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.
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.
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