Immunoregulatory Defects In Inflammatory Bowel Disease

项目I--布劳综合征： 在我们的初步研究中，为了确定BLAU综合征中NOD2突变的功能后果，我们评估了表达带有BLAU突变的NOD2的NOD2构建体在HEK293T细胞中的功能。我们发现，具有这些突变的NOD2表现出低聚、与RIPK2相互作用或磷酸化和/或泛素化以及激活NF-kappaB的能力降低。重要的是，这些研究是在避免NOD2过度表达的条件下进行的，这是之前研究的特点。然后，我们进行了体内研究，确定了过表达的完整NOD2或带有BLAU突变的NOD2(BS-NOD2)(R314W)交叉(下调)调节TNBS结肠炎或DSS结肠炎潜在的TLR反应的能力。尽管给予完整的NOD2构建体可以保护小鼠免受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敲入突变的小鼠的研究证实了这些发现，其中我们发现，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处理的那样，事实上，在杂合子小鼠中，突变的NOD2等位基因对正常NOD2等位基因表现出显性-负效应。最后，我们进行了研究，以确定这些交叉调节缺陷的分子基础。在这里，我们表明，由于上述信号异常，来自BS-NOD2Ki小鼠的MDP刺激细胞未能上调IRF4的表达，IRF4已被证明通过去泛素化NF-kappaB信号成分介导NOD2交叉调节。在体外BLAU KI细胞缺乏IRF4介导的交叉调节功能，这是因为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，尽管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。 项目II--Atg16L1基因多态性与克罗恩病： Atg16L1基因编码一种对自噬功能至关重要的蛋白质，自噬是一种进化上保守的吞噬小体过程，有助于丢弃的细胞内蛋白质的处理，并参与宿主防御的某些方面。十多年前的研究表明，Atg16L1基因(Atg16L1T300A)的单核苷酸多态性(SNP)增加了克罗恩病(CD)的发病风险。由于与多态相关的自噬减少导致上皮细胞凋亡增加，可能是由于细胞内有毒物质的积累，因此多态导致克罗恩病的主要理论是它损害了上皮细胞的存活。然而，目前的研究结果支持一个非常不同的结论，即由该多态性介导的缺陷自噬导致了增强的核因子-kB反应和促炎细胞因子的产生。 在这些研究中，我们首先表明，Atg16L1 T300A多态对巨噬细胞(可能也包括树突状细胞)中TLR或NLR介导的信号转导的影响，可能是克罗恩病炎症的主要因素。支持这一观点的主要发现是，巨噬细胞中的完整自噬调节着核因子-kB的激活，而巨噬细胞中由该基因多态引起的自噬缺陷，或者实际上是由导致自噬丧失的其他分子异常引起的，伴随着TLR或NLR诱导的核因子-kB的激活以及伴随的促炎细胞因子/趋化因子的增加。接下来，我们证明了由于Atg16L1T300A多态导致的缺陷自噬导致了核因子-kB激活因子(TRAF6和RIP2)泛素化的增加，这是细胞积累p62(SQSTM1)的结果，p62(SQSTM1)是一种能够与TRAF6和RIP2结合并泛素化的隔离小体受体分子。最后，我们发现在自噬缺陷细胞中p62的缺失导致这些细胞表现出的增强的核因子-B激活的基本正常化，从而证明了自噬对核因子-B激活的影响主要与p62的积聚有关。从这些发现得出的结论是，TLR或NLR刺激的巨噬细胞自噬缺陷导致核因子-kB介导的促炎反应增加，这本身可能是导致克罗恩病背后的胃肠道共生生物高反应性的原因之一。 非常令人感兴趣的事实是，从携带克罗恩病相关Atg16L1多态的正常人获得的巨噬细胞也显示出同样的NF-kB激活增加。这表明，这种多态是一种遗传决定的宿主防御机制，它赋予生育个体对病原体的增强反应，但代价是有更大的风险出现过度的粘膜反应和克罗恩病。

Project I - Blau Syndrome: In our initial studies to define the functional consequences of mutations of NOD2 in Blau Syndrome we assessed the function of NOD2 constructs expressing NOD2 with a Blau mutations in HEK293T cells. We found that NOD2 with these mutations exhibits a reduced ability to oligomerize, to interact with or phosphorylate and/or ubiquitinate RIPK2 and to activate NF-kappaB. Importantly, these studies were conducted under conditions that avoided effects of NOD2 over-expression that mat have characterized previous studies. We then conducted 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 or DSS-colitis. Whereas administration of intact NOD2 constructs 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 and, in fact, the mutated NOD2 allele exhibited dominant-negative effects on the normal NOD2 allele in heterozygous mice. Finally, we conducted studies to determined the molecular basis of these cross-regulation defects. Here we showed that MDP-stimulated cells from BS-NOD2 KI mice, as a result of the signaling abnormality described above, 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. Lack of IRF4-mediated cross-regulatory function in Blau KI cells was shown in vitro 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. Project II - Atg16L1 Polymorphism and Crohn's disease: The Atg16L1 gene encodes a protein essential to the function of autophagy, an evolutionarily conserved phagosome-like process that facilitates the disposal of discarded intra-cellular proteins and participates in some aspects of host defense. Over a decade ago it was shown that a single nucleotide polymorphism (SNP) in the Atg16L1 gene (Atg16L1T300A) confers increased risk for the development of Crohns disease (CD). Inasmuch as the decreased autophagy associated with the presence of the polymorphism causes increased epithelial apoptosis perhaps due to accumulation of toxic intracellular substances, the main theory of how the polymorphism results in Crohn's disease is that it compromises epithelial survival. Results of the present study, however, support a very different conclusion, namely that the defective autophagy mediated by the polymorphism leads to enhanced NF-kB responses and pro-inflammatory cytokine production. In these studies we showed first that the effect of the Atg16L1 T300A polymorphism on TLR- or NLR-mediated signaling in macrophages (and perhaps in dendritic cells as well), is likely to be a major contributor to Crohns disease inflammation. The key findings reported here supporting this view were that intact autophagy in macrophages regulates NF-kB activation, and that defective autophagy in macrophages caused by the polymorphism or indeed caused by other molecular abnormalities resulting in loss of autophagy is accompanied by increased TLR- or NLR-induced NF-kB activation and a concomitant increase in pro-inflammatory cytokine/chemokines. Next, we showed that defective autophagy due to the Atg16L1T300A polymorphism leads to increased ubiquitination of NF-kB activating factors (TRAF6 and RIP2) as a result of cellular accumulation of p62 (SQSTM1), a sequesterosome receptor molecule that has the capacity to bind to and ubiquitinate TRAF6 and RIP2. Finally, we showed that deletion of p62 in autophagy-defective cells results in substantial normalization of the enhanced NF-B activation otherwise displayed by these cells and thus prove that the autophagy effect on NF-B activation is mainly related to p62 accumulation. The conclusion that emerged from these findings is that defective autophagy in TLR- or NLR-stimulated macrophages causes increased NF-kB-mediated pro-inflammatory responses which itself can be a cause of the hyper-responsiveness to commensal organisms in the GI tract that underlies Crohn's disease. Of very considerable interest was the fact that the same increase in NF-kB activation was displayed by macrophages obtained from normal individuals bearing the Crohn's disease-associated Atg16L1 polymorphism. This suggests that this polymorphism is a genetically determined host-defense mechanism that confers upon the bearing individual an enhanced response to pathologic agents at the expense os a greater risk for excess mucosal responses and Crohn's disease.