Intestinal Mucosal Immunity

描述(由申请人提供)：许多急性病毒感染在形成典型的T细胞依赖的B生发中心之前，诱导早期T细胞非依赖性多克隆B细胞激活和产生抗原特异性的IgA或Ig G。然而，启动早期T细胞非依赖性类开关重组(CSR)所需的信号尚不清楚。这项应用的目的是确定驱动T细胞非依赖性B细胞激活和IgA CSR的基本途径。我们建议利用轮状病毒感染的小鼠模型，在该模型中，在病毒暴露后48小时内诱导Peyer‘s Patch(PP)T细胞非依赖性多克隆B细胞激活，随后产生轮状病毒特异性IgA。轮状病毒体外诱导的B细胞活化需要树突状细胞。在活体内PP树突状细胞中存在轮状病毒抗原，这些细胞在B细胞活化时被激活，提示树突状细胞是B细胞对轮状病毒应答的重要调节器。我们发现B细胞的激活依赖于轮状病毒颗粒的结构完整性，这表明轮状病毒通过模式识别诱导树突状细胞反应的可能性。初步实验表明，MyD88的缺乏表达会导致B细胞活化的消融和肠道IgA水平的大幅降低。我们发现B细胞激活因子(BAFF)信号通路的突变也导致B细胞活化的消融，提示MyD88和BAFF之间存在潜在的联系。PP B细胞活化后数天，PP内的转化生长因子-2的表达和IgA+B细胞数量增加，提示转化生长因子-2在PP对轮状病毒的多克隆应答中也起重要作用。因此，我们假设，树突状细胞通过诱导多克隆B细胞反应来调节PP中快速T细胞非依赖性病原体特异性抗体的产生，该多克隆B细胞反应是由MyD88、BAFF和转化生长因子-2信号导致的，而不依赖于抗原特异性BCR。我们将通过使用体外和体内方法来验证这一假说，以追求以下三个目标：目的1.确定轮状病毒是否诱导树突状细胞MyD88信号，从而驱动B细胞反应。目的2.确定MyD88信号是否导致BAFF和TGF-ss的产生。目的3.检测多克隆B细胞对轮状病毒的反应。本申请中提出的研究对于解决有关肠道T细胞非依赖性抗体诱导的分子机制方面的悬而未决的问题具有重要意义。这项工作将为肠道免疫反应提供新的见解，并有助于改进粘膜疫苗策略。确定病原体如何诱导快速而特异的抗体具有巨大的潜力，可以确定早期抗体在限制病毒复制和传播以及提供保护方面的作用。公共卫生相关性：这项建议旨在了解宿主如何启动和调节肠道抗体对病毒病原体的反应。具体地说，将研究诱导B细胞产生病毒特异性抗体所需的树突状细胞和B细胞之间的最早信号。所获得的信息将通过定义新的信号通路提供对肠道免疫调节的更全面的了解，并将与治疗学和粘膜疫苗的开发相关。

DESCRIPTION (provided by applicant): Many acute viral infections induce early T cell independent polyclonal B cell activation and production of antigen-specific IgA or IgG prior to the formation of classical T cell dependent B germinal centers. However, the necessary signals for initiation of early T cell independent class switch recombination (CSR) are not known. The objective of this application is to determine the essential pathways that drive T cell independent B cell activation and IgA CSR. We propose to utilize the murine model of rotavirus infection in which Peyer's patch (PP) T cell independent polyclonal B cell activation is induced within 48 hours after viral exposure followed by production of rotavirus- specific IgA. Rotavirus induced B cell activation in vitro requires dendritic cells. Rotavirus antigen is present in PP dendritic cells in vivo and these cells are activated at the time B cell activation occurs, suggesting that dendritic cells are important modulators of the B cell response to rotavirus. Our finding that B cell activation is dependent on the structural integrity of the rotavirus particles suggested the possibility that rotavirus induces dendritic cell responses as a result of pattern recognition. Preliminary experiments demonstrate that lack of MyD88 expression results in an ablation of B cell activation and substantial reduction in intestinal IgA levels. We find that a mutation in the B cell activating factor (BAFF) signaling pathway also results in the ablation of B cell activation, suggesting a potential link between MyD88 and BAFF. Several days following PP B cell activation there is an increase of TGF-2 expression and numbers of IgA+ B cells in the PP, suggesting TGF-2 also plays an important role in the PP polyclonal response to rotavirus. Therefore, we hypothesize that dendritic cells modulate the production of rapid T cell independent pathogen-specific antibody in the PP through induction of a polyclonal B cell response that results from MyD88, BAFF, and TGF-2 signaling independent of an antigen-specific BCR. We will test this hypothesis by using both in vitro and in vivo approaches to pursue the following three aims: Aim 1. Determine whether rotavirus induces dendritic cell MyD88 signaling that drives B cell responses. Aim 2. Determine whether MyD88 signaling results in BAFF and TGF-ss production. Aim 3. Characterize the polyclonal B cell responses to rotavirus. The studies proposed in this application are of importance in addressing unanswered questions regarding the molecular mechanisms of T cell independent antibody induction in the intestine. This work will provide new insights into intestinal immune responses as well as contribute to improved mucosal vaccine strategies. Defining how pathogens induce rapid but specific antibody has tremendous potential to define the role of early antibody in limiting viral replication and dissemination and providing protection. PUBLIC HEALTH RELEVANCE: This proposal aims to understand how the intestinal antibody response to a viral pathogen is initiated and regulated by the host. Specifically, the earliest signals between dendritic cells and B cells required to induce B cells to produce viral specific antibodies will be investigated. Information gained will provide a more comprehensive understanding of intestinal immune regulation by defining novel signaling pathways and will be pertinent to development of therapeutics and mucosal vaccines.