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Alterations of leukocyte integrin signaling leading to diabetes and autoimmunity

白细胞整合素信号的改变导致糖尿病和自身免疫

基本信息

批准号：
10502136
负责人：
Mark S Anderson
金额：
$ 64.67万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-08-12 至 2026-07-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10502136
关键词：
Actins Adaptor Signaling Protein Address Adhesions Adoptive Cell Transfers Adoptive Transfer Affect Animal Model Animals Antigen Presentation Antigens Autoantibodies Autoimmune Autoimmune Diseases Autoimmunity B-Lymphocytes Biochemical Biological Breeding CRISPR/Cas technology CTLA4 gene Cell Adhesion Cell Communication Cell Line Cell surface Cells Clone Cells Critical Pathways Data Defect Dendritic Cells Detection Development Diabetes Mellitus Disease Engineering Enrollment Enzyme-Linked Immunosorbent Assay Enzymes Etiology Event Experimental Models Family Female Frequencies Generations Genes Genetic Genetic Polymorphism Genetic Risk Genetic study Guanosine Triphosphate Phosphohydrolases Histologic Human Cell Line Human Genome Hyperactivity Hyperglycemia ITGB2 gene Image Immune Immune Tolerance Inbred BALB C Mice Inbred NOD Mice Incidence Individual Inflammation Inherited Insulin Insulin-Dependent Diabetes Mellitus Integrin Signaling Pathway Integrins Islets of Langerhans Kinetics Knock-in Knock-in Mouse Lead Leukocytes Life Link Lipid Binding Mediating Modeling Mouse Strains Mus Mutagenesis Mutation Myeloid Cells Neutrophil Activation Non obese Open Reading Frames Pathogenicity Pathway interactions Patients Phenotype Phosphatidylinositols Phosphotransferases Pre-Clinical Model Precision therapeutics Protein Region RNA Sequence Analysis Registries Regulator Genes Reporting Risk Factors STAT3 gene Signal Transduction Signaling Molecule T-Lymphocyte T-cell receptor repertoire Testing Therapeutic Transgenic Mice Transgenic Organisms Tumor-infiltrating immune cells Variant adhesion receptor autoreactive T cell diabetic draining lymph node early onset exome sequencing experimental study gain of function gain of function mutation genetic linkage genetic risk factor genome wide association study immune activation immune function insulin dependent diabetes mellitus onset insulitis islet knockin animal leukocyte activation macrophage male mouse model mutant neutrophil prevent two photon microscopy

项目摘要

Project Summary/Abstract The study of the genetic basis for type 1 diabetes (T1D) has benefited tremendously from examination of rare individuals with likely monogenic forms of the disease. Combined with GWAS, a number of polymorphisms in immune regulatory genes have been defined that contribute to genetic risk for T1D. Using whole exome sequencing of individuals with T1D enrolled in a monogenic diabetes registry, we identified an individual with a gain-of-function mutation in the SKAP2 gene, as well as several other T1D patients with potentially pathogenic variants in other leukocyte integrin signaling genes. These patients tend to have a number of autoimmune manifestations in addition to T1D, indicating defects in critical pathways of immune tolerance. Multiple GWAS studies have identified a strong genetic linkage between SKAP2 polymorphisms and T1D (at a frequency of ~20%), however the mechanisms by which alteration of SKAP2 could lead to autoimmune T1D are unknown. SKAP2 is expressed primarily in myeloid cells, where it functions as an adapter protein in the integrin signaling pathway, linking cell surface integrins to WASP and actin rearrangements that occur following leukocyte adhesion. The SKAP2 G153R mutation in our patient resulted in constitutive association of SKAP2 with WASP leading to a hyperadhesive phenotype in macrophages cultured from the patient or macrophages engineered to contain the SKAP G153R substitution. To understand how activation of leukocyte integrin signaling may contribute to T1D, we have generated knock-in (KI) mice containing the G153R substitution in murine Skap2, on the NOD genetic background. Female NOD.SKAP2 KI mice have a higher incidence and earlier onset of T1D than do NOD.WT animals; male NOD.SKAP2 also develop T1D (incidence ~50%) while male NOD.WT do not develop frank hyperglycemia. Initial analysis of these mice reveals evidence of ongoing inflammation early in life with development of a broad spectrum of auto-reactive antibodies. Dendritic cells from NOD.SKAP2 KI mice have increased antigen presenting activity to islet-specific transgenic T-cells while neutrophils from these mice show evidence of increased integrin signaling. These observations demonstrate that the NOD.SKAP2 KI mice appropriately model the autoimmune T1D disease observed in our patient. The project proposes to complete the analysis of these mice, under the hypothesis that increased cell adhesion in dendritic cells leads to prolonged DC-T cell interactions, which drives selection of auto-reactive T-cell clones leading to development of T1D, associated with broad spectrum autoimmunity. We will test this hypothesis in a variety of adoptive cell transfer experiments, by generation of conditional knock-in mice and by imaging of DC-T cell interactions in the inflamed islets. Similar studies will be performed for other candidate leukocyte integrin signaling mutations identified in the monogenic T1D registry. This study will address whether dysregulation of leukocyte integrin signaling may constitute an unrecognized genetic risk factor for T1D, suggesting potential alterative therapeutic approaches for these patients.

项目总结/摘要 1型糖尿病（T1 D）遗传基础的研究从罕见的可能患有单基因型疾病的个体。与GWAS结合，免疫调节基因已被确定为导致T1 D遗传风险的基因。使用整个外显子组通过对登记在单基因糖尿病登记处的T1 D个体进行测序，我们确定了一个具有 SKAP 2基因中的功能获得性突变，以及其他几种具有潜在致病性的T1 D患者，其他白细胞整合素信号基因的变体。这些患者往往有一些自身免疫性除了T1 D之外，还存在其他临床表现，表明免疫耐受的关键途径存在缺陷。多个GWAS 研究已经确定了SKAP 2多态性和T1 D之间的强烈遗传联系（频率为 ~20%），然而SKAP 2的改变可导致自身免疫性T1 D的机制尚不清楚。 SKAP 2主要在髓样细胞中表达，在髓样细胞中它作为整合素信号传导中的衔接蛋白发挥作用。途径，将细胞表面整合素与WASP和肌动蛋白重排联系起来，粘连我们患者的SKAP 2 G153 R突变导致SKAP 2与WASP的组成性关联导致从患者培养的巨噬细胞或工程化的巨噬细胞中的超粘附表型含有SKAP G153 R取代。为了了解白细胞整合素信号的激活如何我们已经产生了在鼠Skap 2中含有G153 R置换的敲入（KI）小鼠， NOD基因背景雌性NOD.SKAP2 KI小鼠具有更高的发病率和更早的发病， T1 D的发生率高于NOD.WT动物;雄性NOD. SKAP 2也发生T1 D（发生率约50%），而雄性NOD.WT 不要出现明显的高血糖。对这些小鼠的初步分析揭示了持续炎症的证据在生命的早期，随着广谱自身反应性抗体的发展。树突状细胞 SKAP 2 KI小鼠对胰岛特异性转基因T细胞的抗原呈递活性增加，来自这些小鼠的嗜中性粒细胞显示出整联蛋白信号传导增加的证据。这些观察证明 SKAP 2 KI小鼠适当地模拟了在我们的患者中观察到的自身免疫性T1 D疾病。的该项目提议在细胞粘附增加的假设下完成对这些小鼠的分析。树突状细胞导致DC-T细胞相互作用延长，这驱动了自身反应性T细胞克隆的选择导致与广谱自身免疫相关的T1 D的发展。我们将测试这个假设，各种过继细胞转移实验，通过产生条件性敲入小鼠和通过成像，炎症胰岛中的DC-T细胞相互作用。将对其他候选白细胞进行类似研究在单基因T1 D登记中鉴定的整合素信号传导突变。本研究将探讨白细胞整联蛋白信号传导的失调可能构成T1 D的未被认识的遗传危险因素，提示对这些患者的潜在替代治疗方法。