Fig. 5
Impact of macrophage depletion on synovial pathology in 4-month-old Prg4 gene-trap (Prg4GT/GT) animals. The knee joints of Prg4GT/GT animals were injected with clodronate liposomes (CLO) (10μL; 18.4 mM) or PBS liposomes (PBS) (10μL) twice weekly for 2 weeks according to the scheme presented in Supplementary Figure 1D. The percentages of CD11b+ F4/80+ MHC class II− CD86+ or CD86−/CD206+ macrophages were determined in cell suspensions from digested joint capsule tissues on day 15 following treatment initiation, and the numbers of CD86+ macrophages were determined using precision counting beads. Cell gating was performed as described in Supplementary Figure 2 and Fig. 1B. In a separate study, synovial pathology measurements were performed on day 30 following treatment initiation and included synovial thickness and staining intensities of fibrosis markers: alpha-smooth muscle actin (α-SMA), PLOD2, and collagen type I (COL-I) as well as the monocyte/macrophage marker CD11b (n=6 per group). Age-matched Prg4 wildtype animals (Prg4+/+, n=3) were used as controls. Analyses of macrophage cell count, synovial thickness, α-SMA, PLOD2, COL-I, and CD11b staining intensities were performed using one-way ANOVA followed by Tukey’s post hoc test. Analysis of CD86+ or CD86−/CD206+ macrophages was performed using Student’s t test. Scale = 10μm. ns non-significant; *p<0.05; **p<0.01; ***p<0.001; and ****p<0.0001. A Representative contour plot of CD86 and CD206 staining in macrophages of synovial tissues from CLO- or PBS-treated joints at 15 days following treatment initiation. The most prominent change with CLO treatment was a reduction in the percentage of CD86+/CD206+ macrophages. B Percentages of CD86+ and CD86−/CD206+ macrophages were reduced with CLO treatment. C Inflammatory macrophages in Prg4GT/GT joints were depleted with CLO treatment. D Representative histological sections (H&E, α-SMA, PLOD2, COL-I, and CD11b) depict thickening of the synovial membrane, immune cell infiltration, positive α-SMA, PLOD2, COL-I, and CD11b staining (all indicated by arrows) in PBS-treated joints. Sections were counterstained with DAPI. Macrophage depletion reduced synovial thickness in Prg4GT/GT joints. E Macrophage depletion reduced α-SMA, PLOD2, COL-I, and CD11b staining in Prg4GT/GT joints