A Role for Connexin Signalling in the Innate Immune Response in the Skin

Abstract

Skin acts as a defence organ in the immune system, presenting a physical barrier to resist micro-organism penetration. Chronic inflammatory skin disorders affect patients and healthcare systems. Many patients are highly susceptible to infection and colonization by Staphylococcus aureus. Cells from the skin’s immune system, including keratinocytes, trigger and control skin inflammation. Currently, immune modulation (such as TNF inhibitors) oragents with anti-proliferative or pro-differentiative effects on keratinocytes, such as vitamin D and retinoids, are effective treatments. Such agents help control hyper proliferative skin conditions; however, none of them help cure these conditions. There are different connexin proteins found in the skin that have been implicated in various skin conditions. Bacterial infections initiate an innate immune response resulting in the induction of inflammatory cytokine expression (e.g. IL-6), which can further modulate connexin expression in diverse cell types. Previous work suggests that this process can in part beinitiated by the activation of connexin hemichannels to release ATP, and that the induction of the innate immune response can be reduced by connexin hemichannel blockers. I reasoned that the connexin mimetic peptide (Gap27) may reduce such pro-inflammatory responses. HaCaT cells, as a model for keratinocytes, were challenged with different bacterial cell wall components extracted from S. aureus and S. epidennidis.RNA was extracted from cells and real-time PCR analysis determined that peptidoglycan (PGN) extracted from S. aureus but not the skin commensalsS. epidermidis induced IL-6 expression. In human adult dermal fibroblasts, an induction of IL-6 expression following exposure to PGN S. aureus wasobserved. Also, Cx43 and Cx26 levels were measured under the effect of PGN S. aureus; this had no effect on Cx43 expression in both cell types,while Cx26 expression was enhanced significantly in HaCaT cells. HaCaT cells, human adult primary keratinocytes, human dermal fibroblasts (adult and neonatal) and neonatal mouse fibroblasts were challenged with 10 pg/ml of PGN S. aureus in the presence or absence of the connexin hemichannel blocker Gap27 (100 pM). Supernatants were harvested and subject to ELISA (IL-6) or a cytokine array (R&D systems). PGN induced a pro-inflammatory response in all cell types. Exposure to Gap27 reduced the level of IL-6 expression in HaCaT, human adult primary keratinocytes and adult fibroblasts, but not in human neonatal fibroblasts or mouse neonatal fibroblasts. Expression of a panel of cytokines was reduced in the cytokinearray in human adult fibroblasts. I conclude that PGN from different Staphylococcal species has tissuespecific effects on connexins function and IL-6 and connexins expression patterns. My data suggest that such pro-inflammatory mediators activate opening of the connexin hemichannel, thereby influencing purinergic signalling pathways, contributing in the induction of an innate immune response. Further, inhibition of connexin hemichannel activity by Gap27 has an impact on the innate immune responses that targets connexins in adult keratinocytes and fibroblasts but not neonatal fibroblasts; suggesting that connexin targeting agents may hold therapeutic utility for adult inflammatory skin disorders.

Date of Award	2016
Original language	English
Awarding Institution	Glasgow Caledonian University
Supervisor	Patricia Martin (Supervisor)