Characterization of IL36, a novel cytokine

描述(申请人提供)：我们基于全基因组的Affymetrix 133 2.0基因阵列，构建了一个包含90多个人类组织和细胞的全面的基因表达数据库，我们称之为身体基因表达指数(BIGE)。鉴于许多人类基因仍未确定特征，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。我们鉴定了许多在T、B细胞、单核细胞或树突状细胞中强表达的基因，其中包括92个编码跨膜蛋白或分泌蛋白的未鉴定基因。在对这些基因进行广泛的生物信息学分析后，我们发现了一种由激活的巨噬细胞和树突状细胞表达的新的分泌蛋白。我们暂时将其命名为白介素36。IL36是一种由311个氨基酸组成的小分子蛋白质，其信号肽由45个氨基酸组成，预测一个由266个氨基酸组成的成熟蛋白质，其相对分子质量约为29 KDa。在人外周血单核细胞或小鼠腹腔巨噬细胞中，它被内毒素激活后表达。有趣的是，它也在人类或小鼠未成熟的树突状细胞中表达，但在这些细胞成熟后下调。CD4T细胞在被抗CD3激活后也会产生它。除白细胞外，IL36在黏膜组织和皮肤中也有表达，提示上皮细胞也有表达。由于一些抗原提呈细胞表达IL36，我们推测IL36影响T细胞的激活，也可能影响其分化。我们表达了重组小鼠和人IL36，并制备了多克隆抗体。在具体目标1中，我们将在体外寻找IL36对T细胞的生物学活性。我们将在胸腺细胞或成熟T细胞上测试IL36的增殖和分化试验。我们还将使用T细胞杂交瘤和抗原诱导的T细胞来研究IL36对T细胞活化的可能影响，以及对巨噬细胞和树突状细胞呈递抗原能力的影响。我们还将研究CD4T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，我们将生产一种IL36基因敲除小鼠。为此，我们已经有了IL36基因失活的ES细胞。我们将通过分析其免疫器官的细胞并通过测量各种免疫功能来表征IL36(-/-)小鼠的表型。在具体目标3中，我们将通过研究IL36在各种疾病患者的存档皮肤样本中的表达，来研究IL36在人类皮肤病中的可能作用。我们还将研究它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。 公共卫生相关性：通过对全面的人类基因表达数据库进行系统筛选，我们确定了一种新的细胞因子，我们将其命名为IL36。在这项建议中，我们旨在通过探索其在体外对白细胞的影响以及在体内的功能来进行其生物学特性的研究。

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.