Impact of the menstrual cycle on granulysin-mediated immunity in the human cervicovaginal tract

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PROJECT SUMMARY/ABSTRACT Granulysin (GNLY) is an understudied human protein necessary for cell-mediated killing of microbes, including intracellular bacteria. We observed that GNLY is abundant in cervicovaginal tract (CVT) secretions during the follicular phase of the menstrual cycle, but nearly absent from the luteal phase. GNLY may play a major role in CVT immunity by killing intracellular pathogens prior to infected cell lysis (thus limiting inflammation) or even without damaging the infected cell. Thus, the change in GNLY over the menstrual cycle may have important consequences for women’s health, particularly susceptibility to sexually transmitted infections (STIs). The bacterial pathogen Chlamydia trachomatis (Ct) has a large disease burden, with more than 125 million annual infections worldwide and about 10% of untreated infections progressing to pelvic inflammatory disease, a major cause of infertility. Because Ct is an intracellular bacterial pathogen, we hypothesize that GNLY- expressing immune cells contribute to control of Ct in humans. There is suggestive evidence that Ct- specific T cells may express GNLY, but GNLY has not been studied in the context of Ct. The lack of studies may be in part due to complete control of infection by IFN-γ-producing CD4 T cells in mice. However, mice lack a GNLY homologue, making inference from mouse studies about the role of GNLY in human Ct infection impossible. In fact, human GNLY-transgenic mice control infection with other bacteria better than wild-type mice that lack GNLY. Additionally, Ct infection is more commonly detected late in the menstrual cycle, when GNLY disappears from CVT secretions. In Aim 1, we will determine how GNLY shifts so dramatically across the menstrual cycle. We will follow women from the follicular to the luteal phase. We will assess GNLY expression in cells from endocervical cytobrushes and cervical and vaginal biopsies at one visit per phase, with detailed cell phenotyping. We will assess GNLY levels in daily, self-collected secretions. In Aim 2, we will investigate GNLY’s contributions to anti-Ct immunity and increased control of CT in the follicular phase. We will perform in vitro experiments using Ct alone and Ct-infected cells; purified GNLY, granzymes, and perforin; and GNLY-expressing cells. These studies will contribute to our understanding of women’s reproductive health and STI susceptibility by elucidating mechanisms of CVT immunity during the menstrual cycle. This knowledge is important for designing clinical trials (such as of hormonal, especially intravaginal, contraceptives) and Ct therapeutics.