Heetaek Yang1, Rajan Bhandari1, Noelle Kosarek2, Jonathan Garlick3, Michael Whitfield4 and Patricia Pioli1, 1Geisel School of Medicine at Dartmouth, Lebanon, NH, 2Dartmouth Geisel School of Medicine, Lebanon, NH, 3Tufts University School of Dental Medicine, Boston, MA, 4Geisel School of Medicine, Lebanon, NH
Background/Purpose: Macrophage (MØ) activation derives from coordination of signals received in local tissue microenvironments. In prior studies, we demonstrated that cocultured MØs and fibroblasts from patients with systemic sclerosis (SSc) engage in reciprocal activation, and MØs and fibroblasts are co-localized in the skin of SSc patients. Thus, we hypothesize that sustained pro-fibrotic activation of dermal SSc MØs derives from SSc fibroblast-derived secreted mediators.
Methods: Fibroblasts from SSc patients (n=9) or healthy age and sex-matched control (HC) donors (n=7) were isolated from skin biopsies following informed written consent and cultured in complete fibroblast media supplemented with exosome-depleted fetal bovine serum (FBS). Fibroblasts were stimulated with 5 ng/ml TGF-b for 24 hours followed by biochemical isolation of exosomes from cell supernatants. Exosomes were quantified using nanoparticle NanoSight NS300 tracking analysis and exosome purity was assessed by immunoblot for canonical markers. HC monocytes were cultured in RPMI/FBS+M-CSF for 3 days; on day 4, media was replaced with RPMI/exosome-free FBS and HC or SSc exosomes for an additional 2 days. MØs were immunophenotyped using flow cytometry, qRT-PCR and multiplex. To identify fibroblast-derived exosome cargo, microRNAs (miRNAs) were isolated from exosomes followed by library preparation and sequencing. To complement sequencing results, potential fibroblast-derived exosome miRNA regulators were computationally inferred using MiRaGE (miRNA Ranking by Gene Expression) and miRSCAPE.
Results: MØs activated with dermal fibroblast-derived exosomes from SSc patients upregulated surface expression of CD163, CD206, and MHC Class II compared with MØs incubated with healthy control fibroblast exosomes. This pattern of expression was consistent with the previously identified surface marker profile established for human SSc MØs. In addition, SSc fibroblast-derived exosomes elicit elevated levels of secreted IL-6, IL-10, IL-12p40 and TNF from MØs. Combining exosomal miRNA sequencing results with computational prediction algorithms identified several candidate miR regulators of SSc MØ activation: miR-16-5p, miR-100-5p, miR-221-3p, and miR-3666.
Conclusion: In this work, we demonstrate for the first time that human SSc dermal fibroblasts can mediate MØ activation through exosomes. Consistent with prior studies, we show that MØs express surface markers and release mediators associated with both alternative and inflammatory MØ activation. Our results implicate miRs in the induction of SSc MØ activation and may provide additional targets for therapeutic intervention in SSc.
Disclosures: H. Yang, None; R. Bhandari, None; N. Kosarek, None; J. Garlick, None; M. Whitfield, Bristol-Myers Squibb(BMS), Celdara Medical LLC; P. Pioli, Celdara Medical, LLC.
