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FOXP3 ΔE2 Isoform in Treg Function and Pathophysiology of Lupus

FOXP3 αE2 亚型在 Treg 功能和狼疮病理生理学中的作用

基本信息

批准号：
10194961
负责人：
BAOHUA ZHOU
金额：
$ 23.78万
依托单位：
INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-04-12 至 2023-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10194961
关键词：
Alleles Allergic Allergic Disease Alternative Splicing Amino Acids Asthma Autoimmune Diseases CD4 Positive T Lymphocytes Cells Clinical Protocols Data Deletion Mutation Development Disease Ectopic Expression Effector Cell Environment Exons FOXP3 gene Female Flow Cytometry Frequencies Functional disorder Grant Health Homeostasis Human Hypersensitivity Immune Immune Tolerance Immune response Immune system In Vitro Inflammation Lead Length Link Lupus Mediating Methods Modeling Mus Mutation Outcome Patients Pharmacology Phenotype Physiological Play Protein Isoforms RNA Splicing Regulatory Element Regulatory T-Lymphocyte Risk Risk Factors Role Severities Symptoms Syndrome Systemic Lupus Erythematosus Testing Therapeutic Thymus Gland X Inactivation autoreactivity base cytokine demethylation design early onset in vivo insight mRNA Precursor novel novel strategies overexpression programs single-cell RNA sequencing systemic autoimmune disease systemic autoimmunity transcription factor transcriptomics treatment strategy

项目摘要

PROJECT SUMMARY FOXP3 is a key transcription factor for the development and function of regulatory T cells (Tregs) and amorphic mutations at this locus in humans and mice lead to systemic autoimmune diseases and allergic symptoms. FOXP3 gene is highly conserved with 87% amino acid identity between humans and mice. Human FOXP3 however has two major isoforms, a full length (FOXP3 FL isoform) and an alternatively spliced isoform lacking exon 2 (FOXP3 ∆E2 isoform), while mouse Foxp3 gene only produce the FOXP3 FL isoform. The roles of these two isoform in health and disease have not been clearly defined. In this grant we present evidence that patients who only express the FOXP3 ΔE2 isoform due to mutations developed autoimmune diseases. We generated mice with Foxp3 exon 2 deletion to study the functionality of FOXP3 ΔE2 isoform in vivo and demonstrated that Foxp3 ΔE2 mice developed a systemic autoimmunity resembling systemic lupus erythematosus (SLE). We further demonstrated that Tregs expressing only the Foxp3 ΔE2 isoform were unstable, transdifferentiated to effector T helpers, and were sufficient to induce SLE-like disease when transferred into Tcrb-/- mice. We hypothesize that FOXP3 ΔE2, one of the two major isoforms in humans, is a risk factor for development and/or severity of SLE. We will test our hypothesis in the following specific aims. (1) Define the role of Foxp3 ΔE2 Tregs on lupus onset and severity. We hypothesize that Foxp3 ΔE2 Tregs have a different transcriptomic program from Foxp3 FL Tregs and render them to be lineage instability. Under lupus inducing environment, Foxp3 ΔE2 Tregs lose lineage identity in the periphery and transdifferentiate into autoreactive effector cells, resulting earlier onset and/or more severe disease even in the presence of Foxp3 FL Tregs. (2) Define the impact of FOXP3 ΔE2 isoform on human Treg phenotypes and disease course of systemic lupus. We hypothesize that the ratio of FOXP3 isoform expression determines human Treg phenotypes and correlates with disease course of systemic lupus. We will determine whether the FOXP3 ΔE2:FOXP3 FL ratio, the existence and frequency of Tregs expressing mainly FOXP3 ΔE2 isoform correlate with disease state of SLE using flow cytometry and a specially designed method of single cell RNA-Seq. We will further test whether cytokine milieu in SLE patients regulates FOXP3 pre-mRNA splicing thus shift the isoform expression. The information learned from these studies will provide novel insights into the physiological functions of the naturally existing human FOXP3 ΔE2 isoform and would pave the way to develop efficient clinical protocols aimed at shifting FOXP3 isoform expression as therapeutic treatment of SLE as well as other autoimmune diseases.

项目概要 FOXP3 是调节性 T 细胞 (Treg) 发育和功能的关键转录因子，人类和小鼠中该位点的无定形突变会导致系统性自身免疫性疾病和过敏症状。 FOXP3基因高度保守，人和小鼠之间的氨基酸一致性为87%。人类然而，FOXP3 有两种主要同种型，全长（FOXP3 FL 同种型）和选择性剪接同种型缺少外显子 2（FOXP3 ΔE2 同种型），而小鼠 Foxp3 基因仅产生 FOXP3 FL 同种型。角色这两种异构体在健康和疾病方面的作用尚未明确定义。在这笔赠款中，我们提供了证据由于突变而仅表达 FOXP3 ΔE2 同工型的患者会患上自身免疫性疾病。我们生成了 Foxp3 外显子 2 缺失的小鼠，以研究 FOXP3 ΔE2 亚型的体内功能证明 Foxp3 ΔE2 小鼠产生了类似于系统性狼疮的系统性自身免疫红斑狼疮（SLE）。我们进一步证明仅表达 Foxp3 ΔE2 亚型的 Tregs 不稳定，转分化为效应 T 辅助细胞，并且足以诱发 SLE 样疾病转移到 Tcrb-/- 小鼠中。我们假设 FOXP3 ΔE2（人类的两种主要亚型之一）是一种 SLE 发展和/或严重程度的危险因素。我们将在以下具体目标中检验我们的假设。 (1) 定义 Foxp3 ΔE2 Tregs 对狼疮发病和严重程度的作用。我们假设 Foxp3 ΔE2 Tregs 具有与 Foxp3 FL Tregs 不同的转录组程序，并使它们具有谱系不稳定性。狼疮下诱导环境下，Foxp3 ΔE2 Tregs 失去外围谱系特征并转分化为即使存在 Foxp3，自身反应性效应细胞也会导致更早发病和/或更严重的疾病 FL 特雷格斯。 (2)定义FOXP3 ΔE2亚型对人类Treg表型和病程的影响系统性狼疮。我们假设 FOXP3 同工型表达的比例决定了人类 Treg 表型并与系统性狼疮的病程相关。我们将确定 FOXP3 是否 ΔE2：FOXP3 FL比率，主要表达FOXP3 ΔE2亚型的Tregs的存在和频率相关使用流式细胞术和专门设计的单细胞 RNA 测序方法来了解 SLE 的疾病状态。我们将进一步测试 SLE 患者的细胞因子环境是否调节 FOXP3 pre-mRNA 剪接，从而改变同种型表达。从这些研究中学到的信息将为生理学提供新的见解天然存在的人类 FOXP3 ΔE2 同工型的功能，将为开发高效的旨在改变 FOXP3 同工型表达作为 SLE 以及其他疾病治疗的临床方案自身免疫性疾病。