A novel role for the inflammasome in the immunopathogenesis of Candida vaginitis

描述(由申请人提供)： 白色念珠菌是真菌性阴道炎的主要病原体，75%的育龄妇女一生中至少会感染一次。通常使用的抗真菌药物的静态活性和慢性感染日益受到关注；因此，迫切需要替代疗法。尽管长期以来人们认为对念珠菌阴道炎的易感性是由于 获得性免疫方面，最近的研究表明，与念珠菌阴道炎相关的临床免疫病理学(即瘙痒、灼热、疼痛、不适、阴道粘膜发红)是由多形核白细胞(PMN)向阴道上皮的强劲募集引发的。使用阴道炎的小鼠模型，S100警报被确定为这一标志性PMN反应中的关键信号分子。然而，最近使用S100警蛋白产生缺陷的小鼠的研究，当受到白色念珠菌的挑战时，仍然以强有力的PMN募集来回应。因此，关于念珠菌阴道炎期间中性粒细胞被招募到阴道上皮的替代和/或补充机制(S)，仍然存在根本的认识空白。 在上皮表面引发炎症的一个可能过程是通过激活炎症小体，炎症小体是一组胞浆细胞受体，发出信号并启动先天免疫反应。因此，这项建议的目的是：1)证明Nlrp3炎症体是先天炎症反应的关键早期介质；2)识别导致阴道上皮炎症性小体激活的特定菌丝相关白念珠菌毒力因子。在强大的初步数据的指导下，这些目标将检验我们的中心假设，即阴道炎期间由白色念珠菌酵母到菌丝的转变诱导的炎症小体的激活会加剧中性粒细胞的流入和阴道上皮中的促炎细胞因子信号。所有AIMS将利用一种成熟的阴道炎小鼠模型来测试我们的假设。在第一个目标下，将利用遗传或药物阻断炎症体信号级联中的离散靶点来阐明导致念珠菌阴道炎的信号通路。炎症体依赖信号的成功阻断将通过测量炎症标志物(PMN、IL-1b、S100警示蛋白)来评估。在第二个目标中，缺乏特定菌丝相关毒力因子(分泌型蛋白酶或粘附素)的形态发生能力的白色念珠菌菌株或锁定在酵母形态但过度表达这些关键的菌丝毒力因子的菌株将被用来评估它们在体内产生强大的先天免疫反应所需的作用。这种方法是创新的，因为它检查了与念珠菌阴道炎相关的免疫反应所需的宿主和真菌过程，并试图确定阴道粘膜中的炎性黏膜功能。这项拟议的工作意义重大，因为它最终将有助于识别新的信号通路，这些信号通路可能成为此类感染的新治疗靶点。 这项工作还将为候选人获得独立的学术研究计划提供坚实的基础。这位候选人的直接职业目标是成功地从一名非终身教职的初级教员过渡到一名终身教职的助理教授职位，研究宿主的先天性免疫反应和与真菌性阴道炎相关的分子机制。他的长期职业目标是获得终身教职。 指导和维持一支资金充足的翻译研究团队，研究粘膜界面上真菌病原体的先天免疫机制。为了实现这些职业目标，除了进行上述研究外，应聘者还将组建并维持一支支持性的指导/咨询团队，以帮助成功完成研究，磨练技能发展以获得有效的资助机会，并加快高影响力数据的发布。因此，这些职业目标实现了 NIAID的使命是寻找新的治疗方法来对抗真菌疾病，并支持初级调查人员在K22奖励机制下向独立教员地位过渡。

DESCRIPTION (provided by applicant): Candida albicans, the primary causative agent of fungal vaginitis, will affect 75% of all women of reproductive age at least once in their lifetime. Static activity of commonly deployed antifungals and chronic infections are of growing concern; therefore, alternative therapies are crucially needed. Despite long-held beliefs that susceptibility to Candida vaginitis resulted from defects in adaptive immunity, it has recently been shown that the clinical immunopathology (i.e. itching, burning, pain, discomfort, redness of the vaginal mucosa) associated with Candida vaginitis is triggered by robust recruitment of polymorphonuclear leukocytes (PMN) to the vaginal epithelium. Using a mouse model of vaginitis, the S100 alarmins were identified as key signaling molecules in in this hallmark PMN response. However, recent studies using mice deficient in S100 alarmin production still responded with vigorous PMN recruitment when challenged with C. albicans. Therefore, a fundamental gap in knowledge still exists as to the alternative and/or complementary mechanism(s) by which neutrophils are recruited to the vaginal epithelium during Candida vaginitis. One possible process by which inflammation can be triggered at epithelial surfaces is by activation of the inflammasomes, a group of cytosolic cellular receptors that signal and initiate innate immune responses. Therefore, the objective of this proposal is to 1) demonstrate that the Nlrp3 inflammasome is a key early mediator of the innate inflammatory response and to 2) identify specific hypha-associated C. albicans virulence factors that result in inflammasome activation in the vaginal epithelium. Guided by strong preliminary data, these aims will test our central hypothesis that activation of the inflammasome induced by the C. albicans yeast-to-hypha transition during vaginitis potentiates exacerbated neutrophil influx and pro-inflammatory cytokine signaling in the vaginal epithelium. All aims will utilize a well-established mouse model of vaginitis to test our hypotheses. Under the first aim, genetic or pharmacologic blockade of discrete targets in the inflammasome signaling cascade will be utilized to elucidate signaling pathways contributing to Candida vaginitis. Successful disruption of inflammasome-dependent signaling will be assessed via measurement of inflammatory markers (PMNs, Interleukin-1b, S100 alarmins). In the second aim, morphogenesis-competent C. albicans strains deficient in specific hypha-associated virulence factors (secreted proteinases or adhesins) or strains locked in the yeast form but over-expressing these key identified hyphal virulence factors will be utilized to assess their required role for generating robust innate immune responses in vivo. This approach is innovative because it examines both host and fungal processes required for immune responses associated with Candida vaginitis and seeks to identify inflammasome function in the vaginal mucosa. This proposed work is significant because it will ultimately aid in the identification of novel signaling pathways that may serve as new therapeutic targets for such infections. This work will also provide a strong foundation on which the candidate can attain an independent academic research program. The candidate's immediate career goal is to successfully transition from a non-tenure track junior faculty member to a tenure-track Assistant Professor position studying the host innate immune response and molecular mechanisms associated with fungal vaginitis. His long-term career goal is to attain a tenured research faculty position to direct and maintain a well-funded, translational research team that studies innate immune mechanisms to fungal pathogens at the mucosal interface. In order to attain these career goals, aside from conducting the research described above, the candidate will assemble and maintain a supportive mentoring/consulting team that will aid in the successful completion of the research, hone the development of grantsmanship skills leading to effective funding opportunities, and accelerate the publication of high-impact data. Thus, these career goals fulfill the NIAID's mission to identify novel therapeutic approaches to combat fungal disease and to support junior investigators in making a career transition to independent faculty status under the K22 award mechanism.