P154 High-density proteomic analysis of skin blister fluid and plasma in systemic sclerosis identifies local and systemic differences for key proteins

Abstract

Abstract Background/Aims Simultaneous analysis of multiple proteins in biological fluids offers insight into the pathogenesis of SSc. Here, we report a proteomic analysis of plasma and dermal interstitial fluid in SSc compared with healthy controls (HC). Methods The prospectively collected BIOPSY cohort recruited 52 SSc patients (21 early dcSSc, 15 established dcSSc,16 lcSSc) and 16 HC. Mean baseline skin score (MRSS) for early dcSSc was 21 (sd 11.2). This analysis utilised forearm skin blister fluid obtained using the dermal suction blister method and paired simultaneous plasma samples from early dcSSc and HC at baseline. These were assayed using the Olink antibody platform(www.olink.com) and reported as normalised protein expression (NPX), corresponding to log2 (expression). T-test with FDR correction (p < 0.05) assessed statistical significance. Pathway analysis was conducted by STRING consortium 2020. Results 1,196 proteins were analysed in paired blister/plasma samples from 14 early dcSSc patients and 16 HC. 447 proteins were significantly different in the blister fluid of early dcSSc patients compared with HC (Table 1), whereas only 183 proteins in plasma. Of these, 130 proteins were simultaneously different in both blister fluid and plasma of early dcSSc including key cytokines associated with fibrosis and vasculopathy such as IL-6,VEGF-1, MCP-1, COL4A1, COMP, Thy1 and THBS4. No correlation was seen between these proteins and MRSS. 310 proteins were significantly elevated in blister fluid alone in early dcSSc patients compared to HC. These included cytokines (IL7,IL18, OSM), chemokines (CCL7,CCL18, CCL3), matricellular proteins (CYR61 and osteopontin). KEGG pathway analysis of the significantly elevated proteins in blister fluid in early dcSSc compared to HC highlighted pathways including cytokine-cytokine receptor interaction, cell adhesion molecules, MAPK signalling pathway and PI3K-AKT signalling pathway (FDR<0.01) P154 Table 1:Protein symbolRaw mean NPXFold ChangeAdjusted p valueDetails of proteinHCdcSScKLK42.69815.9260.004Kallikrein-related peptidase 4IL616.13815.1070.008Interleukin 6NT-proBNP33.60712.8570.018N-terminal pro-brain natriuretic peptideAREG12.74711.9780.017AmphiregulinLTBP24.59311.7990.015Latent-TGF beta-binding protein 2SFRP181.10710.4040.025Secreted Frizzled Related Protein 1TNC18.0128.7280.010Tenascin CCPXM12.1477.4650.002Probable carboxypeptidase X1CYR6110.4936.1060.011Cysteine-rich angiogenic inducer 61 (CCN1)PAPPA10.2675.4800.008Pregnancy-associated plasma protein AEDA2R11.4795.4070.008Ectodysplasin-A2 rceptorNOV61.6385.1190.003Nephroblastoma overexpressed protein (CCN3)GDF-1520.8074.5400.002growth/differentiation factor 15SCARF211.8684.3770.008Scavenger Receptor Class F Member 2CXCL1071.6204.1960.028interferon-γ inducible protein 10THBS4110.6824.1630.008Thrombospondin 4CXCL1324.2564.0470.011C-X-C Motif Chemokine Ligand 13MAPT1.6614.0410.015Microtubule-associated protein tauCOL4A116.7054.0090.003Collagen type IV alpha 1MCP-11128.943.6510.016Monocyte chemoattractant protein-1Top 20 most upregulated proteins in skin blister fluid for early dcSSc (n = 14) compared with healthy control (HC, n = 16). Conclusion Numerous dysregulated proteins were identified in dermal blister fluid and plasma of early dcSSc patients. Substantially more were identified in dermal blister fluid, highlighting its potential for providing detailed information on local pathologic processes. A subset of proteins were dysregulated in both plasma and blister fluid, suggesting these may reflect systemic abnormalities. Further work will utilise this cohort to integrate gene and protein expression across the full spectrum of early dcSSc, established dcSSc and lcSSc Disclosure K.E. Clark: None. C. Campochiaro: None. E. Csomor: Corporate appointments; employee of GSK. A. Taylor: Corporate appointments; employee of GSK. K. Nevin: Corporate appointments; employee of GSK. M.A. Morse: Corporate appointments; employee of GSK. V.H. Ong: None. E. Derrett-Smith: None. N. Wisniacki: Corporate appointments; employee of GSK. S. Flint: Corporate appointments; employee of GSK. C.P. Denton: Consultancies; Actelion, GlaxoSmithKline, Bayer, Sanofi, lnventiva, Boehringer Ingelheim, Roche, Bristol Myers Squibb, CSL Behring, UCB, Leadiant Biosciences, Corbus, Servier, Arxx Therapeutics.