Fig. 3

Impact of endogenous Prg4 recombination on macrophage accumulation and polarization in synovial tissues isolated from the knee joints of Prg4 gene-trap (Prg4^GT/GT) animals and the role of PRG4 in regulating inflammatory macrophage recruitment to the joint in response to Pam3CSK4, a Toll-like receptor 2 (TLR2) agonist. Prg4 gene recombination (Prg4^GTR/GTR) was performed in 3-week-old mice by intraperitoneal administration of tamoxifen (0.1 mg/g) in corn oil vehicle daily for 10 days, using vehicle-only administered age-matched Prg4^GT/GT as controls. Pam3CSK4 (3μg in 10-μL sterile water) or vehicle (10μL) treatments were performed in age-matched 2-month-old Prg4^GT/GT or Prg4^GTR/GTR animals (n=4 per group; 2 males and 2 females), and macrophage polarization and newly infiltrated inflammatory macrophage accumulation were determined in synovial tissues at 72h post-injections. Macrophages in 2- and 6-month-old Prg4^GTR/GTR animals were compared to age-matched Prg4^GT/GT animals (n=4–6 per group). Macrophages in synovial tissues were identified as CD11b+ F4/80+ MHC class II− CD86+ and/or CD206+ (using gating strategies shown in Supplementary Figure 2 and in Fig. 1B). Newly infiltrated inflammatory macrophages were identified as CD11b+ F4/80+ MHC class II+ Ly-6C+ CD86+. Macrophage cell counts were determined using precision counting beads and expressed as numbers per joint. We compared the ratio of M1 (CD11b+ F4/80+/MHC class II− CD86+/CD206−) and M2 (CD11b+ F4/80+ MHC class II− CD86−/CD206+) macrophages in synovial tissues from 6-month-old Prg4^GT/GT and Prg4^GTR/GTR animals. In all experiments, independent biological samples were analyzed in duplicates, and the averages of both technical replicates were included in the analysis. Statistical analyses of total macrophages and percentages of CD86+ and/or CD206+ macrophages were performed using one-way and two-way ANOVA, respectively. Tukey’s post hoc test was used in one- and two-way ANOVAs. ns non-significant; *p<0.05; **p<0.01; ***p<0.001; and ****p<0.0001. A Prg4 gene recombination at 3 weeks reduced macrophage accumulation in 2- and 6-month-old joint tissues. B Prg4^GTR/GTR joints at 6 months contained lower percentages of CD86+/CD206− and CD86+/CD206+ macrophages and a higher percentage of CD86−/CD206+ macrophages compared to age-matched Prg4^GT/GT joints. C M1/M2 ratio was reduced as a consequence of Prg4 recombination. D Gating strategy to identify newly infiltrated inflammatory macrophages. Singlets and viable cells were identified as shown in Supplementary Figure 2. Cells were then gated for CD11b and F4/80 and identified MHC class II+ Ly-6C+ CD86+ population as shown in the representative flow cytometry contour plot. E Representative flow cytometry contour plot depicting stronger CD86 epitope staining in newly infiltrated inflammatory macrophages compared to the existing macrophage populations in synovial tissues of Pam3CSK4-administered Prg4^GT/GT joints. F Pam3CSK4 administration resulted in greater inflammatory macrophage recruitment in 2-month-old Prg4^GT/GT joints compared to Prg4^GTR/GTR joints. G Pam3CSK4 administration resulted in a shift towards an inflammatory CD86+ macrophage, particularly in 2-month-old Prg4^GT/GT joints