Dermal fibroblasts subsets activate Ccr3 to promote allergic inflammation

Summary: Abstract Body: Dermal fibroblasts are involved in various inflammatory diseases, including acne, psoriasis, and S. aureus infections. However, the role of fibroblasts in atopic dermatitis (AD) remains unclear. To investigate this, we analyzed scRNA-seq data from patients with AD (n=5 non-lesional skin, n=4 lesional skin) compared to controls (n=8). Unbiased CellChat analysis revealed that PDGFRa+ fibroblasts exhibit increased communication with T cells and myeloid cells in AD, along with higher expression of CCL5, CCL8, CCL11, CCL13, and CCL26 relative to normal skin. Analysis of scRNA-seq data in a mouse AD model (MC903 +S. aureus) demonstrated a similar response, with certain dermal fibroblast clusters showing elevated levels of several chemokines that interact with the Ccr3 receptor. Furthermore, this response was absent in IL4ra-/- mice, indicating a specific role for IL4/IL13. To test this, cultured 3T3 fibroblasts were treated with and without IL4/IL13 after administration of an inflammatory cocktail (IL1b, IL17A, and TNFa). RNA sequencing and subsequent qPCR validation confirmed that IL4 and IL13 induce fibroblast expression of CCR3 ligands (Ccl2, Ccl7, Ccl8, and Ccl11). This was functionally relevant, as conditioned media from fibroblasts showed increased protein levels of Ccl8 and Ccl11 following the addition of IL4/IL13, and this conditioned media promoted T cell migration, which was inhibited by a Ccr3 antagonist (P<0.05). Targeted siRNA against Ccl8 in fibroblasts also reduced the ability of IL4 and IL13 to stimulate fibroblasts to enhance T-cell migration (P<0.01). Finally, administering a Ccr3 antagonist in the AD mouse model via intraperitoneal injection reduced inflammation and T-cell migration into the skin. These findings demonstrate that fibroblasts are key responders to Th2 cytokines and that Ccr3 ligands, particularly Ccl8 from fibroblasts, may play a role in leukocyte recruitment during allergic inflammation. Tomofumi Numata¹, Michael Shia¹, Yoshiyuki Nakamura¹, Hung M. Chan¹, Fengwu Li¹, Kellen Cavagnero¹, Jared Simmons¹, Teruaki Nakatsuji¹, Richard L. Gallo¹ 1. Dermatology, University of California San Diego, La Jolla, CA, United States. Translational Studies: Preclinical