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. 2021 Sep 14;23:241. doi: 10.1186/s13075-021-02621-9

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© The Author(s) 2021

Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

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Fig. 5 — Impact of macrophage depletion on synovial pathology in 4-month-old Prg4 gene-trap (Prg4^GT/GT) animals. The knee joints of Prg4^GT/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 Prg4^GT/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 Prg4^GT/GT joints. E Macrophage depletion reduced α-SMA, PLOD2, COL-I, and CD11b staining in Prg4^GT/GT joints