Immunoregulatory Defects In Inflammatory Bowel Disease

炎症性肠病的免疫调节缺陷

• 批准号: 10272022
• 负责人: Warren Strober
• 金额: $ 28.15万
• 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/10272022
• 关键词: Acetylmuramyl-Alanyl-Isoglutamine; Affect; Alleles; Arthritis; Binding; Blau syndrome; Cells; Colitis; Collagen Arthritis; Crohn' s disease; Defect; Development; Dimerization; Disease; Distant; Dominant-Negative Mutation; Exanthema; Exhibits; Failure; Family; Functional disorder; Gastrointestinal tract structure; Genes; Genetic Polymorphism; Goals; Granulomatous; IRF4 gene; Immune response; Impairment; In Vitro; Individual; Inflammation; Inflammatory; Inflammatory Bowel Diseases; Innate Immune Response; Intestines; Joints; Knock-in; Knock-in Mouse; Leucine; Ligands; Link; Mediating; Molecular; Molecular Abnormality; Mucosal Immune System; Mus; Mutate; Mutation; NF-kappa B; Nucleotides; Organism; Patient Care; Patients; Procedures; RIPK2 gene; Regulation; Risk; Signal Transduction; Site; Sterility; Subfamily lentivirinae; Tissues; Ubiquitination; Ulcerative Colitis; Uveitis; disease-causing mutation; dysbiosis; gut microbiota; immunoregulation; in vivo; innate immune function; microbiota; microorganism; overexpression; prevent; response; sensor; theories

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.

为了探索由NOD 2的NBD中的突变引起的疾病的机制，我们首先进行体外研究以调查携带各种NBD突变的NOD 2构建体支持NOD 2信号传导功能的能力。我们发现，携带这种突变并在HEK 293 T细胞中表达的NOD 2表现出寡聚化、与RIPK 2相互作用或激活RIPK 2以及激活NF-κ B的能力降低。 这些研究是在避免过度表达效应的条件下进行的，过度表达效应是沿着这些路线的先前研究的特征。 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结肠炎潜在TLR应答的能力。尽管施用完整的NOD 2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，NOD 2配体（胞壁酰二肽，MDP）给药对这些小鼠的DSS-结肠炎保护作用不如经LPS处理的同窝对照小鼠。重要的是，在杂合子小鼠中也注意到这些保护失败，其中突变仅发生在一个等位基因上;因此突变的NOD 2产生显性负效应，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-NOD 2在“交叉调节”TLR诱导的GI炎症的能力方面表现出缺陷。在Blau KI小鼠中进行的抗胶原性关节炎研究中也发现了类似的缺陷。 最后，我们进行了研究，以确定的NOD 2轴承Blau突变介导的交叉调节失败的分子基础。我们表明，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。事实上，KI细胞中缺乏IRF 4介导的交叉调节功能，这一事实表明，这些细胞表现出的增强的TLR应答被IRF 4的慢病毒转导抑制。 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小鼠在类似条件下不能表达IRF 4 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。 因此，影响NOD 2的Blau突变通过影响NOD 2免疫调节功能而引起炎性疾病。