Zoe Chafouleas1, Angelique Cortez1, James Whitley1, Dorothea Barton1, Lin Brown1, Michael Whitfield2, Patricia pioli3 and Sladjana Skopelja-Gardner1, 1Dartmouth Hitchcock Medical Center, Lebanon, NH, 2Dartmouth Geisel School of Medicine, Department of Biomedical Data Science, Lebanon, NH, 3Geisel School of Medicine at Dartmouth, Lebanon, NH
Background/Purpose: With an incidence of 10 cases per million and lack of animal or ex vivo disease models, dermatomyositis (DM) remains a debilitating disease without a cure or the understanding of the pathogenic mechanisms. Recent studies showed that monocytes and macrophages reside in DM skin and demonstrate diverse phenotypes. While keratinocytes in DM skin have an inflammatory gene signature, whether and how macrophages contribute to keratinocyte activation is not known.
Methods: DM and healthy controls (HC) were recruited by informed consent. Keratinocytes were isolated from skin biopsies and cultured in vitro. Monocytes were isolated by CD14+ selection from PBMCs and differentiated into macrophages in RPMI complete medium with M-CSF and 10% autologous serum. RNA was isolated after 5 days of differentiation and RNA-seq performed. 3D skin tissues were constructed from healthy fibroblasts, HC or DM monocytes, and autologous HC or DM keratinocytes over 6 weeks. RNA-seq and pathway analyses (Rosalind) were done on the macrophages, 3D tissues, and the corresponding intact skin biopsies.
Results: Cell type enrichment analyses of RNA-seq data revealed a monocyte/macrophage signature in 3/5 DM patients, relative to HC skin (PanglaoDB Cell Types). The gene-enriched signaling pathways significantly upregulated in DM vs. HC macrophages were: extracellular matrix (ECM)-Receptor interactions and Il1-mediated ECM reorganization. Interestingly, these same pathways were upregulated in the intact skin of the matched DM patients, i.e. only the patients with a skin monocyte/macrophage signature. Several cytokines and proteases with ECM-remodeling ability were significantly upregulated in DM macrophages and the corresponding skin biopsies: Cxcl7, Cxcl5, Ccl13, MMP-11,Il1b, and Il-27. To investigate the pathogenic interactions between DM macrophages and keratinocytes, we established 3D skin tissue models that incorporate autologous HC or DM macrophages and keratinocytes. Gene-set enrichment scores for ECM remodeling pathways uncovered in DM macrophages and intact skin biopsies were also higher in autologous DM 3D tissues compared to the autologous HC tissues (e.g. DM +0.482 vs HC -0.421). Likewise, the 3D tissues demonstrated decreased expression of genes involved in skin keratinization, as seen in the intact biopsies. The 3D skin tissues constructed of healthy fibroblasts and keratinocytes but DM macrophages showed an upregulation of the Il1-mediated ECM reorganization pathway. DM macrophages retained increased expression of cytokines and proteases outlined above. Moreover, healthy keratinocytes had increased surface expression of the activation marker HLA-DR in the presence of DM but not healthy macrophages.
Conclusion: Differentiated macrophages from DM patients upregulate genes involved in the remodeling of ECM. The same transcriptomic signature is found in the skin of a subset of DM patients: enriched for monocyte/macrophage signature. Our novel autologous 3D tissue model of DM mimics transcriptionally disease-specific changes in ECM organization and demonstrates that DM macrophages activate keratinocytes and contribute to the IL-1 mediated remodeling of ECM.
Disclosures: Z. Chafouleas, None; A. Cortez, None; J. Whitley, None; D. Barton, None; L. Brown, None; M. Whitfield, Corbus Pharmaceuticals, Celdara Medical LLC, Bristol-Myers Squibb(BMS); P. pioli, None; S. Skopelja-Gardner, None.