Background/Purpose: Systemic sclerosis (SSc) is a connective tissue disease characterized by dysregulated fibrosis of the skin. During fibrosis, macrophage release of transforming growth factor (TGF-β) and resistin like-alpha (RELM-α) leads to myofibroblast over-activation and excessive extracellular matrix deposition. However, the role of post-translational fucosylation in fibrosis has yet to be examined. Herein, we demonstrate mechanisms by which fucosyltransferase-1 (Fut1) contributes to macrophage driven fibrosis and to TGF-β signaling in murine and SSc DFs.

Methods: Wild type (WT) and Fut1 knockout (Fut1^-/-) mice were intradermally injected with bleomycin or PBS and euthanized at days 1, 3, 5, and 9 to evaluate the longitudinal involvement of macrophages in skin fibrosis. Following euthanasia, fibrotic murine skin was harvested to assess cytokine expression by qPCR or pro-fibrotic macrophage ingress by immunofluorescence. To determine whether Fut1 was critical to macrophage-induced myofibroblast differentiation, WT and Fut1^-/- macrophages were co-cultured with WT DFs for 48h. Co-culture supernatants were collected for ELISAs for pro-fibrotic cytokines TGF-β, RELM-α, IL-6, and MCP-1. DF alpha smooth muscle actin (α-SMA) and type 1 collagen (Col1a1) expression were assessed via qPCR. WT and Fut1^-/- DFs were stimulated with TGF-β and downstream transcription factor expression assessed via qPCR. Fucosylation of TGF-βR1 by Fut1 was confirmed by ulex europpaeus agglutinin 1 (UEA1) immunoprecipitation. Lastly, α-1,2 fucosidase treatment of SSc DFs was performed to determine whether Fut1-added fucose was critical to TGF-βR1 function.

Results: Fut1^-/- mice were resistant to bleomycin-induced fibrosis. At day 5, Col1a1, TGF-β, and RELM-α mRNA were significantly reduced in Fut1^-/- compared to WT fibrotic skin. Additionally, at day 5 and 9, Fut1^-/- mice exhibited significantly fewer F4/80+ RELM-α+ macrophages in skin lesions, suggesting the involvement of Fut1 in pro-fibrotic macrophage function. IL-6, MCP-1, M-CSF, and RELM-α expression in supernatant were significantly reduced in WT DF co-culture with Fut1^-/- compared to WT macrophages. Consequently, WT DF expression of α-SMA and Col1a1 mRNA was significantly diminished in Fut1^-/- compared to WT macrophage co-culture, suggesting the involvement of Fut1 in macrophage-induced myofibroblast differentiation. TGF-βR1 is fucosylated by Fut1 in murine and SSc DFs, as suggested by UEA1 immunoprecipitation. Fut1^-/-DFs exhibit diminished expression of TGF-β associated transcription factors compared to WT, pointing to the involvement of Fut1 in TGF-β-mediated functions. Finally, α-1,2 fucosidase diminished TGF-βR1 expression detected by flow cytometry, indicating that Fut1 regulates TGF-β signaling via the critical addition of α-1,2 fucose to TGF-βR1.

Conclusion: Overall, this work provides novel evidence that Fut1 mediates macrophage-driven fibrosis and that Fut1-catalyzed fucosylation of TGF-βR1 regulates TGF-β signaling. The multi-faceted involvement of Fut1 in fibrosis points to its candidacy as a novel therapeutic target in the treatment of SSc.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/fucosyltransferase-1-mediates-macrophage-driven-myofibroblast-differentiation-and-tgf-%ce%b2-signaling-in-systemic-sclerosis-and-bleomycin-induced-fibrosis/