Extended Data Fig. 1: Spatiotemporal profiling of synovial CX₃CR1⁺ macrophages.

a, BFM of macrophages within the synovial tissue using the macrophage markers F4/80 (left and middle; colour as indicated) and CD68 (right; green) in ColVI^creR26-tdTomato reporter mice (left; tdTomato⁺, red), Cx3cr1^GFP mice (middle; GFP⁺, green), and Cx3cr1^creR26-tdTomato, mice (right; tdTomato⁺, red). Scale bars, 25 µm. b, Flow cytometry analysis of macrophages of dissociated hind-paw joints of Cx3cr1^gfp mice (n = 3) gated for CD45⁺, CD11b⁺, F4/80⁺ and GFP. Data are mean ± s.e.m. c, Representative 3D LSFM showing the spatial distribution of PMNs (Ly6G, green) and mononuclear phagocytes (tdTomato⁺, red) in knee joints of Cx3cr1^creR26-tdTomato mice at indicated time points upon induction of K/BxN STA (AF, grey). Filled arrowheads point towards the macrophage lining layer to highlight changes in its morphology upon induction of STA. Scale bars, 100 µm. d, Exemplary BFM images of the synovial membrane of knee joints of Cx3cr1^creR26-tdTomato mice at day 0, day 2 and 7 after induction of STA. Macrophages are defined as tdTomato⁺ (red) and F4/80⁺ (white) and infiltrating neutrophils as Ly6G⁺ (green) cells. Scale bars, 25 µm. e, Spinning disk confocal microscopy images of the synovial membrane of Cx3cr1^creR26-tdTomato mice at day 2 after induction of K/BxN STA visualizing macrophages (tdTomato⁺, red) and neutrophils (Ly6G, green). Scale bars, 10 µm. f, CLSM scans of the synovial membrane in knee joints of ColVI^creR26-tdTomato reporter mice at the indicated time points after the induction of STA, enabling the visualization of synovial fibroblasts (tdTomato, red) and macrophages (CD68, green) along the synovial cavity (sc). Scale bars, 20 µm. g, LSFM of knee joints of Cx3cr1^creR26-tdTomato mice showing the spatial distribution of macrophages (tdTomato, red) along the synovial cavity at day 21 after the first immunization during collagen-induced arthritis before the onset of arthritis (steady state) (top) and at day 35 after the first immunization after onset of joint inflammation, identifying rearrangement of macrophages in the form of palisade-like structures (filled arrowheads). Scale bars, 500 µm (left), 100 µm (right). AF, grey. h, CLSM images of knee joints of Cx3cr1^creR26-tdTomato mice at day 21 (top; steady state before onset of arthritis) and day 35 (bottom; during active arthritis) of collagen-induced arthritis, illustrating reorganization of lining macrophages (tdTomato, red; CD68, green). Scale bars, 100 µm; scale bar of magnified view, 10 µm.

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