Thymic Stromal Lymphopoietin Interferes with the Apoptosis of Human Skin Mast Cells by a Dual Strategy Involving STAT5/Mcl-1 and JNK/Bcl-xL.

Mast cells (MCs) play critical roles in allergic and inflammatory reactions and contribute to multiple pathologies in the skin, in which they show increased numbers, which frequently correlates with severity. It remains ill-defined how MC accumulation is established by the cutaneous microenvironment, in part because research on human MCs rarely employs MCs matured in the tissue, and extrapolations from other MC subsets have limitations, considering the high level of MC heterogeneity. Thymic stromal lymphopoietin (TSLP)-released by epithelial cells, like keratinocytes, following disturbed homeostasis and inflammation-has attracted much attention, but its impact on skin MCs remains undefined, despite the vast expression of the TSLP receptor by these cells. Using several methods, each detecting a distinct component of the apoptotic process (membrane alterations, DNA degradation, and caspase-3 activity), our study pinpoints TSLP as a novel survival factor of dermal MCs. TSLP confers apoptosis resistance via concomitant activation of the TSLP/ signal transducer and activator of transcription (STAT)-5 / myeloid cell leukemia (Mcl)-1 route and a newly uncovered TSLP/ c-Jun-N-terminal kinase (JNK)/ B-cell lymphoma (Bcl)-xL axis, as evidenced by RNA interference and pharmacological inhibition. Our findings highlight the potential contribution of TSLP to the MC supportive niche of the skin and, vice versa, highlight MCs as crucial responders to TSLP in the context of TSLP-driven disorders.

Apoptotic resistance of human skin mast cells is mediated by Mcl-1.

Mast cells (MCs) are major effector cells of allergic reactions and contribute to multiple other pathophysiological processes. MCs are long-lived in the tissue microenvironment, in which they matured, but it remains ill-defined how longevity is established by the natural habitat, as research on human MCs chiefly employs cells generated and expanded in culture. In this study, we report that naturally differentiated skin MCs exhibit substantial resilience to cell death with considerable portions surviving up to 3 days in the complete absence of growth factors (GF). This was evidenced by kinetic resolution of membrane alterations (Annexin-V, YoPro), DNA degradation (propidium iodide), mitochondrial membrane disruption (Depsipher), and Caspase-3 activity. Because of the high basal survival, further protection by SCF was modest. Conversely, survival was severely compromised by staurosporine, implying functional caspase machinery. Contrary to the resistance of freshly purified MCs, their culture-expanded counterpart readily underwent cell death upon GF deprivation. Searching for the molecular underpinnings explaining the difference, we identified Mcl-1 as a critical protector. In fact, silencing Mcl-1 by RNAi led to impaired survival in skin MCs ex vivo, but not their cultured equivalent. Therefore, MCs matured in the skin have not only higher expression of Mcl-1 than proliferating MCs, but also greater reliance on Mcl-1 for their survival. Collectively, we report that human skin MCs display low susceptibility to cell death through vast expression of Mcl-1, which protects from mortality and may contribute to MC longevity in the tissue.

An efficient method for gene knock-down by RNA interference in human skin mast cells.

Mast cells (MCs) from human skin have been notoriously resistant to gene manipulation, and a method to knock-down gene expression in in situ differentiated MCs is highly desired. The Dharmacon Accell® transfection system proved successful on several "difficult-to-transfect" cells. In the present work, we therefore tested this method on skin-derived MCs using different siRNA entities. The siRNA was readily taken up, followed by pronounced, specific reduction of gene and protein expression. Hence, we present the first efficient technique for the manipulation of gene expression in primary skin MCs ex vivo, which combines high transfection rates with retained cell viability.

Retinoic acid potentiates inflammatory cytokines in human mast cells: identification of mast cells as prominent constituents of the skin retinoid network.

Retinoic acid (RA), the active vitamin-A-metabolite, has well-established functions in skin homeostasis and in the immune system. Skin mast cells (MCs) combine traits of both structures, being of hematopoietic origin, but functional in the skin environment. It remains largely unknown whether mature MCs are targeted by the retinoid network. Here, we demonstrate that human skin MCs display substantial susceptibility to RA by which they are instructed to increase pro-inflammatory mediators (IL-1ß, IL-8, TNF-α) but not histamine release. The effects are observed at physiological RA levels, in different microenvironments, and are largely donor-independent. RA susceptibility is owed to the cells' abundant expression of RARA, the receptor mediating MC cytokine responses. Unexpectedly, bioinformatics calculations on the FANTOM5 expression atlas revealed general enrichment of retinoid network components in MCs against other skin cells, and MCs rapidly upregulated RA responsive genes. In conclusion, MCs are important yet hitherto overlooked retinoid targets in the skin.