Profibrotic Activation of Human Macrophages in Systemic Sclerosis

Abstract

Objective: Genome-wide gene expression studies implicate macrophages as mediators of fibrosis in systemic sclerosis (SSc), but little is known about how these cells contribute to fibrotic activation in SSc. We undertook this study to characterize the activation profile of SSc monocyte-derived macrophages and assessed their interaction with SSc fibroblasts.

Methods: Plasma and peripheral blood mononuclear cells (PBMCs) were obtained from whole blood from SSc patients (n = 24) and age- and sex-matched healthy controls (n = 12). Monocytes were cultured with autologous or allogeneic plasma to differentiate cells into macrophages. For reciprocal activation studies, macrophages were cocultured with fibroblasts using Transwell plates.

Results: The gene expression signature associated with blood-derived human SSc macrophages was enriched in SSc skin in an independent cohort and correlated with skin fibrosis. SSc macrophages expressed surface markers associated with activation and released CCL2, interleukin-6, and transforming growth factor β under basal conditions (n = 8) (P < 0.05). Differentiation of healthy donor monocytes in plasma from SSc patients conferred the immunophenotype of SSc macrophages (n = 13) (P < 0.05). Transwell experiments demonstrated that coculture of SSc macrophages with SSc fibroblasts induced fibroblast activation (n = 3) (P < 0.05).

Conclusion: These data demonstrate that the activation profile of SSc macrophages is profibrotic. SSc macrophages are activated under basal conditions and release mediators and express surface markers associated with both alternative and inflammatory macrophage activation. These findings also suggest that activation of SSc macrophages arises from soluble factors in local microenvironments. These studies implicate macrophages as likely drivers of fibrosis in SSc and suggest that therapeutic targeting of these cells may be beneficial in ameliorating disease in SSc patients.

Figure 1:. The gene expression signature of blood-derived SSc macrophages is significantly enriched in SSc skin.

Monocytes were isolated from peripheral blood of SSc patients or healthy age and gender-matched controls and differentiated in autologous plasma for 7 days. RNA was extracted and analyzed by DNA microarray (1A) to generate the gene expression signature enriched in SSc macrophages (371 genes, FDR<10%) that was used to interrogate an independent cohort. The gene expression signature of SSc blood-derived macrophages was found to be significantly enriched in the skin of dcSSc and lcSSc patients (1B) relative to healthy controls and in the inflammatory molecular subset (1C).

Figure 2:. Immunophenotypic characterization of SSc macrophages.

SSc patient or control monocytes were differentiated in autologous plasma for 7 days and stimulated or not with 10 ng/ml LPS for 24 hours. RNA was extracted and analyzed by qRT-PCR for mRNA cytokine levels (2A); supernatants were collected and analyzed by ELISA for secreted protein production (2B). Supernatants were collected from healthy control or SSc macrophage/fibroblast co-cultures (or monocultures, as indicated), and secreted TGF-β1 levels were measured by ELISA (2C). mRNA levels of TGF-β receptors (2D) or macrophage activation markers (2E) were quantified by qRT-PCR. Surface marker expression was quantified using flow cytometry and is presented in mean fluorescence intensity (MFI) units (2F). Gating of positively stained cells was determined by FMO controls. n=7 controls and 8 SSc patients, p<0.05

Figure 3:. STAT3 is phosphorylated in dcSSc macrophages under basal conditions.

dcSSc or control monocytes were differentiated with autologous plasma for five days, washed, and cultured in fresh media in the absence of plasma for an additional day in the presence of blocking anti-IL-6 or isotype control antibody. Whole cell lysates were prepared and analyzed using a phospho-STAT3 ELISA. Total STAT3 levels were not significantly different between healthy control and dcSSc macrophages (not shown). Data are plotted as absorbance units (450 nM). Data are representative of results obtained with 4 healthy controls and 4 dcSSc patients, p<0.05

Figure 4:. Differentiation of healthy cells in SSc plasma confers SSc macrophage activation.

Healthy donor monocytes (Control) were differentiated in autologous (CP) or SSc patient plasma (SScP) for 7 days, and SSc patient monocytes (SSc) were differentiated in autologous (SScP) or healthy donor (CP) plasma, as indicated. Macrophages were immunophenotyped using flow cytometry (4A), qRT-PCR (4B) and by ELISA (4C). n=3 control and 3 SSc patients (4A); n=4 controls and 4 SSc patients (4B); and n=6 healthy controls and 6 SSc patients (4C), p<0.05

Figure 5:. SSc plasma-differentiated macrophages activate SSc fibroblasts.

Healthy donor monocytes were differentiated in control or SSc plasma and co-cultured with SSc or control dermal fibroblasts using Transwells for 5 days (5A). RNA was extracted from fibroblasts cultured alone (monoculture) or with SSc plasma-differentiated macrophages (co-culture) and assayed for α-SMA expression by qRT-PCR (5B). RNA from monocultured or co-cultured macrophages (5C and 5D) and fibroblasts (5E and 5F) was analyzed by microarray for changes in gene expression and pathway enrichment. Data are representative of 3 independent experiments using cells derived from 3 separate controls and SSc patients, p<0.05