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

项目概要/摘要 颗粒溶素（GNLY）是一种研究不足的人类蛋白质，对细胞介导的微生物杀伤至关重要， 胞内细菌我们观察到GNLY在子宫颈阴道分泌物（CVT）中含量丰富， 月经周期的卵泡期，但几乎没有黄体期。GNLY可能在以下方面发挥重要作用： 通过在感染的细胞裂解之前杀死细胞内病原体（从而限制炎症）或甚至 而不破坏受感染的细胞。因此，GNLY在月经周期中的变化可能具有重要意义。 对妇女健康的后果，特别是对性传播感染的易感性。 细菌性病原体沙眼衣原体（Ct）的疾病负担很大， 每年全球感染，约10%未经治疗的感染进展为盆腔炎， 不孕不育的主要原因因为Ct是一种细胞内细菌病原体，我们假设GNLY- 表达免疫细胞有助于控制人类中的Ct。有证据表明CT- 特异性T细胞可以表达GNLY，但GNLY尚未在Ct的背景下进行研究。缺乏研究 可能部分是由于小鼠中产生IFN-γ的CD 4 T细胞完全控制了感染。然而，老鼠 缺乏GNLY同源物，从小鼠研究中推断GNLY在人类Ct中的作用 不可能感染。事实上，人类GNLY转基因小鼠控制其他细菌感染的效果比 缺乏GNLY的野生型小鼠。此外，Ct感染更常见于月经后期 周期，当GNLY从CVT分泌物中消失时。 在目标1中，我们将确定GNLY如何在月经周期中发生如此显著的变化。我们将遵循 女性从卵泡期到黄体期。我们将评估GNLY在宫颈内膜细胞中的表达， 每个阶段一次访视时进行细胞刷和宫颈及阴道活检，并进行详细的细胞表型分析。我们将 评估每日自我收集的分泌物中的GNLY水平。 在目标2中，我们将研究GNLY对抗Ct免疫和增加CT控制的贡献。 卵泡期我们将使用单独的Ct和Ct感染的细胞进行体外实验;纯化的GNLY， 颗粒酶和穿孔素;以及表达GNLY的细胞。 这些研究将有助于我们了解妇女的生殖健康和性病的易感性， 阐明月经周期中CVT免疫的机制。这些知识对于 设计临床试验（如激素，特别是阴道内避孕药）和Ct治疗。

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.