subsequent experiment, B. parapertussis numbers in the lower respiratory tract and blood were measured for the same mouse strains on day 28 postinoculation. Wild-type C57BL/6 mice reduced B. parapertussis to about 100 CFU in the lower respiratory tract by day 28. However, RAG2 /, µmt, and TCR-α / mice were all unable to reduce bacterial numbers below the maximal levels observed on day 7 in wild-type mice ( 10 6 to 10 7 CFU). Although each of these immunodeficient mouse strains were unable to efficiently reduce colonization in the lungs (Fig.4.1B), Rag2 / mice were the only strain that allowed colonization of the blood by B. parapertussis (Fig.4.1C). The observation that 70

86 only mice lacking both B cells and T cells succumb to lethal, systemic B. parapertussis infection suggests that either B cells or T cells can control the spread from the respiratory tract. Mice deficient in antibody production are unable to clear B. parapertussis. Figure 4.1. Role of adaptive immune response in protecting against B. parapertussis infection. C57BL/6 (squares), -/- -/- RAG2 (X), μmt (diamonds), and TCRα (triangles) mice were inoculated with B. parapertussis. (A) Mice were monitored for a survival curve, moribund mice were euthanized to prevent unnecessary suffering. Groups of mice were sacrificed on day 28 for the quantification of bacteria in the (B) lungs or (C) blood. Bacterial numbers are represented as the Log 10 mean +/- standard deviation. Asterisk represents P-value < Infect Immun 73(10):

87 To assess the role of adaptive immunity in the elimination of B. parapertussis from the lower respiratory tract, the level of colonization was monitored in C57BL/6, RAG2 /, µmt, and TCR-α / mice for 105 days to determine whether they were able to clear the infection. Groups of 24 mice were infected, and 4 mice of each strain were sacrificed on day 7, 14, 28, 49, 70, or 105 post-inoculation. C57BL/6 mice reduced B. parapertussis to low numbers in the lower respiratory tract by day 28, and no bacteria could be recovered by day 49 post-inoculation. RAG2 /, µmt, and TCR-α / mice were all defective in bacterial clearance compared to wild-type mice, and high numbers of B. parapertussis were still present in the lower respiratory tract at 105 days postinoculation (Fig.4.2A). Serum samples were taken from mice Figure 4.2. Mice deficient in antibody production are unable to clear B. parapertussis from the lower respiratory tract. Groups of 24 C57BL/6 (squares), RAG2 / (X), µmt (diamonds), and -/- TCRα (triangles) mice were inoculated with B. parapertussis and sacrificed on day 7, 14, 28, 49, 70, or 105 for (A) the quantification of bacterial numbers. CFU are represented as the Log 10 mean +/- standard deviation. (B) Pooled sera were collected on day 28 post-inoculation to assess B. parapertussis-specific antibody titers for C57BL/6 (black bars), μmt (white bars), and TCRα -/- mice (hatched bars). Infect Immun 73(10): sacrificed on day 28 postinoculation to quantify the titers of anti-b. parapertussis polyclonal antibodies, IgA, IgG, IgG1, IgG2a, IgG2b, IgG3, and IgM by ELISAs. Anti-B. parapertussis IgA and IgG3 were undetectable in the sera of any infected mice (data not shown). While C57BL/6 72

88 mice produced high titers of anti-b. parapertussis antibodies of other isotypes, µmt and TCR-α / mice were defective in the production of all isotypes except for the production of IgM by TCR-α / mice (Fig. 4.2B). Mouse strains that were unable to produce anti-b. parapertussis antibodies were also defective in the clearance of the bacteria from the lower respiratory tract, suggesting that antibodies may be critical to the elimination of B. parapertussis from the lower respiratory tract. Figure 4.3. Mucosal antibodies are not crucial to clearance of B. parapertussis from the lower respiratory tract. Groups of 24 C57BL6 (diamonds) and IgA -/- mice (squares) were inoculated with B. parapertussis and sacrificed on day 7, 14, 28, 49, 70, or 105 to quantify bacteria in the lungs. Bacterial numbers are expressed as the Log 10 mean +/- standard deviation. Infect Immun 73(10): Serum antibodies are required to clear B. parapertussis from the lower respiratory tract, while mucosal antibodies are not. Since B. parapertussis is an extracellular mucosal pathogen, we examined the role of IgA antibodies, which is the predominant isotype within mucosal secretions. C57BL/6 and IgA / mice were inoculated with B. parapertussis, and sacrificed 7, 14, 28, 49, 70, or 105 days later to monitor the colonization level in the lower respiratory tract. While mice that were ineffective in producing B. parapertussis-specific antibodies were unable to clear bacteria from the lower respiratory tract, mice lacking only IgA eliminated the bacteria with kinetics similar to that of wild-type mice (Fig.4.3). This finding indicates that IgA is not required and suggests that other isotypes are sufficient to clear B. parapertussis from the lower respiratory tract. 73

89 Adoptive transfer of serum antibodies clears B. parapertussis from the lower respiratory tract but requires help from T cells. An adoptive transfer model was used to determine whether serum antibodies are sufficient to eliminate B. parapertussis. Groups of four mice were injected I.P. with 200 µl of naïve or immune sera (collected Figure 4.4. T cells are required for the function of from wild-type mice 28 days serum antibodies in clearing B. parapertussis from the lower respiratory tract. Four control C57BL/6 (C57) mice were given an I.P. injection of naïve serum postinoculation), inoculated with B. (NS) at the time of intranasal inoculation with B. parapertussis. Groups of four C57BL/6, RAG2 /, µmt, parapertussis, and sacrificed on day 14 and TCRα -/- mice were given I.P. injections of immune serum (IS) at the time of inoculation. Groups of post-inoculation. Although previous C57BL/6 mice were given I.P. injections of anti-cd4 or anti-cd8 antibody in addition to immune serum at the time of inoculation. All mice were sacrificed on day 14. studies showed that transferring immune Numbers of bacteria are expressed as the Log 10 mean +/- standard deviation. Asterisks represent P-values < serum to naïve wild-type mice upon 0.05 when compared to naïve serum treated mice. Infect Immun 73(10): infection has no effect on bacterial numbers in the first 7 days postinoculation (3), we observed a substantial effect thereafter. C57BL/6 mice given naïve serum had colonization levels similar to those of untreated C57BL/6 mice ( 10 5 CFU) (Fig.4.4). In contrast, both C57BL/6 and µmt mice treated with immune serum reduced bacterial numbers to less than 100 CFU in the lower respiratory tract by day 14, indicating that antibodies are very effective in these mice. However, treatment with immune serum had no effect on the numbers of B. parapertussis in RAG2 / or TCR-α / mice (Fig. 4.4), indicating that αß T cells are required for the function of antibodies. To determine whether cytotoxic T lymphocytes and/or helper T cells are necessary for antibody- 74

90 mediated clearance of B. parapertussis, C57BL/6 mice that were inoculated with B. parapertussis and treated with immune serum were injected I.P. with anti-cd4 or anti-cd8 Abs at days 0 and 7. Mice depleted of CD8 + T cells were able to clear the infection from the lower respiratory tract by day 14, but those lacking CD4 + T cells were unable to do so (Fig. 4.4). These data suggest that not only are T cells required for the production of anti-b. parapertussis antibodies but that CD4 + cells are also involved in their function. C3 is required for antibody-mediated clearance of B. parapertussis, but complement receptor 3 (CR3) and Fc receptors have an intermediate effect. Several host immune factors were analyzed to determine the mechanism by which antibodies facilitate the elimination of B. parapertussis from the lower respiratory tract. Aspects of the complement cascade / / were examined using C3 and C5 mice. In mice lacking C3, antibodies were unable to clear the bacteria by day 14. However, in mice lacking only C5, antibodies greatly reduced B. parapertussis numbers (approximately 99% reduction), although not to the limit of detection (Fig.4.5). The roles of CR3 and Fc receptors, which bind opsonized C3b and antibodies, respectively, Figure 4.5. Complement is required for the function of serum antibodies against B. parapertussis. Four control C57BL/6 (C57) mice were given I.P. injections of naïve serum (NS) upon inoculation with B. parapertussis. Groups of four C57BL/6, C3 /, C5 /, CD11b / /, and FcRγ mice were injected I.P. with immune serum (IS) at the time of inoculation. All mice were sacrificed on day 14. The numbers of bacteria are expressed as the log10 mean ± standard deviations. Asterisks represent P-values < 0.05 when compared to naïve serum treated mice. The dashed line represents the limit of detection. Infect Immun 73(10): were also analyzed using mice lacking CD11b (CD11b / ) and mice lacking all three Fc 75

91 receptors (Fc R2 /, common / ). Both mouse strains were able to significantly decrease numbers of B. parapertussis by day 14 post-inoculation (approximately 99% clearance), although they did not completely clear the infection (Fig.4.5). These results suggest that CR3 and Fc receptors contribute to clearance of the infection, but are not required to reduce B. parapertussis numbers. Together these data indicate that both complement and Fcγ receptors play a role in the function of serum antibodies against B. parapertussis. Neutrophils are required for the antibody-mediated clearance of B. parapertussis. The fact that antibody-mediated clearance of B. parapertussis requires C3, but not C5, suggests that elimination occurs via phagocytosis of C3b- and antibodycoated bacteria. Since we have recently noticed that neutrophils are crucial to controlling B. pertussis and B. bronchiseptica infections (16-17), the role of neutrophils in the antibody-mediated clearance of B. parapertussis was tested by depleting these cells with an I.P. injection of the monoclonal antibody RB6-8C5 24 h prior to and 7 days after infection (10-11). Groups of four µmt mice were given I.P. Figure 4.6. Neutrophils are required for the function of serum antibodies against B. parapertussis. Four control µmt mice were injected I.P. with naïve serum (NS) upon inoculation. Four µmt mice were injected I.P. with immune serum (IS) upon inoculation. Four µmt mice were injected with the monoclonal antibody RB6-8C5 (RB6) to deplete neutrophils 24 h prior to and 7 days after infection and injected with immune serum upon inoculation. All mice were sacrificed on day 14. Numbers of bacteria are expressed as the log10 mean ± standard deviations. Asterisks represent P-values < 0.05 when compared to naïve serum treated mice. The dashed line represents the limit of detection. Infect Immun 73(10): injections of RB6-8C5 or left untreated and then injected with immune serum and inoculated with B. parapertussis. Mice receiving immune serum alone completely cleared the infection from the 76

92 lungs by day 14 post-inoculation. However, in mice depleted of neutrophils, immune serum had no effect on B. parapertussis numbers, which increased to greater than 10 6 CFU in the lungs by day 14 (Fig.4.6). These data indicate that neutrophils are required for serum antibodies to eliminate B. parapertussis from the lower respiratory tract. Discussion: Our results show that antibodies are crucial for the elimination of B. parapertussis from the lower respiratory tract. Interestingly, serum antibodies cleared this extracellular mucosal pathogen from the lower respiratory tract, while IgA was not essential. Our study of serum antibody-mediated clearance of B. parapertussis suggests a mechanism of clearance in which antibody-opsonized bacteria activate the complement cascade, become coated by C3b, and are subsequently phagocytosed by neutrophils via Fcγ receptors and/or CR3. In this model, either Fcγ receptors or CR3 are sufficient for reduction of the bacterial load (about 99% clearance), but the presence of both receptors results in complete elimination of bacteria. Furthermore, our data also suggest that helper T cells, but not cytotoxic T cells, are necessary for the function of antibodies. The role of helper T cells in facilitating the bacterial clearance will be addressed in chapter 6. It appears that both humoral and cellular responses are crucial in immunity to B. parapertussis. Recent clinical surveys indicate that B. parapertussis may be responsible for a large percentage of diagnosed cases of whooping cough (18). Although the immune response to B. pertussis is fairly well characterized, the immune response to B. parapertussis has largely been ignored. Our data show that like B. pertussis, both humoral and cellular immune responses are required for sterilizing immunity to B. parapertussis. Since these two species are closely related, 77

93 it would be expected that protective immunity induced by either one would induce crossprotection against the other. In fact, experimental studies have suggested that these two species may induce a level of cross-protection, but not as high as their levels of self-protection (19-22). This incomplete cross-protection could result from the two species being antigenically distinct in each of their many surface and secreted proteins. Alternatively, a different set of prominent surface molecules could also prevent effective cross immunity. O antigen is the dominant surface antigen of B. parapertussis but is absent from B. pertussis isolates. Our previous work has shown that O antigen is required for efficient colonization of the murine respiratory tract by B. parapertussis (23), but its role in eliciting a protective immune response or conversely, protecting bacteria from the adaptive immune response is largely unknown. O antigen is expressed by several different bacteria and has various functions in vivo and in vitro, including contributing to host colonization, resistance to complement-mediated killing, and the ability to cause sepsis (23-27). Our previous studies have shown that the O antigens of B. parapertussis and B. bronchiseptica protects them from killing by complement in the absence of antibodies, while B. pertussis, which lacks this virulence factor, is naturally sensitive to the effects of naïve serum complement (23). As a major component of the outer surface of B. parapertussis, it is possible that O antigen either shields other protective antigens or presents a decoy antigen. The role of O antigen in immune evasion, particularly the evasion of B. pertussis-induced immunity, will be discussed in chapter 7. The lack of expression of Ptx by B. parapertussis is another key difference between this bacterium and B. pertussis. Compared to wild-type B. pertussis strains, strains deleted of Ptx are not able to colonize the respiratory tract as efficiently (17,28). However, B. parapertussis is able 78

94 to reach high levels in the lower respiratory tract without this toxin. Ptx is a prominent antigen of B. pertussis to which high titers of protective antibodies are produced (28). Thus, it is possible that by not expressing this toxin, B. parapertussis may avoid B. pertussis-induced immunity. Ecological theory holds that two antigenically similar pathogens cannot occupy the same host population indefinitely due to immune-mediated competition; one of the two will ultimately be displaced from the host population (29). The coexistence of B. parapertussis and B. pertussis in the same host population, and occasionally in the same individual (30), indicates that these organisms have some mechanism to avoid immune-mediated competition. Phylogenetic analysis suggests that B. parapertussis emerged from a B. bronchiseptica-like progenitor and adapted to humans more recently than B. pertussis did (31). Thus, it could be predicted that B. parapertussis successfully invaded a human population in which B. pertussis was already prevalent and, consequently, needed to develop mechanisms to avoid B. pertussis-induced immune responses. The presence of B. parapertussis and B. pertussis in the same population is intriguing, and understanding their abilities to coexist could provide an excellent model to study evolution, adaptation, and spread in the emergence of bacterial pathogens. This will be addressed further in chapter 7. Whooping cough has recently been increasing in prevalence in vaccinated populations (32-36). The dangers of B. parapertussis and B. pertussis infections lie in the spread of these bacteria to unvaccinated individuals or those in whom immunity has waned. Both species are quite capable of colonizing B. pertussis-vaccinated individuals, often going undiagnosed (5,37-38), and whooping cough epidemics are frequent and periodic in some populations (39). Control of these pathogens at the population level requires greater understanding of immune mechanisms 79

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101 38. He, Q., M. K. Vijanen, S. Nikkari, R. Lyytikainen, and J. Mertsola (1994) Outcomes of Bordetella pertussis infection in different age groups of an immunized population. J Infect Dis 170: Rohani, P., C. J. Green, N. B. Mantilla-Beniers, and B. T. Grenfell (2003) Ecological interference between fatal diseases. Nature 422:

102 Chapter 5: A Lack of TLR4 Stimulation Enables Bordetella parapertussis to Avoid Rapid Antibody-Mediated Clearance 87

103 Abstract: Although lipid A structures are highly conserved among gram negative bacteria, they are not invariant and can even vary within a genus. The bordetellae provide an excellent example of lipid A variation within a genus; as a result, these bacteria differ greatly in their capacity to stimulate TLR4. This led us to speculate that the low level of TLR4 stimulation by Bordetella parapertussis may help it evade the host immune response. Therefore, we sought to address the effect of TLR4 stimulation on the outcome of B. parapertussis infection in a mouse model. Clearance of this bacterium from the murine respiratory tract correlates with the accumulation of leukocytes in the lungs. However, B. parapertussis does not efficiently stimulate TLR4- dependent pro-inflammatory signaling and the accumulation of leukocytes does not peak until one week post-inoculation. Providing an exogenous source of TLR4 stimulation in the form of a co-inoculation with B. bronchiseptica induced an early recruitment of leukocytes to the lungs. This co-inoculation also allowed for more efficient control and rapid antibody-mediated clearance of B. parapertussis, both of which were dependent on TLR4. Together, these data suggest that by not stimulating pro-inflammatory TLR4 responses, B. parapertussis is able to avoid rapid clearance from the respiratory tract. This strategy may be crucial to the ability of this pathogen to infect immunized hosts. Introduction: For pathogens that transmit directly from host to host, transmissibility is proportional to the infectious period. In order to persist in its host and potentially extend its infectious period, a pathogen must first evade rapid control and clearance by the innate immune response. Many gram negative bacteria have developed mechanisms to evade or modulate the innate immune response. For example, Helicobacter pylori limits the microbicidal effects of reactive oxygen 88

104 intermediates and nitric oxide, rendering it resistant to phagocytic killing (11). Yersinia enterocolitica elicits the production of IL-10 which inhibits the pro-inflammatory TNFα response and limits the number of leukocytes recruited to the site of infection (12). Limiting the recruitment of antimicrobial leukocytes to the site of infection is a strategy used by a number of pathogens to enhance their ability to colonize and persist in the host. B. pertussis, which is very closely related to B. parapertussis (13), inhibits the innate immune response by preventing an early leukocyte migration to the lungs via Ptx (14-15). In doing so, Ptx enables this bacteria to avoid rapid clearance by antibodies (15). Interestingly, B. parapertussis also avoids rapid clearance by antibodies (16-17) but does not express Ptx. Pathogen associated molecular patterns interact with pathogen recognition receptors to induce an innate immune response; a major route of innate immune initiation for gram negative bacteria such as the bordetellae involves the recognition of LPS by TLR4. Although B. parapertussis and B. pertussis are very closely related to each other as well as the animal pathogen B. bronchiseptica (13), there is substantial variation among the LPS structures of these species (18-20). As a result, the LPS s of these species greatly differ in their ability to stimulate pro-inflammatory TLR4 responses. B. bronchiseptica LPS is a strong stimulator of TLR4, B. pertussis LPS is somewhat less stimulatory, and B. parapertussis LPS is the least stimulatory of the three (21). The portion of LPS responsible for TLR4 stimulation, lipid A (22-23), is highly conserved among gram negative bacteria but not invariant (23). While it is not clear if B. parapertussis may trigger signaling through other TLRs, modulation of the lipid A structure could allow the evasion of TLR4-mediated immune responses. Since B. parapertussis induces little TLR4-mediated production of proinflammatory cytokines relative to the other bordetellae (21), we hypothesized that the lack of TLR4 89

105 stimulation may help it avoid rapid clearance from its host. Here we show that few leukocytes are recruited to the lungs within the first few days of a B. parapertussis infection, but leukocyte numbers peak after around seven days post-inoculation, correlating with the time when bacterial numbers begin to decline. B. parapertussis does not appear to actively inhibit TLR4-mediated responses to other bacteria in vivo. This allowed us to test the effect of TLR4 stimulation by B. bronchiseptica on the course of B. parapertussis infection. Stimulating TLR4 with B. bronchiseptica leads to more efficient control and rapid antibody-mediated elimination of B. parapertussis from the lungs. Thus, expressing an LPS that does not efficiently stimulate TLR4 contributes to the ability of B. parapertussis to avoid clearance from its host. Materials and Methods: Bacterial strains and growth. B. parapertussis strain was isolated from German clinical trials (24) and 12822G is a gentamicin-resistant derivative of (17). B. bronchiseptica strain RB50 was originally isolated from a rabbit and has also been previously described (25). Bacteria were maintained on Bordet-Gengou agar (Difco) containing 10 % defibrinated sheep blood (Hema Resources) and appropriate antibiotics. Liquid culture bacteria were grown at 37 C overnight on a roller drum to mid-log phase in Stainer-Scholte broth. Inoculation of mice. C57BL/6, C3H/HEOuJ, and C3H/HEJ were obtained from Jackson Laboratories and bred in our Bordetella-free, specific pathogen-free facilities at The Pennsy lvania State University. Bacteria grown overnight (to an optical density of approximately 0.3) in liquid culture were diluted in PBS to approximately 10 7 CFU/ml. 50 μl of the inoculum (5 x 10 5 CFU) was pipetted on to the external nares of 4-6 week old mice that had been lightly sedated with 5% isoflurane in oxygen. For co-inoculations with B. parapertussis and B. bronchiseptica, inocula were prepared so that both species were present at 10 7 CFU/ml and mice 90

106 were inoculated as above. For co-inoculation with heat-killed B. bronchiseptica, B. bronchiseptica was grown overnight to an optical density of 0.3 and heat-inactivated by incubating in a water bath at 75 o C for 30 minutes. Bacteria in the inoculum were plated before and after heat-inactivation to quantify the number of bacteria present and ensure that the incubation killed B. bronchiseptica. Inocula were prepared so that they contained 10 7 CFU/ml of B. parapertussis and 10 9 CFU/ml of heat-killed B. bronchiseptica and mice were inoculated as above. All protocols were reviewed by the university s IACUC and all animals were handled in accordance with institutional guidelines. Adoptive transfer of serum antibodies. To generate immune serum, C57BL/6 mice were inoculated with 5 x 10 5 CFU of B. parapertussis and allowed to convalesce for 28 days. By this time, these mice have generated high titers of B. parapertussis-specific antibodies (17). Blood was then collected from these mice and the serum portion was isolated and stored at -80 o C until use. 200 μl of immune serum was injected I.P. into mice at the time of inoculation. Serum from uninfected mice (naïve serum) was used as a control. Quantification of bacteria, leukocytes, and cytokines in the lungs. To quantify bacteria, the lungs were excised at day 0, 3, 7, or 14 post-inoculation. Lungs were homogenized in 1 ml of PBS. The lung homogenate was then plated on to Bordet-Gengou agar plates at the appropriate dilutions and CFU were counted 4 days later for B. parapertussis and 2 days later for B. bronchiseptica. To quantify leukocytes, mice were infected for 0, 0.5, 1, 2, 3, 7, or 14 days, sacrificed, and BAL fluid was collected. Red blood cells were lysed by ammonium chloride as previously described (26). Leukocytes were counted on a hemocytometer to quantify total numbers of leukocytes in the BAL fluid. Aliquots of cells were stained with FITC-labeled anti- was Ly-6G or FITC-labeled anti-f4/80, and the percentage of Ly-6G or F4/80 positive cells 91

107 multiplied by the total number of leukocytes to calculate the number of neutrophils or macrophages respectively. For quantification of cytokines in the lungs, wild type mice were inoculated with B. parapertussis, B. bronchiseptica, or both species and sacrificed 2 hours later. Lungs were homogenized in one ml of PBS and samples were run on ELISAs specific for TNFα, KC, JE, or MIP-1α according to the manufacturer s protocols (R&D Systems, Minneapolis, Minnesota, USA). In vitro growth curves of B. parapertussis and B. bronchiseptica. To address whether these bacteria affected each other s growth in vitro, both were grown overnight to an optical density of 0.3. They were then diluted in fresh Stainer-Scholte broth to approximately 10 6 CFU/ml. The liquid cultures were then grown on a roller drum at 37 o C and aliquots were plated at various times to quantify CFU. Statistical Analysis. The mean +/- standard deviation (error bars) was determined for CFU, leukocytes, and cytokines. Two-tailed, unpaired Student s T-tests were used to determine statistical significance between groups. All experiments were performed at least twice with similar results and P-values < 0.05 were taken to be statistically significant. Results: The reduction of B. parapertussis numbers correlates with an accumulation of leukocytes in the lungs. B. parapertussis is a weak stimulator of TLR4; little inflammation is observed during the first few days of infection despite the rapid growth to nearly 10 7 CFU in the lungs (21). Since neutrophils are required to eliminate B. parapertussis from the murine respiratory tract (17), we sought to determine if the reduction of B. parapertussis numbers correlated with an increase in 92

108 leukocyte accumulation in the lungs. C57BL/6 mice were inoculated with B. parapertussis and sacrificed on day 0, 3, 7, or 14 post-inoculation to quantify the numbers of bacteria in the lungs. B. parapertussis numbers peaked at approximately CFU day 3 but began to decline by day 7 and were reduced to 10 4 CFU by day 14 post-inoculation (Fig. 5.1A). Groups Figure 5.1: Numbers of B. parapertussis and leukocytes in the lungs over time. Groups of C57BL/6 mice were inoculated with B. parapertussis and sacrificed on day 0, 3, 7, or 14 post-inoculation. (A) Bacterial numbers in the lungs are represented as the Log 10 mean +/- S.D. (B) Leukocyte and neutrophil numbers in the BAL fluid are represented as the mean +/- S.D and asterisks denote P-values < 0.05 when compared to numbers at day 3 for CFU (the highest observed numbers) or day 0 (naïve mice) for leukocytes. of C57BL/6 mice were also sacrificed on day 0, 3, 7, or 14 post-inoculation to quantify the numbers of leukocytes in the BAL fluid. Low numbers of leukocytes (< 2 x 10 5 leukocytes/ml) were observed on day 3 post-inoculation, but leukocyte accumulation peaked at 7 days postinoculation (~6 X 10 5 leukocytes/ml, ~ 2 X 10 5 neutrophils/ml) (Fig. 5.1B). Together, these data show that the time when B. parapertussis numbers began to decline in murine lungs correlated with increasing numbers of leukocytes in the lungs. B. parapertussis does not induce an early, TLR4-mediated inflammatory response. TLR4 responses do not appear to be important to the elimination of B. parapertussis from murine hosts (21). Since B. parapertussis LPS does not efficiently stimulate TLR4 responses (21), we sought to determine if this pathogen induces any TLR4-dependent recruitment of leukocytes to the lungs. Wild type (C3H/HEOuJ) and TLR4-deficient (C3H/HEJ) mice were 93

109 inoculated with B. parapertussis and sacrificed 0, 2 hours, 1, 3, 7, or 14 days later to quantify the numbers of leukocytes in the BAL fluid. As previously shown, leukocyte numbers peaked in the lungs of wild type mice (~3 x 10 5 cells) on day 7 post- post-inoculation inoculation, and the same was true for TLR4-deficient mice (~6 x 10 6 cells) (Fig. 5.2A). Similarly, neutrophil numbers were low in both wild type and TLR4-deficient mice over the first 3 days Figure 5.2: Numbers of leukocytes in the lungs of wild type and TLR4-deficient mice upon B. but peaked on day 7 in both wild type (~2 x parapertussis infection. Groups of C3H/ HEOuJ (WT) and C3H/HEJ (TLR4-def) mice were 10 5 cells) and TLR4-deficient (~5 x 10 5 inoculated with B. parapertussis and sacrificed 2 hours later or on day 1, 3, 7, or 14 post-inoculation. (A) Total leukocytes and (B) neutrophils in the BAL cells) mice (Fig. 5.2B). By day 14 post- fluid were quantified and are represented as the mean +/- S.D. Asterisks denote P-values < 0.05 inoculation, leukocyte numbers in both when compared to naïve mice. mouse strains had decreased from numbers observed on day 7 post-inoculation (Fig. 5.2A-B). Interestingly, more leukocytes accumulated in the lungs of TLR4-deficient mice compared to wild type mice. Therefore, TLR4 does not measurably contribute to the recruitment of leukocytes to the site of B. parapertussis infection, but may mediate anti-inflammatory signals in response to B. parapertussis. B. parapertussis does not inhibit TLR4-mediated inflammatory responses. Since B. parapertussis did not induce a TLR4-mediated recruitment of leukocytes (Fig. 5.2), we assessed whether or not B. parapertussis actively inhibited TLR4-mediated responses. 94

110 To do so, the effects of B. parapertussis on the TLR4 responses to B. bronchiseptica were analyzed. B. parapertussis and B. bronchiseptica were first grown together in liquid culture to determine if they affected the growth of one another in vitro. B. bronchiseptica grew from approximately 10 6 CFU/ml to CFU/ml in 24 hours and its growth was not affected by a co-inoculation with B. parapertussis (Fig. 5.3). Similarly, B. parapertussis, which grows slower, grew from approximately 10 6 CFU/ml to CFU/ml in 24 hours and its growth was not affected by a co- inoculation with B. bronchiseptica (Fig. 5.3). These data show that B. parapertussis and B. bronchiseptica do not affect the growth of each other in vitro. Figure 5.3: Effect of B. parapertussis on TLR4 signaling induced by other bacteria in vitro. B. parapertussis and B. bronchiseptica were grown alone or with each other in liquid culture over a 24 hour period to determine the effect of a co-inoculation on in vitro growth. B. bronchiseptica induces a high level of TLR4-mediated cytokine production and leukocyte recruitment to the lungs in vivo (21). C57BL/6 mice were inoculated with B. parapertussis, B. bronchiseptica, or both bacteria to examine the effects of a co-inoculation with B. parapertussis on TLR4 responses to other bacteria. These mice were sacrificed 2 hours, 12 hours, 1, 2, or 3 days post-inoculation to see if B. parapertussis inhibited B. bronchisepticainduced, TLR4-mediated cytokine production (at 2 hours) and recruitment of leukocytes (at 12 hours, 1, 2, and 3 days). B. parapertussis induced only 600 pg of TNF-α, 2800 pg of KC, 100 pg of JE, and 1500 pg of MIP-1α by 2 hours post-inoculation (Fig. 5.4A-D). In contrast, B. bronchiseptica induced approximately 1700 pg of TNF-α, 4400 pg of KC, 330 pg of JE, and 3000 pg of MIP-1α by this time (Fig. 5.4A-D) and the production of these cytokines by this time 95

111 is known to be mediated by TLR4 (21). In the lungs of mice that were inoculated with both species, approximately 1900 pg of TNF-α,4000 pg of KC, 340 pg of JE, and 4500 pg of MIP-1α were observed (Fig. 5.4A-D) suggesting that B. parapertussis did not inhibit the TLR4-mediated production of cytokines induced by B. bronchiseptica. Consistent with Figures 5.1 and 5.2, the BAL fluid of B. parapertussis-infected mice contained few neutrophils (< 10 5 /ml of BAL fluid) over the first three days postinoculation (Fig. 5.4E). B. bronchiseptica induced the accumulation of approximately 1.5 X 10 6 neutrophils/ml of BAL fluid over the first two days and cell numbers declined thereafter. This early recruitment of neutrophils to the lungs upon B. bronchiseptica infection is dependent on TLR4 (21). The BAL fluid of co-inoculated mice also contained approximately 1.5 X Figure 5.4: Effect of B. parapertussis on TLR4 signaling induced by other bacteria in vivo. Groups of C57BL/6 mice were inoculated with B. parapertussis, B. bronchiseptica, or both and sacrificed at 2 hours, 12 hours, 1, 2, or 3 days post- inoculation. (A) TNF-α, (B) KC, (C) JE, and (D) MIP-1α levels in the lungs were quantified at 2 hours post-inoculation. (E) Neutrophils in the BAL fluid were quantified at 12 hours, 1, 2, and 3 days post-inoculation. Values are expressed as the mean +/- S.D. Asterisks represent P-values < 0.05 when compared to mice infected only with B. parapertussis neutrophils/ml (Fig. 5.4E), indicating that B. parapertussis did not inhibit the TLR4- mediated recruitment of neutrophils to the lungs of B. bronchiseptica-infected mice. 96

112 Co-inoculation with B. bronchiseptica allows for more efficient control of B. parapertussis. Since the reduction of B. parapertussis numbers correlated with the accumulation of leukocytes in the lungs, we sought to determine if pro-inflammatory TLR4 stimulation by conumbers. Groups of C57BL/6 mice inoculating with B. bronchiseptica affected B. parapertussis were inoculated with B. parapertussis, B. bronchiseptica, or both bacteria and sacrificed 12 hours, 1, 2, or 3 days later to quantify bacterial numbers in the lungs. B. bronchiseptica numbers were not affected by a co-inoculation with B. parapertussis (Fig. 5.5A). When inoculated by itself, B. parapertussis numbers rose over the first three days, peaking at approximately 10 CFU on day 3 post-inoculation. Upon a coinoculation with B. bronchiseptica however, 6.5 B. parapertussis numbers began to decline after one day and were reduced to approximately CFU by day 3 post-inoculation (Fig. 5.5B). These data suggest that TLR4 stimulation may allow for more efficient control of B. parapertussis numbers in murine lungs. Figure 5.5: Effect of B. bronchiseptica on B. parapertussis growth in vivo. Groups of C57BL/6 mice were inoculated with B. parapertussis (Bpp), B. bronchiseptica (Bb), or both and sacrificed 12 hours, 1, 2, or 3 days later. (A) Bb numbers in the lungs were quantified upon inoculation by itself (Bb) or upon a co-inoculation (Bb + Bpp) and (B) Bpp numbers in the lungs were quantified upon inoculation by itself (Bpp) or upon coinoculation (Bpp + Bb) and are expressed as the Log10 mean +/- SD. Asterisks represent P- values <

113 Co-inoculation with B. bronchiseptica allows for rapid antibody-mediated clearance of B. parapertussis. Limiting neutrophil recruitment to the lungs via Ptx enables B. pertussis to delay antibody-mediated clearance. We hypothesized that the lack of TLR4 stimulation by B. parapertussis could similarly enable it to avoid rapid clearance by antibodies. To test this, we examined the effect of TLR4 stimulation on the antibody-mediated clearance of B. parapertussis. Groups of C57BL/6 mice were inoculated with B. parapertussis, given I.P. injections of naïve or immune serum, and sacrificed on day 0, 3, 7, or 14 post-inoculation to assess the effects of antibodies on B. parapertussis. As previously shown, antibodies had no effect on B. parapertussis during the first week of infection (16) but cleared B. parapertussis by day 14 postinoculation (17, Fig. 5.6A) indicating that B. parapertussis avoids rapid clearance by antibodies. C57BL/6 mice were then inoculated with B. parapertussis or both B. parapertussis and B. bronchiseptica. These mice were also given an I.P. injection of naïve serum or immune serum and sacrificed on day 3 or 7 post-inoculation for the enumeration of bacteria in the lungs. Again, immune serum alone had no effect on the level of B. parapertussis colonization at these times (16, Fig. 5.6B). However, immune serum rapidly reduced B. parapertussis numbers upon a co- inoculation with B. bronchiseptica to approximately 100 CFU by day 3 and to undetectable levels by day 7 post-inoculation (Fig. 5.6B). The co-inoculation did not affect the ability of B. bronchiseptica to colonize the lungs of mice treated with naïve serum, but B. bronchiseptica numbers were approximately 500-fold lower in the lungs of mice treated with immune serum (Fig. 5.6C). Because these bacteria are so closely related, this effect may have been due B. parapertussis-induced antibodies being cross-protective against B. bronchiseptica. 98

114 Figure 5.6: Effect of B. bronchiseptica on antibody-mediated clearance of B. parapertussis. (A) Groups of C57BL/6 mice were inoculated with B. parapertussis, given an I.P. injection of naïve serum or immune serum, and sacrificed on day 0, 3, 7, or 14 post-inoculation to quantify bacterial numbers. (B) C57BL/6 mice were inoculated with B. parapertussis (Bpp) or Bpp and B. bronchiseptica (Bb) and given and I.P. injection of naïve (NS) or immune serum (IS). Bpp numbers were quantified 3 or 7 days later. (C) Bb numbers in the lungs of wild type mice were quantified 3 days post-inoculation upon a co-inoculation with Bpp with or without treatment with immune serum. (D) C57BL/6 mice were inoculated with Bpp or Bpp and heat-killed (HK) Bb and given an I.P. injection of NS or IS. Bacterial numbers were quantified 3 days later. Asterisks denote P-values < A similar experiment was performed by co-inoculating with B. parapertussis and heat- killed B. bronchiseptica to determine if the observed effect on B. parapertussis required live B. bronchiseptica. The addition of heat-killed B. bronchiseptica to the inoculum had a similar effect in that adding heat-killed bacteria alone resulted in a 10-fold reduction of B. parapertussis numbers within 3 days compared to untreated mice. Adding heat-killed B. bronchiseptica and treating with immune serum resulted in near complete elimination of the bacteria from the lungs 99

115 by 3 days post-inoculation (Fig. 5.6D). Together, these data indicate that a co-inoculation with B. bronchiseptica allows for more efficient control of B. parapertussis and suggest that not stimulating TLR4 could allow B. parapertussis to evade rapid antibody-mediated clearance. Rapid clearance of B. parapertussis upon a co-inoculation is dependent on TLR4. We hypothesized that the protective effects of adding B. bronchiseptica to the inoculum in vivo were due to the robust TLR4-mediated inflammatory response to this bacterium. To test this, groups of wild type C3H/HEOuJ and TLR4-deficient C3H/HEJ mice were inoculated with B. parapertussis or B. parapertussis and heat-killed B. bronchiseptica. They were then given an I.P. injection of naïve serum or immune serum and sacrificed 3 days later. Again, wild type mice co-inoculated with heat-killed B. bronchiseptica harbored approximately 10-fold fewer bacteria than untreated mice. Wild type mice coinoculated with heat-killed B. Figure 5.7: Effect of TLR4 on the rapid clearance of B. parapertussis. Groups of wild type (C3H/HEOuJ) and TLR4-deficient (C3H/HEJ) mice were inoculated with B. parapertussis (Bpp) or Bpp and heat-killed B. bronchiseptica (Bb) and given I.P. injections of naïve serum (NS) or immune serum (IS). Bacterial numbers were quantified 3 days later and are expressed as the Log 10 mean +/- S.D. Asterisks denote P-values < bronchiseptica and treated with immune serum reduced B. parapertussis in the lungs to nearly undetectable levels within 3 days (Fig. 5.7). However, B. parapertussis numbers were not reduced in the lungs of TLR4-deficient mice that were co-inoculated with B. bronchiseptica. Immune serum also had no effect on bacterial numbers upon the co-inoculation (Fig. 5.7). Thus, the rapid antibody-mediated clearance of B. parapertussis upon a co-inoculation was dependent on TLR4 signaling. Together, these data indicate that the evasion of pro-inflammatory TLR4 responses enables B. parapertussis to delay antibody-mediated clearance from its host. 100

116 Discussion: The elimination of B. parapertussis correlated with the accumulation of leukocytes in the lungs. Expressing an LPS that does not efficiently stimulate TLR4 allowed B. parapertussis to avoid inducing an early inflammatory response, delaying the accumulation of leukocytes. A coinoculation with B. bronchiseptica, which does induce an early inflammatory response, allowed for more efficient control of B. parapertussis numbers. Furthermore, inducing an early inflammatory response led to rapid antibody-mediated clearance of B. parapertussis. In TLR4- deficient mice, co-inoculation with B. bronchiseptica had no effect on B. parapertussis numbers indicating that the rapid clearance upon the co-inoculation was due to the TLR4 response. Together, these results provide an example of how a gram negative bacterium may optimize its ability to infect its host by avoiding the innate TLR4-mediated immune response. Compared to the closely-related animal pathogen, B. bronchiseptica, B. parapertussis and B. pertussis are able to avoid rapid antibody-mediated clearance (15-17). This is likely important to the success of these pathogens in human populations in which they are able to re-infect the same host multiple times. Our lab previously showed that Ptx confers the ability of B. pertussis to evade antibody-mediated clearance. Although B. pertussis LPS may induce proinflammatory TLR4 signaling, this toxin inhibits the migration of neutrophils to the lungs, delaying the clearance of the infection by antibodies (15). The lack of TLR4-mediated recruitment of leukocytes by B. parapertussis appears to have a similar effect on the course of infection by this species. Neutrophils are also required for the antibody-mediated clearance of this pathogen (17), thus, limiting the cellular recruitment to the site of infection delays the clearance of B. parapertussis. 101

117 The evasion of TLR4-mediated immunity has been suggested to contribute to the virulence of some gram negative bacteria (27-29). For example, Yersinia pestis produces a TLR4-stimulatory LPS at 26 o C, but an unstimulatory LPS at 37 o C, the typical mammalian body temperature (30-32). Montminy et.al. genetically modified Y. pestis so that it would produce the stimulatory 26 o C LPS at all times. While wild type Y. pestis causes sepsis and mortality in a mouse model of infection, the expression of the stimulatory LPS resulted in containment of the infection by the innate immune response, less efficient systemic spread of the infection, and an increased lethal dose (33). Similar to Y. pestis, temperature has an influence on the structure of B. parapertussis LPS (18). Unfortunately, we were unable to alter the genes responsible for the lack of TLR4 stimulation by B. parapertussis LPS since it is not known which genes are responsible for this. However, the exogenous stimulation of TLR4 by B. bronchiseptica resulted in an early influx of inflammatory cells and rapid, TLR4-mediated clearance of B. parapertussis from the lungs. These data support our hypothesis that B. parapertussis expresses an unstimulatory LPS in order to maximize its ability to colonize and persist in its host. Avoiding the induction of an inflammatory response allows a pathogen to colonize its niche without the antimicrobial effects of inflammatory cells such as neutrophils. Streptococcus pneumoniae is efficiently eliminated from the murine respiratory tract by neutrophils, but this pathogen does not induce a significant amount of inflammation, allowing it to persist in its host. However, when more neutrophils are recruited to the site of infection due to a concomitant infection, S. pneumoniae is rapidly cleared from its host (34). Similarly, the lack of stimulation of an early inflammatory response may enable a B. parapertussis infection to spread throughout the respiratory tract. While an inflammatory response could limit B. parapertussis to its initial 102

118 site of colonization, the evasion of an early inflammatory response may allow B. parapertussis to spread throughout the lung, optimizing its use of the host. Like B. pertussis, B. parapertussis appears to avoid antibody-mediated clearance by preventing the recruitment of phagocytes to the lungs. However, B. pertussis actively inhibits neutrophil migration while B. parapertussis simply appears to not induce much of a response in the first place. Overcoming the lack of neutrophil migration in response to B. parapertussis may be easier for the host immune response than overcoming the inhibitory effects of B. pertussis infection. This could potentially explain why B. parapertussis disease some times appears to be milder than that of B. pertussis (7-9). For example, if these pathogens colonize lungs in which they have little to no competition, they may behave similarly and cause identical disease. However, if there is a co-infection that induces inflammation, B. pertussis actively suppresses that response while B. parapertussis would remain susceptible to its effects. This could lead to more rapid clearance of B. parapertussis, and what some have reported as a shorter course of disease relative to B. pertussis (7-9). References: 1. Cherry, J.D. and U. Heininger (2004) Pertussis and other Bordetella infections, p In R. D. Feigin, J. D. Cherry, G. J. Demmler, and S. Kaplan (ed.), Textbook of pediatric infectious diseases, 5th ed. The W. B. Saunders Co, Philadelphia, Pa. 2. de Melker, H.E., J.F. Schellekens, S.E. Neppelenbroek, F.R. Mooi, H.C. Rumke, and M.A. Conyn-van Spaendonck (2000) Reemergence of pertussis in the highly vaccinated population of the Netherlands: observations on surveillance data. Emerg Infect Dis 6(4):

119 3. Skowronski, D.M., G. De Serres, D. MacDonald, W. Wu, C. Shaw, J. Macnabb, S. Champagne, D.M. Patrick, and S.A. Halperin (2002) The changing age and seasonal profile of pertussis in Canada. J Infect Dis 185(10): Celentano, L.P., M. Massari, D. Paramitti, S. Salmaso, A.E. Tozzi; EUVAC-NET Group (2005) Resurgence of pertussis in Europe. Pediatr Infect Dis J 24(9): von Konig, C.H., S. Halperin, M. Riffelmann, and N. Guiso (2002) Pertussis of adults and infants. Lancet Infect Dis 2(12): Centers for Disease Control and Prevention (2002) Pertussis--United States, JAMA 287(8): Bergfors, E., B. Trollfors, J. Taranger, T. Lagergard, V. Sundh, and G. Zackrisson (1999) Parapertussis and pertussis: differences and similarities in incidence, clinical course, and antibody responses. Int J Infect Dis 3(3): Heininger, U., K. Stehr, S. Schmitt-Grohe, C. Lorenz, R. Rost, P.D. Christenson, M. Uberall, and J.D. Cherry (1994) Clinical characteristics of illness caused by Bordetella parapertussis compared with illness caused by Bordetella pertussis. Pediatr Infect Dis J 13(4): Mastrantonio, P., P. Stefanelli, M. Giuliano, Y. Herrera Rojas, M. Ciofi degli Atti, A. Anemona, and A.E. Tozzi (1998) Bordetella parapertussis infection in children: epidemiology, clinical symptoms, and molecular characteristics of isolates. J Clin Microbiol 36(4):

120 10. Olson, L.C. (1975) Pertussis. Medicine (Baltimore) 54(6): Baldari, C. T., A. Lanzavecchia, and J.L. Telford (2005) Immune subversion by Helicobacter pylori. Trends Immunol 26(4): Sing, A., A. Roggenkamp, A.M. Geiger, and J. Heesemann (2002) Yersinia enterocolitica evasion of the host innate immune response by V antigen-induced IL-10 production of macrophages is abrogated in IL-10-deficient mice. J Immunol 168(3): Parkhill, J., M. Sebaihia, A. Preston, L. D. Murphy, N. Thomson, D. E. Harris, M. T. G. Holden, C. M. Churcher, S. D. Bentley, K. L. Mungall, A. M. Cerdeño-Tárraga, L. Temple, K. James, B. Harris, M. A. Quail, M. Achtman, R. Atkin, S. Baker, D. Basham, N. Bason, I. Cherevach, T. Chillingworth, M. Collins, A. Cronin, P. Davis, J. Doggett, T. Feltwell, A. Goble, N. Hamlin, H. Hauser, S. Holroyd, K. Jagels, S. Leather, S. Moule, H. Norberczak, S. O'Neil, D. Ormond, C. Price, E. Rabbinowitsch, S. Rutter, M. Sanders, D. Saunders, K. Seeger, S. Sharp, M. Simmonds, J. Skelton, R. Squares, S. Squares, K. Stevens, L. Unwin, S. Whitehead, B.G. Barrell, D.J. Maskell (2003) Comparative analysis of the genome sequences of Bordetella pertussis, Bordetella parapertussis and Bordetella bronchiseptica. Nat Genet 35(1): Carbonetti, N. H., G.V. Artamonova, C. Andreasen, and N. Bushar (2005) Pertussis toxin and adenylate cyclase toxin provide a one-two punch for establishment of Bordetella pertussis infection of the respiratory tract. Infect Immun 73:

121 15. Kirimanjeswara, G. S., L.M. Agosto, M.J. Kennet, O.N. Bjornstad, and E.T. Harvill (2005) Pertussis toxin inhibits neutrophil recruitment to inhibit antibody-mediated clearance of Bordetella pertussis. J Clin Invest 115(12): Kirimanjeswara, G.S., M. Pilione, and E.T. Harvill (2003) Role of antibodies in immunity to Bordetella infections. Infect Immun 71(4): Wolfe, D.N., G. Kirimanjeswara, and E.T. Harvill (2005) Clearance of Bordetella parapertussis from the lower respiratory tractrequires humoral and cellular immunity. Infect Immun 73(10): van den Akker, W.M (1998) Lipopolysaccharide expression within the genus Bordetella: influence of temperature and phase variation. Microbiology 144(Pt 6): Preston, A., B.O. Petersen, J.O. Duus, J. Kubler-Kielb, G. Ben-Menachem, J. Li, and E. Vinogradov (2006) Complete structures of Bordetella bronchiseptica and Bordetella parapertussis lipopolysaccharides. J Biol Chem 281(26): Caroff, M., L. Aussel, H. Zarrouk, A. Martin, J.C. Richards, H. Therisod, M.B. Perry, and D. Karibian (2001) Structural variability and originality of the Bordetella endotoxins. J Endotoxin Res 7(1): Mann, P.B., D.N. Wolfe, E. Latz, D. Golenbock, A. Preston, and E.T. Harvill (2005) Comparative toll-like receptor 4-mediated innate host defense to Bordetella infection. Infect Immun 73(12):

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123 (2006) Lack of in vitro and in vivo recognition of Francisella tularensis subsepecies lipopolysaccharide by Toll-like receptors. Infect Immun 74(12): Trent, M.S., C.M. Stead, A.X. Tran, and J.V. Hankins (2006) Diversity of endotoxin and its impact on pathogenesis. J Endotoxin Res 12(4): Kawahara, K., H. Tsukano, H. Watanabe, B. Lindner, and M. Matsuura (2002) Modification of the structure and activity of lipid A in Yersinia pestis lipopolysaccharide by growth temperature. Infect Immun 70(8): Rebeil, R., R.K. Ernst, B.B. Gowen, S.I. Miller, and B.J. Hinnebusch (2004) Variation in lipid A structure in the pathogenic yersiniae. Mol Microbiol 52(5): Knirel, Y.A., B. Lindner, E.V. Vinogradov, N.A. Kocharova, S.N. Senchenkova, R.Z. Shaikhutdinova, S.V. Dentovskaya, N.K. Fursova, I.V. Bakhteeva, G.V. Titareva, S.V. Balakhonov, O. Holst, T.A. Gremyakova, G.B. Pier, and A.P. Anisimov (2005) Temperature-dependent variations and intraspecies diversity of the structure of the lipopolysaccharide of Yersinia pestis. Biochemistry 44(5): Montminy, S.W., N. Khan, S. McGrath, M.J. Walkowicz, F. Sharp, J.E. Conlon, K. Fukase, S. Kusumoto, C. Sweet, K. Miyake, S. Akira, J.D. Goguen, E. Lien (2006) Virulence factors of Yersinia pestis are overcome by a strong lipopolysaccharide response. Nat Immunol 7(10):

124 34. Lysenko, E.S., A.J. Ratner, A.L. Nelson, and J.N. Weiser (2005) The role of innate immune responses in the outcome of interspecies competition for colonization of mucosal surfaces. PLoS Pathog 1(1):e1. 109

125 Chapter 6: Induction of Interleukin-10 by Bordetella parapertussis Limits the Interferon-γ Response and Bacterial Clearance 110

126 Abstract: For pathogens that spread directly from host to host, transmissibility is proportional to the length of the infectious period. Therefore, transmissibility could be enhanced if the host immune response was modulated to allow longer persistence within the host. Since B. parapertussis, one of the causative agents of whooping cough, spreads directly between hosts via aerosolized droplets, the ability to persist in the respiratory tract may be important to its ability to spread from host to host. Here, we have used a mouse model of infection to determine the immune factors that contribute to the clearance of B. parapertussis and how this bacterium may evade these immune responses. Clearance of this pathogen from the lungs correlated with the expansion of CD4+ T cell numbers in the lungs and IFN-γ production by these cells. IFN-γdeficient mice were defective in reducing B. parapertussis numbers in the lungs. Additionally, this cytokine contributed to the recruitment of phagocytic cells to the site of infection. While IFN-γ proved to be protective, B. parapertussis induced the production of IL-10 which inhibited the generation of an IFN-γ response and the elimination of this bacterium from the respiratory tract. Importantly, these studies suggest that vaccination strategies that induce a Th1-skewed immune response may be beneficial to protecting against B. parapertussis infection and disease. Introduction: IFN-γ is largely produced by natural killer cells and activated T cells and serves to potentiate the immune response to microbial pathogens (1). Thus, the inhibition of IFN-γ responses by a pathogen can substantially affect its course of infection. For example, the inhibition of IFN-γ production by LcrV toxin enhances the lethality of Yersinia pestis in a murine model of infection (2). Additionally, microbes modulate specific aspects of IFN-γinduced signaling pathways to optimize their interactions with the host. The chlamydiae inhibit 111

127 MHC class II upregulation that is induced by IFN-γ, limiting the capacity of CD4+ T cells to recognize these pathogens (3). Despite the protective role of this cytokine in the immune response to a wide range of pathogens (4-5), however, its production must be tightly regulated in order to avoid unnecessary damage to host tissues. IFN-γ potentiates persistent pathology of the cornea caused by Pseudomonas aeruginosa (6) and chronic eczema caused by Staphylococcus aureus (7) among other chronic inflammatory diseases. Produced by a variety of cell types, Interleukin (IL)-10 is an anti-inflammatory cytokine that inhibits the production of certain proinflammatory cytokines including IFN-γ (8-9). In addition to modulating cytokine production via its suppressive effects on antigen presenting cells, IL-10 upregulates SOCS1, the gene responsible for the majority of the physiological inhibition of IFN-γ-induced signaling (10-11). Several bacterial pathogens dampen the IFN-γ response by stimulating the production of IL-10, often resulting in an enhanced ability to grow in numbers or persist within the host (12-16). Bordetella pertussis and B. parapertussis are the etiologic agents of whooping cough, a severe coughing illness that is re-emerging in vaccinated populations (17-21). However, the relative role of B. parapertussis in the recent resurgence of whooping cough is unclear (reviewed in 22). Serological data has suggested that as much as 60 percent of a population had recently been exposed to this pathogen (23), indicating that B. parapertussis is endemic and efficiently transmits among humans. Since B. parapertussis spreads directly from host to host via aerosolized droplets, the ability to persist in the host respiratory tract would likely enhance its transmissibility. We previously documented that CD4+ T cells are important to the elimination of B. parapertussis from the lower respiratory tract (24). Here we show that the reduction in bacterial 112

128 numbers correlates with the expansion of CD4+ T cell populations in the lungs and the production of IFN-γ by these cells. This cytokine appears to facilitate clearance of B. parapertussis, at least in part, by contributing to the inflammatory response. However, B. parapertussis inhibits the IFN-γ response by inducing the production of IL-10 which subsequently results in delayed clearance of the bacteria from the lungs. Thus, B. parapertussis appears to modulate the IFN-γ response in order to persist in its host. Materials and Methods: Bacterial strains and growth. B. parapertussis strain 12822G is a gentamicin-resistant derivative of B. parapertussis strain from the German clinical trials (24) and has previously been described (25). B. parapertussis was maintained on Bordet-Gengou agar (Difco) containing 10 % defibrinated sheep blood (Hema Resources) and appropriate antibiotics. Liquid culture bacteria were grown at 37 C overnight on a roller drum to mid-log phase (an O. D. of approximately 0.3) in Stainer-Scholte broth. Inoculation of mice. C57BL/6, TCRαδ -/-, IFN-γ -/-, and IL-10 -/- mice were obtained from Jackson Laboratories and bred in our Bordetella-free, specific pathogen-free facilities at The Pennsylvania State University. Bacteria grown overnight (to an O.D. of approximately 0.3) in liquid culture were diluted in PBS to approximately 10 7 CFU/ml. Groups of 4-6 week old mice were lightly sedated with 5% isoflurane in oxygen and inoculated by pipetting 50 μl of the inoculum (5 x 10 5 CFU) on to the external nares. All protocols were reviewed by the university s IACUC and all animals were handled in accordance with institutional guidelines. Adoptive transfer of serum antibodies. To generate immune serum, C57BL/6 mice were inoculated with B. parapertussis and allowed to convalesce for 28 days. Blood was then collected from these mice and the serum portion was isolated and stored at -80 o C until use

129 μl of this convalescent phase serum or serum from naïve mice was injected I.P. into mice at the time of inoculation. Quantification of bacteria and leukocytes in the lungs. To quantify bacteria, the lungs were excised on day 0, 3, 7, 14, or 28 post-inoculation. Lungs were homogenized in 1 ml of PBS, and the homogenate was then plated on to Bordet-Gengou agar plates at the appropriate dilutions. CFU were counted after incubating for 4 days at 37 o C. To quantify leukocytes, mice were infected for 0, 3, 7, 14, or 28 days, sacrificed, and BAL fluid was collected. Red blood cells were lysed by treating with 0.83% ammonium chloride. Leukocytes were counted on a hemocytometer to quantify total numbers in the BAL fluid. Aliquots of cells were then analyzed by flow cytommetry to obtain the percentage of leukocytes that were neutrophils or CD4+ T cells. Cells were stained with FITC-labled anti-ly-6g for neutrophils or FITC-labeled anti-cd4, and the percentage of Ly-6G positive or CD4+ cells was multiplied by the total number of leukocytes to calculate the total number of neutrophils or CD4+ T cells, respectively. Splenocyte restimulations. Splenocytes were isolated by homogenizing spleens through a wire sieve, spinning at 400 x g for 5 min at 4 C, lysing the red blood cells with 0.83% ammonium chloride, and then washing the cells with Dulbecco's modified Eagle cell culture medium (HyClone, Logan, Utah, USA). 2 x 10 6 cells were re-suspended in Dulbecco's modified Eagle medium supplemented with 10% fetal calf serum (HyClone), 1 mm sodium pyruvate (HyClone), 100 µg/ml penicillin and streptomycin (HyClone), and 0.005% beta-mercaptoethanol and placed into wells of a 96-well plate. Splenocytes were stimulated with medium or 10 7 heat-killed B. pertussis or B. parapertussis. After three days, the supernatant was collected and analyzed for IFN-γ and IL-10 production as specified by the manufacturers of ELISA kits (R & D Systems, Minneapolis, Minnesota, USA). 114

130 Statistical Analysis. The mean +/- standard deviation (error bars) was determined for CFU, leukocytes, and cytokines. Two-tailed, unpaired Student s T-tests were used to determine statistical significance between groups. All experiments were performed at least twice with similar results and P-values < 0.05 were taken to be statistically significant. Results: Clearance of B. parapertussis correlates with the expansion of CD4+ T cell populations in the lungs. We have previously shown that CD4+ T cells are essential to the clearance of B. parapertussis (24) and sought to determine if the elimination of B. parapertussis from the respiratory tract correlated with increasing numbers of CD4+ T cells. C57BL/6 mice were inoculated with B. parapertussis and sacrificed on day 0, 3, 7, 14, or 28 postinoculation to quantify bacterial numbers in the lungs or numbers of CD4+ T cells in the BAL fluid. B. parapertussis numbers peaked at CFU on day 3 postinoculation and declined thereafter until bacteria were nearly undetectable by day 28 post-inoculation (Fig. 6.1). CD4+ T cells were nearly undetectable on days 0 and 3 post-inoculation but had increased to Figure 6.1. Reduction of B. parapertussis numbers in the lungs correlates with increased CD4+ T cell numbers. C57BL/6 mice were inoculated with B. parapertussis and sacrificed on day 0, 3, 7, 14, or 28 post-inoculation to quantify bacterial numbers in the lungs or CD4+ T cells in the BAL fluid. Bacterial numbers are expressed as the Log10 mean +/- standard deviation. CD4+ T cell numbers are expressed as the mean +/- standard deviation. 115

131 approximately 3 x 10 3 cells/ml of BAL fluid by days 7 and 14 post-inoculation and 2 x 10 4 cells/ml by day 28 post-inoculation (Fig. 6.1). Thus, the reduction of B. parapertussis numbers in the lungs correlated with an increase in numbers of CD4+ T cells. T cells produce IFN-γ which facilitates the clearance of B. parapertussis. Since the production of cytokines by T cells can contribute to protection against microbial pathogens, the cytokine response to B. parapertussis was measured. Specifically, IFNγ production was assessed because this cytokine is protective against B. pertussis (26-27) and B. bronchiseptica (15). To address IFN-γ production by T cells, C57BL/6 and TCRαδ -/- mice were inoculated with B. parapertussis, sacrificed on day 0, 3, 7, 14, or 28 post-inoculation, and their spleens were excised for the restimulation of splenocytes as a representative of the immune response to B. pertussis. IFN-γ was produced at low levels (< 50 pg) by unstimulated splenocytes from either mouse strain. Upon stimulation by B. parapertussis, splenocytes from wild type mice produced low levels of IFN-γ at days 0, 3, and 14 post-inoculation, but produced approximately 1000 pg and 200 pg at days 7 and 28 respectively (Fig. 6.2A). B. parapertussisstimulated splenocytes from T cell-deficient mice produced low levels of IFN-γ at all time points (Fig. 6.2A). These data indicated that T cells produced the majority of this cytokine on days 7 and 28. The IFN-γ production 7 days post-inoculation correlated with the time when B. parapertussis numbers began to decline while the production at day 28 correlated with the time when the bacteria were nearly cleared from the lungs. 116

132 Since T cells produced IFN-γ in response to B. parapertussis and its production correlated with the reduction of bacterial numbers, we hypothesized that IFN-γ is required for efficient clearance of B. parapertussis. C57BL/6 and IFN-γ -/- mice were inoculated with B. parapertussis and sacrificed on day 0, 3, 7, 14, or 28 postinoculation for the quantification of bacterial numbers. B. parapertussis numbers peaked at approximately CFU on day 3 post- inoculation and declined thereafter until they were nearly undetectable by day 28 postinoculation (Fig. 6.2B). Bacterial numbers in the lungs of IFN-γ -/- mice were similar to those of wild type mice on days 3 and 7. Figure 6.2. T cells produce IFN-γ which contributes to bacterial clearance. (A) C57BL/6 and TCRαδ -/- mice were inoculated with B. parapertussis and sacrificed 0, 7, 14, or 28 days later. Splenocytes were exposed to media or heat-killed B. parapertussis (Bpp) and monitored for the production of IFN-γ. (B) C57BL/6 and IFN-γ -/- mice were inoculated with B. parapertussis and sacrificed on day 0, 3, 7, 14, or 28 for the quantification of bacterial numbers. IFN-γ is expressed as the mean +/- standard deviation and CFU are expressed as the Log 10 mean +/- standard deviation. Asterisks represent P-values < However, IFN-γ -/- mice harbored approximately 10-fold more bacteria on days 14 and 28 post- that IFN-γ contributes to the elimination of B. inoculation (Fig. 6.2B). These data indicate parapertussis from the lungs. IFN-γ contributes to the recruitment of leukocytes to the lungs upon B. parapertussis infection. Compared to the other bordetellae, B. parapertussis induces a very low level of inflammation early after inoculation (28), but neutrophils are important to bacterial clearance 117

133 (25) and peak in numbers at around 7 days post-inoculation (Fig. 5.1). Thus, we hypothesized that IFN-γ played an important role in overcoming the lack of an early pro-inflammatory stimulus by contributing to the recruitment of leukocytes to the lungs later in infection. To address the role of IFN-γ in leukocyte recruitment, C57BL/6 and IFN-γ -/- mice were inoculated with B. parapertussis and sacrificed on day 3, 7, 14, or 28 post-inoculation to quantify the numbers of leukocytes in the BAL fluid. As previously shown, few leukocytes had accumulated in the BAL fluid of wild type mice by 3 days (~5 x 10 5 ), but leukocyte numbers peaked around day 7 post-inoculation (~2 x 10 6 ) and declined thereafter (5.1B, Fig. 6.3A). Leukocyte numbers were also low in the BAL fluid of IFN-γ -/- mice on day 3 post-inoculation (~5 x 10 5 ) but remained low even on day 7 post-inoculation (~8 x 10 5 ) (Fig. 6.3A). Like IFN-γ -/- mice, T cell- to deficient mice were also defective in the accumulation of leukocytes in the lungs compared wild type mice (data not shown). Similar results were observed when we specifically quantified neutrophils or macrophages (Fig. 6.3B-C) but not T cells (data not shown). Therefore, IFN-γ contributes to the recruitment of leukocytes to the lungs in response to B. parapertussis. 118

134 Figure 6.3. IFN-γ contributes to leukocyte accumulation in response to B. parapertussis infection. C57BL/6 and IFN-γ -/- mice were inoculated with B. parapertussis and sacrificed on day 3, 7, 14, or 28 to quantify (A) total leukocytes, (B) neutr ophils, or (C) macrophages in the BAL fluid. (D) TCRαδ -/- and IFN-γ -/- mice were inoculated with B. parapertussis (Bpp) or Bpp and heat-killed B. bronchiseptica (HK Bb). Mice were then treated with naïve serum (NS) or immune serum (IS) and sacrificed 3 days later. Cell numbers are expressed as the mean +/- standard deviation and CFU are expressed as the Log 10 mean +/- standard deviation. Asterisk represents P-value < The lack of TLR4 stimulation by B. parapertussis is responsible for the lack of a significant inflammatory response within the first few days post-inoculation (28). Since T cells and IFN-γ may facilitate bacterial clearance by contributing to the inflammatory response to B. parapertussis, we expected that inducing the recruitment of leukocytes by TLR4 stimulation would abolish the need for T cells and IFN-γ. TLR4 was stimulated by adding heat-killed B. bronchiseptica to a B. parapertussis inoculum. TCRαδ -/- and IFN-γ -/- mice were inoculated with B. parapertussis or B. parapertussis and heat-killed B. bronchiseptica, treated with naïve serum or immune serum, and bacterial numbers were quantified three days later. This co-inoculation resulted in more efficient control and rapid antibody-mediated clearance of B. parapertussis from wild type mice (Fig. 5.6B). Compared to being inoculated with only B. parapertussis, the 119

135 co-inoculation led to a 10-fold reduction of bacterial numbers in the lungs of TCRαδ -/- and IFNγ -/- mice. Furthermore, treating these mice with convalescent phase serum had no effect on colonization upon inoculation by only B. parapertussis, but reduced B. parapertussis numbers by 10, ,000-fold upon the co-inoculation with B. bronchiseptica (Fig. 6.3D). This indicated that T cells and IFN-γ were not crucial to the reduction of B. parapertussis numbers if an inflammatory response is induced by some other means (Fig. 6.3D). Together, these data indicate that T cells and IFN-γ are required for efficient reduction of B. parapertussis numbers and suggest that their key role involves the recruitment of other leukocytes to the site of infection. B. parapertussis modulates the IFN-γ response by inducing IL-10 production. IFN-γ is important to the clearance of B. parapertussis from the lungs, but its production can be inhibited by IL-10 (29), an anti-inflammatory cytokine that is induced by B. pertussis and B. bronchiseptica (15,30). Therefore, we investigated whether IL-10 was also produced in response to B. parapertussis infection. C57BL/6 and TCRαδ -/- mice were inoculated with B. parapertussis and sacrificed on day 0, 3, 7, 14, or 28 post-inoculation. Splenocytes were isolated and exposed to B. parapertussis in order to determine if IL-10 was being produced by these cells. IL-10 was nearly undetectable in the supernatants of unstimulated splenocytes. Low levels of IL-10 (< 50 pg) were observed in the supernatants of stimulated splenocytes from mice that had been infected for 7 days. However, large amounts of IL-10 were produced by splenocytes from wild type (200, 700, and 4000 pg) or T cell-deficient (350, 400, and 600 pg) mice that had been infected for 3, 14, or 28 days, respectively (Fig. 6.4). Together, these data indicate that B. parapertussis induced the production of IL-10, and some cells other than T cells are the major source during the first two weeks of infection but T cells contribute more thereafter. 120

136 We then sought to determine if the IL-10 response induced by B. parapertussis limited the IFN-γ response to this pathogen. To do so, the amount of IFN-γ produced by splenocytes from C57BL/6 versus IL-10 -/- mice in response to B. parapertussis was compared. Unstimulated splenocytes, as well as B. parapertussis-stimulated splenocytes from naïve mice produced low levels of both IFN-γ and IL-10. B. parapertussis-stimulated splenocytes from convalescent wild type mice produced approximately 5000 pg of IL-10 but only Figure 6.4. IL-10 production in response to B. parapertussis. C57BL/6 and TCRαδ -/- mice were inoculated with B. parapertussis and sacrificed on day 0, 3, 7, 14, or 28 post-inoculation for the quantification of IL-10 production by splenocytes. P-values < 0.05 when comparing media control to B. parapertussis- stimulated (Bpp) are denoted by (a) for C57BL/6 or (b) for TCRαδ -/- and (c) represents P-value < 0.05 when comparing Bpp stimulated C57BL/6 and TCRαδ -/- mice. The lower limit of detection was 50 pg. 200 pg of IFN-γ (Fig. 6.5). In contrast, B. parapertussis-stimulated splenocytes from convalescent IL-10 -/- mice produced approximately 6000 pg of IFN-γ (Fig. 6.5). Together, these data indicate that IL-10 inhibits the IFN-γ response to B. parapertussis. Figure 6.5. Effect of IL-10 on the IFN-γ response to B. parapertussis. Splenocytes from naïve or convalescent (Bpp imm.) C57BL/6 and IL-10 -/- mice were exposed to media (unstim) or heat-killed B. parapertussis (Bpp stim.) IFN-γ and IL-10 production was quantified. The lower limit of detection was 50 pg and asterisks represent P- values <

137 IL-10 inhibits the clearance of B. parapertussis from the respiratory tract. If inhibiting IFN-γ production via IL-10 induction was a strategy used by B. parapertussis to persist in its host, it would be expected that this pathogen would be eliminated more rapidly from IL-10-deficient hosts. C57BL/6 and IL-10 -/- mice were inoculated with B. parapertussis and sacrificed on day 0, 3, 7, 14, or 28 post-inoculation for the quantification of bacterial numbers in the lungs. B. parapertussis numbers peaked at ~ CFU in the lungs of wild type mice on day 3 post-inoculation and were reduced to ~ and less than 100 CFU by days 14 and 28 respectively (Fig. 6.6). In the lungs of IL-10 - /- mice, bacterial numbers also peaked at ~ CFU on day 3 post-inoculation but were reduced to less than 100 CFU by 14 and were Figure 6.6. Effect of IL-10 on clearance of B. parapertussis from the lungs. C57BL/6 and IL- 10 -/- mice were inoculated with B. parapertussis and sacrificed on day 0, 3, 7, 14, or 28 to quantify bacterial numbers in the lungs. CFU are expressed as the Log 10 mean +/- standard deviation and asterisks represent P-values < undetectable by day 28 (Fig. 6.6). These data indicate that IL-10 inhibits the elimination of B. parapertussis from murine lungs. Discussion: Here, we have uncovered a strategy used by B. parapertussis to persist in the respiratory tract of its host. In a murine model of infection, B. parapertussis numbers increase rapidly over the first few days post-inoculation and decline slowly thereafter. The reduction of bacterial numbers correlates with the expansion of CD4+ T cell numbers in the lungs and the production of IFN-γ by T cells. T cells and IFN-γ appear to facilitate bacterial clearance, at least in part, by contributing to the inflammatory response to B. parapertussis. However, the IFN-γ response to 122

138 this pat hogen is dampened by the induction of an IL-10 response. The inhibition of the IFN-γ response appears to limit the ability of the host to eliminate B. parapertussis from the lungs. IFN-γ has multiple roles in augmenting both the innate and adaptive immune responses. Other models of infection have shown this cytokine to contribute to the recruitment of leukocytes to the site of infection and the activation of the phagocytic capability of these leukocytes (31-32). IFN-γ also upregulates the expression of MHC class I and II molecules, as well as other costimulatory molecules, by antigen presenting cells (33-34) and drives the differentiation of naïve T cells to Th1 cells (35). In the case of B. parapertussis, the most important role that IFN-γ plays in bacterial clearance appears to be the recruitment and/or activation of leukocytes. This is supported by the fact that stimulating TLR4, and consequently an early inflammatory response, allowed for efficient control and clearance of B. parapertussis even in the absence of IFN-γ. Since an IFN-γ response to B. parapertussis is important to protection and IL-12 is involved in the differentiation of naïve T cells to Th1 cells, IL-12 production by macrophages may be an important early step in generating an effective immune response against B. parapertussis. Like B. parapertussis, B. bronchiseptica and B. pertussis also stimulate the production of IL-10. B. bronchiseptica elicits the production of IL-10 via its Type III Secretion System (15), a virulence factor that is not expressed by B. parapertussis. Adenylate cyclase toxin and LPS synergize to promote the production of IL-10 in response to B. pertussis (30). This pathway to IL-10 production may also be used by B. parapertussis as this pathogen expresses adenylate cyclase toxin and LPS. Interestingly, IL-10 inhibits the clearance of B. bronchiseptica and B. parapertussis from the respiratory tract (15, Fig. 6.6), but this cytokine does not appear to affect the course of B. pertussis infection (unpublished data). This could potentially be explained by 123

139 the expression of Ptx by B. pertussis. For B. bronchiseptica and B. parapertussis, IL-10 limits the IFN-γ-mediated inflammatory response. When IL-10 is absent, a stronger inflammatory response may result in more efficient clearance of these pathogens. If IL-10 is absent during an infection by B. pertussis, more IFN-γ may be produced. However, the prevention of the migration of leukocytes to the site of infection by Ptx (36) could render the increased IFN-γ ineffective at enhancing bacterial clearance. Our data suggest that the early IL-1response to B. parapertussis. Macrophages and/or dendritic cells are the likely sources of this may be produced by cells other than T cells in early IL-10. Type I activated macrophages typically produce IL-12 upon exposure to bacteria, but co-stimulation with IL-4 results in Type II activation and the upregulation of IL-4 and IL-10 (reviewed in 37). Similarly, the cross-linking of Fc receptors by immune complexes results in type II activation and the production of large am ounts of IL-10 by macrophages (38). Dendritic cells also have the potential to produce large quantities of IL-10 (39), in some cases serving to dampen the immune response to microbial commensals (40). However, pathogens may also promote IL-10 production by macrophages and dendritic cells in order to dampen the protective immune response to themselves. The effects of IL-10 and IFN-γ on infection by B. parapertussis should be considered when creating the next generation of whooping cough vaccines. The incidence of both B. pertussis and B. parapertussis dropped upon the introduction of the first whooping cough vaccines (41-42). However, the change from whole cell to acellular vaccines has roughly correlated with the recent resurgence of whooping cough. Whole cell vaccines stimulate a relatively balanced Th1/Th2 response, but current acellular vaccines stimulate a Th2-skewed T cell response (43-45). Since IFN-γ is now known to be protective against both B. pertussis (26) 124

140 and B. parapertussis (Fig. 6.2), it seems likely that modifying vaccines to induce more of a Th1 response would help reduce the incidence of whooping cough. Importantly, multiple studies have indicated that acellular vaccines are less effective against B. parapertussis infection and disease than whole cell vaccines (46-47). The inefficacy of acellular vaccines could potentially lead to an increase in the prevalence of B. parapertussis relative to B. pertussis. In fact, there is evidence suggesting that B. parapertussis incidence may have increased since the introduction of acellular vaccines (47). Due to the rarity of differential diagnoses, unfortunately, the relative role of B. parapertussis in the current resurgence of whooping cough remains unknown. Regardless, designing vaccine strategies aimed at inducing a more balanced T cell response would likely be more efficacious against both B. parapertussis and B. pertussis. References: 1. Cooper, A.M., L.B. Adams, D.K. Dalton, R. Appelberg, and S. Ehlers (2002) IFNgamma and NO in mycobacterial disease: new jobs for old hands. Trends Microbiol 10(5): Brubaker, R.R. (2003) Interleukin-10 and Inhibition of Innate Immunity to Yersiniae: Roles of Yops and LcrV (V Antigen). Infect Immun 71(7): Zhong, G., T. Fan, and L. Liu (1999) Chlamydia inhibits interferon-gamma-inducible major histocompatability complex class II expression by degradation of upstream stimulatory factor I. J Exp Med 189(12): Novelli, F. and J.L. Casanova (2004) The role of IL-12, IL-23, and IFN-gamma in immunity to viruses. Cytokine Growth Factor Rev 15(5):

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147 Chapter 7: O antigen Enables Bordetella parapertussis to Avoid Bordetella pertussis-induced Immunity 132

148 Abstract: Whooping cough is re-emerging in human populations and is caused by two different but closely related endemic human pathogens, Bordetella pertussis and B. parapertussis. Despite how closely-related these pathogens are, their co-existence, and the limited efficacy of B. pertussis vaccines against B. parapertussis, suggests a lack of cross protective immunity between the two. We sought to address the ability of infection-induced immunity against one of these pathogens to protect against subsequent infection by the other using a mouse model of infection. Immunity induced by B. parapertussis infection protected against subsequent infections by either species. However, immunity induced by B. pertussis infection prevented subsequent B. pertussis infections but did not protect against B. parapertussis infections. The O antigen of B. parapertussis inhibited binding of antibodies to the bacterial surface and was required for B. parapertussis to colonize B. pertussis-convalescent mice. Thus, the O antigen of B. parapertussis confers asymmetrical cross immunity between the causative agents of whooping cough. We propose that these findings warrant investigation of the relative role of B. parapertussis in the resurgence of whooping cough. Introduction: The co-existence of B. pertussis and B. parapertussis in human populations creates a paradox to the ecological theory that two closely related, immunizing pathogens can not occupy the same host population if immunity is cross protective (1-5). Because B. pertussis and B. parapertussis evolved from a common progenitor, B. bronchiseptica, and share the majority of their virulence factors (6), cross immunity could be expected. Furthermore, genetic comparisons of B. pertussis and B. parapertussis to B. bronchiseptica suggest that B. parapertussis emerged as a pathogen more recently than B. pertussis (6-8). Since the mean age of primary B. pertussis 133

149 infection of unvaccinated children is less than fiv e years (9) and infection-induced immunity lasts approximately 5-10 years (10), it is likely that B. parapertussis successfully emerged in populations that had some level of immunity to B. pertussis (11). Although adapted to humans, both organisms efficiently colonize and rapidly grow throughout the murine respiratory tract, but are ultimately eliminated from the lower respiratory tract by B and T cell-dependent immunity (12-15). Watanabe et al. suggested that an immune response induced by one species conferred efficient protection against infections by either species (16). However, bacterial numbers were quantified only two weeks post-inoculation, when bacterial numbers were significantly reduced even in the respiratory tracts of naïve hosts (12,16). Additionally, the strain used in that study (18-323) is quite distinct from other B. pertussis strains by multi-locus enzyme electrophoresis and multi-locus sequence typing (17-18) and may actually be more closely related to B. bronchiseptica and B. parapertussis strains than other B. pertussis strains (19-20). These problems, along with clinical studies showing that B. pertussis vaccines poorly protect against B. parapertussis and these two organisms co-exist in the same populations, led us to investigate cross protective immunity between these two pathogens. We sought to determine whether or not B. pertussis and B. parapertussis induced effective cross immunity using the sequenced prototype strains Tohama I (B. pertussis) (6) and (B. parapertussis) (6), which are indistinguishable from current isolates by multi-locus enzyme electrophoresis and multi-locus sequence typing (17-18). B. parapertussis-induced immunity protected against both species, however B. pertussis-induced immunity protected only against B. pertussis infection. The asymmetrical cross immunity appeared to be the result of inefficient binding of B. pertussis-induced antibodies to B. parapertussis. O antigen prevented B. 134

150 pertussis-induced antibodies from binding to B. parapertussis in vitro and from clearing the bacteria from the respiratory tract in vivo. Together these data provide a molecular basis for the ability of B. parapertussis to avoid B. pertussis-induced immunity, an ability that may be critical to the co-existence of these pathogens in human populations. Materials and Methods: Bacterial Strains and Growth. B. pertussis strains 536 and 536 NaI (from Dr. Duncan Maskell, University of Cambridge, UK) are streptomycin- and nalidixic acid-resistant derivatives of Tohama I, respectively (21). B. parapertussis strain has been described (22), and 12822G is a gentamycin-resistant derivative of the parent strain (13). B. parapertussis strains CN2591 (wild type) and CN2591Δwbm (O antigen deficient) have previously been described (23). All were maintained on Bordet-Gengou agar (Difco) containing 10 % defibrinated sheep blood (Hema Resources) and appropriate antibiotics. Liquid culture bacteria were grown at 37 C overnight on a roller drum to mid-log phase in Stainer-Scholte broth. Animal experiments. C57BL/6 mice were obtained from Jackson laboratories (Bar Harbor, Maine, USA) and bred in our Bordetella-free, specific pathogen-free breeding rooms at The Pennsylvania State University. 4-6 week old mice were sedated with 5% isoflurane (Abbott laboratories) in oxygen and inoculated by pipetting 50 μl of PBS containing 5 x 10 5 CFU onto the external nares (12). For challenge experiments, mice were inoculated with 5 x 10 5 CFU of antibiotic-resistant strains at 28 or 70 days post-inoculation. For passive transfer of immune serum, 200μL of sera from naïve or convalescent mice (collected 28 days post-inoculation) was I.P. injected just prior to inoculation. All protocols were reviewed by the university IACUC and all animals were handled in accordance with institutional guidelines. 135

151 Bacterial quantification. Mice were sacrificed immediately after or 3 days post-inoculation to quantify bacteria in the nasal cavities, tracheae, and lungs. Tissues were homogenized in PBS, plated at specific dilutions onto Bordet-Gengou agar containing appropriate antibiotics, and colonies were counted four days later. The lower limit of detection was 10 CFU (the lower limit of the Y-axes). Splenocyte re-stimulations. Splenocytes were isolated by homogenizing spleens through a wire sieve, spinning at 400 x g for 5 min at 4 C, lysing the red blood cells with 0.83% ammonium chloride, and then washing the cells with Dulbecco's modified Eagle cell culture medium (HyClone, Logan, Utah, USA). 2 x 10 6 cells were re-suspended in Dulbecco's modified Eagle medium supplemented with 10% fetal calf serum (HyClone), 1 mm sodium pyruvate (HyClone), 100 µg/ml penicillin and streptomycin (HyClone), and 0.005% beta-mercaptoethanol and placed into wells of a 96-well plate. Splenocytes were stimulated with medium or 10 7 CFU of heatkilled B. pertussis or B. parapertussis. After three days, the supernatant was collected and analyzed for IFN-γ and IL-10 production as specified by the manufacturers of ELISA kits (R & D Systems, Minneapolis, Minnesota, USA). Enzyme-linked immunosorbent assays (ELISAs). Bacteria were grown to an optical density of 0.7, heat-inactivated, diluted in carbonate buffer, and used to coat 96-well plates. Plates were stored at 4 C (wells were filled with PBS-T plus 1% bovine serum albumin) until use. For ELISAs using live bacteria at the start of the assay, bacteria were grown to an optical density of 0.7, diluted in carbonate buffer, added to each well of a 96-well plate, and incubated for 1 hour at 37 C prior to use. A 1:25 dilution (for heat-inactivated bacteria) or a 1:10 dilution (for live bacteria) of serum samples was added to the first wells and serially diluted across the plates. Plates were incubated for 2 hours at 37 C in a humidified chamber, washed, and goat anti-mouse 136

152 (Ig H+L) HRP-conjugated antibodies (Southern Biotech, Birmingham, Alabama, USA) were added. Plates were incubated at 37 C in a humidified chamber for 1 h, washed, and 2,2'-Azinobis(3-ethylbenz-thiazoline-6-sulfonic acid) in a phospho-citrate buffer and hydrogen peroxide was added to wells which were incubated at room temperature in the dark for 30 min and read at an absorbance of 405 nm. Western blots. Westerns Blots were performed on lysates containing 1 x 10 8 CFU of heat-killed B. pertussis or B. parapertussis. Lysates were run on 7% SDS-PAGE gels in denaturing conditions. Membranes were probed with serum from B. pertussis or B. parapertussis-infected mice at a 1:100 dilution and goat anti-mouse (Ig H+L) HRP-conjugated (Southern Biotech, Birmingham, Alabama, USA) (at a dilution of 1:15,000) as the detector antibody. The membrane was visualized with ECL Western Blotting Detection Reagents (Amersham Biosciences, Piscataway, NJ). Statistical Analysis. The mean +/- standard deviation (error bars) was determined for CFU, leukocytes, IFN-γ production, and antibody titers. Two-tailed, unpaired Student s T-tests were used to determine statistical significance between groups. Results were also analyzed by nonparametric Mann-Whitney tests with similar significance. All experiments were performed at least twice with similar results. Results: Cross immunity between B. pertussis and B. parapertussis is asymmetrical. The co-existence of B. pertussis and B. parapertussis in humans led us to assess the ability of these pathogens to induce cross protective immunity. Although considered humanadapted pathogens, both species efficiently colonize the murine respiratory tract (24-25) and induce sterilizing protective immunity (12), allowing us to test cross protection in a mouse 137

153 model. Mice were left uninfected or inoculated with B. pertussis or B. parapertussis. 28 days later, when strong immune responses had been generated and bacteria had been reduced to less than 1000 CFU/lung (12-13), these mice were challenged with antibiotic-resistant strains of B. pertussis or B. parapertussis and sacrificed three days later. In comparison to naïve mice, B. pertussis-convalescent mice harbored antibiotic-resistant B. pertussis numbers that were approximately 100-fold lower in the nasal cavity and more than 10,000-fold lower in the trachea and lungs at three days post-challenge (Fig. 7.1A-C). An immune response to B. parapertussis Figure 7.1: B. pertussis colonization of naive or immunized mice. C57BL/6 mice were inoculated with 5 x 10 5 CFU of B. pertussis (Bp) or B. parapertussis (Bpp). Immunized and naïve mice were challenged 28 days later with 5 x 10 5 CFU of a nalidixic acid-resistant strain of Bp and sacrificed 3 days post-secondary inoculation for the quantification of bacterial numbers in the nasal cavity (A), trachea (B), and lungs (C). Values are expressed as the Log 10 mean +/- SD. For day 3 colonization levels, the difference between each individual value and the mean Log 10 CFU approximately 10 minutes after inoculation in the nasal cavity (D), trachea (E), and lungs (F) was calculated. These data are represented as the mean change in Log 10 CFU +/- SD. One asterisk represents P-values < 0.05, two asterisks represent P-values < 0.01 when compared to naïve mice. Infect Immun 75(10):

154 also conferred effective protection against B. pertussis as bacterial numbers were approximately 100-fold lower in the nasal cavity and 100,000-fold lower in the trachea and lungs of B. parapertussis-convalescent mice compared to naïve mice (Fig. 7.1A-C). Groups of mice were also sacrificed 10 minutes after inoculation to determine the amount of bacteria delivered by the inoculation, allowing us to quantify the growth or reduction in bacterial numbers from the initial amount deposited in the respiratory tract to the numbers three days post-inoculation. B. pertussis increased in numbers in the nasal cavities ( CFU), tracheae ( CFU), and lungs ( CFU) of naïve mice (Fig. 7.1D-F). Numbers were reduced in the nasal cavities, tracheae, and lungs of B. pertussis-immune ( , , CFU, respectively) and B. parapertussisimmune ( , , CFU, respectively) mice (Fig. 7.1D-F). 139

155 Upon investigating the ability of B. parapertussis to colonize immunized hosts, it was found that bacterial numbers were approximately 100-fold lower in the nasal cavity and 100,000- fold lower in the trachea and lungs of B. parapertussis-convalescent mice compared to naïve mice (Fig. 7.2A-C). However, B. parapertussis numbers were only 5-fold lower in the nasal cavity, and approximately 20-fold lower in the trachea and lungs (Fig. 7.2A-C), of B. pertussis- convalescent mice compared to naïve mice. B. parapertussis numbers grew over the first three days in the nasal cavities ( CFU), tracheae ( CFU), and lungs ( CFU) of naïve Figure 7.2: B. parapertussis colonization of naive or immunized mice. C57BL/6 mice were inoculated with 5 x 10 5 CFU of B. pertussis (Bp) or B. parapertussis (Bpp). Immunized and naive mice were challenged 28 days later with 5 x 10 5 CFU of a gentamicin-resistant strain of Bpp and sacrificed 3 days post-secondary inoculation for the quantification of bacterial numbers in the nasal cavity (A), trachea (B), and lungs (C). Values are expressed as Log 10 mean +/- SD. For day 3 colonization levels, the difference between each individual value and the mean Log 10 CFU approximately 10 minutes after inoculation in the nasal cavity (D), trachea (E), and lungs (F) was calculated. These data are represented as the mean change in Log 10 CFU +/- SD. One asterisk represents P-values < 0.05, two asterisks represent P-values < Infect Immun 75(10):

156 mice (Fig. 7.2D-F). Bacterial numbers were reduced in the nasal cavities, tracheae, and lungs of B. parapertussis-immune mice ( , , CFU, respectively) but increased in the nasal cavities, tracheae, and lungs of B. pertussis-immune mice ( , , CFU, respectively) (Fig. 7.2D- F). Similar results were obtained when animals were re-challenged on day 70 postinoculation (Fig. 7.3), after the primary infection had been completely cleared from the lower respiratory tract. Thus, B. parapertussis was able to colonize hosts that had previously been infected with, and generated an effective immune response against, B. pertussis. Figure 7.3: Cross-protection between B. pertussis and B. parapertussis upon challenge 70 days post-inoculation. C57BL/6 mice were inoculated with 5 x 10 5 CFU of B. pertussis (Bp) or B. parapertussis (Bpp). Immunized and naïve mice were challenged with Bp or Bpp 70 days later and sacrificed 3 days post-challenge for the quantification of bacterial numbers in the (A) nasal cavity, (B) trachea, and (C) lungs. CFU are expressed as the Log 10 mean +/- SD and the dashed line represents the lower limit of detection. T cell, but not antibody, responses are cross-reactive. The ability of B. parapertussis to infect B. pertussis-immune hosts suggested that the immune response was not effective against B. parapertussis, leading us to examine the cross reactivity of T cell and antibody responses. Mice were left un-inoculated or inoculated with B. pertussis or B. parapertussis and their spleens were excised 28 days post-inoculation. The response of splenocytes to heat-killed B. pertussis or B. parapertussis was assessed by measuring 141

157 the amount of IFN-γ produced, since this cytokine has been implicated in protective immunity to both bacteria (26-27, Fig. 6.2B). Splenocytes from naïve and B. parapertussis-infected animals produced low amounts of IFN-γ in response to either species (Fig. 7.4), although the low amounts of IFN-γ produced by splenocytes from B. parapertussis-infected animals appeared to be protective (Fig ). Splenocytes from B. pertussis-infected mice responded similarly to both species Figure 7.4: IFN-γ production by splenocytes from naïve or B. pertussis- or B. parapertussis-immune 5 hosts. C57BL/6 mice were inoculated with 5 x 10 CFU of B. pertussis (Bp) or B. parapertussis (Bpp). Immunized and naive mice were sacrificed 28 days post-inoculation, spleens were excised, and splenocytes were exposed to media alone (hatched bars), heat-killed Bp (black bars), or heat-killed Bpp (white bars) for 3 days. The supernatant was collected and cytokine ELISAs were performed to quantify the levels of IFN-γ produced by splenocytes. Values are expressed as the mean +/- SD and one asterisk represents P-values < Infect Immun 75(10): by producing large amounts of IFN-γ (~7000 pg) (Fig. 7.4). Similar levels of IL-10 were produced in response to B. pertussis and B. parapertussis, regardless of the primary infection (unpublished data). These data indicate that T cells induced by a B. pertussis infection are able to produce IFN-γ in response to B. parapertussis antigens. Furthermore, IFN-γ can contribute to the recruitment of neutrophils (28), which are important to the clearance of both pathogens (13,15). Therefore, we quantified the numbers of neutrophils in the lungs of mice after a secondary infection with either species. More neutrophils were observed in the lungs of B. pertussis- convalescent mice upon B. parapertussis infection relative to naïve or B. parapertussis- 142

158 convalescent mice (Fig. 7.5), suggesting that these cells were being recruited to the lungs but were not effectively eliminating B. parapertussis. This may be due to the fact that B. pertussis infection induces a robust IFN-γ response, even to B. parapertussis, and this cytokine contributes to the neutrophil response to B. parapertussis (Fig. 6.3). To determine the ability of antibodies induced by one species to recognize the other, sera from B. pertussis- and B. parapertussisconvalescent mice were analyzed by ELISA and Western blots. B. pertussis was recognized by sera raised against Figure 7.5: Leukocyte accumulation in the lungs of mice upon challenge with B. pertussis or B. parapertussis. C57BL/6 mice were inoculated with 5 x 10 5 CFU of B. pertussis (Bp) or B. parapertussis (Bpp) and 28 days later, were challenged with Bp or Bpp. Numbers of (A) total leukocytes and (B) neutrophils wer e quantified in the lungs 1 day post-challenge. Cell numbers are represented as the mean +/- SD. either species, although titers were approximately 5 times higher in B. pertussis-induced sera (titer ~ 1000) compared to B. parapertussis-induced sera ( titer ~ 200) (Fig. 7.6A). B. parapertussis was recognized by B. parapertussis-induced antibodies (titer ~ 7500), but titers in B. pertussis-induced sera were approximately 150-fold lower and near the lower limit of detection (titer ~ 50) (Fig. 7.6B), suggesting that the antibodies were not efficiently binding to B. parapertussis-coated plates. Western blots showed that antibodies induced by B. pertussis infection recognized antigens of both B. pertussis and B. parapertussis (Fig. 7.6C). The 143

159 Figure 7.6: Recognition of B. pertussis and B. parapertussis by antibodies from convalescent phase serum. C57BL/6 mice were inoculated with 5 x 10 5 CFU of B. pertussis (Bp) or B. parapertussis (Bpp) and serum was collected from these mice 28 days later. ELISA was performed on serum from naïve (NS), Bp-infected (Bp IS), and Bppinfected (Bpp IS) mice to quantify titers of antibodies specific for Bp (A) or Bpp (B). The dashed line represents the lower limit of detection. Antibody titers from the ELISA are quantified as the Log 10 mean of the endpoint titer +/- SD. One asterisk represents P-values < 0.05, two asterisks represent P-values < Western blots of pooled Bp-induced (C) and pooled Bpp-induced (D) serum were used to determine if serum raised against one species recognized specific antigens of Bp, Bpp, or B. parapertussisδwbm (BppΔO-ag). Infect Immun 75(10): recognition of denatured, isolated B. parapertussis proteins in this assay is in contrast to the lack of recognition of whole bacteria in the ELISA (Fig 7.6B), suggesting that shared protein antigens may not be exposed on the surface. B. parapertussis-induced antibodies also bound protein antigens of both species, as well as a large region between 17 and 36 kda present in B. parapertussis, but not B. pertussis. This region was absent from an isogenic mutant of B. parapertussis lacking O antigen, and B. pertussis does not express an O antigen, indicating that this smear represents LPS containing O antigen (Fig. 7.6D). These data indicate that antibody responses to B. pertussis and B. parapertussis differ in the recognition of O antigen. 144

160 Transfer of B. parapertussis-specific antibodies to B. pertussis-immune mice inhibits colonization by B. parapertussis. If B. parapertussis escapes cross immunity by avoiding antibody recognition, adding B. parapertussis immune serum should confer protection to B. pertussis-immune mice. Mice were inoculated with B. pertussis and 28 days later were given a passive transfer of naïve serum, B. pertussis immune serum, or B. parapertussis immune serum and challenged with B. parapertussis. Transferring naïve or B. pertussis-induced serum had no measurable effect on B. parapertussis numbers, but transferring B. parapertussis immune serum to B. pertussis-immune hosts resulted in approximately 100-fold reduction in bacterial numbers (Fig. 7.7). Although this treatment resulted in more efficient protection, it did not result in the same level as that conferred by prior B. parapertussis infection, suggesting that the T cell responses may not be completely cross reactive. These data support Figure 7.7: Effect of passive transfer of immune serum to B. pertussis-immune mice on B. parapertussis numbers. C57BL/6 mice were inoculated with 5 x 10 5 CFU of B. pertussis. 28 days later, these mice were passively transferred sera from naïve (Naïve serum), B. pertussis-infected (Bp serum), or B. parapertussis-infected (Bpp serum) mice, then challenged with Bpp. Bpp-convalescent mice (Bpp immune) were also challenged with Bpp for comparison. CFU were quantified 3 days postsecondary inoculation. Numbers of bacteria are expressed as Log 10 mean +/- SD. One asterisk represents P-values < 0.05, two asterisks represent P-values < Infect Immun 75(10):

161 the hypothesis that the lack of recognition by B. pertussis-induced antibodies allows B. parapertussis to avoid the immune response induced by its sister species. O antigen inhibits the binding of B. pertussis-induced antibodies to B. parapertussis. We next sought to determine how B. parapertussis avoids recognition by antibodies induced by such a closely related pathogen. B. parapertussis, but not B. pertussis, expresses O antigen (29) which has been shown to prevent the recognition of surface protein antigens by antibodies in other bacterial models (30-33). We hypothesized that O antigen blocked the binding of B. pertussis-induced antibodies to common antigens. Live bacteria were used in ELISAs to determine if B. pertussis-induced antibodies bound more efficiently to intact B. parapertussis in the absence of O antigen. Sera from both B. pertussis- and B. parapertussis-infected mice recognized B. pertussis (titers ~ 1000 and 600 respectively) (Fig. 7.8A). Titers of B. pertussis-induced antibodies that bound to live B. parapertussis were not Figure 7.8: Antibody recognition of live B. pertussis, B. parapertussis, and O antigendeficient B. parapertussis by immune serum. 5 C57BL/6 mice were inoculated with 5 x 10 CFU of B. pertussis (Bp) or B. parapertussis (Bpp) and serum was collected 28 days later. Sera from Bp- (Bp serum) or Bpp-infected mice (Bpp serum) were examined by ELISA for their ability to bind to live Bp (A), Bpp (B, white bars), or the O antigen-deficient B. parapertussis (BppΔO-ag) (B, hatched bars). The dashed line represents the lower limit of detection. Antibody titers are expressed as the endpoint titer +/- SD. One asterisk represents P-values < 0.05, two asterisks represent P-values < Infect Immun 75(10):

162 significantly higher than the lower limit of detection, but titers binding to live B. parapertussis lacking O antigen were approximately 15-fold higher (Fig. 7.8B). Thus, O antigen inhibits the binding of B. pertussis-induced antibodies to the surface of B. parapertussis. O antigen enables B. parapertussis to colonize B. pertussis-immune hosts. If the blocking of antibody binding by O antigen allowed the evasion of B. pertussisthat induced immunity, it would be expected B. pertussis immunity would confer protection against O antigen-deficient B. parapertussis. Mice were left uninfected or inoculated with B. pertussis or B. parapertussis and 28 days later challenged with wild type or O antigen-deficient B. parapertussis. Again, immunity to B. pertussis did not protect against wild type B. parapertussis (Fig. 7.2, 7.9). The O antigen- deficient strain of B. parapertussis was present at approximately 10 6, 10 6, and 10 5 CFU in the nasal cavities, tracheae, and lungs of naïve mice, respectively, but bacterial numbers were approximately 10- fold lower in the nasal cavities and 100,000-fold lower in the tracheae and Figu re 7.9: Ability of the O antigen-deficient strain of B. parapertussis to colonize B. pertussis- hosts. C57BL/6 mice were inoculated with immune 5 x 10 5 CFU of B. pertussis (Bp) or B. parapertussis (Bpp). Immunized and naive mice were challenged 28 d ays later with 5 x 10 5 CFU of Bpp (white bars) or the O antigen-deficient strain of B. parapertussis (BppΔO-ag, hatched bars). Mice were sacrificed 3 days post-secondary inoculation for the quantification of bacterial numbers in the nasal cavity (A), trachea (B), and lungs (C). All values are expressed as Log 10 mea n +/- SD. One asterisk represents P-values < 0.05, two asterisks represent P-values < Infect Immun 75(10):

163 lungs of B. pertussis-convalescent mice (Fig. 7.9). Thus, O antigen is required for B. parapertussis to evade B. pertussis-induced immunity and colonize B. pertussis-immune hosts. Infection by the O antigen-deficient B. parapertussis does not confer protective immunity against wild type B. parapertussis. Immunity to B. pertussis did not efficiently protect against subsequent B. parapertussis infection. Since B. pertussis infection does not induce antibodies to O antigen (a prominent surface molecule of B. parapertussis), we have begun to address the role of O antigen as a protective antigen. C57BL/6 mice were inoculated with wild type B. parapertussis or O antigen- mice were challenged with the wild type strain deficient B. parapertussis. 28 days later, these and sacrificed 3 days post-challenge to quantify bacterial numbers in the lungs. Again, an immune response to B. parapertussis provided protection against subsequent challenge. However, the immune response to the O antigen-deficient strain did not confer efficient protection against B. parapertussis (Fig. 7.10). The level of protection that was conferred was similar to that conferred by B. pertussis infection. These data suggest that an Figure 7.10: Protection conferred against B. parapertussis by O antigen-deficient B. parapertussis. C57BL/6 mice were inoculated with 5 x 10 5 CFU of B. pertussis (536), B. parapertussis (12822 and 2591), or O antigen-deficient B. parapertussis (2591Δwbm). Mice were challenged with wild type Bpp 28 days later and sacrificed 3 days post-challenge for the quantification of bacterial numbers. Bacterial numbers are expressed as the Log 10 mean CFU +/- SD. immune response to O antigen may be important to protecting against subsequent B. parapertussis infections. 148

164 Discussion: B. parapertussis likely emerged into populations in which B. pertussis was endemic ( 11) making the ability to evade B. pertussis-induced immunity important to invading these populations. Here we have shown that B. parapertussis evades immunity to B. pertussis, but the evasion of cross immunity is asymmetric. O antigen, which has been shown to mask surface antigens in other systems (30-33), inhibits the binding of B. pertussis-induced antibodies and allows B. parapertussis to colonize B. pertussis-immune hosts. The observed asymmetric cross immunity between B. pertussis and B. parapertussis is likely to contribute to their co-existence as endemic human pathogens. Previous work has suggested protective cross immunity between these two species (16) but used a B. pertussis strain that has been suggested to be more similar to B. bronchiseptica and B. parapertussis than other B. pertussis strains (17-18). More recent work has suggested that infection by an attenuated strain of B. pertussis may protect against subsequent B. parapertussis infections (34), but that study also observed that B. parapertussis numbers did not grow even in naïve mice, whereas multiple studies have shown B. parapertussis grows rapidly in naïve mice (12-13,25). The attenuated strain used was also defective in producing Ptx (34), a toxin that inhibits antibody production (35). Since low titers of B. parapertussis-specific antibodies in the serum of B. pertussis convalescent mice enabled the evasion of cross immunity, Ptx-mediated inhibition of antibody production may facilitat e the evasion of immunity. It is important to point out that B. pertussis vaccines show greatly diminished protection against B. parapertussis relative to protection against B. pertussis (36-40). 149

165 B. parapertussis O antigen limits the ability of antibodies from B. pertussis-immunized hosts to recognize surface antigens that are common between these two pathogens. An additional contributor to the asymmetrical cross immunity could be that B. pertussis infection does not induce an antibody response to the appropriate protective antigen(s). B. pertussis does not express O antigen, and thus does not induce O antigen-specific antibodies. O antigen is a key protective antigen for a number of bacteria (41-42) and our data suggest that the same may be true of B. parapertussis. While O antigen variation contributes to the co-existence of related bacterial species (43), we propose that O antigen is not essential to the infection of humans by Bordetella as evidenced by its absence from B. pertussis strains, but the maintenance of O antigen by B. parapertussis may have allowed it to emerge in the niche of a B. pertussis-endemic population. Host immunity has profound effects on the persistence of pathogens within host populations (2,44-45). Cross reactive immunity between two species causes immune-mediated competition that may ultimately displace one from the host population. Despite the apparent asymmetrical cross-immunity between B. pertussis and B. parapertussis, these two species appear to circulate in out-of-phase cycles (46) an d positive antibody titers to each have been observed in more than 50% of a population (47-48). The question is, if there is asymmetrical cross immunity between B. pertussis and B. parapertussis, why has the latter not displaced the former from human populations? Whooping cough caused by B. parapertussis may be milder (49-50), thus, assuming less coughing translates to less transmission, it is possible that B. parapertussis transmits less effectively than B. pertussis. This theory is supported by seroprevalence data indicating that depending on the age group, approximately 60% of a vaccinated population was seropositive for 150

166 B. parapertussis, but over 90% of that population was seropositive for B. pertussis (47-48). Furthermore, immunity induced by B. pertussis wanes after 5-10 years (10). The decay of immunity to B. parapertussis is not known, but cross-reacting immunity is likely to decay more rapidly than autologous immunity, potentially allowing subsequent infections by different strains or species (51). The data presented here are evidence of a novel pattern of asymmetric cross immunity conferred by B. pertussis and B. parapertussis that is consistent with the poor cross protection conferred by vaccines (36-40) and would affect the circulation of these pathogens throughout their host population. The evasion of B. pertussis-induced immunity by B. parapertussis may have allowed the latter to invade a population in which the former was endemic, leading to the observed co-existence. Importantly, it is possible that vaccination has changed, or is changing, the immune-mediated interactions between these two organisms. The effects of vaccines on B. parapertussis are unclear, but some have suggested that the prevalence of this species may be increasing despite, or even because of, vaccination efforts (38). In the face of these epidemiological and immunological complexities, it is important to establish the relative prevalence of B. pertussis and B. parapertussis as etiological agents in the ongoing resurgence of whooping cough. 151

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174 43. Pennington, J.E., G.J. Small, M.E. Lostrom, and G.B. Pier (1986) Polyclonal and monoclonal antibody therapy for experimental Pseudomonas aeruginosa pneumonia. Infect Immun 54(1): Rohani, P., D.J. Earn, and B.T. Grenfell (1999) Opposite patterns of synchrony in sympatric disease metapopulations. Science 286: Restif, O. and B.T. Grenfell (2006) Integrating life history and cross-immunity into the evolutionary dynamics of pathogens. Proc R Soc B 273(1585): Lautrop, H. (1971) Epidemics of parapertussis. 20 years' observations in Denmark. Lancet 1(7711): Maixnerova, M. (2003) The 2001 serological survey in the Czech Republic-- parapertussis. Cent Eur J Public Health 11(Suppl): S Maixnerova, M. (2003) The 2001 serological survey in the Czech Republic pertussis. Cent Eur J Public Health 11(Suppl): S Mastrantonio, P., P. Stefanelli, M. Giuliano, Y. Herrera Rojas, M. Ciofi degli Atti, A. Anemona, and A.E. Tozzi (1998) Bordetella parapertussis infection in children: epidemiology, clinical symptoms, and molecular characteristics of isolates. J Clin Microbiol 36(4): Bergfors, E., B. Trollfors, J. Taranger, T. Lagergard, V. Sundh, and G. Zackrisson (1999) Parapertussis and pertussis: differences and similarities in incidence, clinical course, and antibody responses. Int J Infect Dis 3(3):

175 51. Kawai, H., T. Aoyama, Y. Murase, C. Tamura, and A. Imaizumi (1996) A causal relationship between Bordetella pertussis and Bordetella parapertussis infections. Scand J Infect Dis 28(4):

176 Chapter 8: Summary and Significance 161

177 Whooping cough is re-emerging in vaccinated populations despite the maintenance of high levels of vaccine coverage (1-5). In order to design the most efficacious whooping cough vaccines possible, it is vital to gain a better understanding of the interactions between the endemic bordetellae and the host immune response. This research is focused on these interactions which are discussed in detail below. Impact of Ptx on host immunity and disease: Ptx is unique to B. pertussis and has a variety of effects on the host immune response (6). This toxin inhibits the immune response by preventing the migration of leukocytes to the site of infection (7-8, Fig. 8.1). Leukocyte activities such as phospholipase C stimulation, lysosmal enzyme secretion, and rising intracellular calcium levels upon chemokine stimulation are also inhibited by Ptx (9-10). Ptx affects the adaptive immune response by inhibiting the generation of antibody responses, driving the differentiation of Th1 cells, and eliciting IFN-γ production independent of MHC interactions and IL-12 (11-13). Although Ptx inhibits the early migration of leukocytes to the lungs, the IFN-γ response to B. pertussis, which is due at least in part to Ptx, contributes to the inflammatory response after the first few days of infection as measured by the accumulation of leukocytes in the lungs (Fig. 8.1). Ptx-mediated IFN-γ production likely contributes to the inflammatory response to B. pertussis by inducing the production of a subset of chemokines that act on Ptx-insensitive chemokine receptors (14-16). In contrast, TNF-α limits the accumulation of these cells during B. pertussis infection (Fig. 8.1). TNF-α facilitates the phagocytosis of B. pertussis (17), which could lead to the apoptosis of neutrophils that have phagocytosed bacteria or bacterial products (18-20). Macrophages may then be able to phagocytose the apoptotic neutrophils and resolve the inflammatory response (21). The regulation of the inflammatory response is vital since large amounts of leukocytes in the alveolar 162

178 Figure 8.1: Schematic of clearance of B. pertussis from the respiratory tract. B. pertussis induces an early, TLR4-mediated cytokine and chemokine response. This early response is likely mediated by alveolar macrophages (AM) or dendritic cells (DC). Ptx prevents the effects of these chemokines on the recruitment of neutrophils, allowing B. pertussis to grow to high numbers. B. pertussis induces a T cell response, becoming detectable around day 7 post-inoculation. These T cells produce IFNγ, which induces the production of different chemokines. This overcomes the inhibitory effect of Ptx on neutrophil migration and neutrophils begin accumulating in the lungs. These neutrophils are then able to begin reducing bacterial numbers. TNFα helps limit the accumulation of neutrophils in the lungs, limiting the damage to host tissue caused by these cells. B cells produce antibodies specific for B. pertussis antigens (detectable after about two weeks), and neutrophils are then able to more efficiently phagocytose and kill the antibody-coated bacteria. spaces, recruited in a Ptx- and IFN-γ-dependent manner, could cause damage to the lungs and enhance disease symptoms. This is supported in the mouse model by the fact that depleting CD4+ T cells or neutrophils results in less leukocyte accumulation and an extended lifespan of B. pertussis-infected mice (Fig. 2.7,2.8). Although Ptx enhances the ability of B. pertussis to colonize murine hosts (7-8), its effects are ultimately overcome and this bacterium is eliminated from the respiratory tract. TNFα contributes to the clearance of B. pertussis, but is dispensible for the elimination of a Ptxdeficient mutant of B. pertussis (Fig. 2.3,2.10), suggesting that this cytokine is important to counteracting some effect of this toxin. Antibodies mediate the clearance of B. pertussis from the respiratory tract via Fc-γ receptors and neutrophils, but Ptx prevents the antibody-mediated clearance of this pathogen during the first week of infection by inhibiting neutrophil migration 163

179 (8). T cells produce large amounts of IFN-γ in response to B. pertussis infection (Fig. 3.3), which is crucial for the antibody-mediated clearance of B. pertussis that occurs during the second week of infection. The role of IFN-γ in protection against B. pertussis appears to involve its contribution to leukocyte recruitment to the lungs during infection. Together, these data suggest that IFN-γ may counteract the inhibitory effect of Ptx on bacterial clearance (Fig. 3.4). Our current model of how B. pertussis is eliminated from the respiratory tract involves the following. B. pertussis induces an innate immune response upon colonization of the respiratory tract (as measured by cytokine production in response to infection). Ptx prevents the function of this innate response by inhibiting the migration of leukocytes to the lungs (7-8). However, the production of IFN-γ by T cells eventually overcomes this effect and leukocytes begin migrating to the site of infection (Fig. 8.1). Both IFN-γ and TNF-α are required for the reduction of B. pertussis numbers, and TNF-α is required for the eventual resolution of the inflammatory response. Since antibodies are required for the elimination of B. pertussis, the inhibition of Bordetella-specific antibody generation by Ptx (11) may enhance the ability of B. pertussis to persist in the respiratory tract. With that being said, a protective immune response is ultimately induced by B. pertussis infection that lasts 5-10 years (22). There are a few major implications that arise from the knowledge that TNF-α and IFN-γ are involved in protective immunity against B. pertussis. Anti-cytokine therapies are becoming increasingly common treatments for many autoimmune diseases. For example, over 300,000 patients worldwide are being treated with anti-tnf-α therapies (23). These treatments are associated with increased incidence of tuberculosis, histoplasmosis, listeriosis, aspergillosis, coccidiomycosis, and candidiasis (24) and similar effects may be observed on the frequencies of B. pertussis disease. Defects in TNF-α or IFN-γ responses could also result from opportunistic 164

180 infections such as Pseudomonas aeruginosa, which utilizes mechanisms to alter or inhibit TNFα signaling (25-26). Since secondary infections are commonly observed in B. pertussis-infected individuals, the effects of these secondary infections on cytokine responses could have a significant impact on the severity of B. pertussis disease. Perhaps most importantly, the majority of developed countries have switched to using acellular vaccines because of the reactogenicity of whole cell vaccines. Unfortunately, the current acellular vaccines have been estimated to be less efficacious than whole cell vaccines ( 27). The T cell response induced by the acellular vaccine is skewed towards a Th2 phenotype while that induced by the whole cell vaccine or natural infection consists of a more balanced response (28-30). Thus, the decreased efficacy of acellular vaccines against B. pertussis, relative to whole cell vaccines (31), could be explained by the fact that the Th1-associated cytokine, IFNγ, is involved in protection against B. pertussis. Importantly, the previously mentioned re- stronger IFN-γ and TNF-α responses may be more efficacious against B. pertussis. emergence of whooping cough in countries that have maintained excellent vaccine coverage roughly correlates with the introduction of acellular vaccines. Thus, vaccines designed to induce Evasion of protective immunity by B. parapertussis: Current vaccines, both whole-cell and acellular, consist only of B. pertussis antigens and are fairly effective at limiting disease caused by B. pertussis, but are ineffective against B. parapertussis (32-37). This likely confers a selective, immune-mediated advantage to B. parapertussis that could lead to higher percentages of whooping cough cases being caused by B. parapertussis. Long-term epidemiological studies of both B. pertussis and B. parapertussis are essential for researchers to gain an understanding of the relative roles of these pathogens in the re-emergence of whooping cough. That being said, B. parapertussis can account for anywhere 165

181 from 1% to greater than 95% of cases of whooping cough (reviewed in 38), thus, introducing a B. parapertussis vaccine would likely decrease the overall incidence of whooping cough. In order to develop an effective vaccine against this pathogen, an understanding of protective immunity against B. parapertussis and how this pathogen may evade host immunity is crucial. Despite causing the same disease in the same host as B. pertussis, very little was known about protective immunity against B. parapertussis prior to these studies. It was established that an adaptive immune response was important to protection in a mouse model of infection (39-40) and anti-filamentous hemaglutinin and anti-pertactin antibodies correlated with protection in a clinical study (41). We have since elucidated some of the host immune factors that are required to eliminate this pathogen from the murine respiratory tract. Antibodies are essential to the elimination of this pathogen, but neutrophils, CD4+ T cells, and complement are required for the function of these antibodies against B. parapertussis. Ptx enables B. pertussis to avoid antibody-mediated clearance by preventing the migration of neutrophils to the site of infection (8). Since B. parapertussis does not express Ptx, we sought to determine how this pathogen evaded and/or modulated the host immune response to optimize the infection process. Neutrophils are also important to the antibody-mediated clearance of B. parapertussis (42), thus, preventing their migration to the lungs would be an excellent strategy to avoid immune-mediated clearance. Our lab has previously shown that B. parapertussis LPS is an inefficient stimulator of TLR4 (Fig. 8.2), even compared to the other bordetellae (43). This results in little early recruitment of leukocytes to the site of B. parapertussis infection (35, Fig. 8.2). By not stimulating pro-inflammatory TLR4 responses, this pathogen is able to evade early control by the innate immune response and grow to high 166

182 Figure 8.2: Schematic of clearance of B. parapertussis from the respiratory tract. B. parapertussis grows to high numbers over the first few days of infection, but since its LPS is unstimulatory, there is little TLR4-mediated production of cytokines and chemokines. As a result, very few neutrophils are recruited to the lungs over the first few days of infection. A T cell response to B. parapertussis is generated, becoming detectable around day 7 post-inoculation. These T cells produce IFNγ, which contributes to the recruitment of neutrophils. However, there is an early IL-10 response to B. parapertussis, likely mediated by alveolar macrophages (AM) or dendritic cells (DC), which dampens the IFNγ and neutrophil responses. This results in enhanced persistence of the infection. B cells begin to produce measurable amounts of B. parapertussis-specific antibodies around two weeks post-inoculation. Neutrophils are then able to more efficiently eliminate the antibody-coated bacteria. numbers in the respiratory tract, even in the presence of antibodies. Similarly, the lack of TLR4 stimulation has been shown to contribute to other measures of virulence for bacterial pathogens (44). In addition to evading detection by the innate immune response, B. parapertussis also appears to modulate the T cell response that is induced during infection. IFN-γ is produced by T cells in response to infection by B. parapertussis (Fig. 8.2) and contributes to the inflammatory response to (Fig. 6.3A-C), and elimination of, this pathogen (Fig. 6.2B). However, other cell types produce IL-10 in response to B. parapertussis, which dampens the IFN-γ response (Fig. 8.2). Similarly, B. pertussis and B. bronchiseptica induce the production of IL-10 via synergizing effects of adenylate cyclase toxin and LPS or Type III Secretion System, respectively (45-47). While it is unclear how B. parapertussis elicits IL-10 production, this cytokine does inhibit the clearance of this pathogen from the respiratory tract (Fig. 6.6). Thus, B. 167

183 parapertussis persists in its host by a combination of evading and modulating the host immune response. Although B. parapertussis is able to delay immune-mediated clearance, this bacterium ultimately induces a sterilizing, protective immune response that prevents subsequent infections. Despite how closely related B. pertussis and B. parapertussis are, the immune responses that they induce are not perfectly cross protective. B. parapertussis infection induces immunity that efficiently protects against both pathogens, but B. pertussis infection induces immunity that only efficiently protects against subsequent B. pertussis challenge (Fig. 8.3). However, the immune responses that are induced eventually wane, allowing the reinfection of previously immunized hosts. The current evolutionary and immunological theories regarding the causative agents of whooping cough suggest that B. parapertussis invaded a population in which B. pertussis was already endemic to some degree (48). Thus, some proportion of that population would have generated an immune response to B. pertussis, making the ability to avoid B. pertussis-induced immunity a crucial commodity to the success of B. parapertussis in that population (Restif, et.al. submitted to Journal of Parasitology). 168

184 Figure 8.3: Cross reactivity of immune responses to B. parapertussis and B. pertussis. B. parapertussis infection induces T cell and antibody responses. Upon subsequent challenge by B. parapertussis (A), T cells respond by producing IFNγ, which contributes to the recruitment of neutrophils. The antibodies induced by the primary infection are able bind to B. parapertussis and provide protection. Upon challenge by B. pertussis (B), T cells are again able to respond by producing IFNγ and contribute to neutrophil recruitment. The antibodies induced by B. parapertussis infection are cross reactive with B. pertussis antigens and are able to provide protection against this species as well. B. pertussis infection induces T cell and antibody responses. Upon subsequent challenge by B. pertussis (C), T cells respond by producing IFNγ, which contributes to the recruitment of neutrophils. The antibodies induced by the primary infection are able bind to B. pertussis and provide protection. Upon challenge by B. parapertussis (D), T cells respond by producing IFNγ, but the antibodies induced by B. pertussis infection are not able to bind to the surface of B. parapertussis. The O-antigen of B. parapertussis prevents these antibodies from doing so, resulting in a lack of protection against B. parapertussis. B. pertussis and B. parapertussis evolved from B. bronchiseptica-like progenitors and have undergone a large scale loss of genes over time (49). Although B. bronchiseptica-like progenitors likely expressed an O antigen, B. pertussis strains no longer express an O antigen (50), indicating that this bacterial factor is not essential to its success in human populations. With other Bordetella species, we have observed that O antigen protects the bacteria from complement-mediated killing in naïve animals (51, unpublished data). B. pertussis has developed other mechanisms of inhibiting complement-mediated killing (52), thus, does not need 169

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