Immunoregulatory Defects In Inflammatory Bowel Disease

炎症性肠病的免疫调节缺陷

• 批准号: 10692016
• 负责人: Warren Strober
• 金额: $ 26.04万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10692016
• 关键词: Acetylmuramyl-Alanyl-Isoglutamine; Alleles; Apoptosis; Autophagocytosis; Binding; Blau syndrome; Caspase; Cells; Cellular Stress; Clinical; Colitis; Crohn' s disease; Defect; Dendritic Cells; Development; Disease; Dominant-Negative Mutation; Epithelial; Epithelial Cells; Exhibits; Eye; Gastrointestinal tract structure; Gene Abnormality; Genes; Genetic Polymorphism; Genetic study; Goals; Granulomatous; Host Defense; Host Defense Mechanism; IRF4 gene; In Vitro; Individual; Inflammation; Inflammatory; Inflammatory Bowel Diseases; Inflammatory Response; Joints; Knock-in; Knock-in Mouse; Laboratories; Lentivirus; Ligands; Link; Mediating; Molecular; Molecular Abnormality; Mucous Membrane; Mus; Mutate; Mutation; NF-kappa B; Organism; Paneth Cells; Pathologic; Patients; Pattern; Peptides; Phagosomes; Play; Process; Production; Proteins; RIPK2 gene; Regulation; Reporting; Risk; Signal Transduction; Single Nucleotide Polymorphism; Site; Skin; Sterility; TNF gene; TRAF6 gene; Tissues; Ubiquitination; Ursidae Family; chemokine; cytokine; dextran sulfate sodium induced colitis; disorder risk; genetic linkage; genetic variant; gut inflammation; gut microbiota; ileum; immunoregulation; in vivo; interest; joint inflammation; loss of function; macrophage; overexpression; prevent; receptor; response; sensor; theories

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.

项目I - Blau综合征： 在我们定义Blau综合征中NOD 2突变的功能后果的初步研究中，我们评估了在HEK 293 T细胞中表达具有Blau突变的NOD 2的NOD 2构建体的功能。我们发现具有这些突变的NOD 2表现出降低的寡聚化、与RIPK 2相互作用或磷酸化和/或泛素化以及激活NF-κ B的能力。 重要的是，这些研究是在避免NOD 2过表达的影响的条件下进行的，NOD 2过表达是以前研究的特征。 然后，我们进行了体内研究，其中我们确定了过表达的完整NOD 2或具有Blau突变的NOD 2（BS-NOD 2）（R314 W）交叉（下调）调节TNBS-结肠炎或DSS-结肠炎的TLR应答的能力。尽管施用完整的NOD 2构建体保护小鼠免受TNBS-结肠炎，但BS-NOD 2的过表达未能保护。这些发现得到了携带NOD 2敲入突变的小鼠的研究的证实，这些小鼠与BS患者中的小鼠相似，其中我们表明，这样的小鼠不像用N 0 D2-配体（胞壁酰二肽，MDP）给药的同窝对照小鼠那样受到DSS-结肠炎的保护，事实上，突变的NOD 2等位基因对杂合子小鼠中的正常NOD 2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，我们发现，由于上述信号传导异常，来自BS-NOD 2 KI小鼠的MDP刺激的细胞不能上调IRF 4的表达，IRF 4是一种已被证明通过NF-κ B信号传导组分的去泛素化介导NOD 2交叉调节的因子。 Blau KI细胞中缺乏IRF 4介导的交叉调节功能，这在体外通过IRF 4的慢病毒转导抑制这些细胞表现出的增强的TLR应答的事实来证明。 此外，WT小鼠在MDP给药后在发炎的肠道和关节组织中表达IRF 4，而Blau KI小鼠在类似条件下未能表达。 总之，这些研究表明，携带BS突变的NOD 2缺乏通过其不能激活IRF 4来交叉调节TLR应答的能力。 因此，该突变使BS患者容易受到过度TLR反应的影响，这些反应有可能支持无菌组织部位的炎症。 项目II -Atg 16 L1多态性和克罗恩病： Atg 16 L1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体样过程，有助于处理丢弃的细胞内蛋白质并参与宿主防御的某些方面。十多年前，研究表明Atg 16 L1基因（Atg 16 L1 T300 A）中的单核苷酸多态性（SNP）增加了克罗恩病（CD）的风险。由于与多态性的存在相关的自噬减少导致上皮细胞凋亡增加，这可能是由于有毒细胞内物质的积累，多态性如何导致克罗恩病的主要理论是它损害上皮细胞存活。然而，本研究的结果支持一个非常不同的结论，即由多态性介导的缺陷性自噬导致增强的NF-κ B反应和促炎细胞因子产生。 在这些研究中，我们首先表明Atg 16 L1 T300 A多态性对巨噬细胞（可能也在树突状细胞中）TLR或NLR介导的信号传导的影响可能是克罗恩病炎症的主要贡献者。本文报道的支持这一观点的关键发现是巨噬细胞中完整的自噬调节NF-κ B活化，并且由多态性引起的或实际上由导致自噬丧失的其他分子异常引起的巨噬细胞中的缺陷性自噬伴随TLR或NLR诱导的NF-κ B活化增加以及促炎细胞因子/趋化因子的伴随增加。接下来，我们发现由于Atg 16 L1 T300 A多态性导致的缺陷性自噬导致NF-κ B激活因子（TRAF 6和RIP 2）的泛素化增加，这是由于p62（SQSTM 1）的细胞积累，这是一种隔离体受体分子，具有结合并泛素化TRAF 6和RIP 2的能力。最后，我们发现自噬缺陷细胞中p62的缺失导致这些细胞所显示的增强的NF-B活化的基本正常化，从而证明自噬对NF-B活化的作用主要与p62积累有关。从这些发现中得出的结论是，TLR或NLR刺激的巨噬细胞中的缺陷性自噬引起增加的NF-κ B介导的促炎反应，其本身可能是导致克罗恩病的胃肠道中对肠道微生物的高反应性的原因。 非常令人感兴趣的是，从携带克罗恩病相关Atg 16 L1多态性的正常个体获得的巨噬细胞显示出相同的NF-κ B活化增加。 这表明，这种多态性是一种遗传决定的宿主防御机制，赋予承载个体对病理因子的反应增强，代价是过度粘膜反应和克罗恩病的风险更大。