Role of Gender-associated Microbiota in Organ-specific Autoimmunity

描述(申请人提供)：1型糖尿病是一种器官特有的自身免疫性疾病，需要终身监测和治疗，以防止多个器官的损害。在此提出的研究旨在表征保护性共生菌在非肥胖糖尿病(NOD)小鼠模型中影响1型糖尿病发病机制的特征。这一假设是基于这样一个事实，即在允许共生定居的特定病原体(SPF)条件下，雄性NOD小鼠的1型糖尿病发病率明显低于雌性NOD小鼠。然而，在无菌条件下，微生物区系的缺失导致两种性别的表型都100%外显。据推测，肠道微生物区系通过影响自发的自身免疫反应来调节这种性别偏见。因此，我们计划分析潜在的候选微生物区系，以及雄性、雌性和性激素消融小鼠的微生物群。我们还将剖析两性之间的免疫学差异，重点放在T细胞亚群上，考虑到它们在本病发病机制中的重要性。最后，我们计划研究口服候选微生物区系和抗生素治疗对疾病发展和T细胞功能的影响。我们具体计划实现这些目标如下：具体目标1：在雄性、雌性和性激素消融NOD小鼠中检查候选微生物区系和无偏向微生物群。我们将对NOD小鼠的每个胃肠道节段进行下一代焦磷酸测序。我们将分析从断奶年龄到疾病发展的多个时间点，年轻成年NOD小鼠中可能存在的性别差异的微生物组和选定的候选微生物组。具体目的2：分析NOD雄性小鼠和雌性NOD小鼠小肠和结肠的T细胞亚群。我们将使用流式细胞术和RNA微阵列分析不同的T细胞亚群，以确定男性和女性胃肠道免疫区段的功能差异。具体目标3：给SPF寄居的雌性和无菌雄性NOD小鼠灌胃特定目标1中已确定的潜在保护性微生物群，并用选择性抗生素组合扰乱宿主。我们还将调查这些操作引起的T细胞功能的任何变化。我们预计，这项拟议的研究将导致发现1型糖尿病发病机制中的新机制，并开辟性别相关微生物区系在自身免疫中的新途径。这项研究有望对性别偏见的自身免疫性疾病产生广泛的影响，并有可能开发新的治疗策略。该项目涉及性别相关的共生肠道细菌对非肥胖糖尿病(NOD)小鼠品系1型糖尿病的影响。我们计划识别在标准饲养条件下保护雄性NOD小鼠的有益肠道细菌，并表征它们对高度敏感的无菌NOD小鼠疾病的影响。在这种小鼠模型中探讨共生体对免疫系统的影响和性别偏见将有助于更好地了解女性在人类自身免疫性疾病中的优势，并可能开辟新的治疗策略。

DESCRIPTION (provided by applicant): Type 1 diabetes is an organ-specific autoimmune disease that requires life-long monitoring and treatment to prevent damage of multiple organs. The research proposed herein aims to characterize protective commensal bacteria influencing the pathogenesis of type 1 diabetes in a prototypical murine model, the non-obese diabetic (NOD) mouse. The hypothesis is based on the fact that the incidence of type 1 diabetes is markedly lower in male than female NOD mice under specific-pathogen-free (SPF) conditions that allow colonization with commensals. However, the absence of microbiota under germ-free conditions leads to 100% penetrance of the phenotype in both genders. It is hypothesized that gut microbiota mediate this sex bias by influencing the spontaneous autoimmune response. We therefore plan to analyze potential candidate microbiota as well as the microbiome in male, female and sex-hormone-ablated mice. We will also dissect the immunological differences between both genders, with a focus on T cell subsets, given their importance in the pathogenesis of this disease. Finally, we plan to study the influence of oral gavage with candidate microbiota and antibiotic treatments on disease development and T cell function. We specifically plan to achieve these goals as follows: Specific Aim 1: To examine candidate microbiota and the unbiased microbiome in male, female and sex-hormone-ablated NOD mice. We will perform next-generation pyrosequencing of each gastrointestinal segment from NOD mice. We will analyze the microbiome and selected candidate microbiota for possible gender differences in young adult NOD mice and at multiple time points from weaning age until development of disease. Specific Aim 2: To analysis the T cell compartment in the small intestine and colon of male versus female NOD mice. We will analyze various T cell subsets using flow cytometry and RNA microarray profiling to identify functional differences between male and female gastrointestinal immune compartments. Specific Aim 3: To gavage SPF-housed female and germ-free male NOD mice with potentially protective microbiota that have been identified in Specific Aim 1, and to perturb the host with selective antibiotic combinations. We will also investigate any alterations in T cell functions induced by these manipulations. We anticipate that the proposed research will lead to discovery of new mechanisms in the pathogenesis of type 1 diabetes, and opens the novel avenue of gender-associated microbiota in autoimmunity in general. The research is expected to have broad implications for sex-biased autoimmune diseases with the potential for development of new treatment strategies. The project deals with the influence of gender-associated commensal gut bacteria on type 1 diabetes in the non-obese diabetic (NOD) mouse strain. We plan to identify beneficial gut bacteria that protect male NOD mice under standard housing conditions and characterize their impact on disease in highly susceptible germ-free NOD mice. Exploration of the commensals' effects on the immune system and sex bias in this mouse model will help to better understand the female preponderance of human autoimmune diseases in general, and open possibly new treatment strategies.