Background/Purpose: Genome-wide gene expression studies implicate macrophages (MØs) as mediators of fibrosis in systemic sclerosis (SSc) and our data indicate that MØs constitute the dominant inflammatory signature in SSc tissue. Because MØs are plastic and subject to modulation by local micro-environmental factors, in vivoMØ polarization spans a broad spectrum of activation states. Therefore, SSc MØ activation is likely shaped by the interplay of many factors, resulting in the development of an immuno-phenotype that supports disease development and progression. In this study, we define the activation profile of human SSc MØs as pro-fibrotic, and provide direct evidence that SSc MØs elicit activation of NFkappaB signaling pathways and proliferation in fibroblasts.

Methods: Plasma and PBMCs were obtained from whole blood of 14 SSc patients (disease duration <5 years) and from 5 healthy age and gender-matched control subjects following informed written consent. CD14⁺monocytes were isolated from PBMCs using magnetic bead selection, and were cultured with either autologous or allogeneic plasma for 7 days to differentiate the cells into MØs. Immune activation studies were performed using 10 ng/ml LPS. For reciprocal activation studies, SSc MØs were co-cultured with fibroblasts using Transwells. RNA expression in MØs and fibroblasts was analyzed using genome-wide analysis and RT-PCR, and protein expression and secretion were monitored using flow cytometry and by ELISA.

Results: Genome-wide expression profiling of human SSc MØs shows up-regulation of signaling pathways involved in antigen presentation and extracellular matrix organization. SSc MØs show an alternatively activated phenotype based on expression of cell surface markers, and express higher levels of TGF-beta and IL-6 under both basal and LPS-stimulated conditions. Differentiation of healthy MØs with SSc plasma results in enhanced TGF-beta and IL-6/JAK/STAT3 signaling pathway activation, and SSc-differentiated MØs co-cultured with dermal fibroblasts show enrichment of inflammatory and proliferation expression signatures. Dermal fibroblasts co-cultured with SSc-differentiated MØs show an activated phenotype.

Conclusion: The activation profile of SSc-differentiated MØs is pro-fibrotic. We demonstrate that SSc MØs are activated under basal conditions, and that these cells release mediators associated with both alternative and inflammatory MØ activation. For the first time, we show that co-culture of SSc MØs with fibroblasts induces fibroblast activation and proliferation. Intriguingly, these data suggest that activation of SSc MØs arises from soluble factors in local microenvironments. Collectively, these studies implicate MØs as likely drivers of fibrosis in SSc and suggest therapeutic targeting of these cells may be beneficial in ameliorating disease in SSc patients.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/elucidating-the-activation-profile-of-systemic-sclerosis-macrophages/