A MyD88-Dependent Early IL-17 Production Protects Mice against Airway Infection with the Obligate Intracellular Pathogen Chlamydia muridarum This information is current as of September 25, 2021. Xiaoyun Zhang, Lifen Gao, Lei Lei, Youmin Zhong, Peter Dube, Michael T. Berton, Bernard Arulanandam, Jinshun Zhang and Guangming Zhong J Immunol 2009; 183:1291-1300; Prepublished online 19 June 2009; Downloaded from doi: 10.4049/jimmunol.0803075 http://www.jimmunol.org/content/183/2/1291 http://www.jimmunol.org/ References This article cites 75 articles, 44 of which you can access for free at: http://www.jimmunol.org/content/183/2/1291.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2009 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

A MyD88-Dependent Early IL-17 Production Protects Mice against Airway Infection with the Obligate Intracellular Pathogen Chlamydia muridarum1

Xiaoyun Zhang,*‡ Lifen Gao,* Lei Lei,* Youmin Zhong,* Peter Dube,* Michael T. Berton,* Bernard Arulanandam,† Jinshun Zhang,‡ and Guangming Zhong2*

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-␤, can block the early IL-17 production. Treatment of mice with an anti-IL-17-neutralizing mAb enhanced growth of chlamydial organ- Downloaded from isms 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 Journal of Immunology, 2009, 183: 1291–1300. http://www.jimmunol.org/

he obligate intracellular bacterial species Chlamydia tra- and its signature cytokine IL-17 in C. trachomatis infection has not chomatis, consisting of multiple serovars, can cause many been evaluated. T health problems in humans. Serovars A–C infect human Naive CD4ϩ T cells can be induced to express the transcription ocular epithelial cells, causing trachoma and potentially leading to factor retinoic acid-related orphan receptor ROR␥t and secrete IL- blindness (1). Serovars D–K infect human urogenital tract epithe- 17, developing into the so-called Th17 phenotype, in addition to lial tissues, which, if left untreated, can lead to pelvic inflammatory Th1 and Th2 (18–21). IL-17 is an inflammatory cytokine that diseases, ectopic pregnancy, and infertility (2, 3). The three L or plays a key role in many inflammatory diseases. For example, LGV (lymphogranuloma venereum) serovars (L1–L3) can cause treatment of mice with a neutralizing anti-IL-17 mAb suppressed by guest on September 25, 2021 systemic infections in humans (4–6). The mouse pneumonitis autoimmune inflammation in the CNS (22), and mice deficient in agent strain (designated as MoPn, now classified as a new species, generating Th17 cells were resistant to experimental autoimmune Chlamydia muridarum) can infect mice in both the airway and the encephalomyelitis, collagen-induced arthritis, and inflammatory urogenital tract. Although the C. muridarum organisms cause no bowel disease (22, 23). IL-17 has also been found to play an im- known diseases in humans, these organisms have been used to portant role in host defense against infection by pathogens (24), study C. trachomatis pathogenesis and immunology in various including viruses (25), bacteria (26), and fungi (27, 28). However, mouse models (7–13). Data from the mouse model studies have IL-17 is not always protective, and IL-17-mediated inflammatory shown that the CD4ϩ Th cell (Th1 but not Th2)-dominant and response may even increase host susceptibility and exacerbate pa- IFN-␥-dependent immunity is a major host protective determinant thologies induced by some microbial infections (29–32). In this for controlling chlamydial infection (14), although Abs and CD8ϩ study, we used a mouse model with C. muridarum airway infection T cell-mediated immunity may also contribute to the host resis- to evaluate the role of IL-17 in chlamydial infection. We found that tance to chlamydial infection (15–17). However, the role of Th17 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 rep- lication and MyD88-mediated signaling pathways. Neutralizing *Department of Microbiology and Immunology, University of Texas Health Science Center, San Antonio, TX 78229; †Department of Biology, University of Texas, San the early IL-17 response significantly enhanced replication of C. Antonio, TX 78249; and ‡Department of Biochemistry, Hebei North University, muridarum and decreased mouse survival. These observations rep- Zhangjiakou Hebei, China resent the first demonstration that an early IL-17 response as an Received for publication September 17, 2008. Accepted for publication May 6, 2009. innate immunity component may play an important role in initi- The costs of publication of this article were defrayed in part by the payment of page ating host defense against infection with intracellular bacterial charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. pathogens in the airway. 1 This work was supported in part by grants (to G.Z.) from the National Institutes of Health. Materials and Methods 2 Address correspondence and reprint requests to Dr. Guangming Zhong, Depart- Chlamydial organisms and chlamydial infection in cell culture ment of Microbiology and Immunology, University of Texas Health Science Cen- ter, 7703 Floyd Curl Drive, San Antonio, TX 78229. E-mail address: Zhongg@ C. muridarum Nigg strain (also called MoPn) or C. trachomatis serovar L2 uthscsa.edu organisms were grown, purified, and titrated as previously described (33). Aliquots of the organisms were stored at Ϫ80°C until use. HeLa or L929 Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 cells (both from American Type Culture Collection) were maintained in www.jimmunol.org/cgi/doi/10.4049/jimmunol.0803075 1292 IL-17 IN CHLAMYDIAL AIRWAY INFECTION

DMEM (Life Technologies) with 10% FCS (Life Technologies) at 37°C in 96-well plates at 5 ϫ 106/ml for lymphocyte restimulation experiments

an incubator supplied with 5% CO2. To produce mouse lung fibroblast as described below. The mediastinal lymph nodes and spleen organs cells, whole lungs were removed from exsanguinated female C57BL/6 were also harvested from these mice for in vitro lymphocyte restimu- mice (8–12 wk old), transferred to DMEM, minced into 2- to 3-mm pieces, lation assay. Briefly, the tissues were minced, and single-cell suspen- and subsequently treated with freshly made collagenase type XI (0.7 mg/ sions were made. The nucleated splenocytes or lymph node cells ml) and DNase I type IV (30 ␮g/ml) in DMEM at 37°C for 30 min fol- (mainly lymphocytes) plated at 5 ϫ 106/ml were restimulated with UV- lowed by smashing the lung tissue with stainless steel mesh. Cell suspen- inactivated C. muridarum organisms at 1 ϫ 106 IFUs/ml for 3 days. The sions were filtered through 70-␮m pore size nylon cell strainers (Corning culture supernatants were used for cytokine measurements. Costar), washed, and resuspended in DMEM supplemented with 10% FCS. Titrating live chlamydial organisms in mouse organs Cells were grown at 37°C in a humidified 5% CO2 atmosphere in 24-well ϫ 6 plates with coverslips at a density of 1 10 /ml for 4 days. After detached To quantitate the live C. muridarum organisms in mouse lung, spleen, cells were washed away, the confluent fibroblast cell monolayers were and kidney, the organ homogenates produced as described above were cultured for another 24 h before being used for experiments as indicated titrated on HeLa cell monolayers in duplicates as described previously below. The C. muridarum and L2 organisms or mouse tissue homogenate (13). Briefly, serially diluted homogenate samples were inoculated onto samples were used to infect cells. Briefly, HeLa, L929, or mouse primary HeLa cell monolayers grown on coverslips in 24-well plates. After in- lung fibroblast cells grown on glass coverslips in 24-well plates were pre- cubation for 24 h in the presence of 2 ␮g/ml cycloheximide, the cultures ␮ treated with DMEM containing 30 g/ml DEAE-dextran (Sigma-Aldrich) were processed for immunofluorescence assay as described below. The for 10 min. After the DEAE-dextran solution was removed, chlamydial inclusions were counted under a fluorescence microscope. Five random organisms diluted in DMEM were allowed to attach to the cell monolayers fields were counted per coverslip. For coverslips with Ͻ1 IFU per field, for2hat37°C. The infected cells were continuously cultured in DMEM the IFUs on the entire coverslips were counted. Coverslips showing ␮ with 10% FCS and with or without 2 g/ml cycloheximide (Sigma- obvious cytotoxicity of HeLa cells were not taken into the count. The Aldrich) and processed at various time points after infection as indicated in total number of IFUs per organ was calculated based on the number of individual experiments. IFUs per field, number of fields per coverslip, dilution factors, and Downloaded from Mouse infection and treatment inoculation and total sample volumes. An average was taken from the serially diluted and duplicate samples for any given organ. The calcu- 3 Male or female wild-type (Wt) ; C57BL/6J mice (The Jackson Labo- lated total number of IFUs per organ was converted into log10, and the ratory) or with gene deficiency in MyD88 or Toll/IL receptor domain- log10 IFUs were used to calculate means and SD for each group at each containing adapter-inducing IFN-␤ (TRIF; a gift from Dr. S. Akira, time point. Osaka University, Osaka, Japan) were used at the age of 8–9 wk. When Immunofluorescence assay different groups of mice were compared, both mouse sex and birth date http://www.jimmunol.org/ were matched between groups. For mouse infection, each mouse was HeLa or L929 cells grown on coverslips with or without chlamydial in- inoculated intranasally with live C. muridarum organisms at the appro- fection and other treatments as indicated in individual experiments were priate inclusion-forming units (IFU) as indicated in individual experi- fixed with 2% paraformaldehyde dissolved in PBS for 30 min at room ments in 40 ␮l of sucrose-phosphate-glutamate buffer consisting of 218 temperature, followed by permeabilization with 1% saponin (Sigma- mM sucrose, 3.76 mM KH2PO4, 7.1 mM K2HPO4, and 4.9 mM gluta- Aldrich) for an additional 1 h. After washing and blocking, the cell samples mate, pH 7.2, under light anesthesia with isoflurane. For blocking of were subjected to immunostaining with a rabbit anti-CT395 Ab (this Ab IL-17, the Wt C57BL mice were treated with an anti-IL-17-neutralizing can cross-react with all chlamydial species; our unpublished data) plus a mAb (rat IgG2a, clone 50104.11; R&D Systems) or an isotype-matched goat anti-rabbit IgG conjugated with Cy2 (green; Jackson Immuno- control rat IgG (clone 54447.11; R&D Systems) via i.p. injection every Research Laboratories) to visualize chlamydial inclusions. For phenotyp- other day starting on day 1 before chlamydial infection at 62.5 ␮g/

ing of the mouse primary lung fibroblast cells, the primary cells along with by guest on September 25, 2021 injection (with the exception of the first injection at 125 ␮g). The in- the positive control cells (HeLa epithelial cells, L929 fibroblast cells, and jection continued to day 2 or 8 after infection as indicated in individual RAW macrophages) were grown on coverslips, and the cell monolayers experiments. For IL-17 treatment, the MyD88 knockout (KO) mice were labeled with the following primary Abs: rat anti-mouse CD14 (IgG2a, were given 1.5 ␮g of a rIL-17 (R&D Systems) by i.p. injection 1 day clone 159010; R&D); mouse anti-human cytokeratin (IgG1, clone PCK-26; before infection and the same amount by intranasal inoculation every Sigma-Aldrich; this Ab recognizes both human and mouse cytokeratins); other day after infection. Groups of mice were sacrificed at different mouse anti-human vimentin (IgM, clone VIM-13.2, Sigma-Aldrich; this times after infection as indicated in individual experiments. For some Ab also recognizes both human and mouse vimentin). The primary Ab experiments, the bronchial alveolar lavage fluids (BALF) were col- binding was visualized with Cy3-conjugated goat anti-mouse IgG1 or don- lected as described elsewhere (34). Briefly, mice were anesthetized with key anti-mouse IgM or donkey anti-rat IgG Abs (red; all from Jackson isoflurane, and a triangular syringe device (Three-Way Stopcock; Bax- ImmunoResearch Laboratories). For staining of the cell surface CD14, the ter Healthcare) was inserted into the mouse trachea. PBS (1 ml) was cell samples were only fixed with paraformaldehyde without permeabili- used to gently flush the bronchial alveolar system twice and then cen- zation with saponin. The Hoechst dye (blue; Sigma-Aldrich) was used to trifuged at 100 ϫ g for 10 min at 4°C. The cell-free bronchial alveolar visualize nuclear DNA. The immunolabeled samples were used for image lavage supernatant was used for cytokine measurement. In some exper- analysis and acquisition with an Olympus AX-70 fluorescence microscope iments, the pellet containing the BALF cells was resuspended in 200 ␮l equipped with multiple filter sets and a Hamamatsu digital camera (Olym- of PBS. To count cells, 50 ␮l of the cell suspension were smeared on pus) as described previously (33, 35–37). The images were acquired with a slide precoated with poly-L-lysine (Electron Microscopy Sciences), the software SimplePCI and processed using the Adobe Photoshop pro- and each BALF cell sample was smeared onto three different slides. The gram (Adobe Systems). slides were stained with Wright-Giemsa dye (CS434D; Fisher Diagnos- tics). Neutrophils, macrophages, and lymphocytes were counted using ELISA an upright microscope equipped with a ϫ100 objective oil lens (CH30; To measure the anti-C. muridarum Abs in mouse sera and cytokines in Olympus). At least 200 cells from each slide were counted, and the various mouse samples, a standard ELISA was used as described elsewhere results for each cell type were expressed as the percent of total cells. (38–40). For titrating the mouse anti-C. muridarum Abs, the C. murida- The bronchi-lung tissues with or without the prior collection of BALF rum-infected HeLa cell lysates were used as Ags to coat 96-well ELISA along with the spleen and kidney organs in some experiments were microplates (Nunc). After blocking with 2.5% nonfat milk (in phosphate- harvested for making homogenates as described previously (12). buffered solution), mouse serum samples after serial dilution were applied Briefly, each mouse organ was homogenized in sucrose-phosphate-glu- to the Ag-coated microplates. The serum Ab binding was detected with a tamate buffer (lung in 1.2 ml, spleen and kidney in 0.6 ml, respectively) goat anti-mouse IgG conjugated with HRP (Jackson ImmunoResearch using a 2-ml tissue grinder (Fisher Scientific). The homogenates, after Laboratories) in combination with the soluble substrate ABTS (Sigma- centrifugation to clear residual debris, were used for titrating live chla- Aldrich) and quantitated by reading the OD using a microplate reader mydial organisms and measuring cytokines. Blood was collected for 405 (Molecular Devices). For measuring cytokines from the mouse organ ho- monitoring Ab production. In some experiments, the BALF cells were mogenates or the supernatants of the in vitro stimulated spleen lymphocyte counted for the total number of live nucleated cells and were plated in cultures or Chlamydia-infected L929 cell cultures, standard cytokine ELISA kits were used as instructed by the manufacturer. The commercially ␥ ␣ ␤ 3 Abbreviations used in this paper: Wt, wild type; TRIF, Toll/IL receptor domain- available ELISA kits (mouse IFN- kit, IL-4, IL-5, IL-1 , IL-1 , IL-6, containing adapter-inducing IFN-␤; IFU, inclusion-forming unit; BALF, bronchial IL-8, IL-12, TNF-␣, and IL-17) were all obtained from R&D Systems. alveolar lavage fluid; KO, knockout. Briefly, the mouse samples after the appropriate dilution were applied to The Journal of Immunology 1293 Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 1. Local inflammatory cytokines and Ag-specific Ab and T cell responses to C. muridarum intranasal infection. Wt C57BL mice were intranasally inoculated with 4000 IFUs of C. muridarum organisms. At various time points after the inoculation (x-axis), mice were sacrificed, and the lung homogenates were made for titrating the live organisms and quantitating cytokines (A), spleen cells and sera were collected for measuring circulating chlamydial Ag-specific T cell and Ab responses (B) and BALF infiltrate cells and mediastinal lymph node cells for measuring airway local Ag-specific T cell responses (C). A, The number of live chlamydial organisms was expressed as log10 IFUs per lung whereas the amounts of cytokines as picograms or nanograms per milliliter of the lung homogenates (y-axis). IL-23, IL-17, and IL-12 peaked at 48 h, whereas other cytokines peaked at 72 h or later after infection. B, The splenocyte response was measured for their ability to produce cytokines after restimulating with (f) or without (Ⅺ) UV-inactivated C. muridarum organisms for 3 days, and the cytokines were detected and expressed as picograms or nanograms per milliliter of the culture supernatants (y-axis). The amounts of Abs specifically recognizing C. muridarum Ags in the mouse sera were measured in u an ELISA using the C. muridarum-infected HeLa cell lysates as Ags, and the results were expressed as OD405 ( ) as shown along the y-axis. All mouse sera were used at a dilution of 1/100. C, Both the BALF cells (a and b) and mediastinal lymph node cells (c and d) harvested on various days after infection as indicated along the x-axis were measured for their ability to produce IL-17 (a and b) and IFN-␥ (c and d) after in vitro stimulation with C. muridarum organisms as described in B. There were five to six mice in each time point group, and all data were presented as means Ϯ SD. Mice at time point ϭ 0(x-axis) were not infected. the 96-well ELISA microplates precoated with the corresponding capture Results Abs. The capture antibody-bound cytokines were detected with biotin- IL-17 is produced early in mouse airway upon C. muridarum conjugated anti-cytokine Abs and HRP-conjugated avidin. The cytokine concentrations were calculated based on optical density values, cyto- infection kine standards, and sample dilution factors and expressed as nanograms After an intranasal infection with C. muridarum, we monitored the or picograms per milliliter. levels of infectious organisms and inflammatory cytokines, Statistical analysis chemockines and lymphokines including IL-17 in the mouse lung An ANOVA test (http://www.physics.csbsju.edu/stats/anova.html) was homogenates (Fig. 1A). Infectious chlamydial organisms were re- performed to analyze data from multiple groups and a two-tailed Student t covered starting at day one after infection and the IFU number test (Microsoft Excel) to compare the means between two groups and a log steadily increased along the infection course and reached a plateau rank (Mantel-Cox) test (http://support.sas.com/documentation/cdl/en/ on day 8 (Fig. 1Aa). This infection time course is similar to what statug/59654/HTML/default/statug_seqtest_sect028.htm.) for comparing the mouse survival rates as well as the Chitest (Microsoft Excel) to analyze was previously described in C57 mice (41). When cytokines in the qualitative data between two groups. lung homogenates were compared, we found that IL-23, IL-12, and 1294 IL-17 IN CHLAMYDIAL AIRWAY INFECTION Downloaded from

FIGURE 2. The early IL-17 production is dependent on live chlamydial organism replication. Wt C57BL mice were intranasally infected with 160, 800, 2000, or 4000 IFUs of C. muridarum organisms with or without an- tibiotic treatment (x-axis), and 2 days after infection mice were sacrificed http://www.jimmunol.org/ for measurement of live chlamydial organisms (a) and IL-17 (b) in the lung homogenates (y-axis). For antibiotic treatment, mice were injected i.m. with a combination of rifampin (400 ␮g/injection) and chloramphenicol (400 ␮g/injection) once every day starting 2 days before infection. Each group consisted of four to six mice, and all data were expressed as means Ϯ SD. IL-17 was significantly increased in the lung only when the chlamydial infection dose reached 2000 IFUs or higher, and the antibiotic treatment significantly inhibited chlamydial growth and completely blocked IL-17 production in the lung. FIGURE 4. Effect of IL-17 neutralization on C. muridarum organism by guest on September 25, 2021 growth and spreading in mice. Two groups of mice (seven to nine in each group) treated with either an anti-IL-17 neutralization Ab (f) or an iso- IL-17 peaked at 48 h while other cytokines peaked on day 3 (e.g., type-matched control rat IgG (Ⅺ) as described in the legend to Fig. 3 were TNF-␣, IL-1␣␤, and IL-6, panels e-h respectively) or later (MIP-2, intranasally infected with 2000 IFUs of C. muridarum organisms; and on a mouse homologue of IL-8, Fig. 1Ai). IL-23, IL-17, and IL-12 days 2 and 8 after infection, mice were sacrificed for quantitation of live production was transient. By day 4, IL-23, IL-17, and IL-12 all chlamydial organisms recovered from lung, spleen, and kidney (A) and IL-17 in the lung homogenates (B). The results were expressed as means Ϯ SD. A two-tailed Student t test was used to compare the recovered IFUs between anti-IL-17 Ab- and control IgG-treated groups. Although there was no difference from the day 2 samples, the anti-IL-17 Ab treatment significantly increased the C. muridarum growth in the lung and spreading into other organs compared with the control IgG-treated mice. The anti- IL-17 Ab treatment did effectively reduce the IL-17 levels in the lung (B).

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 CD4ϩ Th17 cells, we also measured other T cell cy- tokines including IL-2, IFN-␥, 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 de- FIGURE 3. Effect of IL-17 neutralization on C. muridarum-induced tected in the supernatants of the cultured mouse BALF cells, me- mouse death. Two groups of Wt C57BL mice with eight in each group diastinal lymph node cells and splenocytes harvested from the Œ were treated with either an anti-IL-17 neutralization Ab ( ) or an isotype- same infected mice after an in vitro restimulation with MoPn or- F matched control rat IgG ( ). Both groups were intranasally infected with ganisms (Fig. 1, B and C, and data not shown). The facts that these 4000 IFUs of C. muridarum live organisms, and mouse death (expressed as cells regardless of their sources did not produce IL-17 on day 2 percent of survival) was recorded daily for up to 40 days after infection (x-axis). Log-rank test (http://support.sas.com/documentation/cdl/en/statug/ even with chlamydial Ag stimulation and only produced IL-17 on 59654/HTML/default/statug_seqtest_sect028.htm) was used to compare the day 8 or later after chlamydial Ag stimulation suggest that the survival rate between the two groups of mice. Starting at day 8 after in- responding cells are Ag-specific and not likely be responsible for fection, the survival rate of the anti-IL-17 Ab-treated group was signifi- the innate IL-17 production peaked on day 2 after chlamydial in- cantly lower than that of the control group (p Ͻ 0.01). tranasal infection. The Ag-specific IL-17 production was detected The Journal of Immunology 1295 Downloaded from http://www.jimmunol.org/

FIGURE 5. Effect of MyD88 deficiency on IL-17 production and C. muridarum infection. Wt C57BL and mice deficient in with MyD88 (MyD88KO) or TRIF (TRIFKO) were intranasally infected with 2000 IFUs of C. muridarum organisms; and on days 2 and 7 after infection, groups of mice were sacrificed for quantitation of IL-17 in the airway (A) and chlamydial IFUs in the lung, spleen, and kidney (B) or observed for mouse death for up to 9 days (C). A, Both BALFs (Ⅺ), and the homogenates made from the remaining lung tissue (u) from Wt or KO mice (x-axis) were measured for IL-17 levels by guest on September 25, 2021 (y-axis). Most IL-17 was secreted into the BALFs harvested from the Wt or TRIFKO mice on day 2 (a) but no obvious IL-17 was detected in either the MyD88KO samples harvested on day 2 (a, columns 3 and 4) or any samples harvested on day 7 (b). B, The live chlamydial organisms recovered from lung, spleen, and kidney of both Wt and MyD88 or TRIF KO mice (x-axis) on days 2 (b)and7(a) after infection were measured and expressed as log10 IFUs per organ (y-axis). The MyD88 KO mice displayed a significant increase in chlamydial growth in both the primary infection organ lung and the distal organs spleen and kidney on day 7 compared with the Wt mice (a), although there was no difference in chlamydial growth in any of the organs between the Wt and MyD88 or TRIF KO mice on day 2 (b). Seven Wt and 7 TRIF KO mice were sacrificed on day 2 and 7, respectively, and 8 MyD88 KO mice sacrificed Ϯ on days 2 and 7 sacrificed on day 7 (one MyD88 mouse died before sacrifice on day 7). The results were expressed as log10 IFUs (mean SD; y-axis) and the Student t test was used to compare the IFUs between Wt and KO mice. C, Eleven Wt mice (f) and 10 MyD88 KO (F) mice were infected with 2000 IFUs as described above and observed for mouse death up to 9 days after infection. The results were expressed as survival rate (y-axis) and the log-rank test was used to analyze the difference in mouse survival rate between the Wt and MyD88 KO mice as described in the legend to Fig. 3. Starting on day 8, the survival rate in MyD88 KO group was significantly lower than that in Wt mice. much earlier in the culture supernatants of the airway infiltrate and protein synthesis are required for the early IL-17 production in cells or draining lymph node cells (Fig. 1C, day 8) than in the the airway. Because IL-12 plays a critical role in mouse resistance splenocytes (Fig. 1B, day 20), which might be caused by the rel- to chlamydial airway infection (43), the parallel early production atively enriched chlamydial Ag-specific Th17 cells at the site of of IL-17 suggests that IL-17 may also play an important role in infection, thus dramatically increasing the detection sensitivity. mouse airway resistance to chlamydial infection. The accompanied Compared with systemic chlamydial Ag-specific IFN-␥ production early production of IL-23 suggests that IL-23 may play a role in (detectable in the splenocyte culture as early as 4 days after infec- the observed early production of IL-17 since IL-23 is a known tion), the systemic IL-17 production is severely delayed, only de- inducer of IL-17. tectable 20 days after infection. The delayed IL-17 production by spleen T cells is consistent with the concept that Th17 is often Neutralization of IL-17 significantly increased mouse fatality accumulated late during infection in the chronic inflammatory tis- and chlamydial organism replication in the lung and spreading sues (42). As expected, chlamydial Ag-specific Ab production was into other organs first detected on day 8 after infection. Since our goal in the current We used a neutralization Ab approach to test whether the early study was to evaluate the role of the early IL-17 in chlamydial IL-17 production in mouse lung plays any role in mouse resistance infection in the airway, we further characterized the early IL-17 to chlamydial infection (Fig. 3). Mice treated with an anti-IL-17 production. We found that the early IL-17 production was infec- neutralization mAb displayed a rapid decrease in survival rate. tion dose dependent and blocked by treatment of mice with anti- Mice in the neutralizing Ab-treated group started to die on day 6 biotics (Fig. 2), suggesting that chlamydial productive infection (with a 75% survival rate), and death continued on day 8 (50% 1296 IL-17 IN CHLAMYDIAL AIRWAY INFECTION Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 6. Effect of IL-17 on C. muridarum growth and cytokine production in cultured cells. A, L929 cells with or without pretreatment with IL-17 or IFN-␥ at the appropriate concentrations for 24 h were infected with or without either C. muridarum or L2 organisms (x-axis) at a multiplicity of infection of 0.5, and the C. muridarum-infected cells were processed at 30 h and the L2-infected cells processed at 48 h after infection for immunofluorescence assay to quantitate the number of inclusions per coverslip (a) and the corresponding culture supernatants were measured for IL-6 (b) and IL-8 (c; y-axis). Both the IL-17 and IFN-␥ treatments were maintained in the cultures throughout the experiments. B, Mouse primary lung fibroblast cells were similarly treated and infected with cytokines and chlamydial organisms and the results were obtained as described in the legend to A. The data from each panel came from four independent experiments with duplicates in each and were expressed as means Ϯ SE. ANOVA Test was performed to analyze data from multiple groups for both A and B and a two-tailed Student t test to compare the means between any two groups if ANOVA test showed p Ͻ 0.05. For example, in A, there was a significant difference in IFUs (Aa) between columns 9 and 11 (p Ͻ 0.01 by t test) but not between columns 9 and 10 (p Ͼ 0.05). C, The purity of the mouse primary lung fibroblast cells used in B was further evaluated using a combination of Abs in an indirect immunofluorescence assay with antivimentin for positively labeling fibroblast cells (a, d, g, and j), anticytokeratin for epithelial cells (b, e, h, and k), and anti-CD14 for macrophages (Møs, c, f, i, and l). L929 cells were used as a positive control of fibroblast cells (d–f), HeLa as epithelial cells (g–i; the mouse anticytokeratin Ab used here can recognize both mouse and human epithelial cells), RAW as Møs (j–l). Most mouse primary lung fibroblast cells were positively labeled with the fibroblast-specific antivimentin Ab. The vimentin-positive cells in the mouse primary lung fibroblast preps were further quantitated by counting 10 random views of each coverslip (a total of 5 coverslips were counted; cells on each coverslip came from a separate mouse; a total of 5 mice were used). Ninety-two % of the cells in the mouse lung fibroblast preparation were labeled positive with the antivimentin, 3% with the anticytokeratin, and 5% with the anti-CD14 Abs. The Journal of Immunology 1297 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 chla- mydial organism replication (Fig. 4A). The neutralization Ab treat- ment significantly increased the IFUs in both lung and spleen or- gans 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 sig- nificant difference in the number of live organisms (IFUs) recov- ered from the mouse organs between the Ab and control IgG treat- FIGURE 7. Effect of IL-17 on other cytokine production during C. ment groups on day 2 (Fig. 4Ab). This might be due to the fact that muridarum infection in MyD88 KO mice. Two groups of MyD88KO (n ϭ a period of 2 days was not long enough for most C. muridarum 7 for each) mice were intranasally infected with live C. muridarum organ- organisms to complete their growth cycle to generate mature EBs isms at 2000 IFUs/mouse. On day 2 after infection, mice were sacrificed Downloaded from in mice. Finally, as expected, the neutralization Ab treatment was for measuring cytokines in BALFs. One group was treated with IL-17 effective in blocking IL-17 in mouse airway because IL-17 was before and after chlamydial infection as described in Materials and Meth- detected only in lung homogenates of mice treated with the control ods. The levels of IL-6 (a) and MIP-2 (b) were measured from the BALFs, Ϯ IgG but the neutralization Ab (Fig. 4B). and the results were expressed as means SD. A two-tailed Student t test was used to compare between the MyD88 mice with or without IL-17 The Chlamydia-induced early IL-17 production is likely treatment. IL-17 treatment significantly increased both IL-6 and MIP-2. http://www.jimmunol.org/ dependent on MyD88-mediated signaling pathways We next compared the Chlamydia-induced early IL-17 in the air- darum or L2 organisms in a mouse L929 cell line (Fig. 6A)or way between MyD88 KO and Wt mice (Fig. 5). Both the IL-17 mouse primary lung fibroblast cells (Fig. 6B), whereas IFN-␥ sig- that was secreted into the airway lumen (detectable in the BALFs) nificantly inhibited L2 organism growth (Fig. 6, Aa and Ba). 92% and the IL-17 that remained in the lung tissues (detectable in the of the cells in the mouse primary lung fibroblast cell preparation homogenates made from the lung tissues after collecting BALF) were confirmed to be fibroblast cells (Fig. 6C). The lack of inhi- were measured separately. On day 2 after infection, Wt mice se- bition of C. muridarum organisms by IFN-␥ in mouse cells is creted much IL-17 into the BALFs although a significant amount thought to be due to the ability of C. muridarum to evade IFN-␥- by guest on September 25, 2021 still remained in the lung homogenates (Fig. 5Aa). However, no induced antichlamydial mechanisms possibly via the expression of significant IL-17 was detected in either BALF or homogenate sam- a large clostridial toxin homolog (44–46). The L2 organisms do ples harvested from the MyD88 KO mice that were similarly in- not carry any toxin homolog gene (47, 48). Nevertheless, the lack fected with C. muridarum. The TRIF KO mice displayed a similar of inhibition of either C. muridarum or L2 organisms by IL-17 IL-17 profile as the Wt mice. These observations have demon- suggests that IL-17 may use a unique mechanism to suppress chla- strated that MyD88-mediated signaling is required for the chla- mydial growth in mice. Surprisingly, we found that both IL-17 and mydial 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 neutral- izing 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. Further- FIGURE 8. Effect of IL-17 on inflammatory cell infiltration during C. more, like the neutralization Ab-treated Wt mice shown in Fig. 3, muridarum infection in MyD88 KO mice. Groups of mice (n ϭ 8 for each the MyD88 but not TRIF KO mice progressively died upon MoPn of the 2 Wt groups, 7 for each of the 4 MyD88 KO groups) were infected infection (Fig. 5C). with live chlamydial organisms and treated with or without IL-17 as de- scribed in the legend to Fig. 7 were sacrificed on days 2 (a)and7(b) after IL-17 can amplify inflammatory responses by enhancing other infection. The BALFs were collected for counting neutrophils (␯, f), mac- cytokine production and inducing neutrophil infiltration during rophages (Mø, u), and lymphocytes (Ly, Ⅺ). The relative amounts of chlamydial infection neutrophils, macrophages, and lymphocytes in each BALF sample were calculated as percent of total cells, and the final results were expressed as To understand the mechanisms of the IL-17 antichlamydial activ- means Ϯ SD (y-axis). A Chitest was used to compare the difference in ity, we evaluated the effect of IL-17 on chlamydial growth in cell percent of neutrophils between MyD88 KO mice with or without IL-17 cultures (Fig. 6). IL-17 did not affect the growth of either C. muri- treatment (p Ͻ 0.01). 1298 IL-17 IN CHLAMYDIAL AIRWAY INFECTION

IFN-␥ significantly exacerbated IL-6 (Fig. 6Cb) and MIP-2 (Fig. source of IL-17 in Mycobacterium tuberculosis infection in mice 6Cc) production by the Chlamydia-infected cells. The ability of (56). However, due to the enormous redundancy in IL-17-produc- IL-17 to enhance cytokine production in response to chlamydial ing cells, the main cellular populations responsible for the early infection was further confirmed in MyD88 KO mice (Fig. 7). IL-17 production during infection with other bacteria including K. MyD88 KO mice produce minimal levels of inflammatory cyto- pneumoniae and M. pneumoniae have not been identified. Regard- kines after infection (49). We found that delivery of exogenous less of the exact cellular basis of the early IL-17 production during IL-17 into mice significantly increased the production of IL-6 and chlamydial infection, the current study has provided convincing MIP-2 upon chlamydial infection compared with the MyD88 KO evidence that the Chlamydia-induced IL-17 during innate immu- mice with mock treatment, confirming that IL-17 and chlamydial nity is dependent on a MyD88-mediated signaling pathway, which infection can synergistically activate inflammatory cytokine genes. is important to understand how the innate immunity cells activate Furthermore, IL-17 also enhanced the infiltration of neutrophils in their IL-17 gene during chlamydial infection. IL-23 is required for the MyD88 KO mice (Fig. 8). Compared with the Wt mice, CD4ϩ Th17 memory cells to produce IL-17 (62), and IL-23 can MyD88 KO mice displayed a much lower level of neutrophil in- also induce IL-17 gene activation in TCR␥␦ T cells via a Tyk2- filtration on day 2 after chlamydial infection. Treatment of the mediated signaling pathway (54). However, it is not clear whether MyD88 mice with exogenous IL-17 significantly enhanced the the early IL-17 production during chlamydial infection is a direct neutrophil population in the BALFs in response to chlamydial in- result of chlamydial invasion of the IL-17-producing cells or in- fection. Thus, IL-17 may exert its antichlamydial effect by enhanc- directly induced by IL-23 secreted from APCs such as dendritic ing inflammatory cytokine production and neutrophil infiltration cells, macrophages, and epithelial cells that are infected with Chla- during the early stage of infection. Downloaded from mydia. These APCs produce IL-23 in response to microbial infec- Discussion tion via their innate immunity receptor-triggered signaling path- IL-17 has been shown to play significant roles in both inflam- ways including the MyD88 pathways (49, 63). It has been shown matory pathologies and host defense against many microbial that the early IL-17 production during K. pneumoniae infection infections. However, its contribution to infection by obligate depends on TLR-mediated induction of IL-23 (64). Chlamydial

intracellular bacterial pathogens such as C. trachomatis was organisms naturally invade epithelial cells but not T cells. Further- http://www.jimmunol.org/ still unknown. In the current study, we found that IL-17 was more, Chlamydia has been shown to activate innate immunity re- induced early in the lung upon chlamydial infection. The early ceptor-mediated signaling pathways in epithelial cells (65, 66). IL-17 production was dependent on MyD88 signaling pathway Thus, it is possible that C. muridarum infection activates the IL- and significantly contributed to the resolution of chlamydial 17-producing cells during the innate immunity by inducing IL-23 infection in the airway. via a MyD88-dependent pathway in the infected-epithelial cells. The early IL-17 production in the lung peaked at 48 h after an This hypothesis is supported by our current finding that airway intranasal infection with C. muridarum represents an innate im- infection with chlamydial organisms induced an early production mune mechanism with which mice might deal with an acute in- of both IL-23 and IL-17. Nevertheless, the precise cellular basis fection. This conclusion is consistent with the general concept that and molecular mechanisms of the Chlamydia-induced early IL-17 by guest on September 25, 2021 adaptive immunity can only take place 4 days after Ag exposure, production require further investigations. which is supported by the observations that the chlamydial Ag- IL-17 is a proinflammatory cytokine with a pleiotropic spectrum specific IFN-␥, Ab, and IL-17 production by lymphocytes was first of biological activity, and its receptors are broadly distributed in detected on days 4, 8, and 20 after the C. muridarum infection, many different types of cells and tissues in both humans and mice respectively (Fig. 1B). Indeed, the early IL-17 production in the (62). The role of IL-17 in airway infection and inflammation has airway was also induced by and contributed to the protection been well recognized (34, 67–71). We found that although IL-17 against other respiratory bacterial pathogens, including Myco- did not directly alter the chlamydial intracellular growth in cell plasma pneumoniae (50), Klebsiella pneumoniae (26, 51), and My- cultures, it synergistically enhanced the production of the inflam- cobacterium bovis (52, 53). These pathogens all triggered a tran- matory cytokine IL-6 and chemokine MIP-2 in L929 cell line and sient IL-17 production, and the peak time appeared to correlate isolated primary lung fibroblasts as well as in the airway of with the speed of the pathogen replication with the IL-17 induced MyD88 KO mice (Figs. 6 and 7) and promoted neutrophil infil- by M. pneumoniae on day 1, K. pneumoniae on day 2, and My- tration in mouse airway upon chlamydial infection (Fig. 8). These cobacterium bovis on day 3, respectively. These previous obser- findings are consistent with the previous observations that IL-17 vations support our current finding that the early IL-17 production can stimulate human airway smooth muscle cells to secrete MIP-2 required chlamydial replication and biosynthesis (Fig. 2). Because (71), mouse neutrophils to secrete matrix metalloproteinase-9 (69), the early IL-17 production significantly contributes to the protec- and human bronchial epithelial cells and keratinocytes to secrete tion against different bacterial pathogen infections, it is important ␤ to identify its molecular and cellular basis. We found that MyD88 -defensins (72). IL-17 is known for its ability to promote gener- but not TRIF was required for the early IL-17 production, sug- ation, chemotaxis, and activation of neutrophils (73, 74). Neutro- gesting that innate immunity receptor-mediated signaling path- phils seem to play a critical role in controlling chlamydial infection ways may be sufficient for activating IL-17 gene. Indeed, besides during the early stage of infection (10), suggesting that IL-17 may the traditional CD4ϩ T cells that are normally involved in adaptive exert its antichlamydial activity via the enhanced neutrophil func- immunity, TCR␥␦ T cells (54–56), NKT-like cells (57, 58), tion. However, other studies have shown that neutrophils may not NK1.1Ϫ iNKT cells (59), neutrophils (60), and Paneth cells (61), be important in the resolution of chlamydial airway infection (75). all of which can participate in the innate immunity, also produce Obviously, further studies are required for understanding the pre- IL-17. These innate immunity cells may form the cellular basis for cise mechanism of the IL-17 antichlamydial activity. Because the the early IL-17 production during chlamydial infection. It has been early IL-17 production by innate immunity cells can affect the shown that NK1.1Ϫ and ␣-galactpsylserine-positive invariant phenotypes of adaptive immunity in the lung (34), future studies NKT cell population is critical for airway neutrophilia in response should also investigate the effect of the early IL-17 production on to endotoxin (59), whereas TCR␥␦ T cells are the predominant the quality of adaptive immunity induced by chlamydial infection. The Journal of Immunology 1299

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