Mechanisms of Chronic Nod2-mediated Effects in Human Macrophages

描述（由申请人提供）：肠道免疫系统面临的一个独特挑战是对肠道细菌的耐受性要求，同时防御病原微生物。这种平衡的失调会导致胃肠免疫系统不受控制的激活，这是克罗恩病和溃疡性结肠炎的一个特征。对微生物的识别和反应部分是由模式识别受体（PRR）介导的，其中包括核苷酸寡聚结构域2 (NOD2)，这是一种细胞内细菌对肽聚糖（PGN）的识别受体，是革兰氏阳性和革兰氏阴性细胞壁的成分。克罗恩病与NOD2的功能缺失多态性相关（CARD15）。然而，导致克罗恩病发展的特异性NOD2功能障碍尚不清楚。在NOD2的急性刺激下，无论是单独刺激还是与其他细菌受体联合刺激，外周源性抗原呈递细胞（APC）都会分泌促炎细胞因子。肠道巨噬细胞和树突状细胞（DC）在很大程度上来源于循环单核细胞，它们迁移到肠道组织并根据肠道环境进行分化。肠道免疫系统是一个导致慢性刺激的环境。因此，通过NOD2了解慢性刺激的后果是至关重要的。我们最近发现，通过NOD2对原代外周单核细胞来源的人巨噬细胞进行慢性刺激，导致通过NOD2或其他PRR再刺激后，促炎细胞因子的产生减少。这种促炎细胞因子分泌的下调与肠道巨噬细胞在细菌产物刺激下表现出的耐受或显著减少促炎细胞因子分泌相似。另一方面，炎症性肠病（IBD）患者的肠道APC产生过多的促炎细胞因子。我们假设，通过NOD2刺激诱导的原发性人类APC耐受是由选择性影响特定信号通路（如促炎细胞因子）的机制组合介导的，其中一些机制与慢性刺激Toll样受体（TLR）介导的机制不同。本研究将明确表达各种克罗恩病相关NOD2突变的人外周巨噬细胞的耐受缺陷，以及通过慢性NOD2与TLR刺激介导外周巨噬细胞耐受的机制，然后将这些发现直接扩展到人肠道巨噬细胞。我们最近发现，通过原代外周单核细胞来源的人巨噬细胞NOD2的慢性刺激可导致通过NOD2或其他模式识别受体再刺激后促炎细胞因子的产生减少。我们试图确定nod2介导的选择性影响这些促炎细胞因子下调的机制组合，并确定这些机制是否与慢性刺激Toll样受体诱导的机制不同。然后，我们将把这些发现直接扩展到人类肠道巨噬细胞中，并确定携带克罗恩病相关NOD2突变的个体在这些机制中是否存在缺陷。

DESCRIPTION (provided by applicant): A unique challenge faced by the intestinal immune system is the requirement for tolerance to luminal bacteria while simultaneously defending against pathogenic organisms. The dysregulation of this balance can lead to uncontrolled activation of the gastrointestinal immune system, a feature of Crohn's disease and ulcerative colitis. The recognition and response to microbial organisms is mediated in part by pattern recognition receptors (PRR), which include nucleotide oligomerization domain 2 (NOD2), an intracellular bacterial recognition receptor for peptidoglycan (PGN), a component of both gram positive and gram negative cell walls. Crohn's disease is associated with loss-of-function polymorphisms in NOD2 (CARD15). However, the specific NOD2 dysfunction(s) leading to the development of Crohn's disease is not yet understood. Upon acute stimulation of NOD2, either alone or in combination with other bacterial receptors, peripherally-derived antigen presenting cells (APC) secrete proinflammatory cytokines. Intestinal macrophages and dendritic cells (DC) are in large part derived from circulating monocytes that migrate into the intestinal tissues and undergo differentiation based on the intestinal environment. The intestinal immune system is an environment which results in chronic stimulation. Therefore, it is critical to understand the consequences of chronic stimulation through NOD2. We have recently found that chronic stimulation through NOD2 in primary peripheral monocyte-derived human macrophages results in reduced production of pro- inflammatory cytokines upon restimulation either through NOD2 or through other PRR. This downregulation in production of pro-inflammatory cytokine secretion is similar to the tolerance, or significantly reduced secretion of pro-inflammatory cytokines, exhibited by intestinal macrophages stimulated with bacterial products. On the other hand, intestinal APC from individuals with inflammatory bowel disease (IBD) have excess production of pro-inflammatory cytokines. We hypothesize that the tolerance induced in primary human APC upon stimulation through NOD2 is mediated by a combination of mechanisms that selectively affect specific signaling pathways (e.g. pro-inflammatory cytokines), and that some of these mechanisms are different than those mediated by chronic stimulation of Toll like receptors (TLR). This proprosal will define the tolerance defects in human peripheral macrophages expressing the various Crohn's disease- associated NOD2 mutations, the mechanisms mediating peripheral macrophage tolerance through chronic NOD2 versus TLR stimulation, and then extend these findings directly into human intestinal macrophages. PUBLIC HEALTH RELEVANCE We have recently found that chronic stimulation through NOD2 in primary peripheral monocyte-derived human macrophages results in reduced production of proinflammatory cytokines upon restimulation either through NOD2 or through other pattern recognition receptors. We seek to define the combination of NOD2-mediated mechanisms that selectively affect the downregulation of these pro-inflammatory cytokines, and determine if these mechanisms are different from those induced upon chronic stimulation of Toll like receptors. We will then extend these findings directly into human intestinal macrophages and determine if individuals harboring Crohn's disease associated NOD2 mutations are defective in these mechanisms.