Clinical and pathogenic significance of S100A4 overexpression in systemic sclerosis.

OBJECTIVES: We have studied the damage-associated molecular pattern protein S100A4 as a driver of fibroblast activation in systemic sclerosis (SSc). METHODS: S100A4 protein concentration was measured by ELISA in serum of SSc (n=94) and healthy controls (n=15). Protein expression in skin fibroblast cultures from diffuse cutaneous SSc (SScF, n=6) and healthy controls (normal fibroblasts (NF), n=6) was assessed. Recombinant S100A4 and a high affinity anti-S100A4 neutralising monoclonal antibody (AX-202) were tested on SScF and NF. RESULTS: Median (range) S100A4 (ng/mL) was higher in serum of SSc (89.9 (15.0-240.0)) than healthy controls (71.4 (7.9-131.8); p=0.027). There was association with SSc-interstitial lung disease (p=0.025, n=55), scleroderma renal crisis (p=0.026, n=4). Median (range) S100A4 (ng/mL) was higher in culture supernatants of SScF (4.19 (0.52-8.42)) than NF controls (0.28 (0.02-3.29); p<0.0001). AX-202 reduced the constitutive profibrotic gene and protein expression phenotype of SScF. Genome-wide RNA sequencing analysis identified an S100A4 activated signature in NF overlapping the hallmark gene expression signature of SScF. Thus, 464 differentially expressed genes (false discovery rate (FDR) <0.001 and fold change (FC) >1.5) induced in NF by S100A4 were also constitutively overexpressed, and downregulated by AX-202, in SScF. Pathway mapping of these S100A4 dependent genes in SSc showed the most significant enriched Kegg pathways (FDR <0.001) were regulation of stem cell pluripotency (4.6-fold) and metabolic pathways (1.9-fold). CONCLUSION: Our findings provide compelling evidence for a profibrotic role for S100A4 in SSc and suggest that serum level may be a biomarker of major organ manifestations and disease severity. This study supports examining the therapeutic potential of targeting S100A4 in SSc.

Effect of Anti-S100A4 Monoclonal Antibody Treatment on Experimental Skin Fibrosis and Systemic Sclerosis-Specific Transcriptional Signatures in Human Skin.

OBJECTIVE: S100A4 is a DAMP protein. S100A4 is overexpressed in patients with systemic sclerosis (SSc), and levels correlate with organ involvement and disease activity. S100A4-/- mice are protected from fibrosis. The aim of this study was to assess the antifibrotic effects of anti-S100A4 monoclonal antibody (mAb) in murine models of SSc and in precision cut skin slices of patients with SSc. METHODS: The effects of anti-S100A4 mAbs were evaluated in a bleomycin-induced skin fibrosis model and in Tsk-1 mice with a therapeutic dosing regimen. In addition, the effects of anti-S100A4 mAbs on precision cut SSc skin slices were analyzed by RNA sequencing. RESULTS: Inhibition of S100A4 was effective in the treatment of pre-established bleomycin-induced skin fibrosis and in regression of pre-established fibrosis with reduced dermal thickening, myofibroblast counts, and collagen accumulation. Transcriptional profiling demonstrated targeting of multiple profibrotic and proinflammatory processes relevant to the pathogenesis of SSc on targeted S100A4 inhibition in a bleomycin-induced skin fibrosis model. Moreover, targeted S100A4 inhibition also modulated inflammation- and fibrosis-relevant gene sets in precision cut SSc skin slices in an ex vivo trial approach. Selected downstream targets of S100A4, such as AMP-activated protein kinase, calsequestrin-1, and phosphorylated STAT3, were validated on the protein level, and STAT3 inhibition was shown to prevent the profibrotic effects of S100A4 on fibroblasts in human skin. CONCLUSION: Inhibition of S100A4 confers dual targeting of inflammatory and fibrotic pathways in complementary mouse models of fibrosis and in SSc skin. These effects support the further development of anti-S100A4 mAbs as disease-modifying targeted therapies for SSc.