Background/Purpose: Activated macrophages (MØs) have been implicated as regulators of fibrosis in patients with systemic sclerosis (SSc), and we have shown that MØs from SSc patients are activated under basal conditions, releasing elevated levels of IL-6, CCL2, and TGF-b. Because MØs are plastic and may be modulated by local micro-environmental factors, it is likely that activation of MØs in SSc is regulated by the interplay of many factors. In this study, we identify a critical role for IL-6 in the regulation of human SSc MØ activation, and provide evidence for a molecular mechanism by which MØ and fibroblast-derived IL-6 supports the pro-fibrotic immuno-phenotype of these cells.

Methods:

Plasma and PBMCs were obtained from whole blood of 6 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. To demonstrate specificity of IL-6-mediated effects on SSc MØ activation, MØ differentiation was performed in indicated cultures in the presence of anti-IL-6 neutralizing antibody or isotype control. 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: Healthy age and gender-matched control MØs differentiated in plasma from SSc patients recapitulate the immuno-phenotype of MØs derived from SSc patients. Intriguingly, when MØs from SSc patients are differentiated in healthy control plasma, the pro-fibrotic activation profile of these cells is abrogated, suggesting SSc MØ activation arises from soluble factors present in the local micro-environment. Analysis of signaling pathway activation in SSc MØs demonstrates constitutive phosphorylation of STAT3 in these cells, consistent with IL-6 activation. Differentiation of SSc MØs in the presence of anti-IL-6 neutralizing antibody results in loss of basal MØ activation—i.e. loss of CCL2 and IL-6 production. To identity the potential mechanism by which IL-6 mediates SSc MØ activation, we assessed expression of regulators of fibrosis and immune activation. We now show that IL-6 upregulates expression of IL-4Ra in SSc MØs, and that stimulation with IL-4/IL-13 results in enhanced production of CCL2 in an IL-6-dependent manner.

Conclusion: IL-6 mediates pro-fibrotic activation of SSc MØs, as blockade of IL-6 abrogates production of CCL2 and IL-6 in these cells. Our results suggest a potential mechanism by which IL-6 mediates these effects, as IL-6 upregulates expression of IL-4Ra, and stimulation of SSc MØs with IL-4/IL-13 results in enhanced CCL2 expression in an IL-6-dependent manner. These results implicate a role for IL-6 in the induction of the pro-fibrotic activation profile of SSc MØs, and suggest targeted intervention against the signaling pathways that underlie MØ activation may be therapeutically beneficial in ameliorating SSc disease.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/il-6-mediates-activation-of-macrophages-in-patients-with-systemic-sclerosis/