Dermal fibroblasts are critical mediators of IL-1 function in the skin

Summary: Abstract Body: IL-1 is essential for host defense and plays key roles in inflammatory disorders, but its mechanism of action remains incompletely understood. As scRNA-seq revealed several fibroblast subsets highly express Il1r1, and CellChatDB ligand-receptor analysis showed major keratinocyte-fibroblast communication during S. aureus infection, we hypothesized that dermal fibroblasts might have an important role in IL-1-mediated inflammation. To test this hypothesis in vitro, human fibroblasts (HPAd) were exposed to conditioned media (CM) from normal human keratinocytes (NHEK). RNA-seq and pathway enrichment analysis showed increased levels of dozens of inflammatory transcripts in NHEK-activated HPAds (fold>2, p<0.05). This result was validated by qPCR (e.g., CXCL8: fold=14.3 p<0.0001) and multiplex immunoassay (CXCL1/5/8, CCL2/20: fold>4, p<0.007). Phosphoproteomic analysis of HPAds also found increased phosphorylation of proteins in several IL-1R-related signaling pathways (e.g., AP-1, NFκB) within 10 minutes of exposure to NHEK-CM. Fibroblast supernatant was then tested in an in vitro migration assay, which showed that NHEK-activated fibroblast products preferentially recruited neutrophils and monocytes from bone marrow and blood (fold>2, p<0.05). These fibroblast responses were dependent on IL-1R, since NHEK-CM was inactivated by the addition of a neutralizing antibody of IL-1α or a competitive inhibitor of IL-1R (anakinra), and fibroblasts derived from Il1r1-KO mice were nonresponsive to keratinocyte CM. We next developed PdgfraΔIl1r1 mice and challenged them with S. aureus to assess the importance of fibroblast IL-1 recognition in vivo. The skin of mice lacking fibroblast IL1R1 had reduced inflammatory transcripts (e.g., Csf3: fold=-1.5, p=0.0097), larger infectious lesions, and increased bacterial load (fold=2.8, p=0.0038). These data show that IL-1 can induce inflammatory activity in fibroblasts and that this response is critical to host defense. Jared Simmons¹, Consuelo Sauceda², Jacquelyn Castaneda², Teruaki Nakatsuji¹, Tomofumi Numata¹, David Gonzalez², Richard L. Gallo¹ 1. Dermatology, University of California San Diego, La Jolla, CA, United States. 2. Pharmacology, University of California San Diego, La Jolla, CA, United States. Cell Communication Networks and Stromal Biology