Characterization of IL36, a novel cytokine

新型细胞因子 IL36 的表征

• 批准号: 8174696
• 负责人: Albert Zlotnik
• 金额: $ 22.24万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-05-15 至 2013-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8174696
• 关键词: Affect; Amino Acids; Antibodies; Antibody Formation; Antigen Presentation; Antigen-Presenting Cells; Antigens; Archives; Atopic Dermatitis; B-Lymphocytes; Bioinformatics; Biological; Biological Assay; CD3 Antigens; CD4 Positive T Lymphocytes; CD8B1 gene; Cell Maturation; Cells; Characteristics; Cytokine Gene; Databases; Delayed Hypersensitivity; Dendritic Cells; Development; Disease; Epithelial Cells; Exhibits; Formalin; Freezing; Gene Expression; Genes; Human; Human Genome; Human body; Hybridomas; Immune; Immune response; Immune system; In Vitro; Inflammation; Inflammatory Response; Interleukins; Knockout Mice; Leukocytes; Mature T-Lymphocyte; Measures; Molecular Weight; Mucous Membrane; Mus; Organ; Paraffin Embedding; Pathogenesis; Patients; Peptide Signal Sequences; Peritoneal Macrophages; Phenotype; Population; Process; Production; Proteins; Psoriasis; Recombinants; Regulation; Role; Sampling; Screening procedure; Skin; Skin Tissue; Source; T cell differentiation; T-Cell Activation; T-Cell Receptor; T-Lymphocyte; T-Lymphocyte Subsets; Testing; Therapeutic; Time; Tissue Sample; Tissues; Vitiligo; base; cytokine; embryonic stem cell; genome-wide; human disease; human tissue; immune function; in vivo; indexing; macrophage; melanoma; monocyte; novel; overexpression; peripheral blood; polyclonal antibody; research study; skin disorder; thymocyte

DESCRIPTION (provided by applicant): We have constructed a comprehensive database of gene expression with more than 90 human tissues and cells, which we call a Body Index of Gene Expression (BIGE), based on the genome-wide Affymetrix 133 2.0 gene array. Given that many human genes remain uncharacterized, we sought to identify novel genes associated with the immune system through a systematic analysis of the BIGE database. We identified many genes that exhibit strong expression in T, B cells, monocytes, or dendritic cells, including 92 uncharacterized genes that encode either transmembrane or secreted proteins. Following extensive bioinformatics analyses of these genes, we identified a novel secreted protein expressed by activated macrophages and dendritic cells. We have tentatively called this protein Interleukin 36. IL36 is a small protein of 311 amino acids and exhibits a signal peptide of 45 amino acids, predicting a mature protein of 266 amino acids, with an estimated molecular weight of ~29KDa. It is expressed in human peripheral blood monocytes or mouse peritoneal macrophages following activation with LPS. Interestingly, it is also expressed in human or mouse immature dendritic cells, but is downregulated upon maturation of these cells. CD4 T cells also produce it upon activation with anti CD3. Besides leukocytes, IL36 is also expressed in mucosal tissues, and in the skin, suggesting that epithelial cells also express it. Since several antigen presenting cells express IL36, we hypothesize that IL36 affects T cell activation and may also influence their differentiation. We have expressed recombinant mouse and human IL36, and produced polyclonal antibodies. In Specific Aim 1, we will search for biological activities of IL36 on T cells in vitro. We will test IL36 on proliferation and differentiation assays on thymocytes, or mature T cells. We will also use T cell hybridomas and antigen-primed T cells to study possible effects of IL36 on T cell activation, and on the ability of macrophages and dendritic cells to present antigen. We will also study the regulation of IL36 expression by CD4 T cells, macrophages and dendritic cells. In Specific Aim 2, we will produce an IL36 knockout mouse. To this end, we already have ES cells where the IL36 gene has been inactivated. We will characterize the phenotype of the IL36 (-/-) mouse by analyzing the cells of its immune organs and by measuring various immune functions. In Specific Aim 3, we will study the possible role of IL36 in human skin diseases, by studying its expression in archived skin samples from patients with various diseases. We will also study its expression in normal skin and mucosal tissues. These experiments will help us characterize IL36 and its role in immune responses and inflammation. PUBLIC HEALTH RELEVANCE: Through a systematic screening of a comprehensive human database of gene expression we identified a novel cytokine which we called IL36. In this proposal, we aim to undertake its biological characterization, by exploring its effects on leukocytes in vitro as well as its function in vivo.

描述（由申请人提供）：我们基于全基因组Affymetrix 133 2.0 Gene Array构建了一个具有90多个人体组织和细胞的基因表达数据库，我们称之为基因表达（BIGE）。鉴于许多人类基因仍然没有表征，我们试图通过对BIGE数据库的系统分析来识别与免疫系统相关的新基因。我们确定了许多在T，B细胞，单核细胞或树突状细胞中表现强的基因，包括92个编码跨膜或分泌蛋白质的未表征的基因。在对这些基因进行广泛的生物信息学分析之后，我们确定了一种新型的分泌蛋白质，该蛋白质由活化的巨噬细胞和树突状细胞表达。我们暂定称这种蛋白质介菌素36。IL36是311个氨基酸的小蛋白质，表现出45个氨基酸的信号肽，预测成熟的蛋白质为266个氨基酸，估计分子量约为29KDA。它以LPS激活后在人外周血单核细胞或小鼠腹膜巨噬细胞中表达。有趣的是，它也在人或小鼠未成熟的树突状细胞中表达，但在这些细胞成熟后被下调。 CD4 T细胞在用抗CD3激活后也会产生它。除白细胞外，IL36在粘膜组织中也表达，在皮肤中也表明上皮细胞也表达了它。由于几种抗原呈递细胞表达了IL36，因此我们假设IL36会影响T细胞的激活，并可能影响其分化。我们表达了重组小鼠和人IL36，并产生了多克隆抗体。在特定目标1中，我们将在体外搜索IL36对T细胞的生物学活性。我们将对胸腺细胞或成熟T细胞的增殖和分化测定进行测试IL36。我们还将使用T细胞杂交瘤和抗原培养基的T细胞来研究IL36对T细胞激活的可能影响，以及巨噬细胞和树突状细胞呈现抗原的能力。我们还将研究CD4 T细胞，巨噬细胞和树突状细胞对IL36表达的调节。在特定的目标2中，我们将产生IL36敲除鼠标。为此，我们已经拥有ES细胞，其中IL36基因已被灭活。我们将通过分析其免疫器官的细胞和测量各种免疫功能来表征IL36（ - / - ）小鼠的表型。在特定目标3中，我们将通过研究其在各种疾病患者的存档皮肤样本中的表达来研究IL36在人体皮肤疾病中的可能作用。我们还将研究其在正常皮肤和粘膜组织中的表达。这些实验将帮助我们表征IL36及其在免疫反应和炎症中的作用。 公共卫生相关性：通过对基因表达的全面人类数据库进行系统的筛选，我们确定了一种新型的细胞因子，我们称为IL36。在该提案中，我们旨在通过探索其对体外白细胞的影响以及体内功能来进行生物学特征。