Fig. 3: Capsular MERTK^high macrophages have a modulatory phenotype.

A The heatmap shows the top 20 marker genes for the capsular myeloid cell clusters (two-sided Wilcoxon test, BH adjusted P < 1 × 10⁻⁹. B Geneset over-representation analysis of gene ontology (GO) Biological Processes (BP) in the capsular myeloid clusters (one-sided Fisher tests, BH adjusted P < 0.05. C UMAPs show the expression of modulatory macrophage genes including MERTK, LYVE1, CD163, TREM2, FOLR2 and MRC1 in capsular macrophages, localising these markers to MERTK+LYVE1^high and MERTK+LYVE1^low clusters. D Representative images of 3,3′-diaminobenzidine immunostaining (brown) for MERTK in sections from comparator and frozen shoulder patient tissues, staining is localised to the capsule lining region. Nuclear counterstain is haematoxylin, scale bar=50μm. E Graph shows quantitative analysis of MERTK+ cells in comparator (C, n = 8 donors) and frozen shoulder (FS, n = 9 donors) patient tissues pooled from 2 independent experiments, bars show median values. Statistically significant differences were calculated using a two-sided Mann-Whitney test (P = 0.0055). F Violin plots show expression of MERTK ligands GAS6 and PROS1 in capsular fibroblast sub-populations. G Selected predicted ligand-receptor interactions between sender population (PROS1+ or GAS6+ fibroblasts) and receiver population (MERTK+LYVE1^high or MERTK+LYVE1^low macrophages) are shown. Predictions were generated from comparator (n = 6 donors) and frozen shoulder (n = 4 donors), sub-populations as in Fig. 1G (NATMI specificity score). H Representative immunofluorescence images of the lining region of frozen shoulder patient tissues, showing the topographical proximity of MERTK with associated ligands GAS6 and PROS1. Fibroblast markers include CLIC5, PDPN, AXL. Scale bar=20μm.