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We found that IL-17, a signature cytokine of Th17, was produced early in the innate immunity phase after an intranasal infection with the obligate intracellular pathogen Chlamydia muridarum. The airway IL-17, which peaked at 48 h after infection, was dependent on live chlamydial organism replication and MyD88-mediated signaling pathways. Treatment with antibiotics or knockout of the MyD88 gene, but not Toll/IL receptor domain-containing adapter-inducing IFN-β, can block the early IL-17 production. Treatment of mice with an anti-IL-17-neutralizing mAb enhanced growth of chlamydial organisms in the lung, dissemination to other organs, and decreased mouse survival, whereas treatment with an isotype-matched control IgG had no effect. Although IL-17 did not directly affect chlamydial growth in cell culture, it enhanced the production of other inflammatory cytokines and chemokines by Chlamydia-infected cells and promoted neutrophil infiltration in mouse airways during chlamydial infection, which may contribute to the antichlamydial effect of IL-17. These observations suggest that an early IL-17 response as an innate immunity component plays an important role in initiating host defense against infection with intracellular bacterial pathogens in the airway.
The obligate intracellular bacterial species Chlamydia trachomatis, consisting of multiple serovars, can cause many health problems in humans. Serovars A–C infect human ocular epithelial cells, causing trachoma and potentially leading to blindness (1). Serovars D–K infect human urogenital tract epithelial tissues, which, if left untreated, can lead to pelvic inflammatory diseases, ectopic pregnancy, and infertility (2, 3). The three L or LGV (lymphogranuloma venereum) serovars (L1–L3) can cause systemic infections in humans (4–6). The mouse pneumonitis agent strain (designated as MoPn, now classified as a new species, Chlamydia muridarum) can infect mice in both the airway and the urogenital tract. Although the C. muridarum organisms cause no known diseases in humans, these organisms have been used to study C. trachomatis pathogenesis and immunology in various mouse models (7–13). Data from the mouse model studies have shown that the CD4+ Th cell (Th1 but not Th2)-dominant and IFN-γ-dependent immunity is a major host protective determinant for controlling chlamydial infection (14), although Abs and CD8+ T cell-mediated immunity may also contribute to the host resistance to chlamydial infection (15–17). However, the role of Th17 and its signature cytokine IL-17 in C. trachomatis infection has not been evaluated.
Naive CD4+ T cells can be induced to express the transcription factor retinoic acid-related orphan receptor RORγt and secrete IL-17, developing into the so-called Th17 phenotype, in addition to Th1 and Th2 (18–21). IL-17 is an inflammatory cytokine that plays a key role in many inflammatory diseases. For example, treatment of mice with a neutralizing anti-IL-17 mAb suppressed autoimmune inflammation in the CNS (22), and mice deficient in generating Th17 cells were resistant to experimental autoimmune encephalomyelitis, collagen-induced arthritis, and inflammatory bowel disease (22, 23). IL-17 has also been found to play an important role in host defense against infection by pathogens (24), including viruses (25), bacteria (26), and fungi (27, 28). However, IL-17 is not always protective, and IL-17-mediated inflammatory response may even increase host susceptibility and exacerbate pathologies induced by some microbial infections (29–32). In this study, we used a mouse model with C. muridarum airway infection to evaluate the role of IL-17 in chlamydial infection. We found that IL-17 was transiently produced early in the innate immunity phase after an intranasal infection with C. muridarum, and this early IL-17 production was dependent on live chlamydial organism replication and MyD88-mediated signaling pathways. Neutralizing the early IL-17 response significantly enhanced replication of C. muridarum and decreased mouse survival. These observations represent the first demonstration that an early IL-17 response as an innate immunity component may play an important role in initiating host defense against infection with intracellular bacterial pathogens in the airway.
C. muridarum Nigg strain (also called MoPn) or C. trachomatis serovar L2 organisms were grown, purified, and titrated as previously described (33). Aliquots of the organisms were stored at −80°C until use. HeLa or L929 cells (both from American Type Culture Collection) were maintained in DMEM (Life Technologies) with 10% FCS (Life Technologies) at 37°C in an incubator supplied with 5% CO2. To produce mouse lung fibroblast cells, whole lungs were removed from exsanguinated female C57BL/6 mice (8–12 wk old), transferred to DMEM, minced into 2- to 3-mm pieces, and subsequently treated with freshly made collagenase type XI (0.7 mg/ml) and DNase I type IV (30 μg/ml) in DMEM at 37°C for 30 min followed by smashing the lung tissue with stainless steel mesh. Cell suspensions were filtered through 70-μm pore size nylon cell strainers (Corning Costar), washed, and resuspended in DMEM supplemented with 10% FCS. Cells were grown at 37°C in a humidified 5% CO2 atmosphere in 24-well plates with coverslips at a density of 1 × 106/ml for 4 days. After detached cells were washed away, the confluent fibroblast cell monolayers were cultured for another 24 h before being used for experiments as indicated below. The C. muridarum and L2 organisms or mouse tissue homogenate samples were used to infect cells. Briefly, HeLa, L929, or mouse primary lung fibroblast cells grown on glass coverslips in 24-well plates were pre-treated with DMEM containing 30 μg/ml DEAE-dextran (Sigma-Aldrich) for 10 min. After the DEAE-dextran solution was removed, chlamydial organisms diluted in DMEM were allowed to attach to the cell monolayers for 2 h at 37°C. The infected cells were continuously cultured in DMEM with 10% FCS and with or without 2 μg/ml cycloheximide (Sigma-Aldrich) and processed at various time points after infection as indicated in individual experiments.
Male or female wild-type (Wt)3; C57BL/6J mice (The Jackson Laboratory) or with gene deficiency in MyD88 or Toll/IL receptor domain-containing adapter-inducing IFN-β (TRIF; a gift from Dr. S. Akira, Osaka University, Osaka, Japan) were used at the age of 8 –9 wk. When different groups of mice were compared, both mouse sex and birth date were matched between groups. For mouse infection, each mouse was inoculated intranasally with live C. muridarum organisms at the appropriate inclusion-forming units (IFU) as indicated in individual experiments in 40 μl of sucrose-phosphate-glutamate buffer consisting of 218 mM sucrose, 3.76 mM KH2PO4, 7.1 mM K2HPO4, and 4.9 mM glutamate, pH 7.2, under light anesthesia with isoflurane. For blocking of IL-17, the Wt C57BL mice were treated with an anti-IL-17-neutralizing mAb (rat IgG2a, clone 50104.11; R&D Systems) or an isotype-matched control rat IgG (clone 54447.11; R&D Systems) via i.p. injection every other day starting on day 1 before chlamydial infection at 62.5 μg/injection (with the exception of the first injection at 125 μg). The injection continued to day 2 or 8 after infection as indicated in individual experiments. For IL-17 treatment, the MyD88 knockout (KO) mice were given 1.5 μg of a rIL-17 (R&D Systems) by i.p. injection 1 day before infection and the same amount by intranasal inoculation every other day after infection. Groups of mice were sacrificed at different times after infection as indicated in individual experiments. For some experiments, the bronchial alveolar lavage fluids (BALF) were collected as described elsewhere (34). Briefly, mice were anesthetized with isoflurane, and a triangular syringe device (Three-Way Stopcock; Baxter Healthcare) was inserted into the mouse trachea. PBS (1 ml) was used to gently flush the bronchial alveolar system twice and then centrifuged at 100 × g for 10 min at 4°C. The cell-free bronchial alveolar lavage supernatant was used for cytokine measurement. In some experiments, the pellet containing the BALF cells was resuspended in 200 μl of PBS. To count cells, 50 μl of the cell suspension were smeared on a slide precoated with poly-L-lysine (Electron Microscopy Sciences), and each BALF cell sample was smeared onto three different slides. The slides were stained with Wright-Giemsa dye (CS434D; Fisher Diagnostics). Neutrophils, macrophages, and lymphocytes were counted using an upright microscope equipped with a ×100 objective oil lens (CH30; Olympus). At least 200 cells from each slide were counted, and the results for each cell type were expressed as the percent of total cells. The bronchilung tissues with or without the prior collection of BALF along with the spleen and kidney organs in some experiments were harvested for making homogenates as described previously (12). Briefly, each mouse organ was homogenized in sucrose-phosphate-glutamate buffer (lung in 1.2 ml, spleen and kidney in 0.6 ml, respectively) using a 2-ml tissue grinder (Fisher Scientific). The homogenates, after centrifugation to clear residual debris, were used for titrating live chlamydial organisms and measuring cytokines. Blood was collected for monitoring Ab production. In some experiments, the BALF cells were counted for the total number of live nucleated cells and were plated in 96-well plates at 5 × 106/ml for lymphocyte restimulation experiments as described below. The mediastinal lymph nodes and spleen organs were also harvested from these mice for in vitro lymphocyte restimulation assay. Briefly, the tissues were minced, and single-cell suspensions were made. The nucleated splenocytes or lymph node cells (mainly lymphocytes) plated at 5 × 106/ml were restimulated with UV-inactivated C. muridarum organisms at 1 × 106 IFUs/ml for 3 days. The culture supernatants were used for cytokine measurements.
To quantitate the live C. muridarum organisms in mouse lung, spleen, and kidney, the organ homogenates produced as described above were titrated on HeLa cell monolayers in duplicates as described previously (13). Briefly, serially diluted homogenate samples were inoculated onto HeLa cell monolayers grown on coverslips in 24-well plates. After incubation for 24 h in the presence of 2 μg/ml cycloheximide, the cultures were processed for immunofluorescence assay as described below. The inclusions were counted under a fluorescence microscope. Five random fields were counted per coverslip. For coverslips with <1 IFU per field, the IFUs on the entire coverslips were counted. Coverslips showing obvious cytotoxicity of HeLa cells were not taken into the count. The total number of IFUs per organ was calculated based on the number of IFUs per field, number of fields per coverslip, dilution factors, and inoculation and total sample volumes. An average was taken from the serially diluted and duplicate samples for any given organ. The calculated total number of IFUs per organ was converted into log10, and the log10 IFUs were used to calculate means and SD for each group at each time point.
HeLa or L929 cells grown on coverslips with or without chlamydial infection and other treatments as indicated in individual experiments were fixed with 2% paraformaldehyde dissolved in PBS for 30 min at room temperature, followed by permeabilization with 1% saponin (Sigma-Aldrich) for an additional 1 h. After washing and blocking, the cell samples were subjected to immunostaining with a rabbit anti-CT395 Ab (this Ab can cross-react with all chlamydial species; our unpublished data) plus a goat anti-rabbit IgG conjugated with Cy2 (green; Jackson Immuno-Research Laboratories) to visualize chlamydial inclusions. For phenotyping of the mouse primary lung fibroblast cells, the primary cells along with the positive control cells (HeLa epithelial cells, L929 fibroblast cells, and RAW macrophages) were grown on coverslips, and the cell monolayers were labeled with the following primary Abs: rat anti-mouse CD14 (IgG2a, clone 159010; R&D); mouse anti-human cytokeratin (IgG1, clone PCK-26; Sigma-Aldrich; this Ab recognizes both human and mouse cytokeratins); mouse anti-human vimentin (IgM, clone VIM-13.2, Sigma-Aldrich; this Ab also recognizes both human and mouse vimentin). The primary Ab binding was visualized with Cy3-conjugated goat anti-mouse IgG1 or donkey anti-mouse IgM or donkey anti-rat IgG Abs (red; all from Jackson ImmunoResearch Laboratories). For staining of the cell surface CD14, the cell samples were only fixed with paraformaldehyde without permeabilization with saponin. The Hoechst dye (blue; Sigma-Aldrich) was used to visualize nuclear DNA. The immunolabeled samples were used for image analysis and acquisition with an Olympus AX-70 fluorescence microscope equipped with multiple filter sets and a Hamamatsu digital camera (Olympus) as described previously (33, 35–37). The images were acquired with the software SimplePCI and processed using the Adobe Photoshop program (Adobe Systems).
To measure the anti-C. muridarum Abs in mouse sera and cytokines in various mouse samples, a standard ELISA was used as described elsewhere (38–40). For titrating the mouse anti-C. muridarum Abs, the C. muridarum-infected HeLa cell lysates were used as Ags to coat 96-well ELISA microplates (Nunc). After blocking with 2.5% nonfat milk (in phosphate-buffered solution), mouse serum samples after serial dilution were applied to the Ag-coated microplates. The serum Ab binding was detected with a goat anti-mouse IgG conjugated with HRP (Jackson ImmunoResearch Laboratories) in combination with the soluble substrate ABTS (Sigma-Aldrich) and quantitated by reading the OD405 using a microplate reader (Molecular Devices). For measuring cytokines from the mouse organ homogenates or the supernatants of the in vitro stimulated spleen lymphocyte cultures or Chlamydia-infected L929 cell cultures, standard cytokine ELISA kits were used as instructed by the manufacturer. The commercially available ELISA kits (mouse IFN-γ kit, IL-4, IL-5, IL-1α, IL-1β, IL-6, IL-8, IL-12, TNF-α, and IL-17) were all obtained from R&D Systems. Briefly, the mouse samples after the appropriate dilution were applied to the 96-well ELISA microplates precoated with the corresponding capture Abs. The capture antibody-bound cytokines were detected with biotin-conjugated anti-cytokine Abs and HRP-conjugated avidin. The cytokine concentrations were calculated based on optical density values, cytokine standards, and sample dilution factors and expressed as nanograms or picograms per milliliter.
An ANOVA test (http://www.physics.csbsju.edu/stats/anova.html) was performed to analyze data from multiple groups and a two-tailed Student t test (Microsoft Excel) to compare the means between two groups and a log rank (Mantel-Cox) test (http://support.sas.com/documentation/cdl/en/statug/59654/HTML/default/statug_seqtest_sect028.htm.) for comparing the mouse survival rates as well as the Chitest (Microsoft Excel) to analyze qualitative data between two groups.
After an intranasal infection with C. muridarum, we monitored the levels of infectious organisms and inflammatory cytokines, chemockines and lymphokines including IL-17 in the mouse lung homogenates (Fig. 1A). Infectious chlamydial organisms were recovered starting at day one after infection and the IFU number steadily increased along the infection course and reached a plateau on day 8 (Fig. 1Aa). This infection time course is similar to what was previously described in C57 mice (41). When cytokines in the lung homogenates were compared, we found that IL-23, IL-12, and IL-17 peaked at 48 h while other cytokines peaked on day 3 (e.g., TNF-α, IL-1αβ, and IL-6, panels e–h respectively) or later (MIP-2, a mouse homologue of IL-8, Fig. 1Ai). IL-23, IL-17, and IL-12 production was transient. By day 4, IL-23, IL-17, and IL-12 all returned to their corresponding background levels. However, the rest cytokines remained at high level during the entire infection course (up to 12 days after infection). Because IL-17 is a signature cytokine of CD4+ Th17 cells, we also measured other T cell cytokines including IL-2, IFN-γ, and IL-4/5 and found that none of them was measurable from any of the lung homogenate samples (data not shown). However, these T cell cytokines were all detected in the supernatants of the cultured mouse BALF cells, mediastinal lymph node cells and splenocytes harvested from the same infected mice after an in vitro restimulation with MoPn organisms (Fig. 1, B and C, and data not shown). The facts that these cells regardless of their sources did not produce IL-17 on day 2 even with chlamydial Ag stimulation and only produced IL-17 on day 8 or later after chlamydial Ag stimulation suggest that the responding cells are Ag-specific and not likely be responsible for the innate IL-17 production peaked on day 2 after chlamydial intranasal infection. The Ag-specific IL-17 production was detected much earlier in the culture supernatants of the airway infiltrate cells or draining lymph node cells (Fig. 1C, day 8) than in the splenocytes (Fig. 1B, day 20), which might be caused by the relatively enriched chlamydial Ag-specific Th17 cells at the site of infection, thus dramatically increasing the detection sensitivity. Compared with systemic chlamydial Ag-specific IFN-γ production (detectable in the splenocyte culture as early as 4 days after infection), the systemic IL-17 production is severely delayed, only detectable 20 days after infection. The delayed IL-17 production by spleen T cells is consistent with the concept that Th17 is often accumulated late during infection in the chronic inflammatory tissues (42). As expected, chlamydial Ag-specific Ab production was first detected on day 8 after infection. Since our goal in the current study was to evaluate the role of the early IL-17 in chlamydial infection in the airway, we further characterized the early IL-17 production. We found that the early IL-17 production was infection dose dependent and blocked by treatment of mice with antibiotics (Fig. 2), suggesting that chlamydial productive infection and protein synthesis are required for the early IL-17 production in the airway. Because IL-12 plays a critical role in mouse resistance to chlamydial airway infection (43), the parallel early production of IL-17 suggests that IL-17 may also play an important role in mouse airway resistance to chlamydial infection. The accompanied early production of IL-23 suggests that IL-23 may play a role in the observed early production of IL-17 since IL-23 is a known inducer of IL-17.
We used a neutralization Ab approach to test whether the early IL-17 production in mouse lung plays any role in mouse resistance to chlamydial infection (Fig. 3). Mice treated with an anti-IL-17 neutralization mAb displayed a rapid decrease in survival rate. Mice in the neutralizing Ab-treated group started to die on day 6 (with a 75% survival rate), and death continued on day 8 (50% survival rate), 11 (25% survival rate), and 18 (12.5% survival rate). By day 21, all neutralizing Ab-treated mice died. However, mice similarly treated with an isotype-matched control IgG maintained a survival rate of 75% throughout the experiment and the minimal death occurred on days 13 and 16, respectively. The surviving mice from the control group were sacrificed on day 40 because these mice had fully recovered their body weights and were not likely to die because of the C. muridarum infection. We further evaluated the effect of the neutralization Ab treatment on chlamydial organism replication (Fig. 4A). The neutralization Ab treatment significantly increased the IFUs in both lung and spleen organs on day 8 after infection (Fig. 4Aa), suggesting that IL-17 produced early during chlamydial infection contributes to the host restriction to chlamydial replication. However, there was no significant difference in the number of live organisms (IFUs) recovered from the mouse organs between the Ab and control IgG treatment groups on day 2 (Fig. 4Ab). This might be due to the fact that a period of 2 days was not long enough for most C. muridarum organisms to complete their growth cycle to generate mature EBs in mice. Finally, as expected, the neutralization Ab treatment was effective in blocking IL-17 in mouse airway because IL-17 was detected only in lung homogenates of mice treated with the control IgG but the neutralization Ab (Fig. 4B).
We next compared the Chlamydia-induced early IL-17 in the airway between MyD88 KO and Wt mice (Fig. 5). Both the IL-17 that was secreted into the airway lumen (detectable in the BALFs) and the IL-17 that remained in the lung tissues (detectable in the homogenates made from the lung tissues after collecting BALF) were measured separately. On day 2 after infection, Wt mice secreted much IL-17 into the BALFs although a significant amount still remained in the lung homogenates (Fig. 5Aa). However, no significant IL-17 was detected in either BALF or homogenate samples harvested from the MyD88 KO mice that were similarly infected with C. muridarum. The TRIF KO mice displayed a similar IL-17 profile as the Wt mice. These observations have demonstrated that MyD88-mediated signaling is required for the chlamydial induction of IL-17. On day 7, no IL-17 was detected from any mice (Fig. 5Ab), which is consistent with the conclusion reached from the time course data (Fig. 1) that the early IL-17 production in response to chlamydial infection was transient. The lack of IL-17 in MyD88 KO mice correlated with the increased susceptibility of these mice to C. muridarum organism infection. The MyD88 but not TRIF KO mice displayed an increased level of live chlamydial organisms recovered from mouse organs on day 7 but not day 2 (Fig. 5B). The lack of difference on day 2 between the MyD88 or TRIF KO and Wt mice is consistent with the early observation that similar levels of IFUs were recovered 2 days after infection from the organs of Wt mice treated with either neutralizing Ab or control IgG (shown in Fig. 4Ab). This may be due to the fact that chlamydial organisms have not had enough time to complete the growth cycle to generate infectious progeny. Furthermore, like the neutralization Ab-treated Wt mice shown in Fig. 3, the MyD88 but not TRIF KO mice progressively died upon MoPn infection (Fig. 5C).
To understand the mechanisms of the IL-17 antichlamydial activity, we evaluated the effect of IL-17 on chlamydial growth in cell cultures (Fig. 6). IL-17 did not affect the growth of either C. muridarum or L2 organisms in a mouse L929 cell line (Fig. 6A) or mouse primary lung fibroblast cells (Fig. 6B), whereas IFN-γ significantly inhibited L2 organism growth (Fig. 6, Aa and Ba). 92% of the cells in the mouse primary lung fibroblast cell preparation were confirmed to be fibroblast cells (Fig. 6C). The lack of inhibition of C. muridarum organisms by IFN-γ in mouse cells is thought to be due to the ability of C. muridarum to evade IFN-γ-induced antichlamydial mechanisms possibly via the expression of a large clostridial toxin homolog (44–46). The L2 organisms do not carry any toxin homolog gene (47, 48). Nevertheless, the lack of inhibition of either C. muridarum or L2 organisms by IL-17 suggests that IL-17 may use a unique mechanism to suppress chlamydial growth in mice. Surprisingly, we found that both IL-17 and IFN-γ significantly exacerbated IL-6 (Fig. 6Cb) and MIP-2 (Fig. 6Cc) production by the Chlamydia-infected cells. The ability of IL-17 to enhance cytokine production in response to chlamydial infection was further confirmed in MyD88 KO mice (Fig. 7). MyD88 KO mice produce minimal levels of inflammatory cytokines after infection (49). We found that delivery of exogenous IL-17 into mice significantly increased the production of IL-6 and MIP-2 upon chlamydial infection compared with the MyD88 KO mice with mock treatment, confirming that IL-17 and chlamydial infection can synergistically activate inflammatory cytokine genes. Furthermore, IL-17 also enhanced the infiltration of neutrophils in the MyD88 KO mice (Fig. 8). Compared with the Wt mice, MyD88 KO mice displayed a much lower level of neutrophil infiltration on day 2 after chlamydial infection. Treatment of the MyD88 mice with exogenous IL-17 significantly enhanced the neutrophil population in the BALFs in response to chlamydial infection. Thus, IL-17 may exert its antichlamydial effect by enhancing inflammatory cytokine production and neutrophil infiltration during the early stage of infection.
IL-17 has been shown to play significant roles in both inflammatory pathologies and host defense against many microbial infections. However, its contribution to infection by obligate intracellular bacterial pathogens such as C. trachomatis was still unknown. In the current study, we found that IL-17 was induced early in the lung upon chlamydial infection. The early IL-17 production was dependent on MyD88 signaling pathway and significantly contributed to the resolution of chlamydial infection in the airway.
The early IL-17 production in the lung peaked at 48 h after an intranasal infection with C. muridarum represents an innate immune mechanism with which mice might deal with an acute infection. This conclusion is consistent with the general concept that adaptive immunity can only take place 4 days after Ag exposure, which is supported by the observations that the chlamydial Ag-specific IFN-γ, Ab, and IL-17 production by lymphocytes was first detected on days 4, 8, and 20 after the C. muridarum infection, respectively (Fig. 1B). Indeed, the early IL-17 production in the airway was also induced by and contributed to the protection against other respiratory bacterial pathogens, including Mycoplasma pneumoniae (50), Klebsiella pneumoniae (26, 51), and Mycobacterium bovis (52, 53). These pathogens all triggered a transient IL-17 production, and the peak time appeared to correlate with the speed of the pathogen replication with the IL-17 induced by M. pneumoniae on day 1, K. pneumoniae on day 2, and Mycobacterium bovis on day 3, respectively. These previous observations support our current finding that the early IL-17 production required chlamydial replication and biosynthesis (Fig. 2). Because the early IL-17 production significantly contributes to the protection against different bacterial pathogen infections, it is important to identify its molecular and cellular basis. We found that MyD88 but not TRIF was required for the early IL-17 production, suggesting that innate immunity receptor-mediated signaling pathways may be sufficient for activating IL-17 gene. Indeed, besides the traditional CD4+ T cells that are normally involved in adaptive immunity, TCRγδ T cells (54–56), NKT-like cells (57, 58), NK1.1− iNKT cells (59), neutrophils (60), and Paneth cells (61), all of which can participate in the innate immunity, also produce IL-17. These innate immunity cells may form the cellular basis for the early IL-17 production during chlamydial infection. It has been shown that NK1.1− and α-galactpsylserine-positive invariant NKT cell population is critical for airway neutrophilia in response to endotoxin (59), whereas TCRγδ T cells are the predominant source of IL-17 in Mycobacterium tuberculosis infection in mice (56). However, due to the enormous redundancy in IL-17-producing cells, the main cellular populations responsible for the early IL-17 production during infection with other bacteria including K. pneumoniae and M. pneumoniae have not been identified. Regardless of the exact cellular basis of the early IL-17 production during chlamydial infection, the current study has provided convincing evidence that the Chlamydia-induced IL-17 during innate immunity is dependent on a MyD88-mediated signaling pathway, which is important to understand how the innate immunity cells activate their IL-17 gene during chlamydial infection. IL-23 is required for CD4+ Th17 memory cells to produce IL-17 (62), and IL-23 can also induce IL-17 gene activation in TCRγδ T cells via a Tyk2-mediated signaling pathway (54). However, it is not clear whether the early IL-17 production during chlamydial infection is a direct result of chlamydial invasion of the IL-17-producing cells or indirectly induced by IL-23 secreted from APCs such as dendritic cells, macrophages, and epithelial cells that are infected with Chlamydia. These APCs produce IL-23 in response to microbial infection via their innate immunity receptor-triggered signaling pathways including the MyD88 pathways (49, 63). It has been shown that the early IL-17 production during K. pneumoniae infection depends on TLR-mediated induction of IL-23 (64). Chlamydial organisms naturally invade epithelial cells but not T cells. Furthermore, Chlamydia has been shown to activate innate immunity receptor-mediated signaling pathways in epithelial cells (65, 66). Thus, it is possible that C. muridarum infection activates the IL-17-producing cells during the innate immunity by inducing IL-23 via a MyD88-dependent pathway in the infected-epithelial cells. This hypothesis is supported by our current finding that airway infection with chlamydial organisms induced an early production of both IL-23 and IL-17. Nevertheless, the precise cellular basis and molecular mechanisms of the Chlamydia-induced early IL-17 production require further investigations.
IL-17 is a proinflammatory cytokine with a pleiotropic spectrum of biological activity, and its receptors are broadly distributed in many different types of cells and tissues in both humans and mice (62). The role of IL-17 in airway infection and inflammation has been well recognized (34, 67–71). We found that although IL-17 did not directly alter the chlamydial intracellular growth in cell cultures, it synergistically enhanced the production of the inflammatory cytokine IL-6 and chemokine MIP-2 in L929 cell line and isolated primary lung fibroblasts as well as in the airway of MyD88 KO mice (Figs. 6 and and7)7) and promoted neutrophil infiltration in mouse airway upon chlamydial infection (Fig. 8). These findings are consistent with the previous observations that IL-17 can stimulate human airway smooth muscle cells to secrete MIP-2 (71), mouse neutrophils to secrete matrix metalloproteinase-9 (69), and human bronchial epithelial cells and keratinocytes to secrete β-defensins (72). IL-17 is known for its ability to promote generation, chemotaxis, and activation of neutrophils (73, 74). Neutrophils seem to play a critical role in controlling chlamydial infection during the early stage of infection (10), suggesting that IL-17 may exert its antichlamydial activity via the enhanced neutrophil function. However, other studies have shown that neutrophils may not be important in the resolution of chlamydial airway infection (75). Obviously, further studies are required for understanding the precise mechanism of the IL-17 antichlamydial activity. Because the early IL-17 production by innate immunity cells can affect the phenotypes of adaptive immunity in the lung (34), future studies should also investigate the effect of the early IL-17 production on the quality of adaptive immunity induced by chlamydial infection.
3Abbreviations used in this paper: Wt, wild type; TRIF, Toll/IL receptor domain-containing adapter-inducing IFN-β; IFU, inclusion-forming unit; BALF, bronchial alveolar lavage fluid; KO, knockout.
The authors have no financial conflict of interest.
1This work was supported in part by grants (to G.Z.) from the National Institutes of Health.