UC Davis

The obligate intracellular bacteria Chlamydia trachomatis is the cause of the most common bacterial sexually transmitted infection worldwide. Untreated Chlamydia infections can develop into severe pathological outcomes in the female reproductive tract (FRT). In order to control transmission rates and prevent pathology, the development of an effective vaccine is urgently required. The mouse model of FRT infection with the closely related Chlamydia muridarum species offers a way to determine what would be needed for a protective immune response. Researchers using this model have found that CD4 T helper cells are the critical component of the immune response to clear Chlamydia infection in the epithelium of the FRT. However, the particular polarization state needed for these CD4 T cells and the mechanism used to orchestrate clearance of infected epithelial cells has not been determined. Here we show that, contrary to expectations, classical Th1 cells defined by high T-bet transcription factor expression are not required to mediate clearance of Chlamydia, as T-bet-deficient mice competently clear infection. Wild-type CD4 T cell responses to Chlamydia in this model do not express high levels of T-bet and do not resemble T-bethi Th1 cells that result from Salmonella infection. While the Th1 typical cytokine IFN- is produced during infection, it is not required for control of the majority of FRT infection. Instead, we found using bone marrow chimera mice that IFN- signaling plays an important role in controlling Chlamydia replication outside of the FRT in non-hematopoietic cells to protect mice from morbidity and mortality. Thus, the CD4 T cell response is not characterized by classical T-bet-driven IFN- production aimed at stimulating macrophages for killing intracellular bacteria. Instead, it is a Th1-like response in which IFN- is still a prominent marker despite low T-bet expression and IFN- is not a necessary component of an effective response in the mucosal barrier site of the FRT. We also tested the involvement of Th2 cells in the FRT using a STAT6-deficient mouse model and determined that Th2 cells are not necessary to mediate clearance of Chlamydia. We then tested a requirement for Th17 cells using an RORt mutant mouse and found that these mice experienced only a one week long delay in clearance, indicating that Th17 cells contribute to the early phase of clearance, but non-Th17 mechanisms swiftly recover the capacity for eliminating Chlamydia. Bulk RNA-sequencing and flow cytometry experiments confirmed a significant Th17 shift in T-bet-deficient mice and a high degree of similarity of RORt mutant mice with wild-type, except for a reduction in some Th17 markers. This showed that either Th1-like or Th17 populations as the major responders were capable of controlling FRT infection. In testing the potential for Th1 and Th17 pathways to compensate for each other, we eliminated both populations in multiple mouse models to assay for a delay in FRT clearance. In T-bet-deficient mice either given neutralizing anti-IL-6 and anti-TGF- or T-bet-deficient mice bred to RORt mutant mice, no significant delay was detected, indicating that clearance mechanisms operate outside of classical Th1 and Th17 specific fate programs. We additionally tested IL-12p40-deficient mice, as this cytokine subunit can promote both Th1 and Th17 associated responses, and found a severe delay in clearance multiple weeks beyond wild-type mice, though these mice do eventually clear infection very late. Data from flow cytometry and a scRNA-seq experiment illustrate a high degree of similarity between wild-type responses dominated by Th1-like CD4 T cells and the responding CD4 T cells in IL-12p40-deficient mice. Analysis of differential gene expression in the CD4 T cells from IL-12p40-deficient mice compared to wild-type and T-bet-deficient mice has revealed a set of genes specifically downregulated in IL-12p40-deficient mice. Further work is needed to determine which of these genes is mediating the clearance of Chlamydia from the FRT. These studies have revealed an effector module regulated by IL-12p40 and unrelated to Th1 or Th17 cell fates that is effective against obligate intracellular bacteria in a mucosal epithelial layer. This provides specific information on what CD4 T cell characteristics will need to be elicited by an effective vaccine against Chlamydia reproductive tract infection.