Bordetella bronchiseptica Flagellin Is a Proinflammatory Determinant for Airway Epithelial Cells
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感染与免疫杂志 2005年第11期
Section on Molecular Medicine, Department of Internal Medicine
Department of Microbiology and Immunology, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157
ABSTRACT
Motility is an important virulence phenotype for many bacteria, and flagellin, the monomeric component of flagella, is a potent proinflammatory factor. Of the three Bordetella species, Bordetella pertussis and Bordetella parapertussis are nonmotile human pathogens, while Bordetella bronchiseptica expresses flagellin and causes disease in animals and immunocompromised human hosts. The BvgAS two-component signal transduction system regulates phenotypic-phase transition (Bvg+, Bvg–, and Bvgi) in bordetellae. The Bvg– phase of B. bronchiseptica is characterized by the expression of flagellin and the repression of adhesins and toxins necessary for the colonization of the respiratory tract. B. bronchiseptica naturally infects a variety of animal hosts and constitutes an excellent model to study Bordetella pathogenesis. Using in vitro coculture models of bacteria and human lung epithelial cells, we studied the effects of B. bronchiseptica flagellin on host defense responses. Our results show that B. bronchiseptica flagellin is a potent proinflammatory factor that induces chemokine, cytokine, and host defense gene expression. Furthermore, we investigated receptor specificity in the response to B. bronchiseptica flagellin. Our results show that B. bronchiseptica flagellin is able to signal effectively through both human and mouse Toll-like receptor 5.
INTRODUCTION
During normal growth and infection, many bacteria produce flagellin, the structural component of the bacterial flagellum, and a potent proinflammatory mediator for many cell types (48) with the ability to modulate innate immune responses in the lung (24, 61). Flagellin from different bacterial species elicits a strong inflammatory program in epithelial cells, including the production of the neutrophil chemoattractant interleukin-8 (IL-8) and inducible nitric oxide synthase (iNOS) (13, 15, 19, 59). Furthermore, secretion of flagellin is involved in the activation of proinflammatory signaling pathways and neutrophil transepithelial migration (49). The expression of innate host defense genes in epithelial cells, including matrilysin and human -defensin-2 (hBD2), is up-regulated by flagellin (31, 32, 43). Flagellin plays a role in triggering adaptive immune responses by stimulating chemokine secretion and subsequent migration and maturation of dendritic cells (39, 55) and by modulating T-cell activation in vivo (38). Indeed, flagellin, the ligand of mammalian Toll-like receptor 5 (TLR-5) (21, 41), is a major proinflammatory determinant of Salmonella species and Pseudomonas aeruginosa (8, 25, 70). Altogether, the host responds to flagellin with the production of factors involved in the recruitment of professional phagocytes and antigen-presenting cells, antimicrobial molecules, and inflammatory mediators to orchestrate an environment conducive to successful phagocyte activation and bacterial clearance (48).
Species from the genus Bordetella, namely, Bordetella pertussis, Bordetella parapertussis, and Bordetella bronchiseptica, colonize the respiratory tracts of mammals. While B. pertussis (the causative agent of whooping cough) has adapted exclusively to the human host, B. parapertussis can infect humans (causing a pertussis-like illness) and sheep. By contrast with the more host-restricted Bordetella spp., B. bronchiseptica causes respiratory tract infections in a wide variety of hosts, both human and animal. A two-component signal transduction system, BvgAS, controls a phenotypic transition (Bvg+, Bvg–, and Bvgi or intermediate phase) involving changes in the expression of virulence factors critical in the pathogenesis of bordetellae (6, 12, 27, 30, 54). The Bvg+ phase in bordetellae is characterized by the expression of virulence factors required for colonization of the respiratory tract, including adhesins (10, 18, 26, 28, 37) and toxins (7, 45). By contrast, the Bvg– phase of B. bronchiseptica, which may be advantageous in survival in the environment (9), is characterized mainly by the expression of genes involved in motility, including flagellin (2, 3), and the specific repression of virulence factors typical of the Bvg+ phase (1). Although motility genes are present in B. pertussis and B. parapertussis, inactivation of the flagellar operon results in a lack of motility in these two species (45). Rabbits and rats infected with wild-type B. bronchiseptica do not develop a potent immune response against flagellin, suggesting that flagellin expression upon infection is rapidly and effectively repressed (1, 9). Furthermore, infection with a B. bronchiseptica strain which expresses flagellin ectopically in the Bvg+ phase results in strong antibody responses to flagella and reduces tracheal colonization by B. bronchiseptica (1), suggesting that flagellin repression is important for Bordetella infection.
The wide host range of B. bronchiseptica and the phenotypic modulation by the BvgAS system make this species an excellent model to elucidate the role of virulence factors important in Bordetella pathogenesis. Although the functions of a number of Bvg+ phase-specific genes involved in Bordetella virulence have been studied, there is no information on the specific role of Bordetella flagellin in the regulation of host inflammatory responses. As stated earlier, flagellin has an inhibitory role in B. bronchiseptica infection (1). Because of the demonstrated role of flagellin in the regulation of host responses, we were prompted to investigate mucosal innate host defense modulation by B. bronchiseptica flagellin. For this, we have used models of coculture of human lung epithelial cells and bacteria to determine the role of B. bronchiseptica flagellin in the development of inflammatory responses. In addition, we have used human and mouse Toll-like receptor 5-expressing cells to examine receptor specificity in the response to B. bronchiseptica flagellin. Our results demonstrate that B. bronchiseptica flagellin is a potent proinflammatory virulence factor for human lung epithelial cells, eliciting a response geared to the effective elimination of the pathogen. Our work also shows that B. bronchiseptica flagellin signals efficiently through human and mouse TLR-5.
MATERIALS AND METHODS
Cell culture, bacteria, and other reagents. Normal human primary bronchial epithelial (NHBE) cells were obtained from Clonetics (Walkersville, MD) and grown in bronchial epithelial supplemented growth medium (Clonetics). BEAS-2B immortalized human bronchial epithelial cells were obtained from the American Type Culture Collection (ATCC) (Manassas, VA) and grown in the same medium (Clonetics). The human lung carcinoma cell lines Calu-3 and A549 (ATCC) were routinely maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS). All cells were routinely grown in the absence of antibiotics. Human (HEK293-hTLR-5), and mouse (HEK293-mTLR-5) TLR-5-expressing HEK293 cells and vector-transfected (HEK293-null) cells were obtained from InvivoGen (San Diego, CA) and grown in Dulbecco modified Eagle medium (DMEM) supplemented with 10% FBS and 10 μg/ml blasticidin (InvivoGen). Stably transfected RAW 264.7 cells expressing a murine TLR-5-enhanced yellow fluorescent protein (mTLR-5-EYFP) (63) were kindly provided by S. Mizel (Wake Forest University School of Medicine, Winston-Salem, NC) and grown in DMEM supplemented with 10% FBS and 200 μg/ml G-418 (Sigma). B. bronchiseptica strains used in this work were kindly provided by Jeff Miller (UCLA School of Medicine, Los Angeles, CA) (Table 1). B. bronchiseptica RB50, a wild-type strain (isolated from a New Zealand White rabbit) which alternates among different phases in response to environmental stimuli (9), is the parental strain of the B. bronchiseptica strains used in this work. Strain RB54 (bvgS54) is locked in the Bvg– phase as a result of an in-frame deletion in bvgS (9). The isogenic flagellin mutant RB54flaA contains a deletion in the flagellin structural gene flaA (10). Strain REV1 (frlr [superscrpt "r" designates reversal of expression of frl locus; reference 1]) expresses frlAB (an activator of flagellar genes) from the fhaB promoter resulting in constitutive expression of flagella in the Bvg+ phase (1). The nonflagellated strain RBA5 (frlr-flaA) has an in-frame deletion in the flaA gene (1). The nonmotile strain RBA2 (frlAB) has a deletion in frlAB (1). Strain RB53 (bvgS-C3) is locked in the Bvg+ phase, and thus, it does not express flagellin (9). Strain WD3 (bscN) is a type III secretion mutant, with an in-frame deletion in bscN, a putative ATPase required for the secretion process (68, 69). B. pertussis Bp536 and its Bvg– phase-locked derivative strain Bp537 (52) and B. parapertussis 12822 (22) have been previously described. B. bronchiseptica, B. pertussis, and B. parapertussis were maintained on Bordet-Gengou (BG) agar, containing 7.5% defibrinated sheep blood for the determination of colony morphology and hemolytic activity. Liquid cultures were grown in Stainer-Scholte broth, supplemented with heptakis(2,6-di-O-methyl-)--cyclodextrin (Sigma) for B. pertussis. BvgAS activity in B. parapertussis was modulated by 40 mM MgSO4. Salmonella enterica serovar Typhimurium 14028 and Pseudomonas aeruginosa 10145 were obtained from ATCC and were routinely grown in 3% tryptic soy broth. Gentamicin, polymyxin B, fetal bovine serum, and chemicals were obtained from Sigma Chemical Company (St. Louis, MO). The monoclonal antibody 15D8 (IGEN International, Gaithersburg, MD), directed against an epitope in E. coli flagellin, was used to detect Bordetella flagellin (2, 3).
Infection of epithelial cells for chemokine and cytokine analyses. Epithelial cells were seeded onto six-well plates and grown to about 95% density. Monolayers were infected with 2 x 108 CFU of each bacterial strain (corresponding to a multiplicity of infection [MOI] of 100) in 1 ml of RPMI 1640 medium without serum or antibiotics and incubated at 37°C for a period of 60 min. Conditioned medium samples from the 60-min infection were collected, centrifuged at 10,000 x g for 10 min to remove debris, concentrated 10-fold by lyophilization, and analyzed by Western blotting with flagellin-specific antibodies as described below. Following the 1-h infection, cells were washed extensively with phosphate-buffered saline and further incubated in RPMI 1640 medium supplemented with 10% FBS and 100 μg/ml gentamicin for 24 h. Conditioned media were then collected for the analysis of cytokine and chemokine secretion by enzyme-linked immunosorbent assays (ELISAs) with Quantikine reagents (R&D Systems, Minneapolis, MN) according to the manufacturer's instructions. Data are reported as means ± standard deviations. All determinations of cytokine and chemokine secretion were done in duplicate and repeated in at least three independent experiments. Cytotoxicity was determined as described previously (8), using a lactate dehydrogenase-based in vitro toxicology kit (Sigma) according to the manufacturer's instructions.
Host defense gene expression analysis. Calu-3 human lung epithelial cells were seeded onto six-well plates and grown to about 95% density. Epithelial monolayers were infected for 60 min and then washed as described above. Cells were subsequently incubated in RPMI 1640 medium containing 10% FBS and 100 μg/ml gentamicin, and total RNA was prepared with RNAzol B (Tel-Test, Inc., Friendswood, TX) at 6 h postinfection. Northern hybridization for matrilysin and glyceraldehyde-3-phosphate dehydrogenase mRNAs was done as described previously (32). For the expression analysis of human -defensins, total RNA samples were reverse transcribed using random hexamer primers (Perkin-Elmer, Branchburg, NJ), and cDNAs were then amplified by PCR as described previously (31). The sizes of the amplified products for hBD2 and hBD1 are 241 and 258 bp, respectively. Reactions were analyzed on 6% acrylamide gels.
Purification of flagellin and protein sequencing. Flagellin was purified from overnight culture supernatants of B. bronchiseptica RB54, P. aeruginosa (ATCC 10145), and S. enterica serovar Typhimurium (ATCC 14028), as described previously (32). Flagellin was analyzed by electrophoresis on 12% sodium dodecyl sulfate (SDS)-polyacrylamide gels and stained with Coomassie blue. Flagellin was further purified by using polymyxin B beads (Sigma), and the removal of endotoxin was verified as described previously (8). For the N-terminal sequencing of flagellin, purified protein was visualized by staining with 0.1% Coomassie brilliant blue, excised, and sequenced by automated Edman degradation using a PE Applied Biosystems 492 sequencer as described previously (31).
Immunoblotting. Conditioned medium samples were separated on 12% SDS-polyacrylamide gels, transferred by semidry electrophoretic transfer to nitrocellulose membranes (Hybond ECL; Amersham Pharmacia Biotechology, Buckinghamshire, United Kingdom), and probed with the antiflagellin monoclonal antibody 15D8 as described previously (31).
Statistical analysis. For statistical comparisons, data on chemokine and cytokine expression were analyzed by analysis of variance and Bonferroni-type multiple t test. A P value of <0.01 was considered significant.
RESULTS
Effect of flagellin in the development of proinflammatory responses in human lung epithelial cells exposed to Bordetella bronchiseptica. To examine the role of B. bronchiseptica flagellin in the development of proinflammatory and host defense responses in the airways, we used a model of coinfection of epithelial cells and bacteria (8, 31, 32) and studied the behavior of selected markers of inflammation regulated by flagellin (48). In particular, we compared cytokine and chemokine secretion by human lung epithelial cells exposed to isogenic flagellated and nonflagellated B. bronchiseptica strains. To determine the role of flagellin in the absence of Bvg+ phase-specific factors, we exposed cells to the Bvg– phase-locked motile strain RB54 and the isogenic flagellin mutant RB54flaA (9, 10). As shown in Fig. 1A, secretion of the chemokine macrophage inflammatory protein 3 (MIP-3) (CCL20) by BEAS-2B cells was up-regulated compared to uninfected cells in response to exposure to RB54 but not RB54flaA. A similar result was obtained when we determined IL-8 (CXCL8) secretion by A549, NHBE, BEAS-2B, and Calu-3 cells exposed to B. bronchiseptica (Fig. 1B and C; see Fig. 3C; also data not shown), suggesting that flagellin expression by the bacterium induces these inflammatory markers. Secretion of IL-8 by NHBE cells was reduced compared to the other cell lines examined (Fig. 1), in agreement with previous reports (64), but a similar significant reduction in IL-8 secretion was observed in response to nonmotile bacteria (Fig. 1C). To determine the effect of flagellin expressed ectopically in the Bvg+ phase, we used REV1 (frlr), a strain engineered to express flagella constitutively in a wild-type background (1). As shown in Fig. 1A and B, REV1 induced the secretion of proinflammatory markers by human lung epithelial cells to an extent similar to RB54, while exposure to the isogenic flagellin mutant RBA5 (frlr::flaA) had no significant effect (Fig. 1A and B; also data not shown). No significant induction of cytokines and chemokines was observed in response to exposure to RBA2 (a nonmotile frlAB deletion mutant [1]) or the nonflagellated Bvg+ phase-locked strain (RB53) in any of the human lung epithelial cell lines mentioned above (Fig. 1 and data not shown). Acute exposure to nonflagellated B. pertussis and B. parapertussis did not induce significant IL-8 secretion by A549 cells (Fig. 1B), consistent with previous reports (5). Finally, we used strain WD3 (bscN), a B. bronchiseptica type III secretion mutant (69). Type III secretion has been shown to modulate signaling pathways in host cells (51, 68). As shown in Fig. 1A and B, WD3 induced the secretion of MIP-3 and IL-8 by human lung epithelial cells to an extent similar to the parental strain RB50, suggesting that factors secreted by the type III secretion system are not critical for these responses. Note that when RB50 and its isogenic derivative WD3 are grown in liquid culture, these strains can switch to the Bvg– phase and express flagellin (Fig. 2), resulting in the induction of proinflammatory mediators in lung epithelial cells (Fig. 1). In contrast, we determined that RB50 and WD3 grown on solid agar media did not express flagellin and did not induce proinflammatory markers (data not shown). Altogether, these data show that flagellin expression by B. bronchiseptica results in the induction of a proinflammatory response in a variety of human lung epithelial cell lines and human primary airway cells. In this experimental design, no significant cytotoxicity due to B. bronchiseptica infection was observed at 6 and 24 h postinfection, the time points at which host responses were analyzed (data not shown).
Expression of flagellin by B. bronchiseptica during infection. Consistent with the hypothesis that flagellin triggers the inflammatory response observed in human lung epithelial cells, comparable amounts of flagellin were detected in the conditioned media of the 1-h period of infection by strains RB54, REV1, RB50, and WD3 (Fig. 2A). As expected, no signal was detected in conditioned media of cells exposed to the flagellin-deficient, nonmotile strains RB54flaA, RBA5, RBA2, and RB53 and B. pertussis and B. parapertussis (Fig. 2A). The motility phenotype was confirmed in standard soft-agar motility assays for all B. bronchiseptica strains and correlated with the presence or absence of flagellin as detected by Western blotting of conditioned media and bacterial cell lysates (data not shown). As mentioned before, RB50 and WD3 grown on solid agar media did not express flagellin (data not shown).
B. bronchiseptica flagellin induces host defense gene expression in human lung epithelial cells. In order to examine the role of flagellin in the pathogenesis of B. bronchiseptica, we purified this protein from strain RB54 (32). A unique band migrating at about 40 kDa, consistent with the expected mass of B. bronchiseptica flagellin (1, 46), was detected by Coomassie blue staining in our purified preparations (Fig. 2B), and its identity was further confirmed as flagellin by Western blotting (Fig. 2C) and N-terminal sequencing. We exposed Calu-3 human airway epithelial cells to purified flagellin, as well as to strains RB54 and RB54flaA. As shown in Fig. 3A, expression of the inducible antimicrobial peptide hBD2 was up-regulated by exposure to flagellin and RB54, but not to the flagellin mutant RB54flaA. hBD1, which is constitutively expressed by epithelial cells (31, 44), was used as an internal control. Furthermore, the expression of matrilysin, a matrix metalloproteinase involved in host defense (66), which is specifically up-regulated by P. aeruginosa flagellin (32), was also induced by Bordetella flagellin and RB54, but not by RB54flaA (Fig. 3B). In similar experiments, conditioned media were harvested at 24 h postinfection, and selected proinflammatory mediators were examined by ELISAs. The secretion of IL-8, granulocyte-macrophage colony-stimulating factor (GM-CSF), and MIP-3 was induced by treatment with purified flagellin to an extent similar to exposure to RB54 (Fig. 3C, D, and E). On the basis of the amount of flagellin detected by Western blotting in conditioned medium samples of infected cells, direct exposure to B. bronchiseptica RB54 for 1 h at an MOI of 100 was roughly equivalent to treatment with 10–7 M purified flagellin (data not shown). Altogether, these results indicate that B. bronchiseptica flagellin is an important determinant of expression of these host defense genes and a modulator of airway mucosal defense.
Bordetella flagellin is similar in potency to other bacterial flagellins. We compared the effects of flagellin purified from Bordetella, Pseudomonas, and Salmonella species on IL-8 secretion by human lung epithelial cells. In these experiments, Calu-3 cells were challenged for 24 h with different concentrations of endotoxin-free flagellin, and the accumulation of IL-8 in the conditioned media was determined by ELISA. As shown in Fig. 4, treatment with 10–8 M Bordetella flagellin resulted in an approximately 20-fold induction in IL-8 levels, while the same concentration of S. enterica serovar Typhimurium flagellin resulted in a 24-fold induction. Consistent with recent observations (4, 24), P. aeruginosa flagellin showed slightly reduced bioactivity (about 10-fold induction in IL-8 secretion with exposure to 10–8 M flagellin). Therefore, Bordetella flagellin bioactivity in human airway epithelial cells is comparable to that of flagellin from other gram-negative pathogens.
Bordetella flagellin signals through human and mouse TLR-5. Bordetella spp. that are human pathogens (B. pertussis and B. parapertussis) are nonmotile, while the species with a broad host range, B. bronchiseptica, modulates motility through BvgAS activity. To determine whether the motility-host restriction dichotomy among bordetellae relates to flagellin receptor specificity, we used an in vitro system of TLR-5 receptors expressed on a human epithelial cell line with intrinsic low levels of expression of most TLRs. Thus, we infected human epithelial kidney cells (HEK293) stably transfected with human (HEK293-hTLR5) and mouse (HEK293-mTLR5) TLR-5 with B. bronchiseptica and determined the levels of IL-8 in conditioned media 24 h postinfection (Fig. 5A). For a control, we used HEK293 cells transfected with vector alone (HEK293-null). As shown in Fig. 5A, infection of HEK293-hTLR5 and HEK293-mTLR5 cells with motile strains RB54 and REV1 resulted in similar induction in IL-8 secretion compared to uninfected cells. Furthermore, there was a very limited response to the flagellin mutants RB54flaA and RBA-5 and the nonmotile strain RBA-2 (Fig. 5A). In fact, IL-8 secretion in response to the flagellin mutants was not significantly different from the low level of IL-8 induction observed in HEK293-null cells exposed to B. bronchiseptica (Fig. 5A). This response of HEK293-null cells may be mediated by intrinsic low levels of expression of other receptors in the parental cells (57). In addition, transfected cells were treated for 24 h with 10–8 M endotoxin-free flagellin purified from B. bronchiseptica and S. enterica serovar Typhimurium, and secretion of IL-8 was examined by ELISA as before (Fig. 5B). Results show that treatment with Bordetella flagellin induces IL-8 secretion to a similar extent in cells expressing human and mouse TLR-5, suggesting that it signals effectively through both receptors. Furthermore, Bordetella flagellin bioactivity in these cells was comparable to Salmonella flagellin bioactivity.
Proinflammatory effects of Bordetella exposure in macrophages. To investigate the contribution of TLR-5-dependent signaling to the proinflammatory effects of Bordetella exposure, we used a system of stably transfected RAW 264.7 murine macrophages expressing this receptor (63). Control RAW 264.7 cells (which respond to TLR-2 and -4 ligands but do not express TLR-5 and do not respond to flagellin [40]) and cells expressing TLR-5 (TLR-5-EYFP) were infected with B. bronchiseptica, and cytokine levels were determined in 4-h conditioned media. As shown in Fig. 6A and B, exposure of TLR-5-expressing RAW cells to motile strains RB54 and REV1 resulted in a three- to fivefold increase in tumor necrosis factor alpha (TNF-) and MIP-2 secretion over the isogenic flagellin mutants RB54flaA and RBA-5. By contrast, exposure of control RAW cells to B. bronchiseptica resulted in increased cytokine levels that were not significantly different between motile strains and flagellin mutants. For a control, cells were incubated with purified Bordetella flagellin, and the levels of TNF- were determined in conditioned media (Fig. 6C). Treatment of TLR-5-expressing cells, but not control cells, resulted in a 10-fold increase in TNF-. Altogether, these results confirm the importance of flagellin in the development of proinflammatory responses to B. bronchiseptica infection.
DISCUSSION
Flagella and the motility they confer are critical virulence factors for a variety of microorganisms, and flagellin is a major inducer of host inflammatory responses (48). In this work, we show that Bordetella flagellin is a potent proinflammatory factor for human lung epithelial cells, further shaping a paradigm proposed for Salmonella and Pseudomonas flagellin (8, 25, 70). Thus, the program of epithelial cell activation triggered by flagellin coordinates a response aimed at the effective elimination of the pathogen. We have previously shown that P. aeruginosa flagellin induces the expression of critical cytokines, chemokines, and host defense genes in human lung epithelial cells (8). Here we demonstrate a role for Bordetella flagellin in the induction of proinflammatory mediators. We selected MIP-3, IL-8, and GM-CSF as critical components of the microenvironment generated by human epithelial cells to coordinate inflammatory responses in the airways. IL-8 and GM-CSF are involved in recruitment and survival of neutrophils (53), and MIP-3 (CCL20) participates in the recruitment and activation of immature dendritic cells (50). Flagellin-regulated human host defense genes, matrilysin and -defensin-2 genes (32, 43), were also specifically induced by Bordetella flagellin.
The conserved N- and C-terminal domains of flagellin from different bacteria are essential for bioactivity (14, 16, 42, 56), and the N-terminal D1 domain conserved amino acid residues required for TLR-5 activation are conserved in Bordetella flagellin (4). Indeed, we show here that the proinflammatory and signaling potencies of Bordetella flagellin are comparable to those of S. enterica serovar Typhimurium and P. aeruginosa flagellins, in contrast with the low intrinsic activity (i.e., weak development of inflammatory responses and/or activation of TLR-5) reported for Helicobacter pylori (20, 29) and Campylobacter jejuni (62) flagellins. Altogether, our results using primary and immortalized cells suggest that Bordetella flagellin shapes a potent proinflammatory and innate immune response in lung epithelial cells. In vivo, B. bronchiseptica elicits acute inflammatory responses in the lung, and host factors may contribute to effective bacterial clearance (65). Among other virulence factors important in Bordetella pathogenesis (10, 17), a role for lipopolysaccharide signaling through TLR-4 has been demonstrated recently (23, 34, 35). Interestingly, up-regulation of most inflammatory genes in B. bronchiseptica-exposed macrophages occurs in a TLR-4-independent manner (34). Exposure of TLR-4-deficient macrophages to whole bacteria resulted in an overall transcriptional response to B. bronchiseptica similar to the one observed in wild-type macrophages, while lipopolysaccharide had no significant effect on TLR-4-deficient macrophages (34). While pointing to the importance of TLR-4-mediated signaling in the early production of TNF- during B. bronchiseptica infection, this work suggested that additional bacterial factors are responsible for eliciting the majority of the macrophage inflammatory response to this microorganism (34). Thus, our present work includes flagellin among the bacterial factors that modulate the host response to B. bronchiseptica.
In the context of Bordetella infection and in addition to the lack of flagellin expression by B. pertussis, another difference between B. bronchiseptica and B. pertussis is the expression of pertussis toxin only by the latter species. Thus, the induction of chemokines by pertussis toxin could result in different inflammatory responses to these two species (reviewed in reference 36). Furthermore, with the demonstrated ability of Bordetella flagellin to activate TLR-5, it is likely that flagellin from this microorganism may also activate other immune cells, including macrophages and dendritic cells (39, 40).
Because of the inflammatory activity of flagellin, an emerging theme in bacterial pathogenesis suggests that the infection strategy of some bacteria has evolved to effectively suppress its expression, whether permanently or during the establishment of chronic infection. B. bronchiseptica carries the entire flagellar operon, and it is motile, whereas the flagellar operons of both B. pertussis and B. parapertussis are inactivated by multiple pseudogenes and insertions, resulting in a lack of motility (45). Indeed, flagellin expression is rapidly and effectively repressed upon infection with wild-type B. bronchiseptica (1, 9), and ectopic expression of flagellin in the Bvg+ growth phase inhibits colonization (1). Therefore, our data on Bordetella flagellin bioactivity provide a rationale for immunological pressure to suppress its expression during infection of the host. In addition, B. bronchiseptica flagellin induced similar proinflammatory responses in cells expressing human or mouse TLR-5. Within the limitations of an in vitro system, the data strongly suggest that B. bronchiseptica flagellin signals effectively through both the human and mouse flagellin receptors.
The loss of flagellar expression by B. pertussis and B. parapertussis evokes the lack of motility developed by P. aeruginosa as a specific long-term adaptation to the environment in the cystic fibrosis lung (33, 67) and the loss of motility of Shigella species and Yersinia pestis as a strategy to adapt to a particular host environment (58). Since Bordetella flagellar expression is limited to the Bvg– phase, it has been suggested that these organelles are important in survival in the environment (45). For B. pertussis, a lack of motility may reflect adaptation to a human-restricted niche, supporting the hypothesis that this microorganism does not have an environmental phase. In host-pathogen interactions, surface structures, such as flagella, fimbriae, and polysaccharides, have a dual role in the establishment of infection and modulation of host responses aimed at clearance. Thus, elimination of flagella may have contributed to enhanced B. pertussis virulence in the human host (45, 47). In this context, the inhibition of colonization by the ectopic expression of flagella in the B. bronchiseptica Bvg+ phase underscores the importance of BvgAS-mediated flagellin repression in the establishment of long-term Bordetella infection (1). In summary, our present work includes Bordetella flagellin in the class of bacterial flagellins with potent proinflammatory effects on lung epithelial cells and demonstrates that human and mouse TLR-5-expressing cells respond similarly to Bordetella flagellin. Because of the abilities of bacterial flagellin to induce mucosal immune responses and to act as a potent adjuvant (11, 24, 60), characterizing responses to Bordetella flagellin may be particularly relevant in the context of the development of new vaccines against Bordetella spp.
ACKNOWLEDGMENTS
We are especially grateful to Jeff Miller for providing bacterial strains. We thank S. Mizel and D. Wozniak for helpful suggestions and critical reading of the manuscript and M. Mishra and G. Parise for excellent technical assistance. We also thank S. Mizel for the generous gift of TLR-5-expressing RAW 264.7 cells. We thank M. Lively and M. Morris for protein sequencing.
This work was supported in part by Wake Forest University School of Medicine institutional funds (Y.S.L.-B. and R.D.). We (Y.S.L.-B., L.M.C., and R.D.) declare no conflict of interest.
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Department of Microbiology and Immunology, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157
ABSTRACT
Motility is an important virulence phenotype for many bacteria, and flagellin, the monomeric component of flagella, is a potent proinflammatory factor. Of the three Bordetella species, Bordetella pertussis and Bordetella parapertussis are nonmotile human pathogens, while Bordetella bronchiseptica expresses flagellin and causes disease in animals and immunocompromised human hosts. The BvgAS two-component signal transduction system regulates phenotypic-phase transition (Bvg+, Bvg–, and Bvgi) in bordetellae. The Bvg– phase of B. bronchiseptica is characterized by the expression of flagellin and the repression of adhesins and toxins necessary for the colonization of the respiratory tract. B. bronchiseptica naturally infects a variety of animal hosts and constitutes an excellent model to study Bordetella pathogenesis. Using in vitro coculture models of bacteria and human lung epithelial cells, we studied the effects of B. bronchiseptica flagellin on host defense responses. Our results show that B. bronchiseptica flagellin is a potent proinflammatory factor that induces chemokine, cytokine, and host defense gene expression. Furthermore, we investigated receptor specificity in the response to B. bronchiseptica flagellin. Our results show that B. bronchiseptica flagellin is able to signal effectively through both human and mouse Toll-like receptor 5.
INTRODUCTION
During normal growth and infection, many bacteria produce flagellin, the structural component of the bacterial flagellum, and a potent proinflammatory mediator for many cell types (48) with the ability to modulate innate immune responses in the lung (24, 61). Flagellin from different bacterial species elicits a strong inflammatory program in epithelial cells, including the production of the neutrophil chemoattractant interleukin-8 (IL-8) and inducible nitric oxide synthase (iNOS) (13, 15, 19, 59). Furthermore, secretion of flagellin is involved in the activation of proinflammatory signaling pathways and neutrophil transepithelial migration (49). The expression of innate host defense genes in epithelial cells, including matrilysin and human -defensin-2 (hBD2), is up-regulated by flagellin (31, 32, 43). Flagellin plays a role in triggering adaptive immune responses by stimulating chemokine secretion and subsequent migration and maturation of dendritic cells (39, 55) and by modulating T-cell activation in vivo (38). Indeed, flagellin, the ligand of mammalian Toll-like receptor 5 (TLR-5) (21, 41), is a major proinflammatory determinant of Salmonella species and Pseudomonas aeruginosa (8, 25, 70). Altogether, the host responds to flagellin with the production of factors involved in the recruitment of professional phagocytes and antigen-presenting cells, antimicrobial molecules, and inflammatory mediators to orchestrate an environment conducive to successful phagocyte activation and bacterial clearance (48).
Species from the genus Bordetella, namely, Bordetella pertussis, Bordetella parapertussis, and Bordetella bronchiseptica, colonize the respiratory tracts of mammals. While B. pertussis (the causative agent of whooping cough) has adapted exclusively to the human host, B. parapertussis can infect humans (causing a pertussis-like illness) and sheep. By contrast with the more host-restricted Bordetella spp., B. bronchiseptica causes respiratory tract infections in a wide variety of hosts, both human and animal. A two-component signal transduction system, BvgAS, controls a phenotypic transition (Bvg+, Bvg–, and Bvgi or intermediate phase) involving changes in the expression of virulence factors critical in the pathogenesis of bordetellae (6, 12, 27, 30, 54). The Bvg+ phase in bordetellae is characterized by the expression of virulence factors required for colonization of the respiratory tract, including adhesins (10, 18, 26, 28, 37) and toxins (7, 45). By contrast, the Bvg– phase of B. bronchiseptica, which may be advantageous in survival in the environment (9), is characterized mainly by the expression of genes involved in motility, including flagellin (2, 3), and the specific repression of virulence factors typical of the Bvg+ phase (1). Although motility genes are present in B. pertussis and B. parapertussis, inactivation of the flagellar operon results in a lack of motility in these two species (45). Rabbits and rats infected with wild-type B. bronchiseptica do not develop a potent immune response against flagellin, suggesting that flagellin expression upon infection is rapidly and effectively repressed (1, 9). Furthermore, infection with a B. bronchiseptica strain which expresses flagellin ectopically in the Bvg+ phase results in strong antibody responses to flagella and reduces tracheal colonization by B. bronchiseptica (1), suggesting that flagellin repression is important for Bordetella infection.
The wide host range of B. bronchiseptica and the phenotypic modulation by the BvgAS system make this species an excellent model to elucidate the role of virulence factors important in Bordetella pathogenesis. Although the functions of a number of Bvg+ phase-specific genes involved in Bordetella virulence have been studied, there is no information on the specific role of Bordetella flagellin in the regulation of host inflammatory responses. As stated earlier, flagellin has an inhibitory role in B. bronchiseptica infection (1). Because of the demonstrated role of flagellin in the regulation of host responses, we were prompted to investigate mucosal innate host defense modulation by B. bronchiseptica flagellin. For this, we have used models of coculture of human lung epithelial cells and bacteria to determine the role of B. bronchiseptica flagellin in the development of inflammatory responses. In addition, we have used human and mouse Toll-like receptor 5-expressing cells to examine receptor specificity in the response to B. bronchiseptica flagellin. Our results demonstrate that B. bronchiseptica flagellin is a potent proinflammatory virulence factor for human lung epithelial cells, eliciting a response geared to the effective elimination of the pathogen. Our work also shows that B. bronchiseptica flagellin signals efficiently through human and mouse TLR-5.
MATERIALS AND METHODS
Cell culture, bacteria, and other reagents. Normal human primary bronchial epithelial (NHBE) cells were obtained from Clonetics (Walkersville, MD) and grown in bronchial epithelial supplemented growth medium (Clonetics). BEAS-2B immortalized human bronchial epithelial cells were obtained from the American Type Culture Collection (ATCC) (Manassas, VA) and grown in the same medium (Clonetics). The human lung carcinoma cell lines Calu-3 and A549 (ATCC) were routinely maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS). All cells were routinely grown in the absence of antibiotics. Human (HEK293-hTLR-5), and mouse (HEK293-mTLR-5) TLR-5-expressing HEK293 cells and vector-transfected (HEK293-null) cells were obtained from InvivoGen (San Diego, CA) and grown in Dulbecco modified Eagle medium (DMEM) supplemented with 10% FBS and 10 μg/ml blasticidin (InvivoGen). Stably transfected RAW 264.7 cells expressing a murine TLR-5-enhanced yellow fluorescent protein (mTLR-5-EYFP) (63) were kindly provided by S. Mizel (Wake Forest University School of Medicine, Winston-Salem, NC) and grown in DMEM supplemented with 10% FBS and 200 μg/ml G-418 (Sigma). B. bronchiseptica strains used in this work were kindly provided by Jeff Miller (UCLA School of Medicine, Los Angeles, CA) (Table 1). B. bronchiseptica RB50, a wild-type strain (isolated from a New Zealand White rabbit) which alternates among different phases in response to environmental stimuli (9), is the parental strain of the B. bronchiseptica strains used in this work. Strain RB54 (bvgS54) is locked in the Bvg– phase as a result of an in-frame deletion in bvgS (9). The isogenic flagellin mutant RB54flaA contains a deletion in the flagellin structural gene flaA (10). Strain REV1 (frlr [superscrpt "r" designates reversal of expression of frl locus; reference 1]) expresses frlAB (an activator of flagellar genes) from the fhaB promoter resulting in constitutive expression of flagella in the Bvg+ phase (1). The nonflagellated strain RBA5 (frlr-flaA) has an in-frame deletion in the flaA gene (1). The nonmotile strain RBA2 (frlAB) has a deletion in frlAB (1). Strain RB53 (bvgS-C3) is locked in the Bvg+ phase, and thus, it does not express flagellin (9). Strain WD3 (bscN) is a type III secretion mutant, with an in-frame deletion in bscN, a putative ATPase required for the secretion process (68, 69). B. pertussis Bp536 and its Bvg– phase-locked derivative strain Bp537 (52) and B. parapertussis 12822 (22) have been previously described. B. bronchiseptica, B. pertussis, and B. parapertussis were maintained on Bordet-Gengou (BG) agar, containing 7.5% defibrinated sheep blood for the determination of colony morphology and hemolytic activity. Liquid cultures were grown in Stainer-Scholte broth, supplemented with heptakis(2,6-di-O-methyl-)--cyclodextrin (Sigma) for B. pertussis. BvgAS activity in B. parapertussis was modulated by 40 mM MgSO4. Salmonella enterica serovar Typhimurium 14028 and Pseudomonas aeruginosa 10145 were obtained from ATCC and were routinely grown in 3% tryptic soy broth. Gentamicin, polymyxin B, fetal bovine serum, and chemicals were obtained from Sigma Chemical Company (St. Louis, MO). The monoclonal antibody 15D8 (IGEN International, Gaithersburg, MD), directed against an epitope in E. coli flagellin, was used to detect Bordetella flagellin (2, 3).
Infection of epithelial cells for chemokine and cytokine analyses. Epithelial cells were seeded onto six-well plates and grown to about 95% density. Monolayers were infected with 2 x 108 CFU of each bacterial strain (corresponding to a multiplicity of infection [MOI] of 100) in 1 ml of RPMI 1640 medium without serum or antibiotics and incubated at 37°C for a period of 60 min. Conditioned medium samples from the 60-min infection were collected, centrifuged at 10,000 x g for 10 min to remove debris, concentrated 10-fold by lyophilization, and analyzed by Western blotting with flagellin-specific antibodies as described below. Following the 1-h infection, cells were washed extensively with phosphate-buffered saline and further incubated in RPMI 1640 medium supplemented with 10% FBS and 100 μg/ml gentamicin for 24 h. Conditioned media were then collected for the analysis of cytokine and chemokine secretion by enzyme-linked immunosorbent assays (ELISAs) with Quantikine reagents (R&D Systems, Minneapolis, MN) according to the manufacturer's instructions. Data are reported as means ± standard deviations. All determinations of cytokine and chemokine secretion were done in duplicate and repeated in at least three independent experiments. Cytotoxicity was determined as described previously (8), using a lactate dehydrogenase-based in vitro toxicology kit (Sigma) according to the manufacturer's instructions.
Host defense gene expression analysis. Calu-3 human lung epithelial cells were seeded onto six-well plates and grown to about 95% density. Epithelial monolayers were infected for 60 min and then washed as described above. Cells were subsequently incubated in RPMI 1640 medium containing 10% FBS and 100 μg/ml gentamicin, and total RNA was prepared with RNAzol B (Tel-Test, Inc., Friendswood, TX) at 6 h postinfection. Northern hybridization for matrilysin and glyceraldehyde-3-phosphate dehydrogenase mRNAs was done as described previously (32). For the expression analysis of human -defensins, total RNA samples were reverse transcribed using random hexamer primers (Perkin-Elmer, Branchburg, NJ), and cDNAs were then amplified by PCR as described previously (31). The sizes of the amplified products for hBD2 and hBD1 are 241 and 258 bp, respectively. Reactions were analyzed on 6% acrylamide gels.
Purification of flagellin and protein sequencing. Flagellin was purified from overnight culture supernatants of B. bronchiseptica RB54, P. aeruginosa (ATCC 10145), and S. enterica serovar Typhimurium (ATCC 14028), as described previously (32). Flagellin was analyzed by electrophoresis on 12% sodium dodecyl sulfate (SDS)-polyacrylamide gels and stained with Coomassie blue. Flagellin was further purified by using polymyxin B beads (Sigma), and the removal of endotoxin was verified as described previously (8). For the N-terminal sequencing of flagellin, purified protein was visualized by staining with 0.1% Coomassie brilliant blue, excised, and sequenced by automated Edman degradation using a PE Applied Biosystems 492 sequencer as described previously (31).
Immunoblotting. Conditioned medium samples were separated on 12% SDS-polyacrylamide gels, transferred by semidry electrophoretic transfer to nitrocellulose membranes (Hybond ECL; Amersham Pharmacia Biotechology, Buckinghamshire, United Kingdom), and probed with the antiflagellin monoclonal antibody 15D8 as described previously (31).
Statistical analysis. For statistical comparisons, data on chemokine and cytokine expression were analyzed by analysis of variance and Bonferroni-type multiple t test. A P value of <0.01 was considered significant.
RESULTS
Effect of flagellin in the development of proinflammatory responses in human lung epithelial cells exposed to Bordetella bronchiseptica. To examine the role of B. bronchiseptica flagellin in the development of proinflammatory and host defense responses in the airways, we used a model of coinfection of epithelial cells and bacteria (8, 31, 32) and studied the behavior of selected markers of inflammation regulated by flagellin (48). In particular, we compared cytokine and chemokine secretion by human lung epithelial cells exposed to isogenic flagellated and nonflagellated B. bronchiseptica strains. To determine the role of flagellin in the absence of Bvg+ phase-specific factors, we exposed cells to the Bvg– phase-locked motile strain RB54 and the isogenic flagellin mutant RB54flaA (9, 10). As shown in Fig. 1A, secretion of the chemokine macrophage inflammatory protein 3 (MIP-3) (CCL20) by BEAS-2B cells was up-regulated compared to uninfected cells in response to exposure to RB54 but not RB54flaA. A similar result was obtained when we determined IL-8 (CXCL8) secretion by A549, NHBE, BEAS-2B, and Calu-3 cells exposed to B. bronchiseptica (Fig. 1B and C; see Fig. 3C; also data not shown), suggesting that flagellin expression by the bacterium induces these inflammatory markers. Secretion of IL-8 by NHBE cells was reduced compared to the other cell lines examined (Fig. 1), in agreement with previous reports (64), but a similar significant reduction in IL-8 secretion was observed in response to nonmotile bacteria (Fig. 1C). To determine the effect of flagellin expressed ectopically in the Bvg+ phase, we used REV1 (frlr), a strain engineered to express flagella constitutively in a wild-type background (1). As shown in Fig. 1A and B, REV1 induced the secretion of proinflammatory markers by human lung epithelial cells to an extent similar to RB54, while exposure to the isogenic flagellin mutant RBA5 (frlr::flaA) had no significant effect (Fig. 1A and B; also data not shown). No significant induction of cytokines and chemokines was observed in response to exposure to RBA2 (a nonmotile frlAB deletion mutant [1]) or the nonflagellated Bvg+ phase-locked strain (RB53) in any of the human lung epithelial cell lines mentioned above (Fig. 1 and data not shown). Acute exposure to nonflagellated B. pertussis and B. parapertussis did not induce significant IL-8 secretion by A549 cells (Fig. 1B), consistent with previous reports (5). Finally, we used strain WD3 (bscN), a B. bronchiseptica type III secretion mutant (69). Type III secretion has been shown to modulate signaling pathways in host cells (51, 68). As shown in Fig. 1A and B, WD3 induced the secretion of MIP-3 and IL-8 by human lung epithelial cells to an extent similar to the parental strain RB50, suggesting that factors secreted by the type III secretion system are not critical for these responses. Note that when RB50 and its isogenic derivative WD3 are grown in liquid culture, these strains can switch to the Bvg– phase and express flagellin (Fig. 2), resulting in the induction of proinflammatory mediators in lung epithelial cells (Fig. 1). In contrast, we determined that RB50 and WD3 grown on solid agar media did not express flagellin and did not induce proinflammatory markers (data not shown). Altogether, these data show that flagellin expression by B. bronchiseptica results in the induction of a proinflammatory response in a variety of human lung epithelial cell lines and human primary airway cells. In this experimental design, no significant cytotoxicity due to B. bronchiseptica infection was observed at 6 and 24 h postinfection, the time points at which host responses were analyzed (data not shown).
Expression of flagellin by B. bronchiseptica during infection. Consistent with the hypothesis that flagellin triggers the inflammatory response observed in human lung epithelial cells, comparable amounts of flagellin were detected in the conditioned media of the 1-h period of infection by strains RB54, REV1, RB50, and WD3 (Fig. 2A). As expected, no signal was detected in conditioned media of cells exposed to the flagellin-deficient, nonmotile strains RB54flaA, RBA5, RBA2, and RB53 and B. pertussis and B. parapertussis (Fig. 2A). The motility phenotype was confirmed in standard soft-agar motility assays for all B. bronchiseptica strains and correlated with the presence or absence of flagellin as detected by Western blotting of conditioned media and bacterial cell lysates (data not shown). As mentioned before, RB50 and WD3 grown on solid agar media did not express flagellin (data not shown).
B. bronchiseptica flagellin induces host defense gene expression in human lung epithelial cells. In order to examine the role of flagellin in the pathogenesis of B. bronchiseptica, we purified this protein from strain RB54 (32). A unique band migrating at about 40 kDa, consistent with the expected mass of B. bronchiseptica flagellin (1, 46), was detected by Coomassie blue staining in our purified preparations (Fig. 2B), and its identity was further confirmed as flagellin by Western blotting (Fig. 2C) and N-terminal sequencing. We exposed Calu-3 human airway epithelial cells to purified flagellin, as well as to strains RB54 and RB54flaA. As shown in Fig. 3A, expression of the inducible antimicrobial peptide hBD2 was up-regulated by exposure to flagellin and RB54, but not to the flagellin mutant RB54flaA. hBD1, which is constitutively expressed by epithelial cells (31, 44), was used as an internal control. Furthermore, the expression of matrilysin, a matrix metalloproteinase involved in host defense (66), which is specifically up-regulated by P. aeruginosa flagellin (32), was also induced by Bordetella flagellin and RB54, but not by RB54flaA (Fig. 3B). In similar experiments, conditioned media were harvested at 24 h postinfection, and selected proinflammatory mediators were examined by ELISAs. The secretion of IL-8, granulocyte-macrophage colony-stimulating factor (GM-CSF), and MIP-3 was induced by treatment with purified flagellin to an extent similar to exposure to RB54 (Fig. 3C, D, and E). On the basis of the amount of flagellin detected by Western blotting in conditioned medium samples of infected cells, direct exposure to B. bronchiseptica RB54 for 1 h at an MOI of 100 was roughly equivalent to treatment with 10–7 M purified flagellin (data not shown). Altogether, these results indicate that B. bronchiseptica flagellin is an important determinant of expression of these host defense genes and a modulator of airway mucosal defense.
Bordetella flagellin is similar in potency to other bacterial flagellins. We compared the effects of flagellin purified from Bordetella, Pseudomonas, and Salmonella species on IL-8 secretion by human lung epithelial cells. In these experiments, Calu-3 cells were challenged for 24 h with different concentrations of endotoxin-free flagellin, and the accumulation of IL-8 in the conditioned media was determined by ELISA. As shown in Fig. 4, treatment with 10–8 M Bordetella flagellin resulted in an approximately 20-fold induction in IL-8 levels, while the same concentration of S. enterica serovar Typhimurium flagellin resulted in a 24-fold induction. Consistent with recent observations (4, 24), P. aeruginosa flagellin showed slightly reduced bioactivity (about 10-fold induction in IL-8 secretion with exposure to 10–8 M flagellin). Therefore, Bordetella flagellin bioactivity in human airway epithelial cells is comparable to that of flagellin from other gram-negative pathogens.
Bordetella flagellin signals through human and mouse TLR-5. Bordetella spp. that are human pathogens (B. pertussis and B. parapertussis) are nonmotile, while the species with a broad host range, B. bronchiseptica, modulates motility through BvgAS activity. To determine whether the motility-host restriction dichotomy among bordetellae relates to flagellin receptor specificity, we used an in vitro system of TLR-5 receptors expressed on a human epithelial cell line with intrinsic low levels of expression of most TLRs. Thus, we infected human epithelial kidney cells (HEK293) stably transfected with human (HEK293-hTLR5) and mouse (HEK293-mTLR5) TLR-5 with B. bronchiseptica and determined the levels of IL-8 in conditioned media 24 h postinfection (Fig. 5A). For a control, we used HEK293 cells transfected with vector alone (HEK293-null). As shown in Fig. 5A, infection of HEK293-hTLR5 and HEK293-mTLR5 cells with motile strains RB54 and REV1 resulted in similar induction in IL-8 secretion compared to uninfected cells. Furthermore, there was a very limited response to the flagellin mutants RB54flaA and RBA-5 and the nonmotile strain RBA-2 (Fig. 5A). In fact, IL-8 secretion in response to the flagellin mutants was not significantly different from the low level of IL-8 induction observed in HEK293-null cells exposed to B. bronchiseptica (Fig. 5A). This response of HEK293-null cells may be mediated by intrinsic low levels of expression of other receptors in the parental cells (57). In addition, transfected cells were treated for 24 h with 10–8 M endotoxin-free flagellin purified from B. bronchiseptica and S. enterica serovar Typhimurium, and secretion of IL-8 was examined by ELISA as before (Fig. 5B). Results show that treatment with Bordetella flagellin induces IL-8 secretion to a similar extent in cells expressing human and mouse TLR-5, suggesting that it signals effectively through both receptors. Furthermore, Bordetella flagellin bioactivity in these cells was comparable to Salmonella flagellin bioactivity.
Proinflammatory effects of Bordetella exposure in macrophages. To investigate the contribution of TLR-5-dependent signaling to the proinflammatory effects of Bordetella exposure, we used a system of stably transfected RAW 264.7 murine macrophages expressing this receptor (63). Control RAW 264.7 cells (which respond to TLR-2 and -4 ligands but do not express TLR-5 and do not respond to flagellin [40]) and cells expressing TLR-5 (TLR-5-EYFP) were infected with B. bronchiseptica, and cytokine levels were determined in 4-h conditioned media. As shown in Fig. 6A and B, exposure of TLR-5-expressing RAW cells to motile strains RB54 and REV1 resulted in a three- to fivefold increase in tumor necrosis factor alpha (TNF-) and MIP-2 secretion over the isogenic flagellin mutants RB54flaA and RBA-5. By contrast, exposure of control RAW cells to B. bronchiseptica resulted in increased cytokine levels that were not significantly different between motile strains and flagellin mutants. For a control, cells were incubated with purified Bordetella flagellin, and the levels of TNF- were determined in conditioned media (Fig. 6C). Treatment of TLR-5-expressing cells, but not control cells, resulted in a 10-fold increase in TNF-. Altogether, these results confirm the importance of flagellin in the development of proinflammatory responses to B. bronchiseptica infection.
DISCUSSION
Flagella and the motility they confer are critical virulence factors for a variety of microorganisms, and flagellin is a major inducer of host inflammatory responses (48). In this work, we show that Bordetella flagellin is a potent proinflammatory factor for human lung epithelial cells, further shaping a paradigm proposed for Salmonella and Pseudomonas flagellin (8, 25, 70). Thus, the program of epithelial cell activation triggered by flagellin coordinates a response aimed at the effective elimination of the pathogen. We have previously shown that P. aeruginosa flagellin induces the expression of critical cytokines, chemokines, and host defense genes in human lung epithelial cells (8). Here we demonstrate a role for Bordetella flagellin in the induction of proinflammatory mediators. We selected MIP-3, IL-8, and GM-CSF as critical components of the microenvironment generated by human epithelial cells to coordinate inflammatory responses in the airways. IL-8 and GM-CSF are involved in recruitment and survival of neutrophils (53), and MIP-3 (CCL20) participates in the recruitment and activation of immature dendritic cells (50). Flagellin-regulated human host defense genes, matrilysin and -defensin-2 genes (32, 43), were also specifically induced by Bordetella flagellin.
The conserved N- and C-terminal domains of flagellin from different bacteria are essential for bioactivity (14, 16, 42, 56), and the N-terminal D1 domain conserved amino acid residues required for TLR-5 activation are conserved in Bordetella flagellin (4). Indeed, we show here that the proinflammatory and signaling potencies of Bordetella flagellin are comparable to those of S. enterica serovar Typhimurium and P. aeruginosa flagellins, in contrast with the low intrinsic activity (i.e., weak development of inflammatory responses and/or activation of TLR-5) reported for Helicobacter pylori (20, 29) and Campylobacter jejuni (62) flagellins. Altogether, our results using primary and immortalized cells suggest that Bordetella flagellin shapes a potent proinflammatory and innate immune response in lung epithelial cells. In vivo, B. bronchiseptica elicits acute inflammatory responses in the lung, and host factors may contribute to effective bacterial clearance (65). Among other virulence factors important in Bordetella pathogenesis (10, 17), a role for lipopolysaccharide signaling through TLR-4 has been demonstrated recently (23, 34, 35). Interestingly, up-regulation of most inflammatory genes in B. bronchiseptica-exposed macrophages occurs in a TLR-4-independent manner (34). Exposure of TLR-4-deficient macrophages to whole bacteria resulted in an overall transcriptional response to B. bronchiseptica similar to the one observed in wild-type macrophages, while lipopolysaccharide had no significant effect on TLR-4-deficient macrophages (34). While pointing to the importance of TLR-4-mediated signaling in the early production of TNF- during B. bronchiseptica infection, this work suggested that additional bacterial factors are responsible for eliciting the majority of the macrophage inflammatory response to this microorganism (34). Thus, our present work includes flagellin among the bacterial factors that modulate the host response to B. bronchiseptica.
In the context of Bordetella infection and in addition to the lack of flagellin expression by B. pertussis, another difference between B. bronchiseptica and B. pertussis is the expression of pertussis toxin only by the latter species. Thus, the induction of chemokines by pertussis toxin could result in different inflammatory responses to these two species (reviewed in reference 36). Furthermore, with the demonstrated ability of Bordetella flagellin to activate TLR-5, it is likely that flagellin from this microorganism may also activate other immune cells, including macrophages and dendritic cells (39, 40).
Because of the inflammatory activity of flagellin, an emerging theme in bacterial pathogenesis suggests that the infection strategy of some bacteria has evolved to effectively suppress its expression, whether permanently or during the establishment of chronic infection. B. bronchiseptica carries the entire flagellar operon, and it is motile, whereas the flagellar operons of both B. pertussis and B. parapertussis are inactivated by multiple pseudogenes and insertions, resulting in a lack of motility (45). Indeed, flagellin expression is rapidly and effectively repressed upon infection with wild-type B. bronchiseptica (1, 9), and ectopic expression of flagellin in the Bvg+ growth phase inhibits colonization (1). Therefore, our data on Bordetella flagellin bioactivity provide a rationale for immunological pressure to suppress its expression during infection of the host. In addition, B. bronchiseptica flagellin induced similar proinflammatory responses in cells expressing human or mouse TLR-5. Within the limitations of an in vitro system, the data strongly suggest that B. bronchiseptica flagellin signals effectively through both the human and mouse flagellin receptors.
The loss of flagellar expression by B. pertussis and B. parapertussis evokes the lack of motility developed by P. aeruginosa as a specific long-term adaptation to the environment in the cystic fibrosis lung (33, 67) and the loss of motility of Shigella species and Yersinia pestis as a strategy to adapt to a particular host environment (58). Since Bordetella flagellar expression is limited to the Bvg– phase, it has been suggested that these organelles are important in survival in the environment (45). For B. pertussis, a lack of motility may reflect adaptation to a human-restricted niche, supporting the hypothesis that this microorganism does not have an environmental phase. In host-pathogen interactions, surface structures, such as flagella, fimbriae, and polysaccharides, have a dual role in the establishment of infection and modulation of host responses aimed at clearance. Thus, elimination of flagella may have contributed to enhanced B. pertussis virulence in the human host (45, 47). In this context, the inhibition of colonization by the ectopic expression of flagella in the B. bronchiseptica Bvg+ phase underscores the importance of BvgAS-mediated flagellin repression in the establishment of long-term Bordetella infection (1). In summary, our present work includes Bordetella flagellin in the class of bacterial flagellins with potent proinflammatory effects on lung epithelial cells and demonstrates that human and mouse TLR-5-expressing cells respond similarly to Bordetella flagellin. Because of the abilities of bacterial flagellin to induce mucosal immune responses and to act as a potent adjuvant (11, 24, 60), characterizing responses to Bordetella flagellin may be particularly relevant in the context of the development of new vaccines against Bordetella spp.
ACKNOWLEDGMENTS
We are especially grateful to Jeff Miller for providing bacterial strains. We thank S. Mizel and D. Wozniak for helpful suggestions and critical reading of the manuscript and M. Mishra and G. Parise for excellent technical assistance. We also thank S. Mizel for the generous gift of TLR-5-expressing RAW 264.7 cells. We thank M. Lively and M. Morris for protein sequencing.
This work was supported in part by Wake Forest University School of Medicine institutional funds (Y.S.L.-B. and R.D.). We (Y.S.L.-B., L.M.C., and R.D.) declare no conflict of interest.
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