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要求对腔细菌耐受，同时防御病原体。这种平衡的失调会导致胃肠道免疫系统失控的激活，这是克罗恩病和溃疡性结肠炎的一个特征。对微生物的识别和应答部分是由模式识别受体(PRR)介导的，其中包括核苷酸寡聚化结构域2(NOD2)，这是一种胞内细菌识别肽聚糖(PGN)的受体，是革兰氏阳性和革兰氏阴性细胞壁的组成部分。克罗恩病与NOD2(CARD15)基因功能丧失相关。然而，导致克罗恩病发生的特异性NOD2功能障碍(S)尚不清楚。在NOD2的急性刺激下，无论是单独还是与其他细菌受体结合，外周来源的抗原提呈细胞(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中的单核细胞，这些单核细胞迁移到肠道组织中并根据肠道环境进行分化。肠道免疫系统是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，在原代外周血单核细胞来源的巨噬细胞中，通过NOD2的慢性刺激导致通过NOD2或通过其他PRR重新刺激时促炎细胞因子的产生减少。这种对促炎细胞因子分泌的下调类似于细菌产物刺激的肠道巨噬细胞对促炎细胞因子的耐受或显著减少。另一方面，炎症性肠病(IBD)患者的肠道APC有过量的促炎细胞因子产生。我们假设，NOD2刺激诱导的原代人APC的耐受是由选择性影响特定信号通路(如促炎细胞因子)的多种机制联合介导的，其中一些机制不同于慢性刺激Toll样受体(TLR)所介导的机制。这一命题将确定表达各种克罗恩病相关NOD2突变的人外周巨噬细胞的耐受性缺陷，通过慢性NOD2与TLR刺激介导外周巨噬细胞耐受性的机制，然后将这些发现直接扩展到人肠道巨噬细胞。公共卫生相关性我们最近发现，在原代外周血单核细胞来源的巨噬细胞中，通过NOD2的慢性刺激导致通过NOD2或通过其他模式识别受体重新刺激时促炎症细胞因子的产生减少。我们试图确定NOD2介导的机制的组合，这些机制选择性地影响这些促炎细胞因子的下调，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突变的个体是否在这些机制上存在缺陷。

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.