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. 2024 Nov 25;25(12):2270–2283. doi: 10.1038/s41590-024-02011-8

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© The Author(s) 2024

Open Access This 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/.

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Fig. 3 — a, Illustration of the experimental protocol. b, Principal component analysis (PCA) of three subsets of synovial macrophages by RNA-seq; n = 3 mice for each plot and n = 9 mice for each population. c, Volcano plots showing DEGs between LYVE1⁺CX₃CR1⁺, MHCII⁺CD11c⁻ and MHCII⁺CD11c⁺ macrophages from WT mice; P_adj, adjusted P value. d, Heat map of the expression of canonical macrophage genes (normalized values) and dendritic cell markers in LYVE1⁺CX₃CR1⁺, MHCII⁺CD11c⁻ and MHCII⁺CD11c⁺ macrophages from bulk RNA-seq analysis. e, Heat map of single-sample gene set enrichment analysis (ssGSEA) of three synovial macrophage subsets by RNA-seq. The signature genes from a previously published dataset (Xue et al.²⁷) describing the transcriptional programs activated with 28 different stimuli were used; TPP, TNF+PGE₂+P3C; IFN, interferon; PA, palmitic acid; LPS, lipopolysaccharide; TNF, tumor necrosis factor; GC, glucocorticoid; HDL, high-density lipoprotein; P3C, Pam3CysSerLys4; OA, oleic acid; Lia, linoleic acid; LA, lauric acid; sLPS, standard lipopolysaccharide; upLPS, ultrapure lipopolysaccharide. f, Quantification of ssGSEA scores for signaling pathways of the indicated stimuli for each subset; n = 3 mice for each plot and n = 9 mice for each population. Data represent mean ± s.e.m. g, Heat map of ssGSEAs of three synovial macrophage subsets with KEGG enrichment analysis (scaled normalized values). h, Illustration of the experimental protocol; FCM, flow cytometry. i, Flow cytometric analysis of MS4A3–tdTomato positivity of the indicated macrophage subsets from 10-week-old mice; n = 3 mice for each group. Data represent mean ± s.e.m.; NC, negative control; mono, monocytes; PC, positive control. j, Three-dimensional reconstruction of representative confocal images of whole-mount synovium and density map of MS4A3–tdTomato. Images are representative of three animals with similar results. Data in f were analyzed by one-way ANOVA with Tukey’s post hoc test, and data in c were analyzed by Wald test.

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