Chlamydia trachomatis is a Gram-negative bacterial pathogen and a major cause of sexually transmitted disease and preventable blindness. In women, infections with C. trachomatis may lead to pelvic inflammatory disease (PID), ectopic pregnancy, chronic pelvic pain, and infertility. In addition to infecting the female reproductive tract (FRT), Chlamydia spp. are routinely found in the gastro-intestinal (GI) tract of animals and humans and can be a reservoir for reinfection of the FRT. Whether Chlamydia disseminates from the FRT to the GI tract via internal routes remains unknown. Using mouse-specific C. muridarum as a model pathogen we show that Chlamydia disseminates from the FRT to the GI tract in a stepwise manner, by first infecting the FRT-draining iliac lymph nodes (ILNs), then the spleen, then the GI tract. Tissue CD11c+ DCs mediate the first step: FRT to ILN Chlamydia transport, which relies on CCR7:CCL21/CCL19 signaling. The second step, Chlamydia transport from ILN to the spleen, also relies on cell transport. However, this step is dependent on cell migration mediated by sphingosine 1-phosphate (S1P) signaling. Finally, spleen to GI tract Chlamydia spread is the third critical step, and is significantly hindered in splenectomized mice. Inhibition of Chlamydia dissemination significantly reduces or precludes the induction of Chlamydia-specific serum IgG antibodies, presence of which is correlated with FRT pathology in women. This study reveals important insights in context of Chlamydia spp. pathogenesis and will inform the development of therapeutic targets and vaccines to combat this pathogen.

Chlamydia trachomatis is an obligate intracellular pathogen that causes sexually transmitted disease. In women, chlamydial infections may cause pelvic inflammatory disease (PID), ectopic pregnancy, and infertility. The role of antibodies in protection against a primary Chlamydia infection is unclear and was a focus of this work. Using the C. muridarum mouse infection model, we show that intestinal mucosa is infected via intra-nasal (IN) or per-oral (PO) Chlamydia inoculation and unlike the female reproductive tract (FRT) mucosa, it halts systemic Chlamydia dissemination. Moreover, PO immunization or infection with Chlamydia confers protection against per-vaginal (PV) challenge, resulting in significantly decreased bacterial burden in the FRT, accelerated Chlamydia clearance, and reduced hydrosalpinx pathology. In contrast, sub-cutaneous (SC) immunization conferred no protection against the PV challenge. Both PO and SC immunizations induced Chlamydia-specific serum IgA. However, IgA was found only in the vaginal washes and fecal extracts of PO-immunized animals. Following a PV challenge, unimmunized control and SC-SC immunized animals developed Chlamydia-specific intestinal IgA, yet failed to develop IgA in the FRT indicating that IgA response in the FRT relies on the FRT to gastrointestinal tract (GIT) antigen transport. Vaginal secretions of PO-immunized animals neutralize Chlamydia in vivo, resulting in significantly lower Chlamydia burden in the FRT and Chlamydia transport to the GIT. We also show that infection of the GIT is not necessary for induction of protective immunity in the FRT, a finding that is important for the development of PO subunit vaccines to target Chlamydia and possibly other sexually transmitted pathogens.

In addition to infecting the female reproductive tract (FRT), Chlamydia spp. infect the gastro-intestinal (GI) tract of animals and humans. In the GI tract Chlamydia spp. persists without causing pathology, is more resistant to azithromycin treatment, and can be a reservoir for reinfection of the FRT. To date no effective vaccines have been licensed for use in humans. We have found that sIgA responses in the FRT mirror the responses in the GI tract following feeding with a high dose soluble Ova, leading us to hypothesize that immunity in the FRT may be induced via per-oral (PO) antigen administration. To test this hypothesis, 6 week-old C57BL/6 mice were PO or sub-cutaneously (SC) primed with live or killed C. muridarum, then 28 days later SC boosted with killed C. muridarum. At day 42 mice were per vaginally challenged with 2×105 IFUs of C. muridarum and six weeks following challenge mice were euthanized for determination of hydrosalpinx severity and C. muridarum tissue loads. PO inoculation with live or killed C. muridarum followed by SC boosting significantly reduced hydrosalpinx pathology and C. muridarum titers in ovaries, uterus, and vaginal swabs compared to unimmunized controls and to mice primed and boosted SC with killed EBs. Neither form of immunization affected C. muridarum titers in the GI tract, in spite of relatively high Chlamydia-specific sIgA titers. Further analysis revealed that intestinal sIgA are almost exclusively specific for a single antigen (HSP60, a cytoplasmic antigen), while serum antibodies recognize multiple antigens. Our results indicate that while mucosal priming is essential for induction of protective immunity in the FRT, colonization of the GI tract by live Chlamydia is not necessary for mucosal priming.

One of the major functions of the female reproductive tract (FRT) is the establishment and maintenance of pregnancy, resulting in healthy offspring. Although many pathogens (e.g. Chlamydia spp.) infect the FRT, no vaccines against these pathogens have been licensed to date. This lack of success has been in part attributed to the inability of the FRT to efficiently internalize antigens. We showed that FRT epithelium of mice can internalize nanoparticles (NPs) < 40 nm and that protein coated 20 nm NPs prime the intestinal mucosa for secretion of IgA. This finding led us to hypothesize that NPs reach the GI tract following per-vaginal (PV) instillation. Using multiphoton and fluorescence microscopy, we show that PV-instilled antigens (DAPI, Dextran, NPs) reach the stomach antrum and the small intestine within 1–3 h. It is proposed that Chlamydia spp. colonizes the GI tract following PV instillation by entering the circulation. We show that colonization of the GI tract by C. muridarum is dependent on cell transport. While within 7 days of PV infection Chlamydia colonizes the GI tract of controls, while no Chlamydia can be detected in iliac lymph nodes (ILNs) or GI tracts of CCR7−/− mice. Moreover, treatment of mice with FTY720 inhibits colonization of the GI tract, but it does not preclude infection of ILNs by Chlamydia. This finding indicates that Chlamydia transport from the ILNs to the GI tract depends on sphingosine 1-phosphate-mediated cell egress. Elucidation of mechanisms by which C. muridarum disseminates systemically to colonize the lymphoid tissues and the GI tract will be important for understanding both the immunity to and pathogenesis of Chlamydia spp.

Chlamydia trachomatis is a leading cause of preventable blindness and bacterial sexually transmitted disease (STD) worldwide. C. trachomatis infections in women can result in pelvic inflammatory disease, ectopic pregnancy, chronic pelvic pain, and infertility. In addition to infecting the female reproductive tract (FRT), Chlamydia spp. colonize the gastro-intestinal (GI) tracts of animals and humans and can be a reservoir for reinfection of the FRT. In mice, C. muridarum disseminates from the FRT to the GI tract via an internal, non-oral, non-rectal route. Here we show that C. muridarum disseminates from the FRT to the GI tract in a step-wise manner, by first infecting the FRT-draining iliac lymph nodes (ILNs), then the spleen, inducing extramedullary erythropoiesis and splenomegaly, which peaks at 7 days post per-vaginal (PV) infection, thus splenomegaly is transient. Infection of and proliferation of C. muridarum in the spleen is essential for its dissemination to the GI tract, as colonization of the GI tract by C. muridarum is significantly reduced or completely inhibited in splenectomized mice following per-vaginal inoculation. While intra-venously infected mice clear C. muridarum from ILNs by day 9 of infection, PV-infected mice do not, indicating that infection of ILNs from FRT is the first step of C. muridarum systemic dissemination. Furthermore, inhibition of cell egress with FTY720 significantly inhibits Chlamydia dissemination from ILNs to the spleen and GI tract, indicating a cell-mediated transport of this pathogen. Further elucidation of the mechanisms of C. muridarum systemic dissemination and ensuing immune responses will be essential for developing therapies and vaccination approaches to target this pathogen.