Immunoregulatory Defects In Inflammatory Bowel Disease

为了探讨NOD2基因NBD突变导致疾病的机制，我们首先进行了体外研究，研究了携带各种NBD突变的NOD2构建体支持NOD2信号功能的能力。我们发现，在HEK293T细胞中表达的NOD2具有这样的突变，表现出与RIPK2相互作用或激活RIPK2以及激活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，而过度表达是先前研究的特征。 然后，我们转向了体内研究，在这些研究中，我们确定了过度表达的完整NOD2或带有BLAU突变的NOD2(BS-NOD2)(R314W)交叉(下调)调节TNBS-结肠炎潜在的TLR反应的能力。虽然给予完整的NOD2可以保护小鼠免受TNBS-结肠炎的影响，但过表达BS-NOD2并不能起到保护作用。对携带与BS患者相似的NOD2基因敲入突变的小鼠的研究证实了这些发现，在这些研究中，我们发现，给予NOD2配体(胞壁二肽，MDP)对DSS-结肠炎的保护作用不如对照小鼠治疗的小鼠。重要的是，在杂合子小鼠中也发现了这些保护的失败，在杂合子小鼠中，突变只发生在一个等位基因上；因此突变的NOD2发挥了显性负效应，这解释了BLAU突变具有常染色体显性效应的事实。因此，这些研究表明，BS-NOD2在“交叉调节”TLR诱导的胃肠道炎症的能力方面存在缺陷。在BLAU KI小鼠身上进行的抗胶原性关节炎的研究中也注意到了类似的缺陷。 最后，我们进行了研究，以确定携带BLAU突变的NOD2未能介导交叉调节的分子基础。我们发现，来自BS-NOD2Ki小鼠的MDP刺激的细胞未能上调IRF4的表达，IRF4已被证明通过去泛素化NF-kappaB信号成分来介导NOD2的交叉调节。事实上，在KI细胞中缺乏IRF4介导的交叉调节功能的事实是，这些细胞表现出的增强的TLR反应被慢病毒转导IRF4抑制。此外，虽然WT小鼠在注射MDP后炎症的肠道和关节组织中表达IRF4，但BLAU KI小鼠在类似条件下未能做到这一点 总体而言，这些研究表明，携带BS突变的NOD2缺乏通过无法激活IRF4来交叉调节TLR反应的能力。因此，这种突变使BS患者容易受到过度的TLR反应的影响，这可能会支持无菌组织部位的炎症。因此，影响NOD2的BLAU突变通过影响NOD2的免疫调节功能而导致炎症性疾病。

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.