P130 Prolonged treatment with novel cyclical RP peptides reprogrammes M2 macrophages towards a resolving phenotype in diffuse cutaneous systemic sclerosis

Abstract Background/Aims Alternatively activated (M2) macrophages are believed to promote pathological fibrosis and represent a potential therapeutic target in fibrotic diseases including systemic sclerosis (SSc). Novel 10 amino acid therapeutic peptides targeting M2 macrophages, via binding to the CD206 receptor, represent promising therapeutics by reducing macrophage-stimulated fibrosis in tissue culture and mouse model systems. In this study, we investigate the potential for prolonged treatment with novel cyclical variants of the RP peptides to reprogramme pathogenic SSc macrophages to a pro-resolving phenotype. Methods Macrophages were derived from peripheral blood monocytes in the presence of M-CSF (4ng/ml) for 7 days from patients with diffuse cutaneous SSc (dcSSc) and healthy controls (n=3 dcSSc and 1 HC). On day 7, 9 and 11, macrophages were treated with 10µM of RP peptide (cyclical RP606-30, or comparator RP; RP class) or left untreated in n=6 replicates per treatment group. On day 14, media were removed and cells were collected, washed, lysed for RNA extraction, and profiled by qPCR for CD206 (pro-fibrotic M2-macrophage marker), CD86 (pro-inflammatory M1-macrophage marker) and MERTK (pro-resolution regulatory efferocytosis marker), relative to the reference gene TBP. The ratios of CD86/CD206 were used to assay pro-inflammatory vs. pro-fibrotic and MERTK/CD206 for pro-resolution vs. pro-fibrotic phenotypes. Results For individual patients with dcSSc, cyclical RP606-30, but not comparator RP, reduced CD206 expression (Patient 1: 5.74 vs. 4.10, p=NS; Patient 2: 7.38 vs. 3.92, p = 0.014; Patient 3: 2.17 vs. 0.26, p = 0.021, relative expression level untreated vs. RP606-30) and enhanced the ratio of MERTK/CD206 (Patient 1: 0.22 vs. 0.35, p=NS; Patient 2: 0.18 vs. 0.31, p = 0.0007, relative expression untreated vs. RP606-30). Similar effects were seen in healthy control macrophages treated with RP606-30, with a significant decrease in relative CD206 expression (p = 0.012), and increase in both the MERTK/CD206 (p < 0.0001) and CD86/CD206 (p < 0.0001) ratios. Combining data for dcSSc macrophages indicated that CD206 expression was reduced with RP606-30 treatment compared to no treatment (mean ± SEM: 2.76 ± 0.51 vs. 5.06 ± 0.73, p = 0.079), and MERTK expression was slightly increased (1.24 ± 0.07 vs. 1.13 ± 0.17, p = 0.809). Compared to untreated cells, RP606-30 treatment significantly increased both the CD86/CD206 (M1/M2) ratio (2.04 ± 0.34 vs. 0.86 ± 0.22, p = 0.0036) and MERTK/CD206 ratio (0.33 ± 0.03 vs. 0.20 ± 0.03, p = 0.016). Conclusion We found that prolonged treatment with RP606-30, a cyclical RP peptide, reduced relative CD206 expression, and significantly increased the CD86/CD206 (M1/M2) ratio and the MERTK/CD206 ratio in macrophages derived from patients with dcSSc. These findings indicate that RP peptides reprogramme macrophages from a pro-fibrotic towards a pro-resolution phenotype. To further characterise the phenotypic changes involved in the polarisation of macrophages towards this pro-resolving phenotype, we plan to assay the media for levels of pro-resolving lipid metabolites (LXA4 and RvD1). Disclosure J. Mendall: None. B. Ahmed Abdi: None. K. Shetty: None. S. Lopez Garces: None. V. Ong: None. C. Denton: None. D. Abraham: None. C. Yates: Corporate appointments; Scientific Officer Riptide. J. Jaynes: Corporate appointments; Scientific Officer Riptide. H. Lopez: Corporate appointments; CEO of Murigenics. G. Martin: Corporate appointments; Senior Scientific Officer Riptide. R.J. Stratton: None.