Primordially conserved principles governing mucosal immune responses to pathogens and microbiota

描述（由申请人提供）：脊椎动物的粘膜表面（即肠道、口腔）持续暴露于内源性和外源性微生物中。在整个进化过程中，特异性免疫球蛋白（Ig）同种型已经成为专门保护这些粘膜部位的免疫球蛋白。在哺乳动物和鸟类中，IgA在粘膜表面占主导地位，而IgG和IgM则提供全身保护。虽然粘膜igg被认为是在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专门用于肠道粘膜免疫的igg同型，IgT。此后，我们发现在包括皮肤和鳃在内的其他粘膜部位也发生了非常相似的IgT反应。基于这些结果，我们假设所有鱼类粘膜区域（肠道、鳃和皮肤）的IgT反应都在原始保守原则的指导下运作。因此，这项更新的总体目标是验证病原体特异性IgT反应以T细胞依赖的方式局部诱导（Aim 1） （Aim 2）的假设，并导致产生效应IgT+ B细胞，产生保护性IgT反应并通过产生IL-10维持组织稳态（Aim 3）。此外，由于我们发现来自鳃、肠道和皮肤的细菌微生物群普遍被IgT包裹，我们假设共生微生物群诱导局部t细胞独立的IgT反应（Aim 2），并且这种IgT稳态调节粘膜部位的微生物群（Aim 3）。在我们的模型中描述基于免疫球蛋白的粘膜免疫策略不仅将阐明非四足动物粘膜免疫反应的未解决范例，而且还将提供对反应的见解。更具体地说，我们提出的研究有望：a)提供新的见解，通过这些途径，IgA的产生是在病原体和微生物群的反应中被调节的；b)有助于理解CD4+ T细胞在产生针对病原体和微生物群的IgA反应中的作用；c)提供关于粘膜Igs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：AIM 1。研究IgT反应产生的诱导位点和效应位点，并分析鱼类共同粘膜免疫系统（CMIS）的存在。为了揭示IgT的诱导位点和效应位点，将使用正常鱼和一种新的去脾鱼模型，在所有malt和脾脏中评估体内病原体诱导的B和T细胞增殖反应以及B-T细胞相互作用的可视化。在这里，我们还将通过确定在一个粘膜部位诱导的B/T细胞反应是否会导致其他远处粘膜区域的效应反应来评估共同粘膜免疫系统（CMIS）的存在。目标2。研究IgT对病原体和微生物群的依赖性。利用创新的CD4-T细胞耗竭鱼模型，我们将评估鱼的死亡率、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CD4-T细胞耗损对所有肠道、鳃和皮肤微生物群组成的影响，以及被IgT和IgM包裹的细菌微生物群的百分比。为了深入了解在没有CD4+ T细胞的情况下IgT反应被调节的机制，我们也将

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.