Role of Cul4A in Governing Th2-Type Tolerance

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PROJECT SUMMARY/ABSTRACT The hallmark of allergic diseases is the infiltration/accumulation of Th2 cells at the sites of inflammation; however the mechanisms governing the development of unwanted Th2-driven airway diseases are poorly understood. Thus, today it is becoming clear that understanding the Th2-type immune responses and ways to control them may prove to be invaluable for the treatment of allergic inflammation. Recently, we as well as others, have demonstrated the role of E3 ubiquitin ligases in T cell tolerance. Cullin4A (Cul4A) belongs to the evolutionally conserved E3 ligase family and has been suggested to be involved in hematopoietic cell activation and in chromatin regulation; however, the role of Cul4A in T lymphocytes has not been addressed yet. Interestingly, expression of Cul4A was upregulated in tolerant T cells. Moreover, Cul4A knockout (KO) CD4+ T cells activated in vitro and in vivo under tolerogenic conditions exhibited an enhanced level of proliferation along with IL-2 and IL-4 production compared to wild-type cells, suggesting an important role of Cul4A in regulation of T cell tolerance. Strikingly, we found that Cul4A KO effector T cells and Treg cells expressed higher levels of Th2 but not Th1 and Th17 cytokines, which correlates with increased levels of IgG1 and IgE in the sera of Cul4A KO mice, indicating the selective role of Cul4A in controlling inflammatory Th2-type responses. Importantly, we detected reduced Cul4A expression in CD4+ T cells from asthmatic patients compared to CD4+ T cells from healthy donors, further indicating that Cul4A potentially contributes to airway immune tolerance induction. In addition, shRNA knockdown of Cul4A in human CD4+ T cells resulted in upregulated expression of Th2-specific cytokines. Based on these findings, we hypothesize that regulation of Th2-type immune responses by Cul4A may be an important checkpoint in airway tolerance induction, which is crucial to prevent development of allergic diseases. In Aim 1, we propose to determine the mechanisms whereby Cul4A controls T cell activation by utilizing gene knockdown approaches and in vitro and in vivo Th2 tolerance induction models. In Aim 2, we will determine the role of Cul4A in Th2 cell programming and its underlying mechanisms as well. We will employ cutting edge technologies, including two-hybrid screening and microarray analysis of gene expression, to identify the exact target(s) of Cul4A to determine its function in Th2 development. In addition, we will assess the cellular mechanisms whereby Cul4A controls Th2 type-driven allergic asthma. In Aim 3, we will determine the mechanism by which Cul4A regulates Treg function, particularly in controlling Th2 inflammatory responses. The physiological significance of this finding will be analyzed in different in vivo models. The proposed research will provide new significant insight into characterization of mechanisms underlying Cul4A-mediated Th2-type tolerance that may potentially have therapeutic implications for allergic diseases.