Haematopoietic innate interleukin 17A production drives immunopathology in female mouse genital Chlamydia muridarum infection

Chlamydia trachomatis infection is the leading cause of bacterial urogenital infection and has been demonstrated to drive inflammation and scarring of the reproductive tract. Recent studies have identified key triggers of proinflammatory adaptive immune responses driven by innate leukocytes and epithelia driving immunopathology. Utilizing chimeric mouse models, we investigated the definitive source and role of IL17 and IL17 signalling receptors during early Chlamydia muridarum infection of the female urogenital tract. Bone marrow transplants from wild‐type (WT) and IL17A−/− mice to recipients demonstrated equivocal infection kinetics in the reproductive tract, but interestingly, adoptive transfer of IL17A−/− immune cells to WT recipients resulted in no infertility, suggesting a haematopoietic (as opposed to tissue) source of IL17 driving immunopathology. To further delineate the role of IL17 in immunopathology, we infected WT and IL17 receptor A (IL17RA)−/− female mice and observed a significant reduction in immunopathology in IL17RA−/− mice. WT bone marrow transplants to IL17RA−/− recipient mice prevented hydrosalpinx, suggesting signalling through IL17RA drives immunopathology. Furthermore, early chemical inhibition of IL17 signalling significantly reduced hydrosalpinx, suggesting IL17 acts as an innate driver of disease. Early during the infection, IL17 was produced by γδ T cells in the cervico‐vagina, but more importantly, by neutrophils at the site of infertility in the oviducts. Taken together, these data suggest innate production of IL17 by haematopoietic leukocytes drives immunopathology in the epithelia during early C. muridarum infection of the female reproductive tract.


| INTRODUCTION
Chlamydia trachomatis is one of the most common sexually transmitted bacterial infections, with over 129 million cases reported annually. 14][5] Chlamydial infection of the female genital tract induces innate immune responses, including the production of IL1α and TNFα, particularly from fallopian tube/oviduct epithelia and IL8/MIP-2. 6,7In mouse studies, these responses then trigger an influx of neutrophils that contribute to the development of hydrosalpinx (a marker for infertility in mice). 8,9It has been suggested that in women these inflammatory responses begin with and are sustained by active infection of non-immune epithelial cells, 10 possibly contributing to the sustained tissue damage that occurs.
4][5] Using animal models, in recent years Th17 immunity, including the production of IL17, has been found to contribute to the generation of this Th1 immunity. 11There are six structurally and functionally different IL17 family members, IL17A-IL17F, each with their own receptor or receptor complex (IL17RA-IL17RE), 12,13 and they all have distinct roles in inflammation, immune responses and immune protection. 13IL17A and its receptor, IL17RA, are the best-characterized of the family.
Our own previous studies have demonstrated that IL17A contributes to Chlamydia muridarum pathogenesis, with IL17A −/− mice exhibiting reduced infection levels, shorter infection time and no hydrosalpinx development, 14 but the finer details, such as the cellular source of IL17A, are unknown.During C. muridarum infections, the Th17 family cytokines and receptors are increased, indicating an increase in IL17-mediated signalling, 15 not only in the development of immunopathology but also in the clearance of infection.IL17 is also produced by CD4 + T cells isolated from cervical washings of C. trachomatispositive women, further supporting a possible role of IL17 in chlamydial clearance. 16In addition, responses to infections such as Mycobacterium tuberculosis, 17 Listeria monocytogenes 18 and E. coli 19 IL17A have been found to be not only produced by Th17 cells but also from neutrophils and γδ TCR cells. 17,20During intracellular infections, including chlamydial respiratory infections, γδ TCR cells are a major contributor to IL17 production. 21e exact role of IL17 in chlamydial clearance and pathogenesis is not completely understood.Here we sought to determine the cellular source of IL17A and IL17RA throughout a C. muridarum infection at the site of initial infection (cervicovaginal tissue) and the site of pathological development (oviduct).To further extend our previous studies, we examined how IL17A and IL17RA from haematopoietic cells contribute to the development of upper reproductive tract pathology throughout infection through the use of chimeric IL17A −/− and IL17RA −/− mice.We also confirmed IL17A/RA's contribution to human fallopian tube immune responses to C. trachomatis in vitro.

| Animals
Female Balb/c wild-type (WT) mice were purchased from the Animal Resources Centre (Perth, WA, Australia) at 6-8 weeks of age.IL17A −/− (Balb/c background) and IL17RA −/− (C57BL/6j background) mice were obtained from Prof. Geoff Hill (QIMR Berghofer Medical Research Institute, Queensland, Australia), were bred and housed in QIMR-B animal facilities and were used at 6-8 weeks of age.All experiments were approved by the Queensland University of Technology and QIMR Berghofer animal ethics committees (QUT Ethics: 1500000029 and 1800000018; QIMR-B Ethics: A1712-619M) and carried out in strict accordance with all recommendations.All mice were appropriately housed in ventilated cages and provided with food and water ad libitum.Mice that received irradiation had neomycin included in their water for 14 days to prevent infections while immune suppressed.

| Mouse-chimera generation
Bone marrow was harvested as per JOVE protocol 22 and reconstituted in sterile PBS.Female Balb/c mice were irradiated with 700 cGy and received retro-orbital transfer of either 10 7 WT or IL17 −/− bone marrow cells per mouse under isoflurane anaesthesia.In this situation, gamma irradiation is used to deplete the bone marrow niche of host progenitor cells.This allows for the transfer and engraftment of the donor bone marrow stem cells. 23Mice were divided into four groups: WT receiving WT bone marrow, WT receiving IL17 −/− bone marrow, IL17 −/− receiving IL17 −/− bone marrow and IL17 −/− receiving WT bone marrow, n = 8/group.Female WT or IL17RA−/− C57BL/6 bone marrow transfers were performed as for Balb/c experiments, but with recipient mice being irradiated with 100 cGy prior to retro-orbital transfer of donor BMT cells.

| Infection
The Chlamydia muridarum Weiss strain was grown and purified as previously described. 24Mice received 2.5 mg of saline-suspended medroxyprogesterone acetate (Depo-Provera cat: LYP_DEPO40; Lyppard, QLD, Australia) one week prior to infection to synchronize their estrous cycles.Animals were infected with 5 × 10 4 inclusion-forming units (IFUs) as previously described 24 and control animals received a sham infection with the equivalent volume of sucrose-phosphate-glutamine buffer (SPG).

| Infection clearance and pathology monitoring
Vaginal swabs were collected from mice every 3 days and cultured on McCoy B cells to determine the level of recoverable C. muridarum as previously described. 24Oviduct pathology (hydrosalpinx) was measured at 35 days postinfection as previously described 14,[24][25][26] and involved measuring and recording the diameter of each oviduct with representative images previously published. 15 2.5 | Anti-MOMP IgG ELISA An enzyme-linked immunosorbent assay (ELISA) was used to measure the titre of MOMP-specific IgG in mouse serum. Trminal cardiac bleeds were collected at day 35 post-infection, clotted and centrifuged to separate clot and serum.Serum was used for ELISA as previously described.27

| Reproductive tissue digestion and cell isolation
At the indicated time points post-infection, animals were euthanized and reproductive tracts were collected (n = 10/group).Each region (cervix/vagina, uterine horns and oviducts) was dissected and placed into separate tubes for tissue digestion.Tissue digestion media consisted of RPMI (Life Technologies, North Ryde, Australia) containing 5% foetal bovine serum (FBS, cat: 26140079), 50 μg/mL gentamicin, 4 mM L-glutamine (Thermo Fisher Scientific, cat: 15750060, A2916801 respectively), 500 U/ mL collagenase A and 0.057 KU/μL DNase I (Sigma-Aldrich, North Ryde, Australia.Cat: COLLA-RO, D4263 respectively).Tissues were cut into 1 mm 2 pieces and placed in 15-mL tubes containing digestion media.Tubes were incubated at 37°C, 200 rpm for 1.5 h to allow cells to dissociate.Cells were isolated into single-cell suspensions as previously described.).Cells were incubated with antibodies for 20 min in the dark, washed and fixed in 4% paraformaldehyde to preserve the RNA.Samples were stored at 4°C in the dark until flow cytometry.Samples were analysed and sorted using FACSAria III (BD Biosciences) with cells being collected straight into 300 μL of RLT buffer (Qiagen, Venlo, Netherlands).Cells were vortexed, and then stored at −80°C until processing.

| RNA extraction and gene expression
Cervical/vaginal cells and oviduct cells in RLT buffer were defrosted, vortexed and had 590 μL ultrapure H 2 O (Life Technologies) and 10 μL Proteinase K (200 μg/mL final concentration, cat: RPROTKSOL-RO) added.The samples were then vortexed and incubated at 56°C for 15 min, mixing by inversion every 3min.To release the formalin cross-linking of the mRNA, incubation at 85°C for 15 min was performed, mixing by inversion every 3 min again.Samples were cooled on ice for 3 min.RNA was extracted using RNA extraction kits, including the genomic DNA removal step according to the manufacturer's instructions (Analytik Jena, Jena, Germany, cat: 845-KS-20800250).Samples were eluted into 25 μL of ultrapure H 2 O and quantified using the Qubit RNA HS Assay Kit (Qubit 3.0, Life Technologies, cat: Q32852) and stored at −80°C until use.Reverse transcription was performed on 103.1 pg of RNA for each sample using iScript reverse transcription supermix for RT-qPCR (Bio-Rad, Gladesville, Australia, cat: 1708840) according to the manufacturer's instructions to generate cDNA.
To quantify the level of expression of il17a and il17ra the Bio-Rad digital droplet automated droplet generator (AutoDG) PCR system was utilized.All procedures were performed in strict accordance with the manufacturer's instructions using the 4-plex probe protocol.Briefly, a master mix was made (10 μL 2× ddPCR Supermix for Probes [No dUTP], cat: 1863024, 1 or 0.5 μL 20× target primer/probe [Life Technologies, Waltham, USA; Table 1] and 5 μL ultrapure H 2 O per sample) and 15 μL was aliquoted into each well of a 96-well Eppendorf plate type.Each sample had 2 μL of cDNA added to the appropriate well, with no template controls included in each plate run.Plates were sealed, centrifuged for 2 min at 500 g and placed in the Bio-Rad AutoDG for droplet generation using Auto DG Oil for Probes (Bio-Rad, cat: 1864110).Droplet samples then underwent PCR using the following cycling conditions: (i) 95°C for 10 min; (ii) 94°C for 30 s; (iii) 60°C for 1 min; 50× cycles of steps (ii-iii); and (iv) 98°C for 10 min.Samples were analysed using the Bio-Rad QX200 Droplet Reader and QuantaSoft™ Analysis Pro (version 1.0.596).

| IL17 signalling pathway inhibition
Female 6-week-old C57BL/6 mice (n = 10/group) were obtained from the Animal Resource Centre (Perth, Australia) and infected as above.The retinoic acid receptor-related orphan receptor (ROR) α and RORγ inhibitor, SR1001 (Sigma, MO, USA cat: SML0322), was dissolved in PBS/10% dimethylsulfoxide/10% Tween 80 solution to a final concentration of 5 mg/mL as previously described. 29Mice were injected intraperitoneally twice a day with vehicle control or SR1001 solution (25 μg/g body weight of mice, as described previously 30 ) between day −1 and day 35 post-infection.One group received vehicle control between days −1 and 5 and then SR1001 thereafter.Mice were sacrificed 35 days post-infection for analysis of gross pathology.
Cells were infected with C. trachomatis serovar D (ATCC #VR885) at MOI 0.1 in multiple experiments.For gene transcript detection via PCR, cell monolayers were scraped into microfuge tubes, and RNA was extracted using the QIAGEN RNeasy RNA extraction kit (cat: 74004).cDNA was generated using the Bio-Rad iScript Reverse Transcription Supermix (cat:1708840) as per the manufacturer's instructions.Conventional PCR was performed as per the conditions and cycling conditions in Table 2. Gels (2.5% agarose) were run for 30 min at 120 V and imaged using a BioRad ChemiDoc system.For microscopy, cells were fixed with 100% methanol for 10 min and washed with PBS before C. trachomatis major outer membrane protein (MOMP) was stained using immunocytochemistry techniques as previously described, 32 and counterstained with DAPI (cell nuclei, 1:40,000 v/v in PBS) and AF594-phalloidin (cell cytoskeleton, 1:250 v/v in PBS, Thermo Fisher Scientific, cat: A12381).

| Statistics
GraphPad Prism version 9 was used for all statistical analyses.For chlamydial clearance data, the area under the curve was calculated, followed by a one-way ANOVA to assess the difference in total burden over the time course.The remaining data were normality-tested for an appropriate choice of parametric or non-parametric analysis.A one-way ANOVA was used for ELISA comparisons.Unpaired two-tailed t-tests were used for oviduct diameter comparisons.Two-way ANOVAs using Tukey's multiple comparisons test were used to determine significant differences between the percentages of cell types isolated from the cervix/vaginal and oviduct tissues.

| Haematopoietic IL17A is involved in the chlamydial-induced pathology of the upper female reproductive tract
To further dissect our previous work on the involvement of IL17A 14,15 in chlamydial-induced reproductive tract pathology and infertility, we developed IL17A −/− chimeric mice (Figure 1A).WT and IL17A −/− Balb/c mice were irradiated and had 10 7 bone marrow cells from IL17 −/− or WT mice, respectively, adoptively transferred T A B L E 1 TaqMan® gene expression assay primer/probes.

Gene Assay ID Concentration
Reporter probe 1A).After the recovery period, mice were infected with C. muridarum and the infection time course and pathology development were measured.In WT mice that were reconstituted with IL17A −/− bone marrow (17A −/− /WT), there was no incidence of hydrosalpinx development, with no swelling of the oviducts recorded (Figure 1B), highlighting the role of haematopoietic IL17A in pathology.In comparison, IL17A −/− mice that were reconstituted with WT bone marrow (WT/17A −/− ) had measurable levels of hydrosalpinx (Figure 1B), indicating an alternate mechanism of pathology induction not related to IL17.Clearance levels of C. muridarum measured from vaginal swabs demonstrated that there were no significant differences between any of the groups in the amount of C. muridarum detected or the time to clearance (Figure 1C).The levels of systemic anti-MOMP IgG were also measured (Figure 1D), demonstrating that mice reconstituted with IL17A −/− bone marrow cells (WT and IL17A −/− ) produced higher levels of anti-MOMP IgG.
The WT mice that received IL17A −/− cells produced significantly greater (p < 0.01) levels of anti-MOMP IgG than the IL17A −/− mice that received WT cells.

| Chlamydial-induced pathology is reduced when the IL17 signalling pathway is inhibited
To determine if the IL17 signalling pathway is involved in chlamydial-induced pathology, we first infected C57BL/6 IL17RA −/− and WT mice.The initial levels of chlamydial recovery from vaginal swabs were lower in the T A B L E 2 Human primers and PCR conditions.
IL17RA −/− mice (Figure 2A), but then remained higher than the WT mice from 9 days post-infection.Overall, the levels of C. muridarum recovery and duration of infection were not significantly different between the WT and IL17RA −/− groups.The levels of hydrosalpinx were also measured, which revealed a significant decrease in the oviduct diameter and a decrease in the overall incidence in the IL17RA mice (Figure 2B).To confirm whether haemopoietic or non-haemopoietic cells were involved in the IL17A signalling, we generated IL17RA chimera mice, as illustrated in Figure 2C.The vaginal infection of these mice revealed no significant differences in the levels or duration of infection (Figure 2D).Both WT and IL17RA −/− mice that were reconstituted with IL17RA −/− bone marrow cells displayed a decreased incidence of hydrosalpinx (Figure 2E).

|
The cellular source of IL17A and

IL17RA at the initial site of infection and the site of pathology development throughout infection
To determine the cellular source of IL17A and IL17RA in the reproductive tract cervix/vaginal and oviduct, tissues were removed from mice at 2, 6 and 35 days p.i., digested and stained for several cell types (NK cells, neutrophils, macrophages, epithelial cells, CD4/8, γδTCR cells and NKT cells).These cells were sorted (Figure 3) and collected via flow cytometry (Figure 4), and the expression of il17a and il17ra was determined using ddPCR (Figure 5). Figure 4 shows the temporal changes in each cell type during the acute phase of infection (days 2 and 6 p.i.) and F I G U R E 1 Haematopoietic IL17A is involved in hydrosalpinx formation following a chlamydial infection.Bone marrow (BM) cells from either wild-type (WT) or IL17A knock-out (17A −/− ) mice were adoptively transferred into irradiated mice (A) and mice were infected.Hydrosalpinx measurements (mm) recorded at day 35 post infection (B) indicated that pathology is driven by haematopoietic sources of IL17A, with WT animals who received 17A −/− BM displaying no hydrosalpinx.There was no significant difference in the chlamydial burden or time to clearance in any of the groups (C).The level of systemic anti-major outer membrane protein (MOMP) IgG was measured via ELISA and demonstrated that WT mice who received 17A −/− BM cells had significantly higher levels than 17A −/− mice that received WT BM (D).Graphs were generated in GraphPad Prism (v7).Data are mean ± SD.Un-paired two-tailed t-tests were used for oviduct diameter significance testing.Area under the curve, followed by one-way ANOVA was used to test for significance for the clearance data and oneway ANOVA was used for ELISA comparisons.If data are not significant, there are no significant markers included; only the data that are significant are indicated.N = 8 mice per group, **p < 0.01.
phase when pathology has developed (day 35 p.i.) at the site of initial infection (cervix/vagina) and site of pathology development (oviduct).Significant increases in cervix/vaginal NK cells, neutrophils and macrophages were found during the initial (day 2) and acute (day 6) stages of infection.By day 35 p.i., these cell numbers were similar to those of uninfected controls.While there were increases in CD4, CD8 and γδTCR cells at day 6 p.i., these were not significantly different from the uninfected controls.In the oviduct tissues, significant increases were seen in the numbers of CD4, CD8 and γδTCR cells from day 6 p.i. when compared to cell numbers isolated from non-infected mice.These increases were maintained for CD4 and CD8 cells.There were increases in NK cells, neutrophils and macrophages at day 6 p.i. in infected mice, but these were not significantly different from the noninfected controls.
To determine the cellular source of IL17A and IL17RA ddPCR was used on each cell type from the cervix/vaginal and oviduct tissues.In the cervix/vaginal tissue cells, il17a expression was higher in macrophages, epithelial cells and CD8 T cells from infected mice at day 2 when compared to uninfected controls (Figure 5).On day 6, il17a expression was higher in neutrophils, epithelial cells and CD4 T cells.By 35 days p.i. il17a expression was lower than that seen at earlier timepoints but was still higher than uninfected controls in γδTCR, CD4, CD8 cells and neutrophils.The expression of il17ra in cells isolated from the cervix/ vaginal tissue was only detected in NK cells from infected mice early in infection.However, at 35 days p.i., il17ra was elevated in macrophages, CD4, CD8 and γδTCR cells from infected animals (Figure 5).
In cells isolated from the oviducts, there was increased il17a at day 2 p.i. in CD8 and NK cells (Figure 5).At day 6 p.i., il17a expression was elevated in γδTCR cells and macrophages, and in neutrophils at day 35 p.i., compared to non-infected controls.The overall expression of il17ra in cells isolated from the oviducts was lower; however, increased expression was observed in CD4 and CD8 cells at 6 days p.i. and epithelial, neutrophil and NK cells at 35 days p.i. when compared to non-infected controls.This further confirms that IL17 production from haematopoietic immune cells is a likely source contributing to chlamydial pathogenesis and infertility in mice.

F I G U R E 2
Chlamydial-induced hydrosalpinx is reduced when the IL17A signalling pathway is restricted.IL17 receptor A knock out (IL17RA −/− ) and wild-type (WT) mice received a vaginal chlamydial infection.Clearance was monitored via swab culture (A) and hydrosalpinx was recorded (B) at 35 days post infection.To determine the origin of cells involved in chlamydial induced pathology IL17RA −/− chimera mice were generated and infected (C).Clearance was monitored (D) and hydrosalpinx was recorded at 35 days post infection (E).N = 10 mice/group.Graphs were generated in GraphPad Prism (v7).Data are mean ± SD.Area under the curve, followed by one-way ANOVA, was used to test for significance for the clearance data.If data are not significant, there are no significant markers included; only the data that are significant are indicated.Oviduct diameter comparisons were made with unpaired, two-tailed t-test, *p < 0.05.

| Inhibition of the IL17A signalling pathway decreases chlamydial-induced pathology and immune responses
To confirm that the IL17A signalling pathway was responsible for C. muridarum-induced pathology, mice were given a RORαγ inhibitor to inhibit the IL17A pathway either prior to infection or from day 6 p.i.When delivered prior to infection, there was a decrease in the severity (Figure 6A) and incidence of oviduct inflammation (Figure 6B), compared to both the vehicle control and later delivery.
To extend on this, we examined the role of IL17A/RA in chlamydial-induced immune responses and pathology using immortalized human fallopian tube epithelial F I G U R E 3 Gating strategy used to identify and sort individual cell types from reproductive tract tissues.Reproductive tract tissues were digested into a single-cell suspension.Cells were stained with live/dead stain and a selection of T cell markers (A) or innate immune cell markers (B).T cells were identified using CD3 and sorted/collected based on CD4, CD8 and γδ TCR expression.Innate immune cells were sorted/collected based on CD49b (NK cells), Ly6G (neutrophils), F4/80 (macrophages and CD362 (epithelial)) marker expression.cells (FTEs), which were a kind gift from A/Prof.Ronny Drapkin (University of Pennsylvania).These cells were grown using an air-liquid interface, allowing cells to differentiate into secretory and ciliated cell types.We confirmed that these cells could be infected with C. trachomatis serovar D (Figure 7A) and characterized the cells to confirm they expressed cytokines and cytokine receptors via PCR (Figure 7B).The FTEs constitutively expressed il1α, il1β, il6, il8, il17c, il17ra, il17rb, il17rc and il23a regardless of chlamydial infection.In contrast, il1r and il17e expression was increased at 48 h p.i., and il17a expression increased at 24 and 48 h p.i. Importantly, this highlights that these cells are responsive to C. trachomatis, express il17a in response to infection and constitutively express il17ra, enabling this signalling pathway to be active in response to chlamydial infection.

| DISCUSSION
Chlamydia infection of the female reproductive tract can cause severe pathological damage, resulting in tubal F I G U R E 4 Percentage of cell types sorted and collected from cervix/vaginal or oviduct tissue during chlamydial infection.At days 2 (early infection), 6 (peak infection) and 35 (pathology development stage) cervical/vaginal and oviduct tissues were collected from noninfected (NI) and infected (INF) mice.Tissues were digested; single-cell suspensions were stained for innate and adaptive cell types; and each cell type was analysed and collected into separate tubes using the BD FACS ARIA II.Collected cells were stored in Qiagen RLT buffer until RNA extraction and IL17 gene expression analysis.Data are mean ± SD.N = 10 mice/group and cervical tissues were pooled to 2 tissues/sample and oviduct tissues pooled to 3-4 tissues per sample to ensure enough cells could be analysed and extracted.Data are mean ± SD.A two-way ANOVA using Tukey's multiple comparisons test was used.If the data are not significant, there are no significant markers included; only the data that are significant are indicated.*p < 0.05, **p < 0.01.occlusion, pelvic inflammatory disease and infertility.The intricate details of chlamydial pathogenesis are still not fully understood in the female reproductive tract, and for a successful vaccine to be developed, the details of exactly what immune responses are involved in both protection and pathogenesis need to be known.
IL17 appears to have conflicting effects on chlamydial infections, and this may likely be related to the cellular source of the cytokine and subsequent signalling of additional immune responses.A recent study using mice has demonstrated that there are functionally distinct Th17 cell subsets involved in autoimmunity (Th-1like IFNγ + /IL17 + Th17 cells) and clearance of infection (IL17 + /IFNγ − Th17 cells), 33 and both these cell types are found during murine chlamydial genital infection, 34,35 suggesting roles in both clearance and the development of pathology.Our previous studies have shown that both the duration of a female genital C. muridarum infection and associated pathology due to neutrophil recruitment and matrix metalloproteinase activity were reduced in IL17A −/− mice compared to WT mice. 14Here, we saw no differences in the rate of clearance in any of the mice that had received WT or IL17A −/− -derived bone marrow, indicating that haematopoietic cells expressing IL17A are not responsible for clearance of infection (Figure 1).However, haematopoietic cells expressing IL17A do contribute to the development of oviduct pathology.In respiratory models of chlamydial infection in IL17A −/− mice, neutrophil infiltration in the lungs was impaired 3 days p.i., along with reduced neutrophil-related chemokines MIP-2, IL6 and KC. 36This suggests that IL17A enhances neutrophil infiltration by increasing cytokine and chemokine expression early in infection, 36 likely contributing to the development of pathogenesis through IL17A signalling.Here, we saw an increase in neutrophils across the first week of infection, which coincided with an increase in il17a expression from these cells at day 6 p.i. (Figures 4 and 5).Other mouse studies using C. trachomatis for vaginal infection also demonstrated that depletion of IL17 only marginally impaired clearance rates and that C. trachomatis-specific Th17 immunity promoted immunopathological damage in the reproductive tract. 35he production of IL17A alone is not solely responsible for chlamydial pathogenesis.Here we demonstrated that the IL17A signalling pathway also contributes to hydrosalpinx development (Figure 2).It has been found that the induction of Th1 cells, specifically IFNγ-secreting T cells required for protection against genital C. muridarum infection, following infection of IL17RA −/− mice was significantly reduced compared to WT mice. 11In addition to this, there was a delay in macrophage responses to the site of infection. 11Reduced neutrophil influx into infected tissues has also been found in IL17RA −/− mice, along with decreased cervical IFNγ-producing NK cells during the first week of infection. 11This suggests that not only does IL17A production/signalling inhibit early responses to C. muridarum infection that contribute to subsequent pathology development, but that IL17 plays a major role in anti-chlamydial Th1 development.We also demonstrated that when IL17A is completely removed, there is still some degree of hydrosalpinx development.One possible reason is that, despite the removal of IL17A, the IL17RA signalling pathway is still activated.One study has shown that the adaptor molecule for IL17RA, NF-κB activator 1 (Act1), is able to bind to other molecules such as Src homology 2-containing protein tyrosine phosphatase 2 (SHP2) and induce signalling in the absence of IL17A. 37ere we have demonstrated that IL17A expressed from haematopoietic cells contributes to C. muridarum pathogenesis in the female reproductive tract through the IL17 signalling pathway.
To further characterize the cellular source of IL17A during infection, we examined the expression of il17a and il17ra in key cell types at the site of initial infection (cervix/vaginal tissue) and the site of pathology development (oviducts) early during infection (D2 and 6 p.i.) and at the time when pathology has developed (D35 p.i.).The early production of IL17A by innate immune cells is believed to act on antigen-presenting cells (APCs), such as macrophages.Through the IL17A signalling pathway, including IL17RA, IL17A can induce APC production of proinflammatory cytokines such as IL6, IL1 and TGFβ. 38hese are important factors in the development of pathogenic Th17 cells.This suggests that early production of IL17A from innate immune cells in immune priming could affect the production of antigen-specific Th17 cells and exacerbate pathogenesis 38 during infections such as Chlamydia.Here we saw an increase in il17a expression from macrophages, neutrophils, γδ T cells and epithelial cells from the cervix/vagina or oviducts of infected animals at either 2 or 6 days p.i. (Figure 5).We also saw increased expression of il17ra in macrophages, neutrophils and γδ T cells on day 6 p.i.In a mouse respiratory model of chlamydial infection, the main source of IL17A early in infection was γδ T cells 21 and similarly, during early E. coli and Listeria monocytogenes infection, γδ T cells were the primary source of IL17A. 39,40n these two latter models of infection, γδ T cell-derived IL17A promoted neutrophil accumulation, 39,40 consistent with our previous studies where IL17A −/− mice had reduced neutrophil infiltration. 14During the later stages of infection, after infection is considered cleared from the genital tract of mice, and when pathology has developed, 24 we saw the greatest expression of il17a and il17ra was from CD4 and CD8 T cells in the cervix/vaginal tissue and sustained expression from γδ T cells.By inhibiting the IL17A signalling pathway prior to infection, we demonstrated a significant reduction in oviduct pathology (Figure 6), indicating that early production of IL17A from innate cells is a trigger for sustained reproductive pathology following chlamydial infection.
To confirm the relevance of these findings in women, we infected human fallopian tube cells with C. trachomatis and examined the expression of key cytokines and receptors, and found that these cells produced increased levels of pro-inflammatory cytokines and the key IL17 receptors at 24 and 48 h p.i. (Figure 7).Cervicovaginal secretions of women with C. trachomatis infection have been found to have significantly elevated IL17 levels.These elevated IL17 levels promoted the expression of several inflammatory mediators, including MIP-1α/β. 41Interestingly, CD4 Th17 cell numbers in the female genital tract did not correlate with the soluble IL17 concentrations, suggesting other cellular sources, such as innate and epithelial cells, may be responsible. 41Here we show that fallopian tube cells are responsive to chlamydial infection and that IL17 signalling may also contribute to pathology development in infected women.
Overall, we have shown that the cellular sources of IL17 change throughout the C. muridarum infection course and have demonstrated for the first time that IL17A production and signalling from haematopoietically sourced cells contribute to the development of chlamydial pathogenesis.Preventing IL17A production early in infection may be key to reducing the pathological burden associated with chlamydial infection, supporting vaccine design that targets rapidly responding Th1 cells to prevent infection, not Th17 cells.

F I G U R E 5
Cervix/vaginal and oviduct cellular expression of il17a and il17ra throughout chlamydial infection.Equal concentrations of RNA were reverse transcribed and used in Bio-Rad digital droplet PCR to examine the expression of il17a and il17ra in each of the cell types isolated from the cervix/vaginal and oviduct tissues at days 2, 6 and 35 post-infection (INF: infected; NI: non-infected control).Data are presented as copies of gene/10 pg of RNA.N = 10 mice/group and cervical tissues were pooled to 2 tissues/sample (n = 5 analysed) and oviduct tissues pooled to 3-4 tissues per sample (n = 3 analysed) to ensure enough RNA could be extracted from the relatively low cell numbers.

F I G U R E 6
Chlamydial-induced macroscopic pathology development is reduced in animals that have the IL17A signalling pathway inhibited.Animals received the RORαγ inhibitor, SR001 either prior to or six days post infection, or the vehicle control.At 35 days p.i., hydrosalpinx development was measured (A) and the incidence of unilateral or bilateral hydrosalpinx recorded (B).N = 10 animals/group.Oviduct diameter comparisons were made with unpaired, two-tailed t-test.If data are not significant, there are no significant markers included; only the data that are significant are indicated.***p < 0.001, ****p < 0.0001.
Charles Armitage: Conceptualization, methodology, investigation, visualization and writing -original draft.Emily Bryan: investigation, visualization and

F I G U R E 7
Chlamydial infection and cytokine gene response of primary human fallopian epithelial cells grown in an air-liquid interface (ALI).Secretory and ciliated fallopian tube epithelial cells (FTE) were purified from the fallopian tubes (FTE) of a patient undergoing hysterectomy and grown on 0.4 μm transwells.After incubating for three days, apical media was removed and cells were fed exclusively via the basolateral chamber every second day.(A) Confocal microscopy of primary FTE cells grown in an ALI and infected with Chlamydia trachomatis for 72 h.(B) PCR of cDNA from FTE cells grown in an ALI and infected with C. trachomatis.HeLa cells incubated with 100 ng/mL of LPS.