Comparative Toll-Like Receptor 4-Mediated Innate Host Defense to ...

INFECTION AND IMMUNITY, Dec. 2005, p. 8144–8152 0019-9567/05/$08.00⫹0 doi:10.1128/IAI.73.12.8144–8152.2005 Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Vol. 73, No. 12

Comparative Toll-Like Receptor 4-Mediated Innate Host Defense to Bordetella Infection Paul B. Mann,1 Daniel Wolfe,1 Eicke Latz,2 Douglas Golenbock,2 Andrew Preston,3 and Eric T. Harvill1* Pathobiology Graduate Program, Center for Molecular Immunology and Infectious Disease, Department of Veterinary and Biomedical Science, The Pennsylvania State University, University Park Pennsylvania1; Division of Infectious Diseases and Immunology, University of Massachusetts Medical School, Worcester, Massachusetts2; and Department of Molecular and Cellular Biology, The University of Guelph, Guelph, Ontario, Canada3 Received 31 May 2005/Returned for modification 1 July 2005/Accepted 6 August 2005

Bordetella pertussis, B. parapertussis, and B. bronchiseptica are closely related species associated with respiratory disease in humans and other mammals. While B. bronchiseptica has a wide host range, B. pertussis and B. parapertussis evolved separately from a B. bronchiseptica-like progenitor to naturally infect only humans. Despite very different doubling times in vitro, all three establish similar levels of infection in the mouse lung within 72 h. Recent work has revealed separate roles for Toll-like receptor 4 (TLR4) in immunity to B. pertussis and B. bronchiseptica, while no role for TLR4 during B. parapertussis infection has been described. Here we compared the requirement for TLR4 in innate host defense to these organisms using the same mouse infection model. While B. bronchiseptica causes lethal disease in TLR4-deficient mice, B. pertussis and B. parapertussis do not. Correspondingly, TLR4 is critical in limiting B. bronchiseptica but not B. pertussis or B. parapertussis bacterial numbers during the first 72 h. Interestingly, B. bronchiseptica induces a TLR4-dependent cytokine response that is considerably larger than that induced by B. pertussis or B. parapertussis. Analysis of their endotoxins using RAW cells suggests that B. bronchiseptica lipopolysaccharide (LPS) is 10- and 100-fold more stimulatory than B. pertussis or B. parapertussis LPS, respectively. The difference in LPS stimulus is more pronounced when using HEK293 cells expressing human TLR4. Thus, it appears that in adapting to infect humans, B. pertussis and B. parapertussis independently modified their LPS to reduce TLR4-mediated responses, which may compensate for slower growth rates and facilitate host colonization. sis express overlapping yet distinct subsets of these factors (38). This suggests that each species has unique ways to modify immune responses in order to optimize its ability to infect the host. Toll-like receptors (TLR) are a family of germ line-encoded pattern recognition receptors that mediate the host’s ability to detect pathogen-associated molecular patterns (PAMPs) and activation of host defense mechanisms (3, 23, 35, 36). TLR4 is required for the detection of many bacterial LPSs and thus plays an important role in host defense to certain gram-negative bacteria (8, 39). Interestingly, the described role of TLR4 in host defense against a number of gram-negative organisms appears to vary widely, and in some instances it does not appear to be required (5, 6, 13, 17, 21, 31, 32, 37, 40, 43, 44, 47). The apparently different roles of TLR4 may simply be explained by differences in experimental design or, more interestingly, may be related to differences in host-pathogen interactions. We previously demonstrated that TLR4 is critical for innate immunity during B. bronchiseptica infection and that TLR4deficient mice rapidly die following infection with initial doses as low as 5,000 CFU (32, 33). In this model a critical role for TLR4 is in mediating a protective early elicited tumor necrosis factor alpha (TNF-␣) response. Interestingly, Higgins et al. have described a different role for TLR4 during infection with B. pertussis. In their model TLR4 appears to be more important for developing adaptive immunity, specifically, interleukin-10 (IL-10)-mediated T regulatory cell responses (21). Currently, there are no published observations on a requirement

The bordetellae are aerobic, gram-negative coccobacilli that infect the respiratory tracts of mammals (38, 46). Bordetella pertussis and B. parapertussis are pathogens of humans and the etiologic agents of whooping cough, or pertussis, an acute respiratory disease. B. bronchiseptica, the likely progenitor of the two human pathogens, has a wide host range and often causes persistent asymptomatic infection (38, 46). These three bacteria are closely related species that share a large number of virulence determinants that facilitate colonization of the host’s respiratory tract (34). Mouse models for studying hostpathogen interactions during Bordetella infection are a welldeveloped system in which to compare the role of specific host defense mechanisms during infection with each of these bacteria (19). The ability of bacteria to successfully infect the host is dependent on a variety of factors which, in the case of Bordetella species, are regulated via the BvgAS two-component system (2, 10). This system regulates a number of adhesins, toxins, and lipopolysaccharide (LPS) modifications, some of which may counter host defenses and aid in colonization of the respiratory tract (34). Previous work suggested that certain Bordetella virulence factors synergize to modulate host immunity (42, 45). Interestingly, B. bronchiseptica, B. pertussis, and B. parapertus-

* Corresponding author. Mailing address: Pathobiology Graduate Program, Immunology Research Laboratories, Department of Veterinary Science, The Pennsylvania State University, University Park, PA 16802. Phone: (814) 863-8522. Fax: (814) 863-6140. E-mail: eth10 @psu.edu. 8144

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for TLR4 for host defense during B. parapertussis infection. Here, we used the mouse infection model to compare the requirement for TLR4 in innate host defense to each of these closely related bacteria using strains whose genomes were recently sequenced. Our results indicate that TLR4-mediated immunity is vital to innate host defense against B. bronchiseptica; however, it appears less significant against B. pertussis or B. parapertussis. MATERIALS AND METHODS Animal infections. Wild-type C57BL/10ScSn, TLR-deficient (TLR4def) C3H/ HeJ, and TLR4-deleted (TLR4del) C57BL/10ScNCr mice were obtained from Jackson Laboratories. Wild-type C3H/HeN mice were obtained from Charles River Laboratories. All mice were maintained in the animal care facility at The Pennsylvania State University in accordance with institutional guidelines. Mice were lightly sedated with isofluorane and inoculated intranasally (i.n.) with the indicated CFU of B. bronchiseptica in 50 ␮l of phosphate-buffered saline (PBS; Merck) by pipetting the inoculum onto the tip of the external nares. B. bronchiseptica (RB50), B. pertussis (536), and B. parapertussis (12822) were grown as previously described. (18, 20). For animal experiments, groups of at least four mice were sacrificed at the indicated time point, and colonization of the lungs was quantified as previously described (18). For in vivo cytokine measurements, the lungs were homogenized in 5 ml of PBS and stored at ⫺80°C until assayed. The lung homogenate aliquots were subsequently thawed and assayed by enzyme-linked immunosorbent assay (ELISA) using matched antibody sets and appropriate standards (R&D Systems). For TNF-␣ neutralization experiments, 1 ml of PBS containing 1 mg of anti-TNF-␣, from clone MP6-XT3, was injected intraperitoneally at the indicated time postinoculation (22). For neutrophil depletion experiments, 1 ml of PBS containing 0.5 mg of anti-Ly6, from clone RB6-8C5, was injected intraperitoneally at the indicated time postinoculation (9, 22). Determinations of lung leukocyte and neutrophil numbers were performed as previously described (33). For survival studies, mice were observed for increased signs and symptoms of bordetellosis, including ruffled fur, labored breathing, and diminished responsiveness. Moribund mice were euthanized to alleviate suffering. In vitro LPS experiments. LPS from B. bronchiseptica (RB50), B. pertussis (536), and B. parapertussis (12822) was extracted and purified as previously described (24). Bone marrow-derived macrophages were obtained as previously described (14). Cells were replated in 24-well plates at approximately 106 cells per well in complete Dulbecco’s modified Eagle’s medium and then exposed to 1 ␮g/ml of LPS in overnight culture. RAW 264.7 cells were obtained from ATCC. HEK293 cells stably transfected with human TLR4/MD2 were previously described (28). LPS stimulations were performed by placing cells in 96-well plates at approximately 105 cells per well in complete Dulbecco’s modified Eagle’s medium. LPS was resuspended in sterile PBS at 1 mg/ml, and 10-fold dilutions were made. Each LPS suspension was sonicated for 5 min prior to diluting or mixing with cell culture media. Cells were exposed to the indicated concentration of LPS in overnight culture. The culture medium was removed and assayed by ELISA. Statistics. The mean ⫾ standard error was determined for each treatment group in the individual experiments. Statistical significance was calculated using a paired Student’s t test, with a significance level of P ⬍ 0.05 for a single comparison. Animal infection experiments for cytokine assays or counting bacteria were repeated two or three times.

RESULTS TLR4 is critical to innate host defense to B. bronchiseptica but not B. pertussis or B. parapertussis. The reported roles for TLR4 in immunity against B. bronchiseptica and B. pertussis appear to differ dramatically (21, 32); however, it is possible that differences in experimental conditions may contribute to these disparate findings. Additionally, the role of TLR4 during B. parapertussis infection has not been reported. In order to examine the relative requirement for TLR4 in innate host defense against the bordetellae using the same experimental model, that of limiting acute infection, we inoculated groups of

FIG. 1. Comparative role of TLR4. (A) Survival curve of TLR4deficient C3H/HeJ mice inoculated with the indicated CFU of B. bronchiseptica (circles), B. pertussis (diamonds), or B. parapertussis (squares). (B to D) CFU of B. bronchiseptica (B), B. pertussis (C), or B. parapertussis (D) in the lungs of WT mice (solid lines) or TLR4deficient mice (dashed lines) at the indicated times postinoculation.

wild-type (WT) C3H/HeN mice and TLR4-deficient C3H/HeJ mice with a range of doses of B. bronchiseptica, B. pertussis, or B. parapertussis via the i.n. route. The mice were then observed for signs of morbidity associated with severe bordetellosis. As previously observed, TLR4-deficient mice inoculated with approximately 5 ⫻ 105 CFU of B. bronchiseptica developed lethal disease within 96 h of inoculation; however, these mice exhibited no outward signs of disease when inoculated with the same number of either B. pertussis or B. parapertussis organisms (Fig. 1A). Additionally, TLR4-deficient mice infected with B. pertussis and B. parapertussis survived and eventually eliminated the bacteria (Fig. 1A and data not shown), whereas TLR4-

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FIG. 2. Comparative role of TLR4. (A) Survival curve of TLR4deleted C57BL/10ScNCr mice inoculated with the indicated CFU of B. bronchiseptica (circles), B. pertussis (diamonds), or B. parapertussis (squares). (B) CFU of B. bronchiseptica, B. pertussis, or B. parapertussis in the lungs of WT mice (open bars) or TLR4-deleted mice (solid bars) at 72 h after inoculation.

deficient mice developed lethal disease following inoculation with as few as 5 ⫻ 103 CFU of B. bronchiseptica. Similar mortality patterns were observed in TLR4-deleted C57BL/ 10ScNCr mice, further supporting a critical requirement for TLR4 in innate host defense against B. bronchiseptica but not B. pertussis or B. parapertussis (Fig. 2A). To examine the requirement for TLR4 in limiting bacterial growth within the lungs during the first 3 days, we inoculated wild-type and TLR4-deficient mice as previously described and measured bacterial numbers in the lungs at 0, 2, 6, 12, 24, 48, and 72 h postinoculation. There were no significant differences between the CFU of B. pertussis or B. parapertussis recovered from the lungs of wild-type and TLR4-deficient mice (Fig. 1C and D) at any time point. However, starting at approximately 12 hours postinoculation, there were approximately 10-fold higher numbers of B. bronchiseptica recovered from the lungs of TLR4-deficient mice compared to the lungs of wild-type mice (Fig. 1B). Wild-type mice limited B. bronchiseptica numbers to between 105 and 106 CFU after this time point. However, the bacterial numbers in TLR4-deficient mice continued to increase to approximately 109 CFU at 72 h postinoculation. These data suggest that TLR4-mediated innate immune responses are critical to limiting infection with B. bronchiseptica, but apparently not those with B. pertussis or B. parapertussis. To further compare the requirement for TLR4 during infection with these three species, we examined bacterial numbers in the lungs of WT C57BL/10ScSn mice and TLR4-deleted C57BL/ 10ScNCr mice 3 days postinoculation. The lungs of WT mice contained approximately 106 CFU of B. bronchiseptica, whereas the lungs of TLR4-deleted mice infected with this bacterium harbored approximately 109 CFU (Fig. 2B). Similar

FIG. 3. Role of TLR4 during B. pertussis and B. parapertussis infection. B. pertussis (A) or B. parapertusis (B) CFU were recovered from the lungs of mice at the indicated times postinoculation (closed symbols) or from TLR4-deficient mice (open symbols).

to what was observed in C3H mice, there were no significant differences between the number of CFU of B. pertussis or B. parapertussis found in the lungs of WT and TLR4-deleted mice. This result further indicates a critical requirement for TLR4 in innate host defense against B. bronchiseptica but not B. pertussis or B. parapertussis. TLR4 is involved in eliminating B. pertussis infection but not B. parapertussis infection. Higgins et al. have reported that B. pertussis infection is more severe after day 7 in TLR4-deficient mice (21). To confirm this finding using a sequenced strain of B. pertussis as well as to examine if B. parapertussis infection is more severe at later time points in TLR4-deficient mice, we inoculated groups of WT C3H/HeN mice and TLR4-deficient C3H/HeJ mice with B. pertussis or B. parapertussis via the i.n. route. The mice were then sacrificed on days 1, 3, 7, 14, 28, and 56 postinoculation, and lung bacterial burdens were measured. As previously reported, TLR4-deficient mice infected with B. pertussis had increased numbers of bacteria in the lungs on day 7 and 14 postinoculation compared to wild-type counterparts (Fig. 3A). However, there were no significant differences in the bacterial numbers at any time point measured in the lungs of WT and TLR4-deficient mice infected with B. parapertussis (Fig. 3B). These results suggest that TLR4 plays a role in reducing bacterial numbers during B. pertussis infection, a finding consistent with published reports, but that this receptor is not involved in controlling or eliminating B. parapertussis.

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FIG. 4. TLR4 is required for the response to LPS. TNF-␣, IL-6 (B), and NO (C) levels were measured in the cell culture supernatant of BMM␾ exposed to 1 ␮g/ml of purified LPS from B. bronchiseptica (Bb), B. pertussis (Bp), and B. parapertussis (Bpp). PGN, S. aureus peptidoglycan at 10 ␮g/ml; M, medium blank.

TLR4 is required for the cytokine response to B. bronchiseptica, B. pertussis, and B. parapertussis LPS in vitro. Recently, several groups have observed that the LPSs from several nonenteric pathogens are able to stimulate TLR2 rather than TLR4 (11). Thus, a potential explanation for the differences in the requirement for TLR4 during host defense to these three closely related species is that the LPSs of B. pertussis or B. parapertussis induce innate immune responses in the absence of TLR4. To examine this possibility, we exposed wild-type or TLR4-deficient bone marrow-derived macrophages (BMM␾) to purified LPS (1 ␮g/ml) derived from each of these bacteria. The cells were incubated overnight, and the supernatants were removed and assayed for TNF-␣, IL-6, and nitric oxide (NO) as indicators of LPS responsiveness. Increased levels of TNF-␣, IL-6, and NO were detected in the supernatants of wild-type BMM␾, compared to media controls; however, TLR4-deficient BMM␾ failed to produce increased levels of these inflammatory mediators upon exposure to any of the Bordetella LPSs (Fig. 4). Both macrophage types produced similar levels of TNF-␣, IL-6, and NO in response to 10 ␮g/ml of Staphylococcus aureus peptidoglycan, a TLR2 agonist, suggesting that TLR4 BMM␾ are not generally defective in cytokine production. These results indicate that TLR4 is required


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for cytokine responses to the LPS from all three Bordetella species and suggest that the lack of a critical requirement for TLR4 during innate host defense to B. pertussis or B. parapertussis is not the result of LPS stimulating via an alternative TLR. TNF-␣ and neutrophils are critical to innate host defense against B. bronchiseptica but not B. pertussis or B. parapertussis. We have previously observed that during B. bronchiseptica infection TLR4-deficient mice have impaired early elicited TNF-␣ responses (32). These findings suggest that TLR4 mediates the early TNF-␣ responses to Bordetella infection. Additionally, deletion of early elicited TNF-␣ using anti-TNF-␣ monoclonal antibodies results in the rapid development of lethal disease during B. bronchiseptica infection. To examine the role of this innate host defense factor during infection with each Bordetella species, we inoculated WT C3H/HeN mice that were depleted of TNF-␣ with 5 ⫻ 105 CFU of either B. bronchiseptica, B. pertussis, or B. parapertussis and observed for signs of severe disease. As previously described, TNF-␣-depleted mice developed lethal bordetellosis within 4 days of inoculation with B. bronchiseptica (Fig. 5A). However, TNF␣-depleted mice infected with B. pertussis or B. parapertussis did not display symptoms of disease. These findings suggest that early elicited TNF-␣ is critical to innate host defense against B. bronchiseptica but not B. pertussis or B. parapertussis. To compare the role of TNF-␣ in limiting the growth of these bacteria within the respiratory tract, we excised the lungs of control and TNF-␣-depleted mice and measured the bacterial numbers on day 3 postinoculation. The lungs of control mice infected with B. bronchiseptica harbored between 105 and 106 CFU, whereas the lungs of TNF-␣-depleted mice contained approximately 109 CFU (Fig. 5C), suggesting that TNF-␣ is critical to limiting bacterial numbers during B. bronchiseptica infection. Interestingly, the lungs of control mice and TNF-␣-depleted mice all contained approximately 107 CFU of B. pertussis or B. parapertussis, suggesting that, unlike infection with B. bronchiseptica, early elicited TNF-␣ is not critical for innate host defense to B. pertussis or B. parapertussis. We have previously observed that during B. bronchiseptica infection TLR4-deficient mice have impaired early neutrophil migration to the lungs; Higgins et al. demonstrated a similar result during B. pertussis infection of TLR4-deficient mice (21). These findings suggest that TLR4 mediates early neutrophil recruitment to the lungs in response to Bordetella infection. To examine the role of these cells during infection with each Bordetella species, we inoculated WT C3H/HeN mice and neutrophil-depleted mice with 5 ⫻ 105 CFU of either B. bronchiseptica, B. pertussis, or B. parapertussis and observed for signs of severe disease. Neutrophil-depleted mice developed lethal bordetellosis within 3 days of inoculation with B. bronchiseptica (Fig. 5B). However, these same mice infected with B. pertussis or B. parapertussis did not display increased signs of disease. These findings suggest that neutrophils are critical to innate host defense against B. bronchiseptica but not B. pertussis or B. parapertussis. To examine the role of neutrophils in limiting the growth of these bacteria within the respiratory tract, we excised the lungs of control and neutrophil-depleted mice and measured bacterial numbers on day 3 postinoculation. The lungs of control mice infected with B. bronchiseptica harbored between 105 and

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FIG. 5. Comparative roles of TNF and polymorphonuclear leukocytes. (A and B) Survival curves for TNF-␣ depleted mice (A) or neutrophil-depleted mice (B) inoculated with the indicated CFU of B. bronchiseptica (circles), B. pertussis (diamonds), or B. parapertussis (squares). (C) Bacterial numbers recovered from the lungs 72 h after inoculation with B. bronchiseptica (Bb), B. pertussis (Bp), or B. parapertussis (Bpp).

106 CFU, whereas the lungs of neutrophil-depleted mice contained approximately 109 CFU (Fig. 5C), suggesting that neutrophils are critical to limiting bacterial numbers during B. bronchiseptica infection. Interestingly, the lungs of control mice and neutrophil-depleted mice all contained approximately 107 CFU of B. pertussis or B. parapertussis, suggesting that, unlike infection with B. bronchiseptica, neutrophils are not critical for innate host defense to B. pertussis or B. parapertussis. Infection with B. bronchiseptica induces greater TLR4-dependent early elicited inflammatory responses in mouse lungs compared to infection with B. pertussis or B. parapertussis. We have previously observed that protective early elicited cytokine responses to B. bronchiseptica are mediated via TLR4. Whereas our in vitro observations indicated that the host response to the LPS of each Bordetella species was TLR4 dependent, our in vivo observations raise the possibility that during infection B. pertussis or B. parapertussis induces protective responses independent of TLR4. To investigate this possibility, we inoculated WT and TLR4-deficient mice with B. bronchiseptica, B. pertussis, or B. parapertussis and measured the lung

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FIG. 6. Differential TLR4-mediated cytokine responses in vivo: TNF-␣ (A), MIP-2 (B), or KC (C) levels measured in the lungs at 2 h postinoculation with B. bronchiseptica (Bb), B. pertussis (Bp), or B. parapertussis (Bpp) in WT mice (open bars) or TLR4-deficient mice (solid bars). Sham, PBS inoculated.

TNF-␣, macrophage inflammatory protein 2 (MIP-2), and keratinocyte-derived chemokine (KC), the murine homolog of IL-8, levels at 2 hours postinfection. In wild-type mice, infection with all three species resulted in an early transient increase in the lung levels of TNF-␣, MIP-2, and KC compared to the PBS control (Fig. 6). There was no significant increase in these cytokines in TLR4-deficient mice, suggesting that during infection TLR4 is critical to the early cytokine response to all three bordetellae. Interestingly, infection with B. bronchiseptica induced a significantly higher cytokine response compared to infection with B. pertussis and B. parapertussis. Secretion of cytokines and chemokines is often associated with a rapid leukocyte migration to the site of infection. Additionally, gram-negative bacteria often induce the rapid migration of neutrophils to the site of infection. Recently, LPS stimulation of TLR4 has been demonstrated to regulate neutrophil chemotaxis (12). We examined whether the differences in cytokine responses observed during infection with the three bordetellae resulted in differences in the number of leukocytes present in the lungs shortly after infection. WT and TLR4deficient mice were infected as described above, and the number of lung leukocytes was measured at 6 h postinoculation.

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FIG. 7. Differential TLR4-mediated polymorphonuclear leukocyte recruitment in vivo. Lung leukocyte numbers were determined at 6 h postinoculation with B. bronchiseptica (Bb), B. pertussis (Bp), or B. parapertussis (Bpp) in WT mice (open bars) or TLR4-deficient mice (solid bars). (A) Total leukocytes; (B) PMNs. Sham, PBS inoculated.

The lungs of WT mice inoculated with B. bronchiseptica contained approximately 25 ⫻ 105 lung leukocytes, whereas the lungs of TLR4-deficient mice inoculated with this bacterium contained approximately 5 ⫻ 105 lung leukocytes (Fig. 7A). Additionally, the predominant leukocyte type present in the lungs of TLR4-deficient mice infected with B. bronchiseptica was neutrophils (Fig. 7B). Interestingly, neither WT nor TLR4-deficient mice exhibited an increase in lung neutrophils upon inoculation with B. pertussis or B. parapertussis. These results suggest that TLR4 mediates the recruitment of neutrophils to the lungs during B. bronchiseptica infection and that B. pertussis and B. parapertussis induce very little neutrophil recruitment in the first 6 hours of infection. B. bronchiseptica LPS induces greater inflammatory responses in vitro than LPSs from B. pertussis and B. parapertussis. Previous studies have reported differences in the biological and immunological properties of LPSs of the three Bordetella species (1, 48, 49), and the results of our infection experiments suggest that B. bronchiseptica induces a greater


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inflammatory response via TLR4 than B. pertussis or B. parapertussis. These results suggest that there may be differences in the ability of each LPS to induce TLR4-dependent responses. To investigate this, we exposed RAW 264.7 murine macrophages to increasing concentrations of purified LPS from the three Bordetella species in overnight culture. The supernatants were then assayed for TNF-␣ as an indicator of LPS responsiveness. B. bronchiseptica LPS induced a measurable increase in TNF-␣ at a concentration of 0.1 ng/ml, whereas similar increases in this cytokine required exposure to 10 times as much B. pertussis LPS or 100 times as much B. parapertussis LPS (Fig. 8A). Similar results were observed by measuring IL-6 and nitric oxide levels in the cell supernatant (data not shown). Using a modified purpald assay (30) and the available literature regarding LPS structures of the bordetellae (4), B. parapertussis was calculated to have 1.15 times as many molecules per ␮g of LPS as B. bronchiseptica (data not shown). This indicates that a difference in the number of LPS molecules used in the assay does not account for the differences observed in the stimulation of the RAW264.7 cells by each LPS. Exposure to 100 ng/ml of each LPS resulted in similar levels of cytokine production. These results suggest that at concentrations in the physiologically relevant range, B. bronchiseptica LPS is considerably more stimulatory than LPS from B. pertussis or B. parapertussis. Since B. pertussis and B. parapertussis are human pathogens, we sought to compare the ability of Bordetella LPS to stimulate human TLR4. To examine this, we exposed HEK293 cells expressing human TLR4/MD2 to increasing concentrations of purified LPS from the three Bordetella species in overnight culture. The supernatants were then assayed for IL-8 as an indicator of LPS responsiveness. B. bronchiseptica LPS induced a measurable increase in IL-8 at concentrations of 1 ng/ml, whereas similar increases in this cytokine required exposure to 10 times as much B. pertussis LPS or 100 times as much B. parapertussis LPS (Fig. 8B). Interestingly, exposure to 1,000 ng/ml of each LPS resulted in significantly different levels of IL-8 production. These results indicate that B. bronchiseptica LPS stimulates human TLR4 more than LPS from B. pertussis or B. parapertussis.

DISCUSSION The ability of a pathogen to infect and colonize the host is dependent on the pathogen’s ability to subvert or avoid host defense mechanisms. Here we show that three closely related species of Bordetella are able to efficiently colonize the mouse lung and establish similar bacterial burdens by day 3 postinfection. The ability of the host to limit these bacteria during this period is independent of adaptive immunity. It is now well established that Toll-like receptors are an important component of innate host defenses, and TLR4 is recognized as playing a central role in limiting certain gram-negative bacterial infections (3, 40). However, recent findings suggest that this receptor is not universally important for innate host defense against all gram-negative bacteria (16, 28). The reasons why a single TLR would be critical for protection against some pathogens but not others which belong to the same class of microorganisms is not understood but may be related to dif-

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FIG. 8. Differential LPS-induced responses in vitro. (A) TNF-␣ levels in cell culture supernatant of RAW264.7 murine macrophages exposed to increasing concentrations of purified Bordetella LPS. (B) IL-8 levels in cell culture supernatants of HEK TLR4/MD2 cells exposed to increasing concentrations of purified Bordetella LPS. Solid diamonds, B. bronchiseptica LPS; open circles, B. pertussis LPS; open triangles, B. parapertussis LPS.

ferences in pathogen virulence pattern and PAMP expression patterns, which modulate host innate immune responses. The results of our study show that TLR4 is critical for limiting bacterial numbers within the first 3 days during infection with B. bronchiseptica but not B. pertussis or B. parapertussis. Our observations are consistent with those of Higgins et al. in that TLR4-deficient mice show a transient defect in limiting B. pertussis numbers after day 7. However, our results indicate that TLR4 is not required to limit B. parapertussis infection at any time point, even though this organism is more closely related to B. bronchiseptica than to B. pertussis. These findings suggest that the apparent differences in the role of TLR4 during infection with these organisms are not a result of vari-

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ation between infection models but are likely due to important biological differences between these species that influence the role of this receptor during infection. It is intriguing that there is such a vast difference in the critical need for a single immune receptor during host defense to these three organisms, especially considering the genetic similarity of these species (38, 46). Recently, Faure et al. demonstrated that TLR4 signaling limits the type III secretion system (TTSS)-induced lung pathology during Pseudomonas aeruginosa infection (13). Thus, a possible explanation for the reduced role of TLR4 during host defense to the B. pertussis and B. parapertussis strains used in this study is that they do not express a functional TTSS (38, 50). This hypothesis is also supported by our preliminary infection studies, which suggest that two strains that do express the TTSS, B. pertussis strain 18323 and a B. parapertussis isolate of ovine origin, are more virulent in TLR4-deficient mice than the strains used in this study. Additionally, our unpublished data suggest that deletion of the TTSS significantly reduces the virulence of B. bronchiseptica in TLR4-deficient mice. Interestingly, early elicited TNF-␣ and neutrophils are not required for innate host defense against B. pertussis or B. parapertussis but are critical for protection against B. bronchiseptica. Additionally, B. pertussis and B. parapertussis induce considerably less TNF-␣ production and neutrophil recruitment compared with B. bronchiseptica. It is intriguing that several innate immune functions that are apparently not required are also poorly induced during infection with the two human-adapted bordetellae. This raises the possibility that each species has developed a method to alter or avoid host immune responses to optimize colonization of the host. Kirimanjeswara et al. demonstrated that adoptive transfer of serum antibodies can rapidly eliminate B. bronchiseptica, but not B. pertussis or B. parapertussis, from the lower respiratory tract (26). The ability to avoid rapid antibody-mediated clearance may provide B. pertussis and B. parapertussis the opportunity to reinfect immune hosts, an important aspect in the epidemiology of these organisms (7). Interestingly, the mechanism by which antibodies rapidly clear B. bronchiseptica is dependent on TLR4 (27). This finding suggests that B. pertussis or B. parapertussis is able to avoid rapid antibody-mediated clearance by subverting or avoiding TLR4-mediated immune responses. In the case of B. pertussis this appears to be a result of PTX interfering with TLR4-mediated neutrophil recruitment. B. parapertussis may evade rapid antibody-mediated clearance by avoiding TLR4 recognition. In modulating or subverting TLR4-mediated immune responses, B. pertussis and B. parapertussis may have been able to alter their infectious disease cycle, which potentially contributed to their adaptation to a new host. It is of interest that in evolving from a B. bronchiseptica-like organism to primarily infect humans, both B. pertussis and B. parapertussis have reduced their ability to stimulate either mouse or human TLR4. It is possible that this is a common strategy that facilitates infection of a new host or reflects a switch from a nasal commensal to an acute pathogen of the lower respiratory tract. There is significant evidence that adaptation to humans provides selective pressure to modify LPS structures in several gram-negative bacteria. Hajjar et al. demonstrated that in adapting to the human lung, P. aeruginosa is


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able to alter its LPS acylation to modulate the response of human TLR4 but not murine TLR4 (16). Subsequent studies determined that several species of Salmonella and Yersinia also were able to modify LPS acylation and that this was associated with altered TLR4-mediated cytokine responses (25, 41). Although B. bronchiseptica rarely infects humans, there is evidence that human-adapted isolates of B. bronchiseptica have undergone LPS modification (15, 29). These findings support a hypothesis that LPS modifications by B. pertussis and B. parapertussis facilitated adaptation to humans. Determining the LPS and lipid A structures of the Bordetella species used in this study, as well as the molecular mechanisms by which the bacteria modify LPS, should provide insight into how organisms are able to alter a highly conserved molecule in order to adapt to a new host. Additionally, using purified LPS from specific Bordetella strains with mutations in the LPS synthesis pathway to examine the response mediated by human versus murine TLR4 may also elucidate molecular mechanisms involved in host adaptation.

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