Dendritic Cell Function Bacterial Infection through Modulating ...

The Journal of Immunology

IL-17/Th17 Promotes Type 1 T Cell Immunity against Pulmonary Intracellular Bacterial Infection through Modulating Dendritic Cell Function1 Hong Bai,*† Jianjun Cheng,* Xiaoling Gao,* Antony George Joyee,* Yijun Fan,* Shuhe Wang,* Lei Jiao,* Zhi Yao,† and Xi Yang2*† Although their contribution to host defense against extracellular infections has been well defined, IL-17 and Th17 are generally thought to have limited impact on intracellular infections. In this study, we investigated the role and mechanisms of IL-17/Th17 in host defense against Chlamydia muridarum, an obligate intracellular bacterium, lung infection. Our data showed rapid increase in IL-17 production and expansion of Th17 cells following C. muridarum infection and significant detrimental impact of in vivo IL-17 neutralization by anti-IL-17 mAb on disease course, immune response, and dendritic cell (DC) function. Specifically, IL-17-neutralized mice exhibited significantly greater body weight loss, higher organism growth, and much more severe pathological changes in the lung compared with sham-treated control mice. Immunological analysis showed that IL-17 neutralization significantly reduced Chlamydia-specific Th1 responses, but increased Th2 responses. Interestingly, the DC isolated from IL-17neutralized mice showed lower CD40 and MHC II expression and IL-12 production, but higher IL-10 production compared with those from sham-treated mice. In two DC-T cell coculture systems, DC isolated from IL-17-neutralized mice induced higher IL-4, but lower IFN-␥ production by Ag-specific T cells than those from sham-treated mice in cell priming and reaction settings. Adoptive transfer of DC isolated from IL-17-neutralized mice, unlike those from sham-treated mice, failed to protect the recipients against challenge infection. These findings provide in vivo evidence that IL-17/Th17 plays an important role in host defense against intracellular bacterial infection, and suggest that IL-17/Th17 can promote type 1 T cell immunity through modulating DC function. The Journal of Immunology, 2009, 183: 5886 –5895.

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nterleukin-17 is a proinflammatory cytokine mainly produced by a newly identified subset of activated CD4⫹ T cells (Th17), although some other cell types, including CD8⫹ T cells, ␥␦ T cells, and NKT cells, have also been reported to be able to produce IL-17 in some circumstances (1– 4). Th17 is thought to be a distinct lineage of Th cells producing IL-17A, IL-17F, and IL-22. Th17 does not share developmental pathways with either Th1 or Th2 cells. Several cytokines, including TGF-␤, IL-6, and IL-21, have been found to be important for priming naive CD4⫹ T cells to develop into Th17 cells (5, 6). IL-23, a heterodimeric cytokine consisting of a IL-12/23 p40 and a unique p19 subunit, acts further to induce Th17 development and expansion (7). Upon the

*Laboratory for Infection and Immunity, Departments of Medical Microbiology and Immunology, Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba, Canada; and †Department of Immunology, Tianjin Medical University, Tianjin Key Laboratory of Cellular and Molecular Immunology, Key Laboratory of Educational Ministry of China, Tianjin, People’s Republic of China Received for publication May 20, 2009. Accepted for publication August 31, 2009. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1

This work was supported by grants (to X.Y.) from Canadian Institutes of Health Research, Manitoba Health Research Council, and Manitoba Institute of Child Health, and a grant (to H.B.) from Tianjin Municipal Science and Technology Commission (07JCYBJC10600). X.G. is a trainee in Canadian Institutes of Health Research National Training Program in Allergy/Asthma and a holder of Manitoba Institute of Child Health Studentship. A.G.J. was a trainee in the Canadian Institutes of Health Research/International Centre for Infectious Diseases National Training Program in Infectious Diseases and a holder of Manitoba Health Research Council postdoctoral fellowship. X.Y. is the Canada Research Chair in Infection and Immunity.

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Address correspondence and reprint requests to Dr. Xi Yang, Department of Medical Microbiology, Faculty of Medicine, University of Manitoba, Room 523, 730 William Avenue, Winnipeg, Manitoba, Canada R3E 0W3. E-mail address: yangxi@ cc.umanitoba.ca

www.jimmunol.org/cgi/doi/10.4049/jimmunol.0901584

secretion, IL-17 may bind to specific receptors (IL-17R) expressed on various type of cells, including leukocytes, epithelial cells, vascular endothelial cells, fibroblasts, and mesothelial cells (8). After being activated through IL-17R-mediated signal pathway, cells can produce inflammatory cytokines, IL-6, IL-8, TNF-␣, and CSF (GCSF), and CXC chemokines, which promote the generation, migration, and accumulation of neutrophils (9 –11). Besides its importance in neutrophil homeostasis and recruitments, IL-17 can also induce the production of antimicrobial peptides, including defensins, S100 proteins, and matrix metalloproteinases, thus contributing to host defense against infections. More recently, studies also showed the importance of IL-17 in the induction of Th1- and Th2-type immune response (12). Strongest evidence has been obtained regarding the involvement of IL-17/Th17 in host defense against extracellular bacterial infections, including, but not limited to, Klebsiella pneumoniae (13), Pseudomonas aeruginosa (14), Bacteroides fragilis (15), and Escherichia coli (16). In contrast, it is generally thought that IL17/Th17 responses have limited role in intracellular bacterial infections mainly based on the theoretical insufficiency of neutrophils in killing intracellular bacteria. Indeed, IL-17 gene knockout (KO)3 and IL-17R KO mice are not more susceptible to primary Mycobacterium tuberculosis and Listeria monocytogenes infections than wild-type mice (17–19). However, the involvement of IL-17 in host defense against M. tuberculosis was also reported,

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Abbreviations used in this paper: KO, gene knockout; BAL, bronchoalveolar lavage; BM, bone marrow; BMDC, BM-derived dendritic cell; Cm, Chlamydia muridarum; DC, dendritic cell; EB, elementary body; IFU, inclusion-forming unit; LN, lymph node; p.i., postinfection; rmIL, murine rIL. Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00

The Journal of Immunology especially in inducing recall response following vaccination (20, 21). Therefore, the role of IL-17/Th17 in host defense against intracellular bacterial infection is a controversial issue, thus warranting further investigation. Chlamydiae are obligate intracellular bacteria that contain two species, Chlamydia pneumoniae and Chlamydia trachomatis, which are common causes of human diseases. C. pneumoniae mainly causes acute and chronic respiratory diseases such as pneumonia and bronchitis, whereas C. trachomatis causes ocular, genital tract, and respiratory infection. Chlamydia muridarum (Cm), a natural chlamydial strain of mouse, has been widely used in mouse models of respiratory and genital tract infections (22). Our and others’ reported studies have shown that in respiratory tract chlamydial infection, type 1 CD4 and CD8 T cell immune responses, including IFN-␥ production, are critical for protection against chlamydial infection, whereas Th2-type immune responses are most likely associated with immunopathology (23–27). Therefore, it is important for a host to develop strong type 1 T cell immune responses for an efficient control of the infection and to prevent immunopathology. Dentritic cells (DC) are the most potent APCs that possess an extraordinary ability to prime naive T cells and to direct the type of the T cell responses (28, 29). DC at different maturation stages, especially the ones with differential expression of costimulatory molecules and the production of cytokines, are different in activating Ag-specific T cells, leading to different types of T cell responses. In particular, DC that produce higher IL-12 are more efficient in inducing type 1 T cell responses (29). We recently reported that coculture of higher IL-12-producing DC, compared with lower IL-12-producing DC, induced stronger IFN-␥ production by CD4 and CD8 T cells (30). Moreover, adoptive transfer of the former induced stronger type 1 Chlamydia-specific T cell responses in vivo (30, 31). To date, there is no report on the role of IL-17/Th17 in chlamydial infection. In this study, we investigated the role and mechanisms of Th17 in chlamydial lung infection by administration of anti-IL-17 mAbs to neutralize endogenous IL-17. We found that IL-17-neutralized mice showed significantly delayed clearance of bacteria and more severe disease. More importantly, we found that the IL-17 neutralization led to alteration of phenotype and function of DC, which is associated with a failure in the development of a protective type 1 immunity. These data indicate that IL-17/Th17 plays an important role in host defense against chlamydial infection, at least partially through modulating DC function.

5887 Infection of mice and neutralization of airway IL-17 Mice were anesthetized and inoculated intranasally with 1 ⫻ 103 inclusionforming units (IFU) of Cm in 40 ␮l final volume of PBS. For neutralization of airway IL-17, some mice were intranasally administered with 10 ␮g of anti-mouse IL-17 mAb (R&D System) in 40 ␮l of PBS at 2 h postinfection (p.i.), followed by subsequent administration of the same dose of Ab at 48-h interval. The anti-IL-17 Ab administration was performed three and four times, respectively, for the mice killed at 7 and 14 days p.i. Infected control mice were administered intranasally with either anti-murine IL-17 Ab isotype control (rat IgG2a; R&D System) or 40 ␮l of sterile PBS alone in the same schedule as anti-IL-17 delivery. Mice were killed at designated days after infection. The chlamydial growth in the lung was determined, as described (31).

Isolation of lung and bronchoalveolar lavage (BAL) cells To obtain single lung cell suspensions, lungs were minced into small pieces and incubated in a solution containing collagenase XI (2 mg/ml) and DNase I (100 ␮g/ml) in RPMI 1640 at 37°C for 60 min. EDTA (2 mM, pH 7.2) was added at the last 5 min of incubation. Digested cells were filtered through 70-␮m cell strainers, and erythrocytes were lysed using ACK lysing buffer (150 mM NH4Cl, 10 mM KHCO3, and 0.1 mM EDTA). BAL fluids were collected using 2 ⫻ 1-ml cold sterile PBS through a canula placed in the trachea. BAL fluids were centrifuged, and the supernatants were collected for cytokine analysis. The total inflammatory cells were counted and stained by Diff-Quik Stain reagents (B4132-1A; Dade Behring), according to the manufacturer’s instructions. A differential cell count was performed based on standard morphological criteria and staining properties at ⫻40 magnification.

DC purification and culture The DC purification was performed, as described (33, 34). Briefly, spleens were aseptically removed from different groups of mice, and splenic singlecell suspensions were prepared by 2 mg/ml collagenase D (Roche Diagnostics) digestion in RPMI 1640 for 30 min at 37°C. To disrupt cell aggregations and DC-T cell complexes, EDTA (5 mM, pH 7.2) was added at the last 5 min of incubation. After lysis of RBC by adding ACK buffer, CD11c⫹ DC were then isolated using the MACS (Miltenyi Biotec) system. The purity of the DC was ⬎95%. To detect the cytokine production by DC, the freshly isolated DC were cultured with complete RPMI 1640 medium in the presence of UV-inactivated EBs (1 ⫻ 105 IFUs/ml) in 96-well plates at 5 ⫻ 105 cells/well for 72 h. IL-12 and IL-10 in the culture supernatants were measured by ELISA (32).

Spleen and draining lymph node (LN) cell cultures and cytokine assay Spleen and draining mediastinal LN cells were cultured for Cm-driven cytokine production, as described (30, 32). Briefly, single-cell suspensions were prepared and cultured at a concentration of 7.5 ⫻ 106 and 5 ⫻ 106 cells/well, respectively, with or without UV-inactivated Cm (1 ⫻ 105 IFU/ ml). The supernatants were harvested after 72-h culture, and the cytokine (IFN-␥, IL-12, IL-4, and IL-17) productions were measured by ELISA.

Materials and Methods

Flow cytometry

Animals

For surface marker expression on splenic DC, freshly isolated DC were analyzed by flow cytometry, as described (30, 32, 33). Briefly, the cells were double stained using fluorescent-labeled anti-CD11c allophycocyanin and anti-MHC class II FITC (I-A/I-E), anti-CD40 FITC, anti-CD80 FITC, or anti-CD86 FITC, or with their isotype controls (eBioscience) in a staining buffer (Dulbecco’s PBS without Ca2⫹ and Mg2⫹ containing 2% FBS and 0.09% NaN3). After staining on ice for 20 min in the dark, cells were fixed using 2% paraformaldehyde for 1 h. The data were collected using a FACSCalibur flow cytometer and analyzed by using CellQuest program (BD Biosciences). For analysis of the surface markers of lung, lung CD11c⫹ cells were isolated from lung single-cell suspensions using MACS CD11c column. The cells were stained with allophycocyanin-labeled anti-CD11c mAb, PE-labeled anti-Gr-1 (for excluding macrophages), and FITC-conjugated anti-CD40 or MHC II Abs. The gated lung DC (CD11c⫹ Gr-1⫺) were analyzed for CD40 and MHC II expression. For intracellular cytokine analysis, the single-cell suspensions of spleen, the mediastinal LN, and lung were analyzed, as described (30, 33). Briefly, cells were cultured at 7.5 ⫻ 106 cells/well in 48-well plates with PMA (50 ng/ml) and ionomycin (1 ␮g/ml) for 6 h in complete RPMI 1640 medium at 37°C. For accumulating cytokines intracellularly, 5 ␮g/ml brefeldin A (eBioscience) was added at the last 3 h of incubation. Cultured cells were washed twice using staining buffer and incubated with FcR block Abs

Female BALB/c mice, 6 – 8 wk old, were purchased from Charles River Laboratories. The breeding pairs of I-Ad-restricted DO11.10 TCR-␣␤ transgenic mice (TCR recognizing OVA323–339 peptide) were purchased from The Jackson Laboratory. The mice were maintained at a pathogenfree animal care facility at the University of Manitoba in a laminar flow cabinet. All mice used in this study were under the guidelines issued by the Canadian Council of Animal Care, and the research protocol was approved by the institutional ethical committee.

Organism Cm was used for the study. The propagation and purification of Cm were performed, as described previously (32, 33). Briefly, Cm was grown in HeLa 229 cell monolayer in Eagle’s MEM containing 10% FBS and 2 mM L-glutamine for 48 h. Infected cells were harvested with sterile glass beads, and Cm elementary bodies (EBs) were purified by discontinuous density gradient centrifugation. The purified organisms were resuspended in sucrose-phosphate-glutamic acid buffer, and stored at ⫺80°C until used. UVinactivated EBs were used in cell culture stimulation assays for cytokine analysis (32). The complete inactivation of EBs was confirmed by incubation of UV-treated EBs on HeLa 229 cells.

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Th17 MODULATES DC FUNCTION IN INFECTION

(anti-16/32; eBioscience) for 15 min on ice to block nonspecific staining. Surface marker staining was performed first using fluorescent-labeled antiCD3 FITC, anti-CD4 PE, or anti-CD8a PE mAbs. Following fixation of cells for more than 30 min, cells were permeabilized with permeabilization buffer (BD Pharmingen) and stained intracellularly with allophycocyaninlabeled anti-IFN-␥, anti-IL-4, or anti-IL-17 mAbs (eBioscience), or with corresponding isotype control Abs. Cells were washed twice with staining buffer and analyzed by flow cytometry.

DC-T cell coculture To assay the influence of DC on T cell priming and reactivation, two DC-T cell coculture systems were used, as described (30, 33, 34). For examining T cell priming by DC, CD4⫹ T cells isolated from the spleen of naive DO11:10 OVA peptide-specific TCR-␣␤ transgenic mice (BALB/c background) were cocultured with DC isolated from different groups of mice in the presence of OVA (100 ␮g/ml) in 96-well plates. For examining the Chlamydia-specific T cell reaction by DC, CD4⫹ T cells (1 ⫻ 106 cells/ well) isolated from inactivated Cm-immunized mice were cocultured with the DC (5 ⫻ 105 cells/well) from different groups of mice in the presence of UV-inactivated Cm (1 ⫻ 105 IFUs/ml) in 96-well plates. Enriched Chlamydia-specific T cells were obtained from inactivated Cm-immunized mice, as described (30). Briefly, mice were injected with inactivated 1 ⫻ 106 IFUs of Cm i.p., and 2 wk later boosted with the same dose of infection. One week following the boosting, CD4⫹ T cells were isolated from spleen using MACS CD4 T cell column (Miltenyi Biotec), according to manufacturer’s instructions. Cell culture supernatants were harvested at 48 h for analyzing Th1 (IFN-␥) and Th2 (IL-4) cytokines by ELISA.

Adoptive transfer of DC and challenge infection Freshly isolated splenic CD11c⫹ DC from different groups of mice were injected into the tail vein of syngeneic naive BALB/c recipient mice (5 ⫻ 105 DC/mouse). The recipient mice were then challenged intranasally with 1 ⫻ 103 IFUs of Cm in 40 ␮l of PBS at 2 h following adoptive transfer. Body weights of the mice were recorded daily, and mice were killed at day 14 posttransfer of DC for analysis of bacterial loads and immune responses.

Generation of bone marrow (BM)-derived DC (BMDC) BMDC were generated from naive BALB/c mice, as described (35), with minor modifications. Briefly, murine femurs and tibiae were isolated mechanically from surrounding tissues, and BM cells were flushed with medium using a syringe with a 0.45-mm needle. After spinning down cells and lysing RBCs by ACK, 2 ⫻ 105 cells were cultured in complete medium (RPMI 1640 supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine, 100 U/ml penicillin, 100 ␮g/ml streptomycin, and 50 mM 2-ME) in the presence of either GM-CSF alone (20 ng/ml), or GM-CSF plus different concentrations of recombinant murine IL (rmIL)-17 (10 or 100 ng/ml) in a 100-mm cell culture dish for 8 days. Fresh complete medium containing GM-CSF and rmIL-17 was supplied on days 3 and 6, and nonadherent cells were harvested at day 8. CD11c⫹ DC were purified with the MACS system and were cultured in 24-well culture plates containing 1 ⫻ 106 cells/well in a final volume of 1 ml. The surface marker expression on DC was analyzed by flow cytometry, and the culture supernatants were analyzed for IL-12p40 and IL-12p70 by ELISA.

Histopathological analysis The lung tissues from different groups of mice were removed and fixed in 10% formalin. The tissue sections were stained by H&E, and histological changes were observed under light microscopy, as described (30, 33).

RT-PCR analysis Cytokine RT-PCR was performed, as described (32, 33). Briefly, total RNA was isolated from lung tissues or 1 ⫻ 106 DC using TRIzol (Invitrogen Life Technologies), according to the manufacturer’s instructions. First strand cDNA was generated from 1.2 ␮g of total RNA in a total reaction volume of 15 ␮l using Moloney murine leukemia virus reverse transcriptase (Invitrogen), and the cDNA was then amplified using murine-specific primers for IL-17, IL-12, and IL-10, with a housekeeping gene (␤-actin) as a control. PCR products were run on 1.5% agarose gels, and the bands were analyzed for density on Scion Image software. The PCR primers used in this study were as follows: IL-17, 5⬘-GGTCAACCTCAAAGTCTTTA ACTC-3⬘ (sense) and 5⬘-TTAAAAATGCAAGTAAGTTTGCTG-3⬘ (antisense); the expected size of the IL-17 PCR product was 399 bp. IL-12, 5⬘-CTCACCTGTGACACGCCTGA-3⬘ (sense) and 5⬘-CAGGACACTG AATACTTCTC-3⬘ (antisense); the expected size of the IL-12 PCR product was 435 bp. IL-10, 5⬘-GTGAGGCGCTGTCATCGATT-3⬘ (sense) and 5⬘AGGTCCTGGAGTCCAGACACA-3⬘ (antisense); the expected size of the

FIGURE 1. Cm lung infection induced IL-17 mRNA expression and protein production in the local lung tissues and in the peripheral lymphoid organs. Mice were intranasally infected with Cm (1 ⫻ 103 IFUs) and were sacrificed at the indicated days p.i. A, Total RNA extracted from the lung tissue was assayed for IL-17 mRNA expression by RT-PCR using specific primers. B, Murine IL-17 protein levels in the lung tissue homogenates were determined by ELISA. C, IL-17-producing CD4⫹ T cells in spleen and draining LN in the infected mice at day 7 p.i. and the spleen cells from naive control mice were analyzed by intracellular cytokine staining, as described in Materials and Methods. D, Summary of intracellular cytokine data. E, Spleen and draining LN cells from infected mice and spleen cells from naive mice were cultured in the presence of UV-inactivated Cm, as described in Materials and Methods. IL-17 in 72-h cell culture supernatants was detected by ELISA. Data are presented as the mean ⫾ SD of each group. At least three independent experiments with four mice in each group were performed, and one representative experiment is shown. ⴱⴱ, p ⬍ 0.01; ⴱⴱⴱ, p ⬍ 0.001. IL-10 PCR product was 191 bp. ␤-actin, 5⬘-GTGGGGCGCCCCAG GCACCA-3⬘ (sense) and 5⬘-CTCCTTAATGTCACGCACGATTTC-3⬘ (antisense). The size of the PCR product was 550 bp.

Statistical analysis Unpaired Student’s t test was used to assay the statistical significance in the comparison of two different groups. One-way ANOVA analysis was used for analyzing data from the experiments with multiple groups. A p value less than 0.05 was considered significant.

Results Cm infection induces IL-17 production and Th17 expansion To test the involvement of IL-17/Th17 in the process of chlamydial lung infection, we first analyzed the IL-17 mRNA expression and the protein levels in the lung tissues following intranasal infection with Cm. We found that IL-17 mRNA expression started to be detectable in the lung of the infected BALB/c mice on day 2, peaked on day 5–7, and significantly decreased by day 14 –21, p.i. (Fig. 1A). Measurement of IL-17 protein in the lung homogenates also showed significantly increased levels of IL-17 at days 7 and 14 p.i. (Fig. 1B), representing the earlier and later stages of infection, respectively (23, 33). IL-17 protein levels were significantly higher on day 7 than on day 14 p.i. (Fig. 1B). Next, we determined IL-17 protein production by the spleen and draining LN cells isolated from Cm-infected mice. Intracellular cytokine staining of


FIGURE 2. Greater body weight loss, higher bacterial growth, and more severe pathological changes in the lungs of IL-17-neutralized mice (antiIL-17) compared with that in IgG2a isotype control (IgG2a) and in PBSadministered control BALB/c mice (PBS) following Cm lung infection. Mice were intranasally infected with 1 ⫻ 103 IFUs of Cm. Two hours later, a group of mice (anti-IL-17) was intranasally administered with 10 ␮g of anti-mouse IL-17 mAb in 40 ␮l of sterile PBS, and the same treatment was given at days 2 and 4 p.i. For the mice that were killed at day 13 p.i., one more anti-IL-17 mAb administration was given on day 7 p.i. The control mice were intranasally administered with 40 ␮l of IgG2a isotype control Ab or sterile PBS following the same schedule. Mice were monitored daily for body weight change (A). The original body weights of the two groups of mice were similar. Mice were sacrificed on day 7 or 13 p.i., and the lungs were collected and analyzed for in vivo chlamydial growth, as described in Materials and Methods (B). C, Lung sections were stained by H&E for histological analysis in ⫻400 magnification under light microscopy at day 7 (D7) and day 13 (D13) p.i. D, BAL cells were stained and counted for infiltrating cell differentials at day 7 p.i. Results are shown as mean ⫾ SD of 12 mice. ⴱⴱ, p ⬍ 0.01; ⴱⴱⴱ, p ⬍ 0.001; PBS vs anti-IL-17.

the freshly isolated spleen and draining LN cells showed significantly enhanced IL-17 production by CD4⫹ T cells on day 7 p.i. (Fig. 1, C and D). Some non-CD4⫹ cells also produce IL-17, although in a lower proportion (Fig. 1C). Bulk culture of either spleen or draining LN cells isolated from infected mice in the presence of killed Cm showed increased IL-17 production with higher levels in the early phase (day 7) than in the later phase (day 14) of infection (Fig. 1E). The culture of the spleen and LN cells isolated from naive and infected mice in the absence of killed Cm stimulation only showed marginal levels of IL-17 production (data not shown). Taken together, the data demonstrated that chlamydial lung infection induced IL-17 production not only in local lung tissues, but also in peripheral lymphoid organs, suggesting its role in the involvement of host defense against chlamydial infection. IL-17/Th17 contributes to protection against chlamydial lung infection To assess the role played by IL-17 in host defense against chlamydial infection, a group of BALB/c mice was intranasally administered with 10 ␮g of anti-mouse IL-17 mAb at 2 h and every other day after Cm lung infection to neutralize endogenous IL-17 at the site of infection. The results showed that the IL-17-neutralized mice (anti-IL-17) suffered more severe disease compared with sham-treated control mice following Cm infection (Fig. 2). The body weight loss was much greater in IL-17-neutralized mice, with no sign of recovery at day 14 when the mice were killed (Fig. 2A). Consistently, the IL-17-neutralized mice showed significantly higher bacterial loads in the lung than sham-treated mice at both early (day 7) and late (day 13) stages of infection (Fig. 2B). In

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FIGURE 3. Lower Th1-related, but higher Th2-related Cm-driven cytokine production in anti-IL-17 mice than in IgG2a isotype control and PBS control mice. Mice (four mice per group) were infected intranasally with Cm, as described in the legend to Fig. 2, and sacrificed at day 7 or 14 p.i. Spleen (A, C, and E) and draining mediastinal LN (B, D, and F) cells were cultured with UV-inactivated EBs, as described in Materials and Methods. IFN␥ (A and B), IL-12 (C and D), and IL-4 (E and F) levels in 72-h culture supernatants were determined by ELISA. Data are presented as the mean ⫾ SD of each group. One representative experiment of three independent experiments is shown. ⴱ, p ⬍ 0.05; ⴱⴱ, p ⬍ 0.01; ⴱⴱⴱ, p ⬍ 0.001; PBS vs anti-IL-17 Ab-treated mice.

particular, at the later phase of the infection (day 13 p.i.), shamtreated mice showed a dramatic decrease in the pulmonary bacterial load. In contrast, the IL-17-neutralized mice still carried bacterial burden in lungs, which was ⬃2000-fold higher than that in the sham-treated mice, suggesting a failure to clear infection (Fig. 2B). The lung histological analysis showed much more tissue damage and pathological change in the IL-17-neutralized mice than in sham-treated mice. Large amounts of cellular infiltration and inflammatory exudates were seen in the lung at day 7, and even at day 13 in the IL-17-neutralized mice (Fig. 2C). There were significantly less neutrophils in the IL-17-neutralized mice than sham-treated mice in the lung shown by BAL cell analysis (Fig. 2D). There was no difference in disease process between the mice with the two different sham-treatment approaches, PBS and isotype control Ab. The data suggested that IL-17/Th17 played a critical protective role in host defense against chlamydial lung infection. IL-17-neutralized mice exhibit reduced type 1, but increased type 2, Cm-specific immune responses Because previous studies have demonstrated the association of protection with type 1 T cell responses and more severe disease severity/ pathology with type 2 T cell responses (23, 24, 27), we further analyzed the Cm-driven type 1 and type 2 cytokine production by spleen and draining LN cells from the infected, IL-17-neutralized mice in comparison with the infected, sham-treated (IgG2a isotype control or PBS control) mice. The results showed that both spleen and LN cells isolated from IL-17-neutralized mice produced significantly lower levels of IFN-␥- and Th1-promoting cytokine (IL-12) than

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FIGURE 4. Less IFN-␥, but more IL-4 production by T cells in IL-17-neutralized mice compared with control mice following Cm lung infection. A, Spleen and LN cells collected at day 7 p.i. were analyzed for cytokine production by intracellular cytokine staining, as described in Materials and Methods. a, IFN-␥producing CD4⫹ T cells. c, IFN-␥-producing CD8⫹ T cells. e, IL-4-producing CD4⫹ T cells. b, d, and f, Summary graphs represent IFN-␥ production by CD4 (b) and CD8 (d) T cells and IL-4 production by CD4⫹ T cells (f), respectively, in each group. B, Lung CD4⫹ T cell and BAL analysis. a, IFN-␥-producing CD4⫹ T cells; b, summary graph for lung IFN-␥-producing CD4⫹ T cells; and c, IFN-␥ levels in the BAL fluids. Data show the mean ⫾ SD. At least three independent experiments with four mice in each group were performed, and one representative experiment is shown. ⴱ, p ⬍ 0.05; ⴱⴱ, p ⬍ 0.01; ⴱⴱⴱ, p ⬍ 0.001.

sham-treated mice at days 7 and 14 p.i. (Fig. 3, A–D). Intracellular cytokine-staining analyses also showed significant reduction of IFN␥-producing CD4⫹ and CD8⫹ T cells in both spleen and LN of the IL-17-neutralized mice (Fig. 4A, a– d). In contrast, IL-17-neutralized mice showed significantly enhanced levels of IL-4 production by spleen and LN cells at day 7 or 14 p.i. (Fig. 3, E and F). Consistently, intracellular cytokine analysis also showed significantly increased IL4-producing CD4⫹ T cells in LN compared with that in the control mice (Fig. 4A, e and f). Lung cells and BAL fluid from IL-17-neutralized mice also produced significantly lower levels of IFN-␥ (Fig. 4B) compared with IgG2a control mice. These results showed a significant contribution of IL-17/Th17 in enhancing type 1 cytokine responses of both CD4⫹ and CD8⫹ T cells during Cm infection. IL-17-neutralized mice exhibit an altered DC phenotype and cytokine production pattern To explore the basis for the promoting role of IL-17/Th17 in type 1 T cell responses, we further examined the effect of IL-17 neutralization on costimulatory molecule expression and cytokine production by DC

during chlamydial lung infection. We isolated DC from the spleen of infected, IL-17-neutralized mice and sham-treated control mice to analyze for surface marker expression. As shown in Table I, compared with naive mice, Cm-infected sham-treated mice showed dramatically increased CD40, CD80, and CD86 and MHC II expression on their DC (naive vs Cm/PBS), whereas IL-17-neutralized mice with the same infection exhibited significantly reduced CD40 and MHC II expression (Cm/PBS vs Cm/anti-IL-17). Similar changes in surface-expressed CD40 and MHC II were also observed in the analysis of DC isolated from lung tissues (Table I). The data demonstrated that IL-17 neutralization in chlamydial-infected mice resulted in altered DC phenotype. Because cytokine production by DC greatly contributes to the modulating effect of DC on T cell responses, we further tested whether IL-17 neutralization influenced DC cytokine production pattern during Cm infection. The freshly purified ex vivo splenic DC were either directly tested by semiquantitative RT-PCR for messages of IL-12 and IL-10 or further cultured in vitro for 72 h to test IL-12 and IL-10 protein production using ELISA (Fig. 5).


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Table I. Summary of DC surface costimulatory molecule expression in PBS- and anti-IL-17-administered mice following Cm infection in spleen and lunga Tissues

Surface Markers

CD40 CD80 Spleen CD86 MHC II CD40 Lung MHC II

(%) MFI (%) MFI (%) MFI (%) MFI (%) MFI (%) MFI

Naive

Cm/PBS

Cm/anti-IL-17

32.69 ⫾ 2.89 16.76 ⫾ 0.24 14.45 ⫾ 0.47 11.20 ⫾ 0.36 11.90 ⫾ 0.19 6.33 ⫾ 0.08 98.44 ⫾ 0.19 345.55 ⫾ 16.27

62.01 ⫾ 2.80 23.31 ⫾ 0.22 60.04 ⫾ 2.25 23.93 ⫾ 0.99 51.41 ⫾ 1.15 9.32 ⫾ 0.36 99.39 ⫾ 0.23 452.30 ⫾ 3.10 48.35 ⫾ 1.45 76.85 ⫾ 0.72 74.72 ⫾ 3.53 386.30 ⫾ 1.04

48.70 ⫾ 3.71* 18.14 ⫾ 0.66** 59.10 ⫾ 1.31 26.58 ⫾ 1.39 46.37 ⫾ 3.84 8.95 ⫾ 0.16 97.39 ⫾ 0.35** 386.70 ⫾ 23.32* 43.68 ⫾ 0.39* 61.86 ⫾ 2.95** 69.01 ⫾ 3.41 267.7 ⫾ 8.19**

a Mice were treated as described in the legend to Fig. 2, and sacrificed on day 7 p.i. Splenic and pulmonary DC were isolated using MACS CD11c microbeads, as described in Materials and Methods. Splenic DC were double stained with allophycocyaninconjugated anti-CD11c mAb and FITC-conjugated Abs specific for other surface markers and were analyzed by flow cytometry. Pulmonary DC were stained with allophycocyanin-labeled anti-CD11c mAb, PE-labeled anti-Gr-1 (for excluding macrophages), and FITC-conjugated anti-CD40 or MHC II Abs. The gated CD11c⫹ Gr-1⫺ DC were analyzed for CD40 and MHC II expression. The percentages of positive cells and the mean fluorescence intensity (MFI) for each marker are shown. Data are presented as the mean ⫾ SD of four mice in each group. One representative of three independent experiments with similar results is shown. ⴱ, p ⬍ 0.05; ⴱⴱ, p ⬍ 0.01; PBS- vs anti-IL-17 Ab-administered mice.

The results showed that the DC isolated from IL-17-neutralized mice expressed significantly lower levels of mRNA for IL-12, but higher levels of IL-10 compared with control mice (Fig. 5, A and B). Consistently, IL-12 and IL-10 levels in the culture supernatants matched the pattern of mRNA expression (Fig. 5, C and D). Taken together, the data suggest that, in Cm infection, IL-17 could significantly influence the DC costimulatory molecule expression and cytokine production, which are closely related to DC function.

FIGURE 5. Altered cytokine production pattern of DC in IL-17-neutralized mice (anti-IL-17) compared with IgG2a isotype control and PBS control (PBS) mice following Cm infection. Mice were killed at day 7 p.i., and splenic DC were isolated using MACS CD11c microbeads, as described in Materials and Methods. The freshly purified CD11c⫹ DC were subjected to RNA extraction for analyses of IL-12 and IL-10 messages by RT-PCR (A and B). DC were cultured at 5 ⫻ 105 cells/well in 96-well plates for 72 h, followed by ELISA analysis of IL-12 (C) and IL-10 (D) protein levels in the culture supernatants, as described in Materials and Methods. One representative experiment of three independent experiments with similar results is shown. Data are shown as the mean ⫾ SD. ⴱ, p ⬍ 0.05; ⴱⴱ, p ⬍ 0.01.

IL-17 neutralization influences the functional ability of DC to direct T cell responses in vitro To confirm whether IL-17 neutralization leads to changes in DC function to direct T cell responses, we used two DC-T cell coculture systems to examine the function of DC from the different groups of mice in modulating Ag-specific T cell responses. First, we cocultured DC with CD4⫹ T cells isolated from Cm-immunized mice in the presence of UV-killed EBs to test the ability of DC in modulating Chlamydia-specific T cell cytokine responses (Fig. 6, A and C). Second, considering the T cells used in the first system were in vivo primed T cells, thus the response most likely reflecting reactivation of T cells, we also cocultured the DC with CD4⫹ T cells isolated from naive DO11:10 transgenic mice (TCR specific for OVA peptide) in the presence of OVA to test the function of DC in priming Ag-specific naive T cells (Fig. 6, B and D).

FIGURE 6. DC from IL-17-neutralized mice show reduced Th1-promoting activity in T cell priming and reactivation. DC (105 cells/well) isolated from anti-IL-17- or PBS-treated mice at day 7 p.i. were cocultured with T cells (106 cells/well) isolated from Cm-immunized mice (A and C) or CD4⫹ T cells from naive DO11:10 transgenic mice (TCR specific for OVA peptide) (B and D) in the presence of UV-killed EBs or OVA, respectively, as described in Materials and Methods. The concentrations of Th1 (IFN-␥) and Th2 (IL-4) cytokines in the culture supernatants were measured by ELISA. Data show the mean ⫾ SD. ⴱ, p ⬍ 0.05; ⴱⴱⴱ, p ⬍ 0.001.

5892


As shown in Fig. 6, DC isolated from IL-17-neutralized mice showed significantly reduced ability to promote Ag-specific IFN-␥ production than those from infected sham-treated control (PBS) mice (Fig. 6, A and B) in both coculture systems. In contrast, DC isolated from IL-17-neutralized mice induced higher IL-4 production compared with PBS-DC in both coculture systems (Fig. 6, C and D). These in vitro data demonstrated that IL-17 indeed influenced the function of DC in modulating Ag-specific, including Chlamydia-specific, T cell cytokine patterns. Adoptive transfer of DC from IL-17-neutralized mice fails to induce type 1 protective immunity against challenge infection To further confirm the effect of IL-17 on DC function in vivo, adoptive transfer experiments were performed to examine the ability of the DC from different groups of mice in inducing protective immunity against challenge Cm infection. DC were isolated from the spleens of IL-17-neutralized and PBS-treated mice, respectively, at day 7 p.i., and then adoptively transferred to naive syngeneic recipients by i.v. injection. Control mice were given PBS i.v. 2 h after transfer of DC or PBS alone; the recipient mice were intranasally challenged with Cm and were killed at day 14 postchallenge. The data in Fig. 7 show that the mice that received DC isolated from sham-treated, Cm-infected mice (DC (PBS)) exhibited much milder body weight loss (Fig. 7A) and significantly lower bacterial loads (Fig. 7B) than the mice without DC transfer (no DC), demonstrating the capacity of the DC from sham-treated, infected mice in generating protective immunity to challenge infection. In contrast, the mice that received DC isolated from IL17-neutralized mice (DC (anti-IL-17)) exhibited similar levels of bacterial loads as those without DC transfer (no DC) (Fig. 7B), indicating a failure of these DC in inducing protective immunity. We further analyzed the IFN-␥ production in the spleen and draining LN by intracellular cytokine staining (Fig. 7, C–F). The data showed that the mice that received DC from IL-17-neutralized mice exhibited reduced levels of IFN-␥-producing CD4⫹ and CD8⫹ T cells in the spleen (Fig. 7, C and D) and the draining LN (Fig. 7F) compared with those that received DC from infected sham-treated mice (DC (PBS)). RT-PCR analysis of major outer membrane protein genes of Cm and measurement of recoverable organisms from the DC preparations from both of the groups showed negative result (data not shown). Thus, it is unlikely that the transfer of the splenic DC from either group caused live infection in the recipients. These findings demonstrated that IL-17 contributed to the function of DC to induce protective type 1 immune responses against chlamydial infection in vivo. Exposure to IL-17 during DC development changed DC phenotype and cytokine production The data above provided evidence on the modulating effect of IL-17 on DC function during chlamydial lung infection. To further test whether IL-17 could act on DC directly, we cultured BM cells in the presence or absence of rmIL-17 and examined the phenotype and cytokine production of BMDC. As shown in Fig. 8A, IL-17 addition in the BM cell culture either in low (10 ng/ml, IL-17(L)) or high (100 ng/ml, IL-17(H)) concentrations up-regulated CD40 and MHC II expression on BMDC in a dose-dependent manner. Moreover, IL-17(L) DC produced significantly higher levels of IL-12p40 and IL-12p70 than those generated with GM-CSF stimulation alone (Fig. 8, B and C). Interestingly, unlike the effect on surface molecule expression, the higher concentration of IL-17 was not as effective as the lower concentration of IL-17 in promoting IL-12 production by DC. Taken together, our results demonstrate that IL-17 has a direct promoting effect on DC maturation

FIGURE 7. Adoptive transfer to evaluate DC function in vivo. DC isolated from the spleens of PBS-treated mice (DC (PBS)) or IL-17-neutralized mice (DC (anti-IL-17)) at day 7 p.i. were adoptively transferred (i.v.) to naive BALB/c mice recipients and intranasally challenged with 1 ⫻ 103 IFUs of Cm at 2 h following DC transfer. Mice that received PBS alone were used as controls (no DC). Mice were killed at day 14 p.i., and the bacterial loads in the lungs and IFN-␥ production by CD4 and CD8 cells in spleen and draining LN were analyzed by intracellular cytokine staining, as described in Materials and Methods. A, Body weight changes in three groups of mice. B, Chlamydial growth in the lung. C and D, Splenic cells were examined for IFN-␥-producing CD4 and CD8 cells by intracellular cytokine staining. E and F, Draining LN cells were tested for IFN-␥ production by CD4 and CD8 T cells by intracellular cytokine staining. Data are expressed as mean ⫾ SD. ⴱ, p ⬍ 0.05; ⴱⴱ, p ⬍ 0.01; ⴱⴱⴱ, p ⬍ 0.001.

and IL-12 production, which are critical for promoting type 1 T cell immunity.

Discussion In the present study, we demonstrated a critical role of IL-17/Th17 in host defense against lung infection with Cm, an obligate intracellular bacterium. Our results showed that IL-17 responses and Th17 cells were elicited not only in local infected tissues (lung), but also in peripheral immune organs, including draining LN and the spleen following intranasal infection with Cm. More importantly, we found that the IL-17/Th17 responses contributed significantly to the development of type 1 protective immunity to the infection because the IL-17-neutralized mice showed significantly higher body weight loss, greater organism growth, and much more severe pathological changes in the lung compared with shamtreated mice, which were associated with reduced IFN-␥ production by CD4⫹ and CD8⫹ T cells and increased IL-4 production in IL-17-neutralized mice. This report depicts a significant contribution of IL-17/Th17 in a primary obligate intracellular infection. The most interesting finding in the present study is the critical modulating effect of IL-17/Th17 response on the phenotype, cytokine production, and, more importantly, function of DC in a real


FIGURE 8. rmIL-17 directly enhanced CD40 expression and IL-12 production by BMDC in vitro. Freshly isolated BM cells were cultured in complete medium containing 20 ng/ml murine rGM-CSF in the presence or absence of rmIL-17 at 10 ng/ml (IL-17(L)) or 100 ng/ml (IL17(H)), respectively, for 8 days. CD11c⫹ DC were purified using MACS CD11c microbeads, as described in Materials and Methods. A, DC were double stained with allophycocyanin-conjugated anti-CD11c and FITC-conjugated Abs for specific surface markers to analyze by flow cytometry. Staining with specific Abs (filled histograms) or matched isotype Ab control (dotted line) is shown. MFI, mean fluorescence intensity. Purified DC were further cultured for 48 h in complete medium, and the cultured supernatants were analyzed for IL-12p40 (B) and IL-12p70 (C) concentrations by ELISA. Data are shown as mean ⫾ SD. ⴱ, p ⬍ 0.05; ⴱⴱ, p ⬍ 0.01; ⴱⴱⴱ, p ⬍ 0.001.

infection model, and the significant impact of this modulation on in vivo T cell responses and protection. The modulating effect was shown by several lines of experiments. First, DC isolated from IL-17-neutralized mice exhibited significantly lower CD40 expression and IL-12 production, but higher IL-10 production than shamtreated mice during Cm infection. Second, coculture of the DC isolated from IL-17-neutralized mice with either primed Chlamydia-specific T cells or OVA peptide-specific naive CD4 T cells from TCR transgenic mice showed reduced IFN-␥ production, but increased IL-4 production, in comparison with DC from shamtreated mice,. These in vitro experiments indicated that the DC from IL-17-neutralized mice were not efficient in inducing Th1 responses in the stages of both T cell priming and T cell reactivation. Due to the lack of commercially available transgenic mice expressing Chlamydia-specific TCR, the experiment was only done using naive OVA peptide-specific TCR transgenic mice. However, the results most likely reflected the function of DC in priming T cells specific for chlamydial Ag, although the speculation needs to be confirmed in future studies. More importantly, we showed that DC isolated from IL-17-neutralized mice, unlike those from sham-treated mice, failed to generate protective type 1 CD4 and CD8 T cell responses in vivo and failed to protect the recipient mice against challenge infection. A similar change in pulmonary DC phenotype was also found. To our knowledge, this is the first report showing a significant modulating effect of IL-17/Th17 responses on DC in an infection model with a real pathogen. How do IL-17/Th17 responses modulate DC function in vivo? Both direct and indirect mechanisms are most likely involved. Our

5893 data showed that rmIL-17 had a direct modulating effect on the cell surface CD40 expression and IL-12 production by BMDC. This is in line with the report by Antonysamy et al. (36) showing a promoting role of IL-17 in allogeneic T cell proliferation possibly through inducing DC maturation. They examined the influence of human rIL-17 on the differentiation and function of BMDC in the presence of GM-CSF with or without IL-4. Their results showed that IL-17R was expressed on a proportion of BM-derived CD11c⫹ DC and that IL-17 promoted the maturation of DC progenitors by enhancing the expression of CD11c, costimulatory molecules, and MHC class II Ag. Based on the reported study and our own data, we think it is possible that, in the beginning of infection, IL-17 may influence DC function through a direct effect. Subsequently, the DC IL-12 production and type 1 immune responses promoted by IL-17/Th17 may further enhance the functional ability of DC to augment type 1 T cell responses. In addition, because IL-17 is particularly powerful in inducing the differentiation and recruitment of neutrophils through including G-CSF and CXCL8/IL-8 chemokines and because it has been reported that neutrophils can modulate the function of DC (37), neutrophils may also partly contribute to the IL-17/Th17-mediated modulation of DC function in chlamydial infection. Indeed, we have observed less neutrophils in the lungs of IL-17-neutralized mice (Fig. 2D). However, the neutrophil-mediated mechanism is unlikely a dominant one, because the role of neutrophils in protective immunity against chlamydial infection has been found to be limited. In particular, we previously reported that the CXCR2 gene KO mice deficient of neutrophils had similar level of infection and disease process as that of wild-type controls following Cm lung infection (38), and Rodriguez et al. (39) has reported promoting role of neutrophils in the lung infection of C. pneumoniae, another species of Chlamydia. One question remaining is why intranasally delivered anti-IL-17 Ab has significant effect on splenic DC. The answer is not clear. However, because it has been demonstrated that the sources of splenic DC include residual DC and circulating DC, it is likely that some pulmonary DC from infection site can traffic to the spleen. Moreover, the intranasally delivered Ab may circulate systemically because of the rich blood circulation in the lung, thus leading to alteration of DC in the spleen. Our data provide new insight into the mechanism by which IL17/Th17 plays a role in host defense against intracellular bacterial infection. Instead of mainly enhancing the differentiation and migration of neutrophils through induction of cytokines and chemokines, often seen in extracellular bacterial infections, IL-17/Th17 was found in the present study to be critically important for the development of type 1 responses of CD4 and CD8 T cells. Previous studies in our laboratory (23, 24, 40) and those of others (27, 41– 43) have shown that Th1-type immune response induced by Cm infection is associated with protection against chlamydial infection. Our data in this study showed more severe infection and diseases in IL-17-neutralized mice, which correlated with reduced type 1 immune responses, including lower levels of Cm-driven IFN-␥ and local IL-12 production, and alteration of DC function. In addition to chlamydial infection, our data may provide insight into the mechanism on the role of IL-17 in promoting type 1 T cell responses reported in other models. A study on pulmonary infection of Mycobacterium bovis bacille Calmette-Guerin has shown a reduced IFN-␥ production by T cells and a decreased delayed-type hypersensitivity in IL-17-deficient mice (17). Moreover, the studies that showed the involvement of IL-17 in host defense against M. tuberculosis in recall responses also demonstrated the importance of IL-17 in inducing type 1 immune responses (21). More

5894 recently, the involvement of IL-17 in host defense against M. tuberculosis infection was demonstrated in a human study (44). Notably, in these reported studies, the mechanism underlying the promoting effect of IL-17/Th17 on Th1 responses was not elucidated. Our finding on the modulating effect of IL-17/Th17 on DC function in directing type 1 T cell immunity suggests a direction to further explore the mechanism by which IL-17 modulates Th1 cell responses in other intracellular bacterial infections. Further studies on the role of IL-17/Th17 responses in chlamydial pathogenesis are warranted. First of all, although we have shown a significant enhancement of IL-17 at gene expression and protein levels and the expansion of Th17 cells following chlamydial infection, Th17 cell is not the only source of IL-17 in this infection model. Our data showed that some IL-17-producing cells are not CD4⫹ T cells (Fig. 1C). It has been reported that ␥␦ T and NKT cells can also produce IL-17 (4, 5), and we have shown that both types of these cells are activated by Cm infection (45– 47). Therefore, it is worthwhile to test whether these cells also produce IL-17 and what cell(s) is the major source of the IL-17 produced during chlamydial infection. This may be particularly important for understanding the mechanism by which IL-17 modulates DC during chlamydial infection. Second, and more importantly, the role of IL-17/Th17 at different stages of chlamydial infection and also the host in different genetic backgrounds need to be carefully examined. Notably, the present study has focused on the role of IL-17 in early stages of infection, because the administration of neutralizing Ab started at the beginning of the infection. Although it is apparent that this early blockade of IL-17 activity showed a critical role of IL-17 in protection, it does not necessarily mean that IL-17 responses at late stages of infection are also beneficial. In particular, we and others have shown that C3H mice, the mouse strain that is extraordinarily susceptible to Cm infection, exhibited massive neutrophil responses especially in the later stages of infection (38, 48). Therefore, it is important to elucidate the role of IL-17/Th17 in later stages of chlamydial infection. This point could be further emphasized based on the numerous reports that show a significant pathological role of Th17 in other models of inflammatory diseases (5, 7). In conclusion, the present study has demonstrated that IL-17/ Th17 plays an important role in host defense against chlamydial infection. Furthermore, the data suggest that IL-17/Th17 can promote type 1 T cell-protective immunity to chlamydial infection by modulating DC function possibly through direct and indirect mechanisms. Further studies on the cellular sources of IL-17 and the effect of IL-17 production on protective immunity and pathological reactions in different stages of chlamydial infection will probably provide new insight into the immunoregulation and immunobiology of Chlamydia. Because the current data showed beneficial effects of IL-17 on the development of protective immunity against chlamydial infection, the possibility to use IL-17 as an immune adjuvant in chlamydial vaccination is worth exploring.

Disclosures The authors have no financial conflict of interest.

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