Immunoregulatory Defects In Inflammatory Bowel Disease

项目一：布劳综合症。为了确定Blau综合征中NOD2的NBD （domain domain）突变引起的免疫异常，我们首先评估了HEK293T细胞中表达带有Blau突变的NOD2构建体的功能。我们发现，具有这些突变的NOD2表现出低聚化、与NOD2的直接下游信号成分RIPK2相互作用或磷酸化和泛素化的能力降低；反过来，这与NF-kappaB激活能力的降低有关。接下来，我们进行了体内研究，确定了完整NOD2或带有Blau突变的NOD2 (BS-NOD2) （R314W）交叉（下调）调节TLR反应的能力，这是tnbs -结肠炎或dss -结肠炎中结肠炎症发展所必需的。然而，通过多种策略过表达完整NOD2构建体的小鼠表现出tnbs -结肠炎的显著减轻（如先前广泛的研究所示），过表达BS-NOD2的小鼠没有表现出这种tnbs -结肠炎的减轻。这些发现得到了对携带NOD2敲入突变的小鼠的研究的证实，这些小鼠与BS患者相似，在研究中我们发现，NOD2配体（muramyl dipeptide， MDP）给药对dss -结肠炎的保护作用不像对照组小鼠那样明显；事实上，在杂合小鼠中也观察到这一点，表明突变的NOD2等位基因对正常的NOD2等位基因表现出显性负向作用。最后，在确定这些交叉调控缺陷的分子基础的研究中，我们发现，由于上述信号异常，dp刺激的BS-NOD2 KI小鼠细胞不能上调IRF4的表达，IRF4是一种通过NF-kappaB信号成分的去泛素化介导NOD2交叉调控的因子。体外实验表明，Blau KI细胞缺乏IRF4介导的交叉调节功能，这些细胞所表现出的增强的TLR反应被IRF4的慢病毒转导抑制。此外，在MDP处理后，WT小鼠在炎症的肠道和关节组织中表达IRF4，而Blau KI小鼠在相似的条件下没有表达IRF4。总的来说，这些研究表明，携带BS突变的NOD2通过无法激活IRF4而缺乏交叉调节TLR反应的能力。因此，这种突变使BS患者容易受到TLR反应过度的影响，而TLR反应有可能在无菌组织部位诱发炎症。

Project I - Blau Syndrome. To define the immunologic abnormalities that arise from mutations of NBD (donain) of NOD2 in Blau Syndrome we first 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 and to interact with or phosphorylate and ubiquitinate the immediate down-stream signaling component of NOD2, RIPK2; this, in turn, was associated with a reduced ability to activate NF-kappaB. We next conducted in vivo studies in which we determined the capacity of intact NOD2 or NOD2 with a Blau mutation (BS-NOD2) (R314W) to cross (down)-regulate TLR responses necessary for the development of colonic inflammation in TNBS-colitis or in DSS-colitis. Whereas mice over-expressing intact NOD2 constructs by several strategems exhibited greatly reduced TNBS-colitis (as shown in extensive previous studies) over-expression of BS-NOD2 didn't exhibit such reduced TNBS-colitis. 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; in fact, this was also observed in heterozygous mice, indicating that the mutated NOD2 allele exhibited dominant-negative effects on the normal NOD2 allele. Finally, in studies to determined the molecular basis of these cross-regulation defects 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 suggest 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 induce inflammation at sterile tissue sites. Project II - Atg16L1 T300A Polymorphism and Crohn's disease: The Atg16L1 gene encodes a protein essential to the development 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 cell survival. Results of the present study, however, support an alternative 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 autophagy in macrophages regulates NF-kB activation, in 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/chemokine production. In addition, enhanced autophagy is accompanied by decreased TLR and NLR-induced NF-kB responses and a concomitant decrease in pro-inflammatory cytokine/chemokine production. Thus, the level of autophagy emerged as a regulator of inflammation. Next, we showed that defective autophagy due to the Atg16L1 T300A polymorphism leads to increased ubiquitination of TLR/NLR 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-kB activation otherwise displayed by these cells and thus proved that the autophagy effect on NF-kB activation is mainly related to p62 accumulation. The conclusion that can be drawn 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. Another conclusion that arises from this finding is that the Atg16L1 polymorphism associated with Crohn's disease must be accompanied by other genetic abnornalities to cause this disease.