Primordially conserved principles governing mucosal immune responses to pathogens and microbiota

描述（由申请人提供）：粘膜表面（即，肠、口）持续暴露于内源性和外源性微生物。在整个进化过程中，特异性免疫球蛋白（IG）同种型已变得专门保护这些粘膜部位。在哺乳动物和鸟类中，伊加在粘膜表面占主导地位，而IgG和IgM提供全身保护。虽然粘膜免疫球蛋白被认为是在四足动物（四足动物）物种中进化的，但在之前的资助期间，我们发现鱼类含有一种IG同种型，IgT，专门用于肠道粘膜免疫。此后，我们发现非常相似的IgT反应发生在其他粘膜部位，包括皮肤和鳃。基于这些结果，我们假设IgT反应在所有的鱼粘膜领域（肠道，鳃和皮肤）的指导下运作的原始保守的原则。因此，该更新的首要目标是检验病原体特异性IgT应答以T细胞依赖性方式（Aim 2）局部诱导（Aim 1），并导致产生效应IgT+ B细胞的假设，所述效应IgT+ B细胞产生保护性IgT应答并通过产生IL-10维持组织稳态（Aim 3）。此外，由于我们已经发现来自鳃、肠道和皮肤的细菌微生物群被IgT包被，我们假设肠道微生物群诱导局部T细胞非依赖性IgT应答（目的2），并且该IgT稳态调节粘膜部位的微生物组（目的3）。在我们的模型中，描述基于免疫球蛋白的粘膜免疫的策略不仅将阐明非四足动物中粘膜免疫应答的未解决范例，而且还将提供对应答的见解。更具体地说，我们提出的研究有望：a）提供对伊加球蛋白A产生响应病原体和微生物群的调节的滤泡外途径的新见解; B）有助于理解CD 4 + T细胞在产生对病原体和微生物群的伊加应答中的作用; c）提供关于粘膜Ig在多个粘膜部位控制微生物群及其组成（微生物组）的机制的突破性知识。因此，这次更新的目标是：目标1。研究鱼类IgT反应的诱导位点和效应位点，分析鱼类是否存在共同的粘膜免疫系统（CMIS）。为了揭示IgT诱导和效应位点，将使用正常鱼和新的脾切除鱼模型在所有MALT和脾中评估体内病原体诱导的B和T细胞增殖应答沿着B-T细胞相互作用的可视化。在这里，我们也将评估一个共同的粘膜免疫系统（CMIS）的存在，通过确定是否B/T细胞反应诱导在一个粘膜部位导致效应器反应在其他遥远的粘膜区域。AIM 2.研究IgT应答对病原体和微生物群的T细胞依赖性。使用创新的CD 4-T细胞耗竭鱼类模型，我们将评估鱼类死亡率，病原体负荷，B细胞增殖和病原体特异性IG反应。我们还将评估CD 4-T细胞耗竭对所有肠道、鳃和皮肤微生物组组成以及IgT和IgM包被的细菌微生物群百分比的影响。为了深入了解IgT应答在缺乏CD 4 + T细胞的情况下被调节的机制，我们还将 研究CD 4 + T细胞耗竭对未感染和感染鱼的所有粘膜部位的IgT序列库的影响。AIM 3.研究IgT+ B细胞控制病原体和细菌微生物群的需求。这一目标的实现将使用一个强大的和新的IgT+ B细胞耗竭鱼模型。在IgT+ B细胞耗竭的鱼中，在病原体攻击后，评估鱼死亡率、组织损伤、病原体负荷、补体激活和病原体特异性全身和粘膜IG应答。使用相同的鱼类模型，我们还将评估所有粘膜部位的微生物组组成的变化以及IgT和IgM包被的细菌微生物群的百分比。

DESCRIPTION (provided by applicant): The mucosal surfaces (i.e., gut, mouth) of vertebrates are continuously exposed to endogenous and exogenous microbes. Throughout evolutionary time, specific immunoglobulin (Ig) isotypes have become specialized to protect such mucosal sites. In mammals and birds, IgA predominates at mucosal surfaces, whereas IgG and IgM provide systemic protection. Although mucosal Igs were thought to evolve in four legged (tetrapod) species, during the previous funding period, we discovered that fish contain an Ig isotype, IgT, that is specialized for gut mucosal immunity. Thereafter we discovered extraordinarily similar IgT responses occurred in other mucosal sites, including the skin and gills. Based on these results, we hypothesize that IgT responses in all fish mucosal areas (gut, gills and skin) operate under the guidance of primordially conserved principles. Thus, the overarching goal of this renewal is to test the hypothesis that pathogen-specific IgT responses are locally induced (Aim 1) in a T cell-dependent fashion (Aim 2), and lead to the generation of effector IgT+ B cells that generate protective IgT responses and maintain tissue homeostasis through the production of IL-10 (Aim 3). In addition, as we have found bacterial microbiota from gill, gut and skin prevalently coated by IgT, we hypothesize that commensal microbiota induce local T-cell independent IgT responses (Aim 2) and, that this IgT homeostatically regulates the microbiome at mucosal sites (Aim 3). Delineating strategies of immunoglobulin-based mucosal immunity in our model will not only illuminate unresolved paradigms of mucosal immune responses in non-tetrapods, but will also provide insights into responses. More specifically, our proposed studies are expected to: a) provide novel insights into the extrafollicular pathways by which IgA production is regulated in response to pathogens and microbiota; b) contribute towards the understanding of the roles of CD4+ T cells in generating IgA responses to pathogens and microbiota; c) provide ground-breaking knowledge on the mechanisms by which mucosal Igs control microbiota and its composition (microbiome) at multiple mucosal sites. Thus, the aims of this renewal are: AIM 1. To investigate the inductive and effector sites involved in the generation of IgT responses and, to analyze the existence of a common mucosal immune system (CMIS) in fish. To uncover the IgT inductive and effector sites, in vivo pathogen-induced B and T cell proliferative responses along with the visualization of B-Tcell interactions will be assessed in all MALTs and the spleen, using normal fish and a novel splenectomized fish model. Here we will also assess the existence of a common mucosal immune system (CMIS) by determining whether B/T cell responses induced at one mucosal site lead to effector responses at other distant mucosal areas. AIM 2. To investigate the T-cell dependency of IgT responses to pathogens and microbiota. Using an innovative CD4-T cell depletion fish model, we will assess fish mortality rate, pathogen load, B cell proliferative and pathogen-specific Ig responses. We will also evaluate the effect of CD4-T cell depletion on the composition of all gut, gill and skin microbiomes and the percentage of bacterial microbiota coated with IgT and IgM. To gain insight into the mechanisms by which IgT responses are modulated in the absence of CD4+ T cells, we will also investigate the effect of CD4+ T cell depletion on the IgT sequence repertoire of all mucosal sites of na�ve and infected fish. AIM 3. To investigate the requirement of IgT+ B cells for the control of pathogens and bacterial microbiota. The goals of this aim will be accomplished using a powerful and novel IgT+ B cell depletion fish model. Fish mortality rates, tissue damage, pathogen load, complement activation and pathogen-specific systemic and mucosal Ig responses will be assessed in IgT+ B cell depleted fish upon pathogen challenge. Using the same fish models we will also evaluate changes in the microbiome composition of all mucosal sites and the percentage of bacterial microbiota coated with IgT and IgM.