Oviduct Pathology Research Articles

Protective anti-chlamydial vaccine regimen-induced CD4+ T cell response mediates early inhibition of pathogenic CD8+ T cell response following genital challenge.

We have demonstrated previously that TNF-α-producing CD8+ T cells mediate chlamydial pathogenesis, likely in an antigen (Ag)-specific fashion. Here we hypothesize that inhibition of Ag-specific CD8+ T cell response after immunization and/or challenge would correlate with protection against oviduct pathology induced by a protective vaccine regimen. Intranasal (i.n.) live chlamydial elementary body (EB), intramuscular (i.m.) live EB, or i.n. irrelevant antigen, bovine serum albumin (BSA), immunized animals induced near-total protection, 50% protection, or no protection, respectively against oviduct pathology following i.vag. C. muridarum challenge. In these models, we evaluated Ag-specific CD8+ T cell cytokine response at various time-periods after immunization or challenge. The results show protective efficacy of vaccine regimens correlated with reduction of Ag-specific CD8+ T cell TNF-α responses following i.vag. chlamydial challenge, not after immunization. Depletion of CD4+ T cells abrogated, whereas adoptive transfer of Ag-specific CD4+ T cells induced the significant reduction of Ag-specific CD8+ T cell TNF-α response after chlamydial challenge. In conclusion, protective anti-chlamydial vaccine regimens induce Ag-specific CD4+ T cell response that mediate early inhibition of pathogenic CD8+ T cell response following challenge and may serve as a predictive biomarker of protection against Chlamydia -induced chronic pathologies.

The role of an enzymatically inactive CPAF mutant vaccination in Chlamydia muridarum genital tract infection

Chlamydia trachomatis urogenital tract infection causes pelvic inflammatory disease and infertility, increases the risk of co-infection with HPV and HIV. Chlamydial vaccination is considered the most promising approach to prevent and control its infection. Among various chlamydial vaccine candidates, chlamydial protease-like activity factor (CPAF) have been reported to provide robust protective immunity against genital chlamydial infection in mice with reduced vaginal shedding and oviduct pathology. However, CPAF is a serine protease which has enzymatical activity to degrade a large number of substrates. In order to increase the safety of CPAF vaccine, in this study, we used a mutant CPAF that is deficient in enzymatical activity to determine whether proteolytic activity of CPAF affect its vaccine efficacy. The wild type or mutant CPAF immunization causes a significant lower chlamydial shedding from the vaginal and resolve the infection as early as day 20, compared to day 28 in adjuvant control mice. More important, reduced upper reproductive tract pathology were also observed in these two groups. The mutant or wild type CPAF immunization induced not only robust splenic IFN-γ and serum IgG2a but also sIgA secretion in the vaginal fluids. Furthermore, neutralization of chlamydia with immune sera did not provide protection against oviduct pathology. However, adoptive transfer of CD4+ splenocytes isolated from the mutant or wild type CPAF immunized mice resulted in a significant and comparable reduced oviduct pathology. Our results indicate mutant CPAF vaccination is as same efficacy as wild type, and the protection relies on CD4+ T cells, which will further promote the development of CPAF as clinical chlamydial vaccine.

IL-1α Is Essential for Oviduct Pathology during Genital Chlamydial Infection in Mice.

Chlamydia trachomatis infection of the female genital tract can lead to irreversible fallopian tube scarring. In the mouse model of genital infection using Chlamydia muridarum, IL-1R signaling plays a critical role in oviduct tissue damage. In this study, we investigated the pathologic role of IL-1α, one of the two proinflammatory cytokines that bind to IL-1R. Il1a-/- mice infected with C. muridarum cleared infection at their cervix at the same rate as wild-type (WT) mice, but were significantly protected from end point oviduct damage and fibrosis. The contribution of IL-1α to oviduct pathology was more dramatic than observed in mice deficient for IL-1β. Although chlamydial burden was similar in WT and Il1a-/- oviduct during peak days of infection, levels of IL-1β, IL-6, CSF3, and CXCL2 were reduced in Il1a-/- oviduct lysates. During infection, Il1a-/- oviducts and uterine horns exhibited reduced neutrophil infiltration, and this reduction persisted after the infection resolved. The absence of IL-1α did not compromise CD4 T cell recruitment or function during primary or secondary chlamydial infection. IL-1α is expressed predominantly by luminal cells of the genital tract in response to infection, and low levels of expression persisted after the infection cleared. Ab-mediated depletion of IL-1α in WT mice prevented infection-induced oviduct damage, further supporting a key role for IL-1α in oviduct pathology.

Antigen-Specific CD4 T cells Protect Against Reproductive Pathology by Subverting Chlamydia-specific CD8 T cell Response

Abstract We have demonstrated previously that CD4 T cells are protective whereas CD8 T cells cause reproductive pathology following genital chlamydial infection. In this study, we explored the interactions of these cell types in context of vaccine-mediated protection. We evaluated three immunization regimens: live-chlamydial elementary body intranasal (EB-i.n.), live-EB i.m. (EB-i.m.), and irrelevant antigen bovine serum albumin i.n. (BSA-i.n.) that induced near-total (90%), partial (50%), and no (0%) protection, respectively against oviduct pathology following i.vag. C. muridarum challenge in C57BL6/J animals. We found no differences in Chlamydia-specific IFN-g, TNF-α, or IL-17 response from purified splenic CD8 T cells on days 7, 14, and 21 after immunization. We further evaluated Chlamydia-specific splenic CD8 T cell responses on days 3, 6, 9, and 12 following i.vag. chlamydial challenge. All groups of challenged animals displayed comparable induction of Ag-specific CD8 T cell cytokine responses on day 6 after challenge. As early as day 9 after challenge, Ag-specific IFN-g and TNF-α production from CD8 T cells was significantly reduced in EB-i.n.-immunized mice, and on day 12, both EB-i.n.- and EB-i.m.-immunized animals displayed minimal Ag-specific TNF-a production, compared to enhanced TNF-a production from CD8 T cells in BSA-i.n. immunized animals. Furthermore, adoptive transfer of Chlamydia-specific CD4 T cells at the time of genital infection induced significant reduction in Chlamydia-specific splenic CD8 T cell response on day 12 after challenge. In summary, these results suggest that Ag-specific CD4 T cells subvert a pathogenic Chlamydia-specific CD8 T cell response to protect against reproductive pathology.

Antigen-specific CD8 T cell response following genital chlamydial challenge is a predictive biomarker of vaccine efficacy in protection against reproductive tract pathology

Abstract TNF-α producing antigen (Ag)-specific CD8 T cells induce upper genital tract (UGT) pathology following primary C. muridarum infection; therefore, we hypothesized that a vaccine regimen must subvert Ag-specific CD8 T cell response following immunization, and/or challenge to attenuate Chlamydia-induced UGT pathology. We evaluated three immunization regimens: live-chlamydial elementary body intranasal (EB-i.n.), live-EB i.m. (EB-i. m.), and irrelevant antigen bovine serum albumin i.n. (BSA-i.n.) that induced near-total (90%), partial (50%), and no (0% ) protection, respectively against oviduct pathology following i.vag. C. muridarum challenge in C57BL6/J animals. Live-EB-specific IFN-γ, TNF-α, and IL-17 response from purified splenic CD8 T cells was evaluated on days 7, 14, and 21 after immunization. There was no detectable reduction of Ag-specific CD8 T cell response following immunization with protective vaccine regimens. We evaluated splenic live-EB-specific CD8 T cell responses on days 3, 6, 9, and 12 following i.vag. chlamydial challenge. All groups of challenged animals displayed comparable induction of Ag-specific CD8 T cell cytokine responses on day 6 after challenge. As early as day 9 after challenge, Ag-specific IFN-γ and TNF-α production from CD8 T cells was significantly reduced in EB-i.n.-, compared to the EB-i.m.- or BSA-i.n.-immunized animals. On day 12, both EB-i.n.- and EB-i.m.-immunized animals displayed minimal Ag-specific TNF-α production, compared to enhanced TNF-α production from CD8 T cells in BSA-i.n. immunized animals. These results suggest that TNF-α producing CD8 T cell response could serve as a predictive biomarker of anti-chlamydia vaccine efficacy against reproductive pathology.

OR18: Interleukin‐1 alpha drives oviduct pathology by enhancing immune cell recruitment, cell death, and bacterial ascension during Chlamydia muridarum infection

OR18: Interleukin‐1 alpha drives oviduct pathology by enhancing immune cell recruitment, cell death, and bacterial ascension during Chlamydia muridarum infection

Chlamydia muridarum plasmid induces mouse oviduct pathology by promoting chlamydial survival and ascending infection and triggering host inflammation.

Infection with plasmid-free Chlamydia trachomatis and Chlamydia muridarum fails to induce severe pathology, however, the mechanisms underlying this phenotype are unclear. To elucidate the mechanisms of chlamydial plasmid-mediated pathology in mouse oviducts. BALB/c mice were intravaginally infected with either plasmid-competent or plasmid-free C. muridarum strains. To compare the survival and ascending infection of these strains, vaginal swabs and genital tract tissues were collected and cultured with HeLa cells to monitor the recovery of live organisms. In addition, Chlamydia strains were intrabursally inoculated into the oviducts of mice to assess pathogenicity. Cytokine levels in the vaginal swabs collected from both the plasmid-competent and plasmid-free C. muridarum-infected mice were detected using Bio-Plex Pro Mouse Cytokine, Chemokine, and Growth Factor Assays. The plasmid-competent C. muridarum strain induced hydrosalpinx formation in mouse oviducts following intravaginal inoculation, however, this was not the case for the plasmid-free C. muridarum strain. The lack of hydrosalpinges in response to the plasmid-free C. muridarum strain correlated with its significantly reduced ability to survive and disseminate to the upper genital tract. Furthermore, the plasmid-free C. muridarum failed to induce hydrosalpinx formation in mice, even when the strain was intrabursally injected into oviducts. A comparison of the cytokine levels in mouse vaginal secretions showed that the plasmid-free C. muridarum strain induced less IL-15, LIF, MIP-2, IL-1α, IL-1β, TNF-α, and RANTES. C. muridarum plasmid contributes to oviduct pathology by promoting bacterial survival and ascending infection, and triggering host inflammatory responses.

Gap Junction Mediated Antigen Transport in Epithelium Promotes Amplification of CD8 T Cell Mediated Bystander Pathology Following Chlamydial Infection

Abstract Chlamydia-specific CD8 T cells play a significant role in oviduct pathology, not bacterial clearance. We hypothesized that gap junction mediated antigen transport (GMAT) from infected to uninfected cells could explain the apparent targeting of uninfected epithelial cells by Chlamydia-specific CD8 T cells. Since gap junction protein connexin 43 is highly expressed in oviduct epithelium, we generated Foxj1Cre-Cx43flox mice with a conditional deficiency of CX43 only in ciliated columnar epithelia in oviducts. Foxj1Cre-Cx43flox mice displayed similar chlamydial infection, but reduced splenic Ag-specific CD8 T cell response and oviduct pathology when compared to WT mice, suggesting that CX43-mediates chlamydial pathogenesis. To evaluate the role of GMAT via CX43 in pathogenesis, HeLa S3 cells were engineered to express functional CX43 or a closed channel point-mutant CX43 (T154A). Chlamydial peptides were observed in neighboring uninfected cells at mid-developmental cycle upon infection of HeLa-CX43, not HeLa S3 or HeLa-CX43-T154A cells. Naïve mouse antigen-presenting cells (APC) optimally activated Chlamydia-specific mouse CD8 T cells upon co-culture with Chlamydia-infected HeLa-CX43, when compared to HeLa S3 or HeLa-CX43-T154A cells, demonstrating that GMAT is involved in activation of CD8 T cells. Furthermore, naïve mouse APC optimally activated Chlamydia-specific mouse CD8 T cells upon co-culture with a 1:4 mixture of Chlamydia-infected HeLa-CX43 and uninfected HeLa-CX43 cells, when compared to mixtures of infected HeLa-CX43 with uninfected HeLa-S3 or HeLa-CX43-T154A cells. These results demonstrate that GMAT via uninfected epithelial cells amplifies pathogenic Chlamydia-specific CD8 T cell response.

The major contribution of IL-1 alpha and caspase-11 in oviduct pathology during Chlamydia infection in the mouse model

Abstract The mouse model of genital Chlamydia trachomatis infection simulates the human chlamydial disease resulting in oviduct damage leading to hydrosalpinx. Using this model, we have shown that signaling by IL-1R is a major contributor of oviduct disease during infection. In the current study, we have investigated the specific contribution of IL-1 alpha and IL-1 beta that signal thorough IL-1R. Further, the contribution of caspase11-mediated cell-death that results in the release of IL-1 alpha and IL-1 beta were investigated. IL-1 alpha KO mice developed significantly less incidence (5%) of hydrosalpinx compared to WT mice (75%) (p=<0.001) upon infection. IL-1 beta KO mice had a reduced incidence in hydrosalpinx than WT mice, but significantly higher than IL-1 alpha KO mice. To determine the mechanism of IL-1 alpha release, infection course and pathology in caspase-1-11 KO mice and caspase-11 KO mice were determined. Both caspase-11 and caspase-1-11 KO mice displayed a reduced incidence of hydrosalpinx. Since the caspase-1 adaptor, ASC KO mice do not show a reduced incidence in pathology, our data suggest that caspase-11 activation is sufficient to drive the pathology. Oviduct pathology is a result of excessive PMN infiltration. Flow cytometric analyses demonstrate significantly reduced PMN infiltration to the genital tracts in IL-1 alpha and caspase-11 KO mice. However, T cell infiltration to the genital tracts is unchanged in both the groups, which reflected in normal infection clearance in IL-1 alpha and caspase-11 KO mice. These data suggest that activation of cell death pathways and local release of IL-1 alpha causes long-term damage to the oviducts during Chlamydia infection. [Supported by PHS funding NIAID AI067678 (UN)]

Visualization of IFN-β mRNA in Chlamydia-infected and adjacent uninfected cells and the contribution of CX43 in intercellular transfer of cGAMP

Abstract Background IFNβ has been implicated a key effector of oviduct pathology after genital chlamydial infection in mice. We reported that the host DNA sensor cGAS (cGAMP synthase) is essential for IFNβ expression during Chlamydia trachomatis infection. Sensing during infection leads to generation of the second messenger, 2′3-cGAMP, which can migrate to uninfected adjacent cells via gap-junction mediated transfer. cGAMP binding to central adaptor molecule, STING drives IFNβ induction. The goals of this study were to visualize the mechanism of c-GAMP transfer and to identify the DNA source triggering this response. Methods cGAMP production was detected by mass spectrometry. The contribution of gap junction proteins, connexins CX43 and CX45 in intercellular transfer of cGAMP was assayed by siRNA depletion and subsequent co-culture experiments. view RNATM methodology was used to detect IFNβ mRNA transcripts in Hela cells. Mitochondrial DNA as IFNβ inducer was assessed in mtDNA depleted cell lines. Results Mass spectroscopy confirmed cGAMP in infected host cytosol. Connexin depletion studies revealed that CX43 but not CX45 was essential for optimal IFNβ expression during infection. IFNβ mRNA transcripts were visualized in infected and adjacent, uninfected cells. In contrast, distal uninfected cells lacking cell-cell contact were negative. IFNβ induction after chlamydia infection was unchanged in mtDNA depleted cells. Conclusion Overall, our results confirm the production of cGAMP in chlamydia-infected cells and its transfer via CX43 to adjacent cells resulting in IFNβ production. Our studies exclude mtDNA as a potential source initiating this response. We are currently investigating the role of Chlamydial-DNA as an initiator.

The contribution of Chlamydia-specific CD8⁺ T cells to upper genital tract pathology.

Genital chlamydial infections lead to severe upper reproductive tract pathology in a subset of untreated women. We demonstrated previously that TNF-α producing CD8+ T cells contribute significantly to chlamydial upper genital tract pathology in female mice. Additionally, we observed minimal chlamydial oviduct pathology develops in OT-1 transgenic (OT-1) mice, wherein CD8+ T cell repertoire is restricted to recognition of the ovalbumin peptide Ova257–264, suggesting that non-Chlamydia-specific CD8+ T cells may not be responsible for chlamydial pathogenesis. In the current study, we evaluated whether antigen-specific CD8+ T cells mediate chlamydial pathology. Groups of wild type C57BL/6J (WT), OT-1 mice, and OT-1 mice replete with WT CD8+ T cells (1×106 cells/mouse intravenously) were infected intravaginally with C. muridarum (5 × 104 IFU/mouse). Serum total anti-Chlamydia antibody and total splenic anti-Chlamydia IFN-γ and TNF-α responses were comparable among the three groups of animals. However, Chlamydia-specific IFN-γ and TNF-α production from purified splenic CD8+ T cells of OT-1 mice was minimal, whereas responses in OT-1 mice replete with WT CD8+ T cells were comparable to those in WT animals. Vaginal chlamydial clearance was comparable between the three groups of mice. Importantly, the incidence and severity of oviduct and uterine horn pathology was significantly reduced in OT-1 mice but reverted to WT levels in OT-1 mice replete with WT CD8+ T cells. Collectively, these results demonstrate that Chlamydia-specific CD8+ T cells contribute significantly to upper genital tract pathology.

Tumor Necrosis Factor (TNF) Receptor Superfamily Member 1b on CD8+ T Cells and TNF Receptor Superfamily Member 1a on Non-CD8+ T Cells Contribute Significantly to Upper Genital Tract Pathology Following Chlamydial Infection.

We demonstrated previously that tumor necrosis factor α (TNF-α)-producing Chlamydia-specific CD8(+) T cells cause oviduct pathological sequelae. In the current study, we used wild-type C57BL/6J (WT) mice with a deficiency in genes encoding TNF receptor superfamily member 1a (TNFR1; TNFR1 knockout [KO] mice), TNF receptor superfamily member 1b (TNFR2; TNFR2 KO mice), and both TNFR1 and TNFR2 (TNFR1/2 double KO [DKO] mice) and mix-match adoptive transfers of CD8(+) T cells to study chlamydial pathogenesis. TNFR1 KO, TNFR2 KO, and TNFR1/2 DKO mice displayed comparable clearance of primary or secondary genital Chlamydia muridarum infection but significantly reduced oviduct pathology, compared with WT animals. The Chlamydia-specific total cellular cytokine response in splenic and draining lymph nodes and the antibody response in serum were comparable between the WT and KO animals. However, CD8(+) T cells from TNFR2 KO mice displayed significantly reduced activation (CD11a expression and cytokine production), compared with TNFR1 KO or WT animals. Repletion of TNFR2 KO mice with WT CD8(+) T cells but not with TNFR2 KO CD8(+) T cells and repletion of TNFR1 KO mice with either WT or TNFR1 KO CD8(+) T cells restored oviduct pathology to WT levels in both KO groups. Collectively, these results demonstrate that TNFR2-bearing CD8(+) T cells and TNFR1-bearing non-CD8(+) T cells contribute significantly to oviduct pathology following genital chlamydial infection.

The DNA sensor, cyclic GMP-AMP synthase, is essential for induction of IFN-β during Chlamydia trachomatis infection.

IFN-β has been implicated as an effector of oviduct pathology resulting from genital chlamydial infection in the mouse model. In this study, we investigated the role of cytosolic DNA and engagement of DNA sensors in IFN-β expression during chlamydial infection. We determined that three-prime repair exonuclease-1, a host 3' to 5' exonuclease, reduced IFN-β expression significantly during chlamydial infection using small interfering RNA and gene knockout fibroblasts, implicating cytosolic DNA as a ligand for this response. The DNA sensor cyclic GMP-AMP synthase (cGAS) has been shown to bind cytosolic DNA to generate cyclic GMP-AMP, which binds to the signaling adaptor stimulator of IFN genes (STING) to induce IFN-β expression. We determined that cGAS is required for IFN-β expression during chlamydial infection in multiple cell types. Interestingly, although infected cells deficient for STING or cGAS alone failed to induce IFN-β, coculture of cells depleted for either STING or cGAS rescued IFN-β expression. These data demonstrate that cyclic GMP-AMP produced in infected cGAS(+)STING(-) cells can migrate into adjacent cells via gap junctions to function in trans in cGAS(-)STING(+) cells. Furthermore, we observed cGAS localized in punctate regions on the cytosolic side of the chlamydial inclusion membrane in association with STING, indicating that chlamydial DNA is most likely recognized outside the inclusion as infection progresses. These novel findings provide evidence that cGAS-mediated DNA sensing directs IFN-β expression during Chlamydia trachomatis infection and suggest that effectors from infected cells can directly upregulate IFN-β expression in adjacent uninfected cells during in vivo infection, contributing to pathogenesis.

CD43-, but not CD43+, IL-10-producing CD1dhiCD5+ B cells suppress type 1 immune responses during Chlamydia muridarum genital tract infection.

Regulatory B (Breg) cells are known to modulate immune responses through predominantly interleukin-10 (IL-10)-dependent mechanisms and can be hypothetically divided into innate and adaptive subsets based on the nature of their activating signals. However, the specific role of different Breg subsets in modulating immune responses remains ambiguous. Here we have shown that Chlamydia induces IL-10-producing splenic B-cell populations consisting of CD43(+) and CD43(-) subsets of IgM(hi)IgD(lo) innate-like B (ILB) cells in vitro. While CD43(+)IL-10-producing B cells displayed innate type features and were readily induced by Chlamydia via Toll-like-receptor (TLR) signaling, CD43(-)IL-10-producing B cells required additional B-cell activating factor (BAFF)-mediated signals from dendritic cells (DCs) for their differentiation and activation, thereby classifying them as adaptive type Bregs. Importantly, CD43(-), but not CD43(+), IL-10-producing ILB cells displayed bona fide Breg activity by potently suppressing interferon-γ (IFN-γ) production in vitro in an IL-10-dependent manner. Furthermore, a novel CD43(-)CD1d(hi)CD5(+) IL-10-producing Breg population was predominantly induced by Chlamydia genital infection in vivo. Correspondingly, mixed bone marrow chimeric mice with B-cell-specific IL-10 deficiency exhibited significantly increased type 1 immune responses, decreased bacterial burden, and reduced oviduct pathology upon infection. Our data demonstrate for the first time a distinct role for CD43(-)CD1d(hi)CD5(+)-adaptive Bregs over CD43(+) innate counterparts in controlling mucosal responses against intracellular bacterial infection.

TNF receptor 2 on CD8 T cells and TNF receptor 1 on non-CD8 T cells mediate upper genital tract pathology following primary genital Chlamydia muridarum infection (MUC7P.764)

Abstract We recently demonstrated that pathogenesis in the upper genital tract (UGT) following primary vaginal Chlamydia muridarum infection in mice is dependent upon TNF-alpha production and antigen-specific CD8 T cells. In this study, we evaluated the contribution of TNF receptor 1 (TNFR1) and TNFR2 to UGT pathogenesis following primary intravaginal C. muridarum infection (5X104 IFU/mouse), using depo-provera ® treated mice deficient in TNFR1 (TNFR1 KO), TNFR2 (TNFR2 KO), or TNFR1 and 2 (TNFR1/2 DKO), and the corresponding C57BL/6J wild type (WT) mice. All groups (n=8) of mice displayed comparable chlamydial clearance, antigen-specific cytokine response, and serum anti-chlamydial antibody following primary bacterial inoculation. On day 80 after primary inoculation, TNFR1 KO, TNFR2 KO, and TNFR1/2 DKO mice displayed significantly reduced oviduct pathology (hydrosalpinx), when compared to WT mice, suggesting that both TNFR1 and TNFR2 contribute significantly to oviduct pathology. Repletion of wild type or TNFR1 KO CD8 T cells did not enhance pathology significantly in TNFR1 KO mice. On the other hand, repletion of WT, but not TNFR2 KO, CD8 T cells enhanced oviduct pathology to WT levels in TNFR2 KO mice. Collectively, these results suggest that TNFR2 on CD8 T cells and TNFR1 on non-CD8 T cells mediate UGT pathology following primary genital chlamydial infection.

The role of gap junction protein connexin 43 in Chlamydial pathogenesis (MUC7P.752)

Abstract We have shown that Chlamydia-specific CD8 T cells do not significantly affect bacterial clearance but cause oviduct pathology, suggesting that they do not target infected cells efficaciously but cause pathology presumably by targeting uninfected cells. This suggests the possibility that chlamydial peptides are presented to CD8 T cells by uninfected cells, but begs the question: “How do uninfected cells acquire chlamydial peptides”? We hypothesized that gap junction mediated antigen transport (GMAT) via channels formed by connexin (Cx) proteins, predominantly Cx43 expressed on oviduct epithelium, contributes to this effect. We have found that HeLa cells engineered to express Cx43, but not those without, can efficaciously transfer Ova257-264 to co-cultured mouse APC, which subsequently activate CD8+ T cells from Ova primed OT-1 mice. Additionally, using Cre-Lox technology, we generated mice (Foxj1Cre-Cx43flox mice) with a conditional deficiency of Cx43 only in ciliated columnar epithelia, including oviducts. The Foxj1Cre-Cx43flox mice displayed comparable kinetics of vaginal chlamydial clearance, comparable splenic antigen-specific cytokine and serum antibody response, but significantly reduced oviduct pathology compared to WT mice on day 80 after intravaginal chlamydial infection. We have thus demonstrated the role of GMAT in immunopathogenesis of microbial infections.

Chlamydia muridarum used in the mouse model of C. trachomatis, contains multiple variants that lead to differential immune responses and disease development. (MPF2P.807)

Abstract C. muridarum Nigg is commonly used in the mouse model of C. trachomatis genital tract infection. Plaque-purified isolates derived from a C. muridarum Nigg stock were compared in the murine model of genital tract infection. C57BL/6 mice were intravaginally infected with strains CM001, CM002, CM005, CM006, CM012, or the parental AR Nigg and differences in infection course and disease outcome were observed. We identified CM001 as hyper-virulent because infection course in the lower tract was extended, bacterial load was elevated (P=0.02) and we observed increased frequency of hydrosalpinx (P<0.01) and oviduct dilatation scores when compared to AR Nigg. CM012 was attenuated, with reduced lower genital tract course of infection (P=0.05), decreased pyosalpinx early, and decreased hydrosalpinx (P<0.001) and oviduct dilatation (P<0.01) at resolution. Infection with CM002, CM005, and CM006 yielded similar bacterial burden and oviduct pathology to the parental stock. Inoculation of immune deficient mice revealed that infection with CM001 resulted in the death of rag1-/- mice in contrast to both CM012 and AR Nigg, which established chronic infections. Furthermore, CM001 caused enhanced oviduct pathology in tlr2-/- mice compared with AR Nigg while CM012 infection resulted in even milder pathology than the parent. Importantly, differences between chlamydial strains used for animal modeling of human disease should be considered as a cause for observed differences in disease phenotype.

The DNA sensor, cyclic GMP-AMP synthase (cGAS) is essential for induction of IFN beta during Chlamydia trachoma tis infection (INM6P.407)

Abstract IFNβ has been implicated as an effector of oviduct pathology resulting from genital Chlamydia infection in the mouse model. In this study, we investigated the role of cytosolic DNA and engagement of DNA sensors in IFNβ expression during Chlamydia infection. Using siRNA technique and gene knock out fibroblasts, we determined that TREX-1, a host 3’exonuclease reduced IFNβ expression significantly during Chlamydia infection, implicating cytosolic DNA as a ligand for this response. STING is a signaling adaptor required for IFNβ expression and the DNA sensor, cGAS binds cytosolic DNA to generate the STING ligand cGAMP. We establish that cGAS is required for IFNβ expression during Chlamydia infection in multiple cell types. Infected cells knocked down for STING or cGAS alone failed to induce IFNβ. Interestingly, IFNβ expression was rescued by co-culture of cells depleted for either STING or cGAS suggesting that cGAMP produced in cGAS+STING- cells migrated to adjacent cells via gap junctions to bind to STING in cGAS-STING+ cells. Further, cGAS was localized in punctate regions on the cytosolic side of the chlamydial inclusion membrane, indicating that chlamydial DNA is likely sensed outside the inclusion as infection progresses. These novel data provides evidence that cGAS-mediated-DNA sensing directs IFNβ expression during C.trachomatis infection and suggest that uninfected cells adjacent to infected cells can also induce IFNβ during in vivo infection, leading to oviduct pathology.

Steroids Alone or as Adjunctive Therapy with Doxycycline Fail To Improve Oviduct Damage in Mice Infected with Chlamydia muridarum

In women, Chlamydia trachomatis can ascend from the cervix to the fallopian tubes, where an overly aggressive host inflammatory response can cause scarring that leads to chronic pelvic pain, infertility, or ectopic pregnancy. Although screening and treatment programs for women have resulted in decreased rates of sequelae, morbidities associated with oviduct scarring continue to occur. Since corticosteroids have anti-inflammatory and antifibrotic effects, we tested the ability of dexamethasone to inhibit inflammation and prevent oviduct scarring in mice genitally infected with Chlamydia muridarum. The administration of 1 or 2.5 mg/kg of body weight of dexamethasone on days 7 to 21 of infection resulted in reduced accumulation of inflammatory cells in the oviducts compared to that in controls. However, a concomitant increase in bacterial burden was observed, and chronic oviduct disease was not reduced. Adjunctive administration of a prolonged (21-day) or short (3-day) course of dexamethasone in combination with the antibiotic doxycycline also failed to reduce chronic oviduct pathology compared to antibiotic treatment alone. Steroids administered alone or adjunctively with antibiotics failed to prevent oviduct damage in this murine model of C. trachomatis infection.

Immunity against a Chlamydia infection and disease may be determined by a balance of IL‐17 signaling

Most vaccines developed against Chlamydia using animal models provide partial protection against a genital tract infection. However, protection against the oviduct pathology associated with infertility is highly variable and often has no defining immunological correlate. When comparing two adjuvants (CTA1-DD and a combination of Cholera toxin plus CpG-oligodeoxynucleotide-CT/CpG) combined with the chlamydial major outer membrane protein (MOMP) antigen and delivered via the intranasal (IN), sublingual (SL) or transcutaneous (TC) routes, we identified two vaccine groups with contrasting outcomes following infection. SL immunization with MOMP/CTA1-DD induced a 70% reduction in the incidence of oviduct pathology, without significantly altering the course of infection. Conversely, IN immunization with MOMP/CT/CpG prevented an ascending infection, but not the oviduct pathology. This anomaly presented a unique opportunity to study the mechanisms by which vaccines can prevent oviduct pathology, other than by controlling the infection. The IL-17 signaling in the oviducts was found to associate with both the enhancement of immunity to infection and the development of oviduct pathology. This conflicting role of IL-17 may provide some explanation for the discordance in protection between infection and disease and suggests that controlling immunopathology, as opposed to the rapid eradication of the infection, may be essential for an effective human chlamydial vaccine that prevents infertility.