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The specific contribution of interleukin-17/interleukin-17 receptor (IL-17/IL-17R)-mediated responses in regulating host susceptibility against obligatory intracellular Chlamydia infection was investigated in C57BL/6 and C3H/HeN mice during Chlamydia muridarum respiratory infection. We demonstrated that Chlamydia stimulated IL-17/IL-17R-associated responses in both Chlamydia-resistant C57BL/6 and Chlamydia-susceptible C3H/HeN mice. However, C3H/HeN mice developed a significantly greater IL-17/IL-17R-associated response than C57BL/6 mice did. This was reflected by an increase in IL-17 mRNA expression, a higher recall IL-17 production from splenocytes upon antigen restimulation, and higher production of Th17-related cytokines (IL-23 and IL-6) and chemokines (chemokine [C-X-C motif] ligand 2 [CXCL1]/keratinocyte-derived chemokine [KC] and CXCL2/macrophage inflammatory protein 1 [MIP2]) in C3H/HeN mice than in C57BL/6 mice. Furthermore, C3H/HeN mice displayed a massive accumulation of activated and preactivated neutrophils in the airway and lung parenchyma compared to their C57BL/6 counterparts. We further demonstrated that the skewed IL-17/Th17 profile in C3H/HeN mice was predisposed by a higher basal level of IL-17 receptor C (IL-17RC) expression and then further amplified by a higher inducible IL-17RA expression in lungs. Most importantly, in vivo delivery of IL-17RA antagonist that resulted in a 50% reduction in the neutrophilic infiltration in lungs was able to reverse the susceptible phenotype of C3H/HeN mice to respiratory Chlamydia infection. Thus, our data for the first time have demonstrated a critical role for the IL-17/IL-17R axis in regulating host susceptibility to Chlamydia infection in mice.
Chlamydia trachomatis is an obligate intracellular gram-negative bacterium that primarily infects epithelial cells lining the ocular, respiratory, and urogenital tract surfaces and causes many human diseases including trachoma, pneumonia, and pelvic inflammatory disease (2). Although effective antibiotics are available, the incidence of C. trachomatis infections continues to increase worldwide (41). In the United States alone, it is estimated that there are approximately 2.8 million new cases of urogenital C. trachomatis infection each year (58). An effective and safe Chlamydia vaccine is needed to address the global C. trachomatis epidemic, and a comprehensive understanding of the means of protective immunity and immunopathology of C. trachomatis infection is essential for vaccine development.
C. trachomatis infection results in a wide variety of clinical manifestations, ranging from asymptomatic to mild or severe symptoms, acute or chronic inflammatory responses, and a wide range of chronic complications (5, 47, 59). Host genetic factors appear to be important in determining the outcome of Chlamydia infections. It has been reported that the increased incidence of Chlamydia-induced chronic diseases, such as tubal infertility and scarring trachoma, is correlated with certain human leukocyte antigen (HLA) haplotypes and polymorphism of genes encoding interleukin-10 (IL-10), CD14, and tumor necrosis factor alpha (6, 7, 16, 25, 44, 54). However, how these specific genes are involved in shaping the specific immune responses during Chlamydia infection in humans remains unclear. As in humans, inbred mouse strains, such as C57BL/6 (H-2b), BALB/c (H-2d), C3H/HeN (H-2k), and DBA/2J (H-2d) mice respond to respiratory (1, 39, 40, 63), genital (9-12, 52), and intraperitoneal (i.p.) (36) Chlamydia infections differently. C57BL/6 mice are regarded as a resistant strain, whereas BALB/c, DBA/2, and C3H/HeN mice are reported as susceptible strains with higher mortality, more prolonged bacterial burden, more severe tissue inflammatory responses (such as neutrophil infiltration), and higher rates of infertility following Chlamydia infection. Thus, these inbred mouse strains have been used extensively for identification of specific host factors that regulate immune responses and immune mechanisms underlying the pathogenesis of Chlamydia infection.
Based on animal models (5, 8, 38, 50) and human studies (20, 21), T-cell-mediated immunity is essential in host defense against Chlamydia infection. Anti-Chlamydia immunity requires induction of potent Th1 cellular immunity that is characterized by production of IL-12 and gamma interferon (IFN-γ) (5, 37, 61). Mice deficient in IL-12 (17), IFN-γ (17, 23), or IFN-γ receptors (23, 24), or mice treated with anti-IL-12 or anti-IFN-γ antibody all demonstrated a marked inability to control Chlamydia infection, highlighting an unequivocal role of Th1 cellular immune response in host defense against Chlamydia infection. In contrast, skewed induction of Th2 immunity or a high level of IL-10 production increases susceptibility to Chlamydia infection in BALB/c mice by inhibiting IFN-γ production (9, 62, 63). While the theory of Th1/Th2 immune regulation has significantly advanced our understanding about anti-Chlamydia immunity, it does not appear to explain all genetically determined susceptibilities to Chlamydia infection. Susceptible C3H/HeN mice are able to mount comparable, if not higher, levels of Th1 immunity, or a similar ratio of Th1/Th2 responses as seen in resistant C57BL/6 mice (9, 39, 40). Thus, it is likely that other immune mechanisms are also involved in regulating differential susceptibilities to Chlamydia infection in different hosts.
In the past few years, significant advances have been made in our understanding of T-cell immunity with the discovery of a unique αβ+ CD4+ T helper lineage termed Th17 cells (13, 19) that differ from the classic Th1 and Th2 lineages. Th17 cells produce novel proinflammatory cytokines including IL-17 (also called IL-17A) and IL-17F—two members of the IL-17 cytokine family, which has a total of six family members (IL-17A to IL-17F) and five receptors (IL-17 receptor A [IL-17RA] to IL-17RE) (27). In mice, Th17 lineage differentiation requires transforming growth factor beta and IL-6 for initiation and IL-23 for further expanding and becoming an established population (3, 30, 31, 35, 53). Although IL-17-producing CD4+ T cells represent one lineage of adaptive immunity, the IL-17/Th17 response functions as a classic innate immune component. Specifically, IL-17 and IL-17F interact with IL-17R, consisting of IL-17RA and IL-17RC subunits, to induce production of other proinflammatory cytokines (e.g., IL-1 and tumor necrosis factor), chemokines (e.g., chemokine [C-X-C motif] ligand 1 [CXCL1], CXCL2, and chemokine [C-C motif] ligand 2 [CCL2]) and growth factors (e.g., IL-6, granulocyte colony-stimulating factor, and granulocyte-macrophage colony-stimulating factor) from tissue structural cells including fibroblasts and epithelial and endothelial cells. As a result, IL-17/Th17 response leads to an accumulation of neutrophils at the sites of infection and inflammation (27, 28). In addition to Th17 cells, γδ+ T cells and neutrophils are also capable of producing IL-17 in response to infectious agents including Mycobacterium tuberculosis (34), Escherichia coli (49), Aspergillus fumigatus (43), and Listeria monocytogenes (18). While a growing body of evidence indicates that IL-17-mediated neutrophilic response plays a critical role in host defense against extracellular bacteria (65, 66), IL-17-mediated responses are also responsible for severe tissue damage in other infection models (15, 45, 46), due largely to their potent effect on neutrophils. To date, however, the specific contribution of the IL-17/Th17 response in host susceptibility to Chlamydia infection remains unclear.
In the present study, we investigated the role of the IL-17/Th17 response in host resistance against respiratory infection caused by the mouse pneumonitis biovar of Chlamydia muridarum in resistant C57BL/6 and susceptible C3H/HeN mice. The objectives of our study were (i) to characterize the IL-17/Th17 response in conjunction with well-characterized Th1 immunity, clinical evaluation, and bacterial clearance during C. muridarum infection; (ii) to investigate whether the IL-17/Th17 immune profile had any correlation with Chlamydia resistance and/or Chlamydia susceptibility, and (iii) if this does occur, to determine the mechanism(s) by which the IL-17/Th17 response modifies host immune responses to C. muridarum infection.
Six- to 8-week-old female C57BL/6 and C3H/HeN mice were purchased from Charles River Laboratory (Senneville, Quebec, Canada) and maintained under specific-pathogen-free conditions at the InVivo Laboratory of the IWK Health Centre. All animal procedures were approved by the University Committee on Laboratory Animal in accordance with the guidelines of the Canadian Council on Animal Care.
The mouse pneumonitis biovar, C. muridarum, was originally obtained from Xi Yang (University of Manitoba, Winnipeg, Manitoba, Canada) and propagated in McCoy cells (ATCC, Manassas, VA) according to procedures described previously (63, 32). Briefly, McCoy cells were infected for 48 to 72 h and harvested with sterile glass beads. The cell suspensions were subjected to brief sonication on ice, and cellular debris was removed by centrifugation at 500 × g for 10 min. The supernatants were collected and spun at 30,000 × g for 30 min to pellet the organism. The elementary bodies (EBs) were then further purified by discontinuous density gradient centrifugation using 30% Isovue-370 (Bracco Diagnostics, Princeton, NJ) and 50% sucrose (Sigma, Oakville, Ontario, Canada) as described previously (32). Purified EB preparations were stored in sucrose-phosphate-glutamic acid (SPG) buffer in small aliquots and frozen at −80°C until needed. To determine the titer of purified EB stocks, McCoy cells grown in 96-well plates were inoculated with serial dilutions of the EB stock. The plates were centrifuged at 1,300 × g for 30 min at 37°C to facilitate the infection. Each well was then washed with Hank's buffer, and 200 μl of minimal essential medium containing 10 μg/ml gentamicin and 1 μg/ml cycloheximide was added to each well and incubated for 72 h at 37°C in 5% CO2. After the incubation, the cell monolayer was fixed with 100% methanol, stained with a genus-specific rabbit anti-Chlamydia antibody (Biodesign International, Saco, ME) and horseradish peroxidase-conjugated anti-rabbit immunoglobulin G (IgG) secondary antibodies (Sigma, Oakville, Ontario, Canada) and developed in 1% 4-chloro-1-naphthol (Sigma, Oakville, Ontario, Canada) (63). The number of inclusion bodies was determined by counting them by looking for their brownish staining using a microscope. The titer of the EB stocks, expressed as the inclusion-forming units (IFU), was calculated according to the number of inclusion bodies of the inoculum and the dilution factor. Aliquots of purified C. muridarum were heat inactivated by incubating the organism at 65°C for 30 min and used as heat-killed crude C. muridarum antigen for in vitro lymphocyte restimulation assay.
RPMI 1640 medium was supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, 100 mg/ml streptomycin, and 2 mM of l-glutamine (Invitrogen, Oakville, Ontario, Canada). IL-17-containing supernatants were generated by transducing A549 cells with a replication-deficient adenoviral gene transfer vector expressing murine IL-17 (AdIL-17) (64). The concentration of murine IL-17 in the supernatants was determined by an enzyme-linked immunosorbent assay (ELISA) (R&D Systems, Minneapolis, MN). A control virus Addl70-3 without the transgene was used to generate control supernatants as described previously (56, 57). All recombinant adenoviral vectors were amplified, purified, and titrated following protocols described previously (57).
Purified C. muridarum EBs were used to establish an in vivo model of pulmonary Chlamydia infection following procedures described previously (56, 63). Briefly, C. muridarum stocks were diluted in SPG buffer and passed through a 30-gauge needle 10 times to ensure proper dispersion of the organisms. A dose of 2 × 103 IFU C. muridarum was diluted in 25 μl of SPG buffer and delivered intranasally into isoflurane-anesthetized mice with a fine pipette tip (55, 56). The mice were monitored daily for body weight changes with 25% loss of original body weight established as the end point. At selected days after infection, groups of mice were sacrificed by exsanguination, and various tissues, including lung and spleen tissues, were isolated and processed aseptically. Bronchoalveolar lavage (BAL) fluid was collected using 500 μl of phosphate-buffered saline (PBS) as described previously (55, 56). BAL fluids were centrifuged at 500 × g for 10 min, and supernatants were stored at −80°C until cytokine/chemokine measurement. The lung tissue samples were homogenized in 3 ml SPG buffer; half of the lung homogenates were centrifuged at 500 × g at 4°C for 15 min, and the supernatants were collected for determination of the Chlamydia load in lungs by counting the number of IFU on McCoy cells or quantitative PCR as described below. The other half of the lung homogenates were centrifuged at 2,000 × g at 4°C for 30 min, and the supernatants were stored at −80°C until cytokine and chemokine measurement. The pellets were combined and resuspended in 0.5% cetyltrimethylammonium chloride (CTAC) (Sigma, Oakville, Ontario, Canada) and stored at −80°C until myeloperoxidase (MPO) assays. Single-cell suspensions were prepared from spleens and restimulated with or without heat-inactivated C. muridarum antigen for 72 h at 37°C in 5% CO2. The culture supernatants were stored at −80°C until cytokine/chemokine measurement.
Lung MPO activity was quantified as described previously (48). Briefly, the lung homogenate pellets in 0.5% CTAC were thawed and heated for 2 h at 60°C and then centrifuged at 10,000 × g for 10 min. The clear supernatants were collected and serially diluted to react with the substrate containing 3 mM tetramethylbenzidine dihydrochloride, 120 μM resorcinol, and 2.2 mM H2O2 (Sigma, Oakville, Ontario, Canada). The reaction was stopped by adding 50 μl of 2 M H2SO4, and the absorbance was determined at an optical density at 450 nm. The recombinant human myeloperoxidase (VWR, Mississauga, Ontario, Canada) was used as a standard.
To quantify the bacterial burden in the lungs of C. muridarum-infected mice, total nucleic acid from 100 μl of each lung homogenate sample was extracted using DNAzol (Invitrogen, Oakville, Ontario, Canada) according to the manufacturer's instructions. The level of bacterial burden in each sample was determined by quantitative PCR with Chlamydia-specific primers for 16S rRNA using SYBR green supermix (Invitrogen, Oakville, Ontario, Canada) in a 7900H fast real-time PCR machine (Applied Bioscience, Foster City, CA). The sense primer was 5′-CGCCTGAGGAGTACACTCGC-3′, and the antisense primer was 5′-CCAACACCTCACGGCACGAG-3′. The Chlamydia load in each mouse lung was calculated using known copy numbers of C. muridarum DNA standards that were extracted from a purified C. muridarum preparation using the same procedure and expressed as log10 value of copies of 16S rRNA per ml. This assay is highly consistent with the traditional IFU assay but more sensitive at detecting low antigen loads (60).
In separate experiments, total RNA was isolated from fresh or frozen lung tissues derived from naïve or C. muridarum-infected C3H/HeN and C57BL/6 mice using TRIzol reagent (Invitrogen, Oakville, Ontario, Canada) according to the manufacturer's protocol. Four micrograms of total RNA was then treated with DNase I (Invitrogen, Oakville, Ontario, Canada) to remove trace amounts of genomic DNA contamination. The first strand of cDNA was synthesized using SuperScript III reverse transcriptase and oligo(dT)20 primer (Invitrogen, Oakville, Ontario, Canada). The expression of IL-17 (IL-17A), IL-17F, IL-17RA, and IL-17RC before and during the course of C. muridarum infection was quantified using SYBR green supermix (Invitrogen, Oakville, Ontario, Canada) in a 7900H fast real-time PCR machine. The expression levels of the target gene were normalized to the reference gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression level using a standard curve with known copy numbers of plasmid DNA encoding GAPDH. The primer pairs for real-time PCR were as follows: for IL-17, the forward primer was 5′-GCTCCAGAAGGCCC TCAGACT-3′ and the reverse primer was 5′-CCAGCTTTCCCTCCGCATTGA-3′; for IL-17F, the forward primer was 5′-CTGGAGGATAACACTGTGAGAGT-3′ and the reverse primer was 5′-TGCTGAATGGCGACGGAGTTC-3′; for IL-17RA, the forward primer was 5′-GTGGCGGTTTTCCTTCAGCCACTTTGTG-3′ and the reverse primer was 5′-GATGCTGTGTGTCCAAGGTCTCCACAGT-3′. All primers except IL-17RC were synthesized by Integrated DNA Technologies, Inc. (Montreal, Quebec, Canada). IL-17RC primer pair was purchased from SA Bioscience Co. (Frederick, MD).
Primary lung fibroblasts were isolated from C3H/HeN and C57BL/6 mice according to procedures described previously (26). Briefly, the lungs were removed aseptically from naïve mice, cut into small pieces, and seeded into 100-mm tissue culture dishes with RPMI 1640 medium containing 20% FBS. The lung pieces were removed after 3 to 5 days after the fibroblasts were attached to the dish. Fibroblasts were then passaged as needed and used at passage 3 to 5. On the day before the assay, fibroblasts were seeded into 96-well flat-bottom tissue culture plates at a density of 1 × 105 cells/well overnight. The cells were then stimulated with different concentrations of IL-17-containing A549 cell supernatants or control A549 cell supernatants for 24 h. The culture supernatants were collected and stored at −80°C until cytokine and chemokine measurement. The data were normalized by subtracting the background level induced by control A549 cell supernatant stimulation for each condition.
We previously demonstrated that a recombinant adenovirus expressing a fusion protein consisting of the murine IL-17RA extracellular domain with the murine IgG CH2 and CH3 domains (AdIL-17R:Fc) was able to block IL-17 receptor A signaling (65). Groups of C3H/HeN mice were i.p. injected with 1 × 109 PFU of AdIL-17R:Fc or empty control viral vector Addl170-3 or with PBS alone 24 h after C. muridarum infection. Mice were monitored daily for body weight changes and sacrificed at 12 days after C. muridarum infection. All samples were collected and processed as described above.
To phenotype infiltrated cellular components in lung parenchyma, the lungs were removed from mice aseptically on days 4, 6, and 12 postinfection and total lung-derived cells were prepared following collagenase digestion as described previously (55). The cell pellets derived from BAL fluid were used to phenotype cellular components in the airway. Approximately 106 cells were first blocked by an anti-mouse CD16/CD32 antibody (clone 2.4G2) for 15 min and then labeled with monoclonal antibodies in a combination of fluorescein isothiocyanate (FITC)-labeled CD11b (FITC-CD11b) (clone M1/70), PercpCy5.5-labeled CD11c (PercpCy5.5-CD11c) (clone N418), Alexa Fluor 647-labeled bone marrow stroma cell antigen 2 (BST2) (also called pDCA-1) (Alexa Fluor 647-labeled BST2) (clone eBio927), and a phycoerythrin (PE)-labeled antibody, such as PE-Gr1 (clone RB6-8C5) or PE-B220 (clone RA3-6B2) or isotype control PE-rat IgG2a (all purchased from eBioscience, San Diego, CA). For standard intracellular cytokine staining (ICCS), splenocytes were isolated from C. muridarum-infected C57BL/6 and C3H/HeN mice on day 12 postinfection and restimulated with or without heat-inactivated C. muridarum antigen for 16 h and then treated with GolgiPlug (BD Pharmingen, San Diego, CA) for an additional 6 h. The cells were washed with PBS containing 1% FBS, and ICCS was conducted by using a Cytofix/Cytoperm kit (BD Pharmingen, San Diego, CA) according to the manufacturer's instructions as described previously (55). The cells were first surface labeled with PercpCy5.5-CD3 (clone 145-2C11), allophycocyanin-labeled T-cell receptors αβ (TCRαβ) (clone H57-597), and FITC-CD4 (clone RM4-5) (all purchased from eBioscience), then fixed, permeabilized, and intracellularly labeled with PE-IL-17A (clone TC11-18H10) or PE-rat IgG1 (purchased from BD Pharmingen). For direct ICCS, naïve or C. muridarum-infected mice were treated with 500 μl of 0.5 mg/ml of brefeldin A i.p. and sacrificed 6 h later. Splenocytes were quickly processed, surface stained with FITC-TCRδγ (clone GL3), then fixed, permeabilized, and intracellularly labeled with PE-IL-17A and Alexa Fluor 647-IFN-γ (clone XMG1.2) (33). A total of 100,000 to 300,000 events were collected on a FACScaliber (BD Biosciences, Sunnyvale, CA), and the data were analyzed using WinMDI 2.8 software (Scripps Institute, La Jolla, CA).
The cytokine contents in BAL fluids and culture supernatants were determined by using antibody pairs specific for mouse IFN-γ, IL-12, IL-17, IL-4, IL-6, IL-13, CXCL1/keratinocyte-derived chemokine (KC), and CXCL2/macrophage inflammatory protein 2 (MIP2) (all purchased from R&D Systems) and IL-23 (purchased from eBioscience).
In most cases, the differences between two mouse strains were analyzed using an unpaired, two-tailed Student t test. The changes in body weight and the mortality rate over the course of infection in C3H/HeN and C57BL/6 mice or in mice treated with different adenoviral vectors were analyzed using two-way analysis of variance (ANOVA) test or log rank survival test using GraphPad Prism software when applicable. In all cases, a P value of <0.05 was considered statistically significant.
The C. muridarum respiratory infection model was established in C3H/HeN and C57BL/6 mice following intranasal delivery of C. muridarum (2,000 IFU/mouse). Both C57BL/6 and C3H/HeN mice remained relatively healthy until day 6 postinfection. C57BL/6 mice showed marginal body weight loss starting on day 7 postinfection (Fig. (Fig.1A).1A). Only 7% of C57BL/6 mice reached the end point after this dose of infection (Fig. (Fig.1B).1B). In contrast, C3H/HeN mice had significantly more body weight loss than that observed in C57BL/6 mice starting on day 8 postinfection onwards. Consequently, 30 to 46% of C3H/HeN mice reached the end point starting day 11 onwards after infection (Fig. (Fig.1B),1B), consistent with previous reports (39, 40). We further examined the kinetics and bacterial load in the lungs of these two strains of mice on days 2, 4, 6, 12, and 21 (Fig. (Fig.1C).1C). While we observed a statistically significant higher bacterial burden in C3H/HeN mice on days 4 and 6 postinfection than that in C57BL/6 mice, the overall bacterial growth curve was similar in the two mouse strains; bacterial loads increased dramatically as early as day 2 postinfection and reached a plateau around day 6 to 12 postinfection, decreasing by day 21 (Fig. (Fig.1C).1C). It is of note that although C3H/HeN mice had reached peak bacterial burden in lungs by day 6, they did not reach the end point until day 11. Furthermore, we found that mice reaching the end point had comparable bacterial loads in lungs with the mice that survived the infection (Fig. (Fig.1D),1D), suggesting that the bacterial load per se was not the ultimate cause or the only cause of the severe clinical illness and higher mortality rate observed in C3H/HeN mice.
To understand whether IL-17/Th17-mediated response is involved in regulating differential host responses to Chlamydia infection, we characterized IL-17/Th17 response in conjunction with well-characterized protective Th1 immunity in lungs and lymphoid organs of C3H/HeN and C57BL/6 mice at multiple time points postinfection. Quantitative RT-PCR was first used to examine the kinetics of IL-17 and IL-17F mRNA expression in lungs after C. muridarum infection. We found that C3H/HeN mice had higher IL-17 mRNA expression in lungs than the C57BL/6 mice did at multiple time points and that this difference reached statistical significance on day 6 (Fig. (Fig.2A).2A). In contrast, IL-17F expression was comparable in both strains of mice throughout the entire course of infection (Fig. (Fig.2B).2B). Therefore, we focused on IL-17 rather than IL-17F in our study. Subsequently, the levels of IL-17 and IFN-γ in BAL fluids were measured by ELISAs. To our surprise, we did not detect any difference in IL-17 levels from C3H/HeN and C57BL/6 mice on days 0, 2, 4, 6, and 12 postinfection (Fig. (Fig.2C).2C). Similarly, IFN-γ production in the BAL fluids from the two strains was also comparable (Fig. (Fig.2D).2D). However, it was observed that larger amounts of IL-17 were produced from splenocytes upon in vitro restimulation with heat-inactivated C. muridarum antigen in C3H/HeN mice on days 4, 6, and 12, while the level of IFN-γ production from the same culture remained similar for the two mouse strains (Fig. 2E and F). This trend, as expressed by the ratio of IL-17/IFN-γ, was consistent in all experiments carried out at different time points postinfection (data not shown). To identify whether γδ+ T cells are the major cellular source of IL-17 production as observed in many other infection models (18, 34, 43, 49), direct intracellular cytokine staining was carried out with splenocytes isolated from naïve or C. muridarum-infected mice for 12 days (33). As demonstrated in Fig. Fig.2G,2G, C. muridarum infection resulted in 17.78% and 3.47% of splenocytes producing IFN-γ and IL-17, respectively, compared to 2.65% basal level of IFN-γ-producing cells and 2.05% basal level of IL-17-producing splenocytes observed in naïve mice. While the majority of IFN-γ-producing cells were found to be non-γδ+ T cells (78% in naïve mice and nearly 92% in C. muridarum-infected mice), approximately 50% of IL-17-producing cells in naïve mice were δγ+ T cells. However, only 23% of IL-17-producing cells in C. muridarum-infected mice were δγ+ T cells, suggesting that C. muridarum infection stimulated more non-γδ+ T cells producing IL-17. Further analysis using standard intracellular cytokine staining indicated that there were more Chlamydia-specific Th17 cells (CD4+ TCRβ+) and CD4− TCRβ+ cells in the spleens of C. muridarum-infected C3H/HeN mice than in the spleens of C. muridarum-infected C57BL/6 mice (Fig. (Fig.2H).2H). Taken together, our results demonstrate a differential induction of IL-17/Th17, but not Th1, responses in C3H/HeN and C57BL/6 mice, which is vigorous in the former and moderate in the latter, during respiratory C. muridarum infection. Further supporting this skewed IL-17/Th17 profile in C3H/HeN mice, we also observed significant differences in the production of Th17-related cytokines and chemokines including IL-23, IL-6, CXCL1/KC, and CXCL2/MIP2 in the BAL fluids of C3H/HeN and C57BL/6 mice on day 6, which remained high in C3H/HeN mice on day 12 postinfection with the exception of IL-23 (Fig. 3A to D).
Since we did not detect any difference in the level of IL-17 in the BAL fluid in the two strains of mice by ELISA whereas a greater IL-17/Th17 response in C3H/HeN mice was evident, we hypothesized that C3H/HeN mice might have a higher level of IL-17 receptor expression, which would bind more IL-17 at the tissue site and lead to low measurement of unbound IL-17 by ELISA. To test this hypothesis, we examined the level of both subunits of IL-17 receptor, IL-17 receptor A and IL-17 receptor C, in the lungs of C57BL/6 and C3H/HeN mice before and after C. muridarum infection by quantitative RT-PCR. Interestingly, while IL-17RA was expressed at similar levels in naïve C57BL/6 mice and C3H/HeN mice (Fig. (Fig.4A),4A), IL-17RC was found to be expressed at a significantly higher level in naïve C3H/HeN mice than in C57BL/6 mice (approximately 1.7-fold higher) (Fig. (Fig.4A).4A). A similar observation was also seen in primary lung fibroblasts isolated from naïve C57BL/6 and C3H/HeN mice and subcultured in vitro (Fig. (Fig.4A).4A). To investigate whether a higher IL-17RC gene expression profile also correlates with greater biological function of the IL-17R complex, these fibroblasts were used to compare their responsiveness to IL-17 stimulation. Consistent with the IL-17RC gene expression profile, lung fibroblasts derived from C3H/HeN mice produced significantly larger amounts of CXCL1/KC, CXCL2/MIP2, and IL-6 than cells from C57BL/6 mice upon IL-17 stimulation (Fig. 4B to D). These results suggest that a higher basal level of IL-17RC predisposes C3H/HeN mice to mount a vigorous proinflammatory response in the early phase of IL-17 production in vivo.
When the expression profiles of IL-17RA and IL-17RC during the course of infection were examined, we found that IL-17RA was consistently induced at higher levels in C3H/HeN mice throughout the course of infection than in C57BL/6 mice, which showed only marginal changes of IL-17RA following C. muridarum infection (Fig. (Fig.5A).5A). In contrast, IL-17RC expression levels fluctuated in both mouse strains following C. muridarum infection (Fig. (Fig.5B).5B). Nevertheless, the overall level of IL-17RC in C3H/HeN mice appeared to be greater than or at least equal to that in C57BL/6 mice (Fig. (Fig.5B).5B). Collectively, our in vitro and in vivo data demonstrate that both IL-17 and IL-17 receptors are involved in regulating differential immune responses in C3H/HeN and C57BL/6 mice.
One of the major biological activities of IL-17 in the host immune system is to stimulate neutrophil-mobilizing chemokine production through IL-17RA or the IL-17RA/IL-17RC heterodimeric complex, which, in turn, recruits neutrophils into inflammatory sites (51, 65). Having examined the kinetic changes of IL-17 and IL-17R expression and their potential contribution to host responses to respiratory C. muridarum infection, we further examined the profile of neutrophil infiltration in these two mouse strains. The level of MPO activity was first used as an indirect measurement of neutrophil infiltration in C57BL/6 and C3H/HeN mice on days 2, 4, 6, and 12 postinfection. Consistent with a higher profile of IL-17/Th17 response, C3H/HeN mice had significantly higher MPO activities in BAL fluid and lung homogenate samples than C57BL/6 mice did on days 4, 6, and 12 postinfection (Fig. 6A and B). Of note, both C3H/HeN and C57BL/6 mice demonstrated a biphasic neutrophil response, with a first peak on day 4 and a second peak on day 12 following C. muridarum infection. We then further examined the phenotype of infiltrated neutrophils in lungs by FACS on days 4, 6, and 12 postinfection. In agreement with the MPO data, there was a dramatic increase in a cell population with a granulocyte profile in C3H/HeN mice compared to C57BL/6 mice (an average 24.72% of total lung-derived cells in C3H/HeN mice versus an average 10% in C57BL/6 mice) on day 6 postinfection (Fig. (Fig.6C).6C). Further analyses indicated that 65 to 70% of this cell population in C3H/HeN mice possessed a neutrophil phenotype (CD11b+ Gr1+), whereas only 40 to 44% of these cells possessed a neutrophil phenotype in C57BL/6 mice. A promiscuous granulocyte activation marker, BST2 (also called pDCA-1), was used to examine the relative maturity and activation status of neutrophils in two mouse strains (4). As presented in Fig. Fig.6D,6D, C3H/HeN mice had more neutrophils with an activated phenotype of CD11b+ Gr1+ BST2+ than C57BL/6 mice did. However, there was an even more striking difference in the accumulation of neutrophils with a nonactivated phenotype of CD11b+ Gr1+ BST2− in the lungs of C3H/HeN mice compared to C57BL/6 mice. Similar results were also obtained on days 4 and 12 postinfection (Fig. (Fig.6E).6E). Taken together, our results demonstrate that a skewed induction of IL-17/Th17 response results in a massive infiltration of not only activated but also preactivated neutrophils at the infection site, which is associated with an increased susceptibility to Chlamydia infection in C3H/HeN mice.
Having demonstrated a close correlation between the IL-17R expression level, a skewed IL-17/Th17 profile, and massive accumulation of neutrophils in lungs during the course of infection in susceptible C3H/HeN mice, we hypothesized that blocking IL-17R signaling would be able to ameliorate the clinical symptoms of C. muridarum infection in C3H/HeN mice. To this end, a recombinant adenovirus expressing a fusion protein of the IL-17RA extracellular domain and murine Ig Fc portion (AdIL-17RA:Fc) was used for in vivo delivery 24 h following C. muridarum infection. Mice receiving control empty virus Addl70-3 or PBS were used as controls. While Addl70-3 treatment was not able to change the course of C. muridarum infection compared to PBS-treated mice, AdIL-17R:Fc delivery significantly prevented the body weight loss in Chlamydia-infected C3H/HeN mice (Fig. (Fig.7A).7A). An approximately 50% reduction in the number of infiltrating Gr1+ neutrophils was observed in mice receiving AdIL-17R:Fc treatment compared to PBS-treated mice using FACS analysis (Fig. (Fig.7B).7B). As expected, there was no significant effect on the level of neutrophil infiltration in lungs of mice treated with Addl70-3 (Fig. (Fig.7B).7B). Similar results were also obtained using the MPO assay (data not shown). Of note, all mice had comparable bacterial loads (Fig. (Fig.7C)7C) and IFN-γ levels produced (Fig. (Fig.7D)7D) in the lung. Taken together, our data collectively support a pathological role of an uncontrolled IL-17/Th17-induced neutrophilic response in mediating the severity of clinical manifestation of respiratory Chlamydia infection in mice.
The IL-17 family is emerging as an important regulator of the cytokine networks that coordinate innate and adaptive immunity under a variety of immune inflammatory conditions. In the present study, the role of IL-17/Th17-mediated host responses during respiratory C. muridarum infection was investigated in Chlamydia-resistant C57BL/6 and Chlamydia-susceptible C3H/HeN mice. While γδ+ T cells are often found as the major cellular source of IL-17 in response to many infectious agents (18, 34, 43, 49), the non-γδ+ T cells including typical Th17 cells (αβ+ CD4+ T cells) appear to be the major IL-17-producing cells induced by C. muridarum infection in both C57BL/6 and C3H/HeN mice. However, there were substantial differences with regard to the magnitude of IL-17/Th17 response in these two mouse strains. C3H/HeN mice naturally express a higher basal level of IL-17RC, which predisposed C3H/HeN mice to develop a more vigorous IL-17-mediated response during the early phase of C. muridarum infection compared to C57BL/6 mice. C3H/HeN mice also expressed a higher inducible level of IL-17RA upon C. muridarum infection, which likely further amplified IL-17/Th17 responses in the later phase of C. muridarum infection compared to C57BL/6 mice. Such differences were demonstrated by an increase in IL-17 mRNA expression, a higher IL-17 recall response, and higher production of Th17-related cytokines (e.g., IL-23 and IL-6) and chemokines (e.g., CXCL1/KC and CXCL2/MIP2). Furthermore, C3H/HeN mice displayed a massive accumulation of activated and preactivated neutrophils in the airway and lung parenchyma compared to C57BL/6 mice as demonstrated by both the MPO assay and FACS analyses. Most importantly, in vivo delivery of IL-17RA antagonist that resulted in a 50% reduction in the neutrophilic infiltration in lungs was able to reverse the susceptible phenotype of C3H/HeN mice to respiratory Chlamydia infection. Thus, our data for the first time demonstrate a critical role of the IL-17/IL-17R axis in regulating host susceptibility to Chlamydia infection in mice.
In contrast to other studies in which neutrophils were identified as the major cellular component in mediating IL-17-dependent host resistance against extracellular bacterial infection (49, 64-66), our data suggest that an augmented neutrophil infiltration as a result of a heightened IL-17/Th17 response and higher IL-17R activity enhances host susceptibility to intracellular Chlamydia infection in C3H/HeN mice. The differential IL-17/Th17 responses to Chlamydia infection in C57BL/6 and C3H/HeN mice are likely controlled at multiple points. First, C3H/HeN mice naturally express a higher basal level of the IL-17RC subunit than C57BL/6 mice do, leading to a more vigorous IL-17R-mediated immune response upon IL-17 stimulation. Both IL-17RA and IL-17RC are implicated in IL-17-mediated cytokine and chemokine production via the IL-17RA/IL-17RC heteromeric complex, which is believed to have at least two preformed IL-17RA subunits and one IL-17RC subunit (28, 29, 51). Consistent with this model, our data suggest that IL-17RA may exist in excess and that IL-17RC likely plays a more direct role in controlling the density of functional IL-17R complex on the cell membrane in response to IL-17 stimulation. Although we did not examine how fibroblasts respond to IL-17F stimulation in this study, it is likely that C3H/HeN mice would also have more robust responses to IL-17F stimulation since IL-17RC alone serves as a high-affinity receptor for IL-17F (29). Such robust cytokine production, particularly IL-6 production in the early phase of C. muridarum infection will significantly promote Th17 differentiation in C3H/HeN mice to a much greater extent (3). Second, in addition to the differences under basal conditions, there are substantial differences in the expression profiles of IL-17RA and IL-17RC in C3H/HeN and C57BL/6 mice following Chlamydia infection (Fig. 5A and B). Although the precise roles of IL-17RA and IL-17RC in the host responses against Chlamydia infection remain unclear, our kinetic results suggest that IL-17RA and IL-17RC have nonredundant roles in regulating IL-17/Th17 response during Chlamydia infection. We believe that a higher basal level of IL-17RC and a higher inducible level of IL-17RA may be responsible for the early peak and late peak of neutrophilic responses observed in the lung, respectively (Fig. (Fig.6).6). Finally, the higher bacterial burden observed in C3H/HeN mice (39, 40) may also contribute to the skewed IL-17/Th17 profile at later time points (after day 4) in C3H/HeN mice.
Neutrophils have been reported to promote intracellular growth of Chlamydia pneumoniae (41) and Mycobacterium tuberculosis (14). Furthermore, IL-17 has been reported to antagonize IFN-γ-induced neutrophil activation against fungal infection (67) and IFN-γ-induced NK activity (22). Thus, it is possible that an augmented neutrophilic response may be responsible for higher bacterial loads in C3H/HeN mice in our study. However, systemic delivery of IL-17RA antagonist did not have any impact on the bacterial load (Fig. (Fig.7C).7C). Therefore, the precise immune mechanism responsible for the impaired ability in controlling Chlamydia intracellular growth in C3H/HeN mice remains to be determined. Nevertheless, our data do suggest that the bacterial load does not seem to be the ultimate cause leading to high mortality in C3H/HeN mice. This notion is supported by the comparable level of bacterial burden observed in C3H/HeN mice that either survived or succumbed to infection (Fig. (Fig.1D).1D). It is likely that the soluble mediators released from neutrophils at the infection site have more direct impact on body weight changes given the observation that systemic delivery of the IL-17R antagonist was able to attenuate neutrophil infiltration in lungs and weight loss simultaneously (Fig. (Fig.77).
We have demonstrated a novel role of the IL-17/IL-17R axis in regulating host susceptibility to Chlamydia infection. We believe that a higher basal level of IL-17RC has a critical role in initiating the whole cascade of skewed immune profile and cellular responses in C3H/HeN mice. This may directly or indirectly link to an unidentified immune mechanism that leads to an impaired immune defense and increased bacterial burden in C3H/HeN mice. The high bacterial burden further stimulates immune responses and results in marked induction of IL-17RA, which likely leads to further amplification of skewed neutrophilic response and causes significant tissue damage in the host. To our knowledge, this is the first report demonstrating the IL-17R as a genetic determinant in regulating host responses to intracellular bacterial infection.
We thank Janet Feng for technical support and Garry Qi for purifying AdIL-17R:Fc.
This study was supported by funds from the IWK Health Centre, the Nova Scotia Health Research Foundation, the Canadian Institutes of Health Research (CIHR) Regional Partnership Program, and the Canadian Foundation for Innovation. J.M. is a recipient of an IWK Graduate Studentship Award. J.W. is a recipient of a CIHR/NSHRF New Investigator Award.
Editor: J. L. Flynn
Published ahead of print on 8 September 2009.