Immunoregulatory Defects In Inflammatory Bowel Disease

为了探究NOD2 NBD突变引起疾病的机制，我们首先进行了体外研究，研究了承载多种NBD突变的NOD2构建体支持NOD2信号功能的能力。我们发现，携带这种突变并在HEK293T细胞中表达的NOD2表现出低聚、与RIPK2相互作用或激活NF-kappaB的能力降低。这些研究是在避免过度表达影响的条件下进行的，这是以前研究的特点。

To probe the mechanism of disease caused by mutations in the NBD of NOD2 we first conducted in vitro studies to investigate the ability of NOD2 constructs bearing a variety of NBD mutations to support NOD2 signaling function. We found that NOD2 bearing such mutations and expressed in HEK293T cells exhibit a reduced ability to oligomerize, to interact with or activate RIPK2 and activate NF-kappaB. These studies were conducted under conditions that avoided effects of over-expression that had characterized previous studies along these lines. We then turned ti in vivo studies in which we determined the capacity of over-expressed intact NOD2 or NOD2 with a Blau mutation (BS-NOD2) (R314W) to cross (down)-regulate TLR responses underlying TNBS-colitis. Whereas administration of intact NOD2 protected mice from TNBS-colitis, over-expression of BS-NOD2 failed to protect. These findings were corroborated by studies of mice bearing a knock-In mutation of NOD2 similar to that in patients with BS in which we showed that such mice were not as protected from DSS-colitis by NOD2-ligand (muramyl dipeptide, MDP) administration as was comparably treated littermate control mice. Importantly, these failure to protect was also noted in heterozygous mice in which the mutation occurred on only one allele; thus the mutated NOD2 exerted a dominant negative effect which explained the fact that the Blau mutation has an autosomal dominant effect. These studies thus showed that BS-NOD2 displays a defect in the ability to "cross-regulate" the TLR-induced GI inflammation. A similar defect was noted in studies of anti-collagen arthritis also conducted in Blau KI mice. Finally, we conducted studies to determine the molecular basis of the failure of NOD2 bearing a Blau mutation to mediate cross-regulation. WE showed that MDP-stimulated cells from BS-NOD2 KI mice fail to up-regulate expression of IRF4, a factor that has been shown to mediate NOD2 cross-regulation by de-ubiquitination of NF-kappaB signaling components. Indeed, lack of IRF4-mediated cross-regulatory function in KI cells was shown by the fact that enhanced TLR responses exhibited by these cells are suppressed by lentivirus transduction of IRF4. In addition, whereas WT mice expressed IRF4 in inflamed gut and joint tissue following MDP administration, Blau KI mice failed to do so under similar conditions Overall, these studies indicate that NOD2 bearing a BS mutation lacks the ability to cross-regulate TLR responses via its inability to activate IRF4. The mutation thus renders BS patients susceptible to excessive TLR responses that have the potential to support inflammation at sterile tissue sites. Thus, the Blau mutation affecting NOD2 causes inflammatory disease by affecting NOD2 immunoregulatory function.