The discovery and validation of disease specific proteins in corticotropin-releasing hormone treated regulatory T cells in atopic dermatitis

Abstract

Atopic dermatitis (AD) is a highly pruritic, chronic relapsing inflammatory skin disease characterized by abnormal skin barrier and immune dysregulation. Most of AD patients were triggered or exacerbated by psychological factors, such as stress. Stress induces corticotropin-releasing hormone (CRH) in the CNS as well as in peripheral sites. Regulatory T cells (Tregs) are a subset of T cells with strong immunosuppressive activity. However, the role of Tregs in AD with stress has not been investigated. Therefore, the aim of this study was to discover CRH-regulated protein in AD Tregs.Female NC/Nga mice were applied with Dermatophagoides farinae body extract (DfE) ointment for six weeks. After sensitizing NC/Nga mice for six weeks, I observed AD-like skin lesions and elevated serum IgE levels in mice. The percentages of CD4+ IL-4+ Th2 cells and CD4+CD25+ Tregs were increased in the spleen of these mice. Splenocytes from AD mice were treated with 10 nM CRH for 24 and 48 hours, and then Tregs were sorted. Differential protein expression between CRH-treated group and un-treated group was compared by quantitative proteome analysis using Tandem Mass Tag (TMT) labeling method. Initially, I identified 1353 proteins in CRH-treated and un-treated Tregs. Afterwards, I attempted to identify proteins that either increased or decreased depending on time after stimulation with CRH. Finally, I identified 20 proteins as candidates for further investigation. Among these proteins, 5 proteins were up-regulated and 15 proteins were down-regulated. Among down-regulated proteins, I paid special attention to dedicator of cytokinesis 8 (DOCK8) because of its role in various immune diseases. Using Western blot and flow cytometry, I confirmed decrease of DOCK8 in CRH-treated Tregs in AD mice and AD patients.To study the role of DOCK8 in Tregs, I first detected inhibitory cytokines TGF-βand

IL-10 in DOCK8 siRNA-treated Tregs. I found that both TGF-β and IL-10 were deceased after DOCK8 siRNA treatment compared to scrambled siRNA. Next, I cocultured CD4+ T cells with control Tregs or Tregs transfected with scrambled siRNA or DOCK8 siRNA. CD4+ T cells cocultured with control Tregs showed decreased proliferative activity and decreased release of TNF-a and IFN-r. However, knockdown of DOCK8 in Tregs caused restored proliferation and release of TNF-a and IFN-r in CD4+ T cells. In summary, CRH-induced down-regulation of DOCK8 in Tregs might result in the impairment of Treg function.In conclusion, I suggest that DOCK8 play an important role in Tregs which may contribute to alleviating stress induced aggravation of AD symptom.