权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Immunoregulatory Defects In Inflammatory Bowel Disease

炎症性肠病的免疫调节缺陷

基本信息

批准号：
10927727
负责人：
Warren Strober
金额：
$ 28.06万
依托单位：
NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
依托单位国家：
美国
项目类别：
财政年份：
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/10927727
关键词：
Acetylmuramyl-Alanyl-Isoglutamine Alleles Apoptosis Autophagocytosis Binding Blau syndrome CASP3 gene Cell Survival Cells Cellular Stress Clinical Colitis Colonic inflammation Crohn&apos s disease Defect Dendritic Cells Deubiquitination Development Disease Dominant-Negative Mutation Epithelial Cells Epithelium Exhibits Eye Gastrointestinal tract structure Gene Abnormality Gene Expression Genes Genetic Genetic Polymorphism Goals Heterozygote Host Defense Host Defense Mechanism IRF4 gene Immunologics In Vitro Individual Inflammation Inflammatory Inflammatory Bowel Diseases Inflammatory Response Innate Immune Response Joints Knock-in Knock-in Mouse Laboratories Ligands Link Macrophage Mediating Molecular Molecular Abnormality Mucous Membrane Mus Mutate Mutation NF-kappa B Organism Other Genetics Outcome Paneth Cells Pathologic Patients Pattern Peptides Phagosomes Phosphorylation Play Predisposition Process Production Proteins RIPK2 gene Regulation Risk Signal Transduction Single Nucleotide Polymorphism Site Skin Sterility Stimulus TNF gene TRAF6 gene Tissues Ubiquitination autosome chemokine cytokine dextran sulfate sodium induced colitis gene function genetic linkage genetic variant genome wide association study gut inflammation gut microbiota ileum immunoregulation in vivo interest joint inflammation lentivirally transduced loss of function overexpression receptor response sensor theories

项目摘要

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.

I计划- Blau综合征。为了确定由Blau综合征中NOD 2的NBD（donain）突变引起的免疫学异常，我们首先评估了在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，NOD 2与这些突变表现出降低的能力，寡聚化和相互作用或磷酸化和泛素化的直接下游信号成分的NOD 2，RIPK 2，这反过来，与降低的能力，激活NF-κ B。我们接下来进行了体内研究，其中我们确定了完整的NOD 2或具有Blau突变的NOD 2（BS-NOD 2）（R314 W）交叉（下调）调节TLR应答的能力，所述TLR应答是TNBS-结肠炎或DSS-结肠炎中结肠炎症发展所必需的。然而，通过几种策略过表达完整NOD 2构建体的小鼠表现出极大降低的TNBS-结肠炎（如广泛的先前研究所示），BS-NOD 2的过表达没有表现出这种降低的TNBS-结肠炎。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患者中的突变相似，我们在这些研究中表明，NOD 2配体不能保护这些小鼠免受DSS-结肠炎的影响（胞壁酰二肽，MDP）给药，如经顺铂处理的同窝对照小鼠;事实上，这也在杂合子小鼠中观察到，表明突变的NOD 2等位基因对正常的NOD 2等位基因表现出显性负效应。最后，在确定这些交叉调节缺陷的分子基础的研究中，我们表明，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是一种已显示通过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，这有可能在无菌组织部位诱导炎症。项目II -Atg 16 L1 T300 A多态性和克罗恩病： Atg 16 L1基因编码一种对自噬发展至关重要的蛋白质，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，可能是由于细胞内有毒物质的积累，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，本研究的结果支持另一个结论，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，我们首先表明巨噬细胞中的自噬调节NF-κ B活化，因为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着TLR或NLR诱导的NF-κ B活化的增加以及促炎细胞因子/趋化因子产生的伴随增加。此外，增强的自噬伴随着TLR和NLR诱导的NF-κ B应答的降低以及促炎细胞因子/趋化因子产生的伴随减少。因此，自噬水平成为炎症的调节因子。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TLR/NLR 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，这是一种螯合体受体分子，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-κ B活化的基本正常化，从而证明自噬对NF-κ B活化的作用主要与p62积累有关。从这些发现中可以得出的结论是，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，其本身可以是导致克罗恩病的胃肠道中对肠道生物体的高反应性的原因。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，它赋予携带个体对病理因子的反应增强，但代价是过度粘膜反应和克罗恩病的风险更大。从这一发现中得出的另一个结论是，与克罗恩病相关的Atg 16 L1多态性必须伴有其他遗传异常才能引起这种疾病。

项目成果

期刊论文数量（18）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Blau syndrome NOD2 mutations result in loss of NOD2 cross-regulatory function.

DOI：
10.3389/fimmu.2022.988862
发表时间：
2022
期刊：
FRONTIERS IN IMMUNOLOGY
影响因子：
7.3
作者：
Mao, Liming;Dhar, Atika;Meng, Guangxun;Fuss, Ivan;Montgomery-Recht, Kim;Yang, Zhiqiong;Xu, Qiuyun;Kitani, Atsushi;Strober, Warren
通讯作者：
Strober, Warren

Adherent-invasive E. coli in Crohn disease: bacterial "agent provocateur".

克罗恩病中的粘附侵袭性大肠杆菌：细菌“挑衅者”。