Background/Purpose: Systemic Sclerosis (SSc) is a rare autoimmune disease with multiple clinical and pathological manifestations including vascular involvement, immune activation, oxidative stress, and fibrosis. In prior work, we have shown that SSc macrophages (MØs) have a pro-fibrotic activation profile that is conferred, at least in part, by crosstalk with SSc dermal fibroblast-derived exosomes, resulting in upregulation of secreted mediators of fibrosis and inflammation. Because MØs are plastic, we hypothesized that reshaping the immunophenotype of SSc MØs would alleviate inflammation and fibrotic activation in SSc.

Methods: To redirect MØ activation, we used CDDO-methyl ester (CDDO-Me), a synthetic triterpenoid derived from oleanoic acid that targets multiple signaling pathways implicated in SSc pathogenesis, including NFkB, STAT3, and Nrf2. Initial studies were performed by incubating SSc MØs with 300 nM CDDO-Me, which we have shown in previous studies alters MØ activation, for 24 hours.To assess effects on exosome-mediated activation, an established Transwell co-culture system was used. Dermal fibroblasts were isolated from SSc patients and healthy control (HC) donors following informed written consent and cultured in complete media supplemented with exosome-depleted fetal bovine serum (FBS) to isolate exosomes. Fibroblasts were stimulated with 5 ng/ml TGF-b for 24 hours prior to exosome harvest. Exosomes were harvested from cell-free supernatants followed by quantification and characterization by immunoblot and NanoSight NS300 tracking analysis. HC monocytes were differentiated into MØs in followed by activation with SSc fibroblast-derived exosome (SSc FB-Exo) in RPMI/exosome-depleted FBS for additional 2 days. After MØ activation, SSc fibroblasts were placed into Transwell inserts and 300 nM CDDO-Me was added to cultures. MØs and fibroblasts were evaluated using flow cytometry, qRT-PCR, and multiplex.

Results: Consistent with prior reports, SSc MØs upregulated expression of IL-10, IL-6, IL-1b, and CCL2 compared with HC MØs. Treatment with CDDO-Me resulted in significant attenuation of these inflammatory and fibrotic mediators.CDDO-Me induced activation of Nrf2 signaling in SSc MØs, as evidenced by increased expression of HO-1 and KEAP1. Surface expression of CD163, CD206, and HLA-DR and pro-fibrotic mediator production, including CCL2, from SSc FB-Exo activated MØs was inhibited by incubation with CDDO-Me. In Transwell experiments, SSc fibroblasts co-cultured with CDDO-Me-treated SSc FB-Exo activated MØs showed decreased expression of fibrosis-related genes, including FN1 and ⍺-SMA.

Conclusion: In this work, we demonstrated that CDDO-Me reshapes the pro-fibrotic immunophenotype of SSc activated MØs, leading to a decrease in release of cytokines implicated in SSc pathogenesis and attenuated SSc fibroblast activation. CDDO-Me targets multiple signaling pathways including oxidative stress and inflammation. Our results suggest CDDO-Me may have therapeutic utility in the reduction of oxidative stress, inflammation, and fibrosis associated with SSc.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/redirecting-macrophage-immunophenotype-attenuates-inflammation-and-fibrotic-activation-in-systemic-sclerosis/