Mechanism of action of cyclosporine A in atopic eczema and role of cyclophilin B as a regulator of human keratinocyte growth and differentiation

Abstract

Atopic eczema has a profound negative impact on patients and their families and improved therapeutic options are required. Although, the pathogenesis is still not fully understood, evidence of a primary barrier defect predisposing to increased stimulation of the innate and adaptive immune system increases. Cyclosporine A (CsA) is an effective therapy for inflammatory skin disease. CsA binds to cyclophilin B (CypB) with high affinity, mediating T-cell immunosuppression. CsA also exerts T-cell independent effects in skin. The Reynolds lab has previously shown that CypB is expressed and secreted by normal human epidermal keratinocytes (NHEK). In this work we aimed to investigate the functional role of CypB in the epidermis. Confocal analysis of normal human skin revealed CypB expression within the granular cell layer. Transduction of NHEK with retroviral vectors containing CypBWT-GFP, CypBW128A-GFP (a mutant reducing CsA binding by 97%) positively regulated keratinocyte differentiation (transglutaminase promoter luciferase and enzyme activity). In addition, transduction of NHEK with CypBWT and CypBW128A significantly increased colony formation and keratinocyte proliferation compared to empty vector. Furthermore, conditioned medium from NHEKs transduced with CypBWT or CypBW128A increased proliferation of naïve NHEK. Conversely, knockdown of CypBWT reduced NHEK proliferation. Moreover, NHEK transduced with CypBWT formed thicker epidermal equivalents compared with empty vector controls. Analysis of eczematous skin treated with CsA, showed changes in expression of CypB before and following 2 weeks of CsA treatment. An increase in filaggrin expression in eczematous skin during these early phases of treatment with CsA, suggests that CsA may act by positively repairing impaired barrier function. These studies provide an increased understanding of the physiological and pathological role of CypB in keratinocytes, further insight into the mechanism of action of CsA and may identify CypB as a novel drug target for atopic eczema.