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.

项目概要/摘要 1 型糖尿病 (T1D) 遗传基础的研究极大地受益于对罕见疾病的检查。可能患有该疾病的单基因形式的个体。结合GWAS，发现多个多态性免疫调节基因已被确定为导致 T1D 遗传风险的因素。使用全外显子组通过对单基因糖尿病登记处登记的 1 型糖尿病患者进行测序，我们确定了一名患有 SKAP2 基因的功能获得性突变，以及其他几位具有潜在致病性的 T1D 患者其他白细胞整合素信号基因的变异。这些患者往往患有多种自身免疫性疾病除 T1D 之外的其他表现，表明免疫耐受的关键途径存在缺陷。多重GWAS 研究已发现 SKAP2 多态性与 T1D 之间存在很强的遗传联系（频率为 ~20%），然而 SKAP2 的改变可能导致自身免疫性 T1D 的机制尚不清楚。 SKAP2 主要在骨髓细胞中表达，在整合素信号传导中充当衔接蛋白途径，将细胞表面整合素与白细胞后发生的 WASP 和肌动蛋白重排联系起来附着力。我们患者的 SKAP2 G153R 突变导致 SKAP2 与 WASP 的组成型关联导致从患者培养的巨噬细胞或工程巨噬细胞出现过度粘附表型包含 SKAP G153R 替代品。了解白细胞整合素信号传导的激活如何可能有助于 T1D，我们已经产生了含有鼠 Skap2 中 G153R 取代的敲入 (KI) 小鼠，关于NOD遗传背景。雌性 NOD.SKAP2 KI 小鼠的发病率较高且发病较早 T1D 比 NOD.WT 动物好；男性 NOD.SKAP2 也会发展 T1D（发病率约 50%），而男性 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早期，随着广泛的自身反应性抗体的发展。树突状细胞来自 NOD.SKAP2 KI 小鼠对胰岛特异性转基因 T 细胞的抗原呈递活性增加，同时这些小鼠的中性粒细胞显示出整合素信号增强的证据。这些观察表明 NOD.SKAP2 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 T1D 疾病。这该项目建议完成对这些小鼠的分析，假设细胞粘附增加树突状细胞导致 DC-T 细胞相互作用延长，从而推动自身反应性 T 细胞克隆的选择导致 T1D 的发展，与广谱自身免疫相关。我们将在通过生成条件敲入小鼠并通过成像进行各种过继细胞转移实验发炎胰岛中的 DC-T 细胞相互作用。将对其他候选白细胞进行类似的研究单基因 T1D 登记中发现的整合素信号突变。这项研究将解决是否白细胞整合素信号传导失调可能构成 T1D 的一个未被识别的遗传风险因素，为这些患者提出潜在的替代治疗方法。