Characterization of IL36, a novel cytokine

描述（由申请人提供）：我们以全基因组Affymetrix 133 2.0基因阵列为基础，构建了一个包含90多个人体组织和细胞的基因表达综合数据库，我们称之为基因表达体指数（Body Index of gene expression， BIGE）。鉴于许多人类基因仍未被表征，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。我们发现了许多在T细胞、B细胞、单核细胞或树突状细胞中表现出强烈表达的基因，包括92个编码跨膜或分泌蛋白的未表征基因。在对这些基因进行了广泛的生物信息学分析后，我们发现了一种由活化的巨噬细胞和树突状细胞表达的新型分泌蛋白。我们暂时把这种蛋白称为白介素36。IL36是一个311个氨基酸的小蛋白，具有45个氨基酸的信号肽，预测成熟蛋白有266个氨基酸，估计分子量约为29KDa。LPS激活后在人外周血单核细胞或小鼠腹腔巨噬细胞中表达。有趣的是，它也在人类或小鼠未成熟树突状细胞中表达，但在这些细胞成熟时下调。CD4 T细胞在被抗CD3激活后也会产生它。除了白细胞外，IL36也在粘膜组织和皮肤中表达，表明上皮细胞也表达它。由于几种抗原提呈细胞表达IL36，我们假设IL36影响T细胞活化，也可能影响它们的分化。我们已经表达了重组小鼠和人IL36，并产生了多克隆抗体。在Specific Aim 1中，我们将在体外寻找IL36对T细胞的生物活性。我们将在胸腺细胞或成熟T细胞的增殖和分化试验中测试IL36。我们还将使用T细胞杂交瘤和抗原启动T细胞来研究IL36对T细胞活化以及巨噬细胞和树突状细胞递呈抗原能力的可能影响。我们还将研究CD4 T细胞、巨噬细胞和树突状细胞对IL36表达的调控。在Specific Aim 2中，我们将产生IL36敲除小鼠。为此，我们已经有了IL36基因失活的胚胎干细胞。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Specific Aim 3中，我们将通过研究各种疾病患者存档皮肤样本中IL36的表达，研究IL36在人类皮肤病中的可能作用。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描述IL36及其在免疫反应和炎症中的作用。

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.