Systemic Sclerosis (SSc) is a complex auto-immune connective tissue disease combining inflammatory, vasculopathic and fibrotic manifestations. Skin effectively recapitulates the main pathogenic processes and therefore is a good organ to decipher the disease pathophysiology, which remains unclear. However, culturing primary skin cells is SSc can be a major issue due to small sample size combined to skin fibrosis. Here, we present a protocol allowing to isolate and culture the four main types of skin cells: dermal cells (microvascular dermal endothelial cells-HDMECs-and fibroblasts) and epidermal cells (keratinocytes and melanocytes), from a single 4 mm-punch biopsy, at a low cost. The present protocol has been optimized to fit SSc skin cells particularities. Such technique allows to culture primary cells, crucial to study the disease pathophysiology, as well as to isolate cells in order to perform immediate molecular biology experiments such as single-cell transcriptomic. Cells grown from biopsies are also suitable for various types of experiments such as immunocytochemistry, Western blot, RT-qPCR or functional in vitro assays (angiogenesis, migration, etc.). Ultimately, they can be used for experimental 3D cell culture models such as reconstructed skin.

ObjectiveInnate lymphoid cells-2 (ILC2) were shown to be involved in the development of lung or hepatic fibrosis. We sought to explore the functional and phenotypic heterogeneity of ILC2 in skin fibrosis within systemic sclerosis (SSc).MethodsBlood samples and skin biopsies from healthy donor or patients with SSc were analysed by immunostaining techniques. The fibrotic role of sorted ILC2 was studied in vitro on dermal fibroblast and further explored by transcriptomic approach. Finally, the efficacy of a new treatment against fibrosis was assessed with a mouse model of SSc.ResultsWe found that ILC2 numbers were increased in the skin of patients with SSc and correlated with the extent of skin fibrosis. In SSc skin, KLRG1− ILC2 (natural ILC2) were dominating over KLRG1+ ILC2 (inflammatory ILC2). The cytokine transforming growth factor-β (TGFβ), whose activity is increased in SSc, favoured the expansion of KLRG1- ILC2 simultaneously decreasing their production of interleukin 10 (IL10), which regulates negatively collagen production by dermal fibroblasts. TGFβ-stimulated ILC2 also increased myofibroblast differentiation. Thus, human KLRG1- ILC2 had an enhanced profibrotic activity. In a mouse model of SSc, therapeutic intervention-combining pirfenidone with the administration of IL10 was required to reduce the numbers of skin infiltrating ILC2, enhancing their expression of KLRG1 and strongly alleviating skin fibrosis.ConclusionOur results demonstrate a novel role for natural ILC2 and highlight their inter-relationships with TGFβ and IL10 in the development of skin fibrosis, thereby opening up new therapeutic approaches in SSc.

HIF-1α exerts both detrimental and beneficial actions in atherosclerosis. While there is evidence that HIF-1α could be pro-atherogenic within the atheromatous plaque, experimental models of atherosclerosis suggest a more complex role that depends on the cell type expressing HIF-1α. In atheroma plaques, HIF-1α is stabilized by local hypoxic conditions and by the lipid microenvironment. Macrophage exposure to oxidized LDLs (oxLDLs) or to necrotic plaque debris enriched with oxysterols induces HIF-1α -dependent pathways. Moreover, HIF-1α is involved in many oxLDL-induced effects in macrophages including inflammatory response, angiogenesis and metabolic reprogramming. OxLDLs activate toll-like receptor signaling pathways to promote HIF-1α stabilization. OxLDLs and oxysterols also induce NADPH oxidases and reactive oxygen species production, which subsequently leads to HIF-1α stabilization. Finally, recent investigations revealed that the activation of liver X receptor, an oxysterol nuclear receptor, results in an increase in HIF-1α transcriptional activity. Reciprocally, HIF-1α signaling promotes triglycerides and cholesterol accumulation in macrophages. Hypoxia and HIF-1α increase the uptake of oxLDLs, promote cholesterol and triglyceride synthesis and decrease cholesterol efflux. In conclusion, the impact of HIF-1α on cholesterol homeostasis within macrophages and the feedback activation of the inflammatory response by oxysterols via HIF-1α could play a deleterious role in atherosclerosis. In this context, studies aimed at understanding the specific mechanisms leading to HIF-1α activation within the plaque represents a promising field for research investigations and a path toward development of novel therapies.