Nilotinib Treatment of Patients Affected by Chronic Graft-versus-Host Disease Reduces Collagen Production and Skin Fibrosis by Downmodulating the TGF-β and p-SMAD Pathway

The present study was conducted to investigate cellular and molecular features of chronic graft-versus-host disease fibroblasts (GVHD-Fbs) and to assess the effectiveness of nilotinib as a fibrosis modulator. Growth kinetics, phenotype, and differentiation of cultured skin biopsy-derived GVHD-Fbs were compared with normal fibroblasts from both a dermal cell line (n-Fbs) and healthy individuals undergoing cosmetic surgery (n-skin-Fbs). Collagen genes (COL1 alpha 1/COL1 alpha 2) and p-SMAD2 expression were assessed by real-time PCR and immunofluorescence. The in vivo effects of nilotinib on chronic GVHD (cGVHD)-affected skin were investigated by immunohistochemistry; the relationship to TGF-beta plasma levels was assessed. Although the morphology, phenotype, and differentiation of cultured GVHD-Fbs were comparable to normal fibroblasts, growth was slower and senescence was reached earlier. The expression of COL1 alpha 1 and COL1 alpha 2 mRNAs was respectively 4 and 1.6 times higher in cGVHD-Fbs (P = .02); the addition of TGF-beta increased n-Fbs, but not GVHD-Fbs, collagen gene expression. Compared with the baseline, the addition of 1 mu M nilotinib induced 86.5% and 49% reduction in COL1 alpha 1 and COL1 alpha 2 expression in cultured GVHD-Fbs, respectively (P< .01). In vivo immunohistochemistry analysis of skin biopsy specimens from patients with cGVHD showed strong baseline staining for COL1 alpha 1 and COL1 alpha 2, which decreased sharply after 180 days of nilotinib; immunofluorescence revealed TGF-beta inhibition and p-Smad2 reduction at the intracellular level. Of note, nilotinib treatment was associated with normalization of TGF-beta levels both in culture supernatants and in plasma. In general, the data show that cGVHD fibroblasts promote fibrosis through abnormal collagen production induced by hyperactive TGF-beta signaling. TGF-beta inhibition at the intracellular and systemic level represents an essential antifibrotic mechanism of nilotinib in a clinical setting. (C) 2020 American Society for Transplantation and Cellular Therapy. Published by Elsevier Inc.