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.

项目摘要 FOXP3是调节性T细胞（Tregs）和在人类和小鼠的该基因座的非态突变导致系统性自身免疫性疾病和过敏性疾病症状。 Foxp3基因高度保守，人类和小鼠之间具有87％的氨基酸身份。人类 FOXP3有两个主要的同工型，一个全长（FOXP3 FL同工型）和一个剪接的同工型缺少外显子2（FOXP3 ∆E2同工型），而鼠标FOXP3基因仅产生FOXP3 FL同工型。角色在这两种健康和疾病中的同工型中，尚未明确定义。在这笔赠款中，我们提供证据由于突变引起的自身免疫性疾病，仅表达FOXP3ΔE2同工型的患者。我们用Foxp3外显子2缺失产生的小鼠，研究体内FOXP3ΔE2同工型的功能证明FOXP3ΔE2小鼠发展出一种系统性自身免疫性类似于系统性狼疮红斑（SLE）。我们进一步证明了仅表达FOXP3ΔE2同工型的Treg是不稳定，转化为效应的助手转移到TCRB - / - 小鼠中。我们假设FOXP3ΔE2是人类的两个主要同工型之一，是一个 SLE开发和/或严重程度的风险因素。我们将在以下特定目标中检验我们的假设。（1）定义FOXP3ΔE2Tregs在狼疮发作和严重程度上的作用。我们假设Foxp3Δe2Tregs具有 FOXP3 FL Tregs的不同转录组程序使它们成为谱系不稳定。在狼疮下诱导环境，FOXP3ΔE2Tregs在外围遗传身份中失去了谱系身份，并转变为自动反应性效应细胞，即使存在FOXP3，也会引起早期和/或更严重的疾病 fl tregs。（2）定义FOXP3ΔE2亚型对人Treg表型和疾病过程的影响系统性狼疮。我们假设FOXP3同工型表达的比率决定了人类Treg 表型，与全身性狼疮的疾病过程相关。我们将确定是否Foxp3 ΔE2：FOXP3 FL比，Tregs的存在和频率主要表达FOXP3ΔE2同工型使用流式细胞术和一种特殊设计的单细胞RNA-seq的疾病状态。我们将进一步测试SLE患者中的细胞因子环境是否调节FOXP3前MRNA剪接，从而改变同工型表达。从这些研究中学到的信息将为生理提供新颖的见解天然现有的人类Foxp3ΔE2同工型的功能，并将为发展有效的方式铺平道路旨在将FOXP3同工型表达转移为SLE以及其他治疗的临床方案自身免疫性疾病。