Female Genital Innate Immune Response to Vaginal Microbiota

描述（由申请人提供）：细菌性阴道病（BV）是一种阴道综合征，其特征为阴道厌氧菌过度生长，并与几种不良临床结局相关，包括盆腔炎、早产和HIV-1感染风险增加。个体之间的细菌种类不同，临床症状，严重后遗症的发生率和细胞因子反应的模式也不同。我们推测，临床差异是由个体物种和宿主免疫反应之间的相互作用引起的。我们计划测试以下假设：1）某些细菌物种下调先天宿主反应，通过降低阴道细胞因子水平和/或抗菌肽的产生来允许高水平的定植（β-防御素、抗菌肽、分泌性白细胞蛋白酶抑制剂[SLPI]、乳铁蛋白）; 2）由于那些抗微生物肽的低基线水平，一些个体更容易受到定植; 3）乳杆菌属物种和BV相关病原体通过模式识别受体如TLR-2不同地激活先天免疫应答;和4）由于存在破坏宿主免疫应答的毒力因子或机制，一些细菌物种更可能定殖上生殖道。我们将使用16 S rRNA基因的qPCR表征前瞻性收集的30名女性每日阴道拭子的单个细菌种类水平，并将其与细胞因子和抗菌肽水平的时间响应相关联（目的1）。阴道上皮细胞培养物中的反应将用于确定乳酸杆菌和BV相关微生物是否不同地激活Toll样受体，从而导致不同的细胞因子谱，以及TLR-2激活是否与更高水平的抗微生物肽相关（Aim 2）。在子宫切除术时采集的子宫内膜样本将通过宽范围16 S rRNA PCR和焦磷酸测序进行表征，以检测上生殖道定植，并与相同女性的阴道微生物群进行比较，确定微生物群。此外，还将测量宫颈细胞因子和防御分子的水平，以评估其与上尿路定植的关系（目的3）。本申请的主要研究者Caroline Mitchell（医学博士）是一名妇产科医生，其目标是了解女性生殖道如何防御细菌病原体，最终目的是开发更好的治疗和预防干预措施。她的导师大卫弗雷德里克博士是一位全国公认的研究人员，以使用先进的分子技术更好地表征阴道微生物群而闻名，最近因其出色的指导而受到认可。华盛顿大学是研究经费最多的公立大学，拥有国际知名的传染病项目和对BV的研究历史悠久。科学咨询委员会在免疫技术、临床试验和数据分析方面具有专长。 叙述：细菌性阴道病是一种常见的临床综合征，在美国影响高达30%的妇女，并与早产、盆腔炎和HIV-1感染的风险增加2倍有关。该项目将增加我们对某些细菌物种如何改变获得细菌性阴道病的风险以及免疫反应的哪些成分对预防细菌性阴道病最重要的理解。这将有助于制定有针对性的预防和治疗干预措施，以减少不良临床结局。

DESCRIPTION (provided by applicant): Bacterial vaginosis (BV) is a vaginal syndrome characterized by an overgrowth of anaerobic vaginal bacteria, and is associated with several adverse clinical outcomes, including pelvic inflammatory disease, preterm birth and increased risk of HIV-1 acquisition. The individual bacterial species differ between individuals, as do the clinical symptoms, the incidence of serious sequelae, and patterns of cytokine response. We hypothesize that clinical differences result from the interactions between individual species and host immune responses. We plan to test the following hypotheses: 1) that some bacterial species downregulate the innate host response, allowing high levels of colonization by decreasing levels of vaginal cytokines and/or production of antimicrobial peptides(beta-defensins, cathelicidin, secretory leukocyte protease inhibitor[SLPI], lactoferrin); 2) that some individuals are more vulnerable to colonization due to low baseline levels of those antimicrobial peptides; 3) that Lactobacillus species and BV-associated pathogens activate the innate immune response differently via pattern-recognition receptors like TLR-2; and 4) that some bacterial species are more likely to colonize the upper genital tract due to the presence of virulence factors or mechanisms for subverting the host immune response. We will characterize prospectively collected daily vaginal swabs from 30 women for levels of individual bacterial species using qPCR for the 16S rRNA gene, and correlate these with the temporal response in levels of cytokines and antimicrobial peptides (Aim 1). Responses in vaginal epithelial cell culture will be used to determine whether lactobacilli and BV-associated microbes activate toll-like receptors differently resulting in different cytokine profiles, and whether TLR-2 activation is associated with higher levels of antimicrobial peptides (Aim 2). Endometrial samples collected at hysterectomy will be characterized by broad range 16S rRNA PCR and pyrosequencing to detect upper genital tract colonization and define the microbiota in comparison to the vaginal microbiota in the same women. In addition, levels of cervical cytokines and defense molecules will be measured to assess their relationship with upper tract colonization (Aim 3). The PI for this application, Caroline Mitchell, MD is an OB/Gyn whose goal is to understand how the female genital tract defends against bacterial pathogens, with the ultimate endpoint of developing better therapeutic and preventive interventions. Her mentor, Dr. David Fredricks, is a nationally recognized investigator known for using advanced molecular techniques to better characterize the vaginal microbiota, and has recently been recognized for excellence in mentoring. The University of Washington is the top public university for research funding, with an internationally-renowned Infectious Diseases program and a long history of studies on BV. The scientific advisory committee has expertise in immunologic techniques, clinical trials and data analysis. Narrative: Bacterial vaginosis is a common clinical syndrome that affects up to 30% of women in the United States and is associated with a 2-fold increase of the risk of preterm birth, pelvic inflammatory disease and HIV-1 acquisition. This project will increase our understanding how certain bacterial species change the risk of acquiring bacterial vaginosis and what components of the immune response are most important for protection against bacterial vaginosis. This will allow development of targeted preventive and therapeutic interventions to reduce the adverse clinical outcomes.