Multiplex cytokine analysis of dermal interstitial blister fluid defines local disease mechanisms in systemic sclerosis

Kristina EN Clark, Henry Lopez, Bahja Ahmed Abdi, Sandra G Guerra, Xu Shiwen, Korsa Khan, Oseme Etomi, George R Martin, David J Abraham, Christopher P Denton, Richard J Stratton

Clinical diversity in systemic sclerosis (SSc) reflects multifaceted pathogenesis and the effect of key growth factors or cytokines operating within a disease-specific microenvironment. Dermal interstitial fluid sampling offers the potential to examine local mechanisms and identify proteins expressed within lesional tissue. We used multiplex cytokine analysis to profile the inflammatory and immune activity in the lesions of SSc patients. Dermal interstitial fluid sample from the involved forearm skin, and synchronous plasma samples were collected from SSc patients (n = 26, diffuse cutaneous SSc (DcSSc) n = 20, limited cutaneous SSc (LcSSc) n = 6), and healthy controls (HC) (n = 10) and profiled by Luminex® array for inflammatory cytokines, chemokines, and growth factors. Luminex® profiling of the dermal blister fluid showed increased inflammatory cytokines (median interleukin ( IL)-6 in SSc 39.78 pg/ml, HC 5.51 pg/ml, p = 0.01,median IL-15 in SSc 6.27 pg/ml, HC 4.38 pg/ml, p = 0.03), chemokines (monocyte chemotactic protein (MCP)-3 9.81 pg/ml in SSc, 7.18 pg/ml HC, p = 0.04), and profibrotic growth factors (platelet derived growth factor (PDGF)-AA 10.38 pg/ml versus 6.94 pg/ml in HC, p = 0.03). In general dermal fluid and plasma cytokine levels did not correlate, consistent with predominantly local production of these factors within the dermal lesions, rather than leakage from the serum. In hierarchical clustering and network analysis IL-6 emerged as a key central mediator. Our data confirm that an immuno-inflammatory environment and aberrant vascular repair are intimately linked to fibroblast activation in lesional skin in SSc. This non-invasive method could be used to profile disease activity in the clinic, and identifies key inflammatory or pro-fibrotic proteins that might be targeted therapeutically. Distinct subgroups of SSc may be defined that show innate or adaptive immune cytokine signatures.