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

项目摘要/摘要 颗粒溶素(Granulysin，GNLY)是一种尚未被研究的人类蛋白质，是细胞介导的杀灭微生物所必需的，包括 胞内细菌。我们观察到GNLY在子宫颈阴道部(CVT)的分泌物中含量丰富。 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 CVT免疫，在感染细胞溶解之前杀死细胞内病原体(从而限制炎症)，甚至 而不会破坏受感染的细胞。因此，GNLY在月经周期中的变化可能对 对妇女健康的影响，特别是对性传播感染(STI)的易感性。 细菌沙眼衣原体(Ct)的疾病负担很大，超过1.25亿 全世界每年的感染和大约10%的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是一种细胞内的细菌病原体，我们假设GNLY- 表达免疫细胞有助于控制人类的CT。有提示性证据表明CT- 特异性T细胞可表达GNLY，但尚未在Ct背景下对GNLY进行研究。缺乏研究 部分原因可能是完全控制了小鼠体内产生干扰素-γ的CD4T细胞的感染。然而，老鼠 缺乏GNLY同源基因，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转基因小鼠比其他细菌更好地控制感染 缺乏GNLY的野生型小鼠。此外，CT感染更常在月经后期被发现。 周期，当GNLY从CVT分泌物中消失。 在目标1中，我们将确定GNLY如何在月经周期中如此戏剧性地变化。我们会跟随 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 细胞刷检和宫颈及阴道活检，每期一次，并有详细的细胞表型。我们会 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在抗CT免疫和增加CT控制中的作用。 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；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传播感染易感性 阐明月经周期中CVT免疫的机制。这方面的知识对 设计临床试验(如荷尔蒙，特别是阴道内避孕药)和CT疗法。

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.