Macrophages are considered to contribute to chronic inflammatory diseases such as rheumatoid arthritis1. However, both the exact origin and the role of macrophages in inflammatory joint disease remain unclear. Here we use fate-mapping approaches in conjunction with three-dimensional light-sheet fluorescence microscopy and single-cell RNA sequencing to perform a comprehensive spatiotemporal analysis of the composition, origin and differentiation of subsets of macrophages within healthy and inflamed joints, and study the roles of these macrophages during arthritis. We find that dynamic membrane-like structures, consisting of a distinct population of CX3CR1+ tissue-resident macrophages, form an internal immunological barrier at the synovial lining and physically seclude the joint. These barrier-forming macrophages display features that are otherwise typical of epithelial cells, and maintain their numbers through a pool of locally proliferating CX3CR1− mononuclear cells that are embedded into the synovial tissue. Unlike recruited monocyte-derived macrophages, which actively contribute to joint inflammation, these epithelial-like CX3CR1+ lining macrophages restrict the inflammatory reaction by providing a tight-junction-mediated shield for intra-articular structures. Our data reveal an unexpected functional diversification among synovial macrophages and have important implications for the general role of macrophages in health and disease.
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The data that support the plots within this paper and other findings of this study are available from the corresponding author upon request. The bulk and single-cell RNA-seq data are available as part of the Gene Expression Omnibus (GEO) SuperSeries GSE134691.
Udalova, I. A., Mantovani, A. & Feldmann, M. Macrophage heterogeneity in the context of rheumatoid arthritis. Nat. Rev. Rheumatol. 12, 472–485 (2016).
Firestein, G. S. & McInnes, I. B. Immunopathogenesis of rheumatoid arthritis. Immunity 46, 183–196 (2017).
Orr, C. et al. Synovial tissue research: a state-of-the-art review. Nat. Rev. Rheumatol. 13, 463–475 (2017).
Croft, A. P. et al. Distinct fibroblast subsets drive inflammation and damage in arthritis. Nature 570, 246–251 (2019).
Barrera, P. et al. Synovial macrophage depletion with clodronate-containing liposomes in rheumatoid arthritis. Arthritis Rheum. 43, 1951–1959 (2000).
Haringman, J. J. et al. Synovial tissue macrophages: a sensitive biomarker for response to treatment in patients with rheumatoid arthritis. Ann. Rheum. Dis. 64, 834–838 (2005).
Misharin, A. V. et al. Nonclassical Ly6C− monocytes drive the development of inflammatory arthritis in mice. Cell Rep. 9, 591–604 (2014).
Zhang, F. et al. Defining inflammatory cell states in rheumatoid arthritis joint synovial tissues by integrating single-cell transcriptomics and mass cytometry. Nat. Immunol. 20, 928–942 (2019).
Ginhoux, F. et al. Fate mapping analysis reveals that adult microglia derive from primitive macrophages. Science 330, 841–845 (2010).
Schulz, C. et al. A lineage of myeloid cells independent of Myb and hematopoietic stem cells. Science 336, 86–90 (2012).
Yona, S. et al. Fate mapping reveals origins and dynamics of monocytes and tissue macrophages under homeostasis. Immunity 38, 79–91 2013).
Hashimoto, D. et al. Tissue-resident macrophages self-maintain locally throughout adult life with minimal contribution from circulating monocytes. Immunity 38, 792–804 (2013).
Rosas, M. et al. The transcription factor Gata6 links tissue macrophage phenotype and proliferative renewal. Science 344, 645–648 (2014).
Okabe, Y. & Medzhitov, R. Tissue-specific signals control reversible program of localization and functional polarization of macrophages. Cell 157, 832–844 (2014).
Lavin, Y. et al. Tissue-resident macrophage enhancer landscapes are shaped by the local microenvironment. Cell 159, 1312–1326 (2014).
Chakarov, S. et al. Two distinct interstitial macrophage populations coexist across tissues in specific subtissular niches. Science 363, eaau0964 (2019).
Aziz, A., Soucie, E., Sarrazin, S. & Sieweke, M. H. MafB/c-Maf deficiency enables self-renewal of differentiated functional macrophages. Science 326, 867–871 (2009).
Armulik, A. et al. Pericytes regulate the blood–brain barrier. Nature 468, 557–561 (2010).
Cronan, M. R. et al. Macrophage epithelial reprogramming underlies mycobacterial granuloma formation and promotes infection. Immunity 45, 861–876 (2016).
Uderhardt, S., Martins, A. J., Tsang, J. S., Lämmermann, T. & Germain, R. N. Resident macrophages cloak tissue microlesions to prevent neutrophil-driven inflammatory damage. Cell 177, 541–555.e17 (2019).
Armaka, M. et al. Mesenchymal cell targeting by TNF as a common pathogenic principle in chronic inflammatory joint and intestinal diseases. J. Exp. Med. 205, 331–337 (2008).
Krljanac, B. et al. RELMα-expressing macrophages protect against fatal lung damage and reduce parasite burden during helminth infection. Sci. Immunol. 4, eaau3814 (2019).
Dithmer, S. et al. Claudin peptidomimetics modulate tissue barriers for enhanced drug delivery. Ann. NY Acad. Sci. 1397, 169–184 (2017).
Pfeifle, R. et al. Regulation of autoantibody activity by the IL-23–TH17 axis determines the onset of autoimmune disease. Nat. Immunol. 18, 104–113 (2017).
Casanova-Acebes, M. et al. Rhythmic modulation of the hematopoietic niche through neutrophil clearance. Cell 153, 1025–1035 (2013).
Dobin, A. et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15–21 (2013).
Liao, Y., Smyth, G. K. & Shi, W. featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics 30, 923–930 (2014).
Love, M. I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550 (2014).
Ipseiz, N. et al. The nuclear receptor Nr4a1 mediates anti-inflammatory effects of apoptotic cells. J. Immunol. 192, 4852–4858 (2014).
Katsuno, T. et al. Deficiency of zonula occludens-1 causes embryonic lethal phenotype associated with defected yolk sac angiogenesis and apoptosis of embryonic cells. Mol. Biol. Cell 19, 2465–2475 (2008).
Ohtsuki, S., Yamaguchi, H., Katsukura, Y., Asashima, T. & Terasaki, T. mRNA expression levels of tight junction protein genes in mouse brain capillary endothelial cells highly purified by magnetic cell sorting. J. Neurochem. 104, 147–154 (2008).
Butler, A., Hoffman, P., Smibert, P., Papalexi, E. & Satija, R. Integrating single-cell transcriptomic data across different conditions, technologies, and species. Nat. Biotechnol. 36, 411–420 (2018).
Trapnell, C. et al. The dynamics and regulators of cell fate decisions are revealed by pseudotemporal ordering of single cells. Nat. Biotechnol. 32, 381–386 (2014).
Qiu, X. et al. Single-cell mRNA quantification and differential analysis with Census. Nat. Methods 14, 309–315 (2017).
Klingberg, A. et al. Fully automated evaluation of total glomerular number and capillary tuft size in nephritic kidneys using lightsheet microscopy. J. Am. Soc. Nephrol. 28, 452–459 (2017).
We thank C. Stoll, A. Klej, L. Seyler, R. Palmisano and the Optical Imaging Center Erlangen for technical assistance. M. Mroz and U. Appelt provided help during cell sorting and W. Baum and U. Baschant helped to generate K/BxN serum. This work was supported by the Deutsche Forschungsgemeinschaft (DFG – FG 2886 “PANDORA” - B01/A03 to G.K. and G.S., the CRC1181-A03/A01/A02/Z2 to G.K., G.S., D.V. and T.B. and the GK 1660 to G.K.), the Emerging Field Initiative (EFI) of the Friedrich-Alexander University Erlangen-Nürnberg (FAU) and the STAEDTLER Stiftung (EFI_Verbund_Med_05_MIRACLE to G.K. and T.B.), the Bundesministerium für Bildung und Forschung (BMBF) (METARTHROS to G.K. and G.S.) and the European Union (Horizon 2020 ERC-2014-StG 640087 - SOS to G.K and Horizon 2020 ERC-2018-SyG nanoSCOPE and RTCure to G.S.). J.Á.N.-Á. was supported by fellowship SVP-2014-068595 and A.H. by grant SAF2015-65607-R from Ministerio de Ciencia, Investigacion y Universidades (MCIU), and co-funding by Fondo Europeo de Desarrollo Regional (FEDER). The CNIC is supported by the MCIU and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (MCIU award SEV-2015-0505). ColVIcre mice were provided by G. Kollias.
The authors declare no competing interests.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data figures and tables
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 ColVIcreR26-tdTomato reporter mice (left; tdTomato+, red), Cx3cr1GFP mice (middle; GFP+, green), and Cx3cr1creR26-tdTomato, mice (right; tdTomato+, red). Scale bars, 25 µm. b, Flow cytometry analysis of macrophages of dissociated hind-paw joints of Cx3cr1gfp 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 Cx3cr1creR26-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 Cx3cr1creR26-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 Cx3cr1creR26-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 ColVIcreR26-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 Cx3cr1creR26-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 Cx3cr1creR26-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.
a, Histological CLSM analysis of embryonic mouse knee joints at E15.5 and E16.5 visualizing CD68 (red) and F4/80 (white) expressing embryonic macrophages (filled arrowheads) within the newly formed synovial lining. Scale bars, 50 µm (top), 10 µm (bottom). b, BFM showing expression of CSF1R and the distribution of macrophages (F4/80, red) within the synovial tissue of CSF1RGFP mice (GFP, green; left). c, Representative CLSM scans of Cx3cr1GFP (green) knee joints confirming the expression of CSF1R (red) by antibody-mediated staining on interstitial CX3CR1− macrophages. Scale bars, 25 µm, scale bar of magnified view, 10 µm. d, Gating strategy for analysis of synovial macrophages isolated from hind paws of parabiotic DsRed/wild-type mice. Synovial macrophages were defined as DAPI− living, CD45+, Ly6G−, CD11b+, F4/80+ cells. DsRed expression discriminates the origin. e, Gating strategy for blood monocytes of parabiotic DsRed/wild-type mice. Blood monocytes were defined as DAPI− living, CD45+, CD11b+, Ly6G−, CD115+ and SSClow. DsRed expression discriminates the origin. f, Representative BFM of the synovial membrane of knee joints of a wild-type mouse sharing circulation with a DsRed mouse, six weeks after establishment of parabiosis (n = 3; DsRed, red; F4/80, green). Scale bars, 25 µm. g, BFM images of parabiotic wild-type (top) and DsRed (bottom) mice after nine weeks of parabiosis. In the wild-type mice, DsRed+ partner-derived macrophages are visible in the bone marrow (bm), but not detected in the macrophage (F4/80, green) lining layer. Scale bars, 25 µm. h, Flow-cytometric analysis of the percentage of partner-derived blood monocytes and synovial macrophages of DsRed/wild-type parabionts after 9 weeks of parabiosis. Mean ± s.e.m. Blood, n = 8; synovial joint, n = 8. i, Chimerism ratio of blood monocytes and synovial macrophages in DsRed/wild-type parabionts after six weeks and nine weeks of parabiosis, calculated as the quotient of content of partner-derived tissue macrophages to partner-derived blood monocytes. A chimerism ratio of one represents the chimerism observed in blood monocytes. Mean ± s.e.m. Monocytes 6 weeks, n = 6; synovial macrophages 6 weeks, n = 6; monocytes 9 weeks, n = 4; synovial macrophages 9 weeks, n = 8. j, k, Flow-cytometric analysis of parabiotic hind paws of DsRed/wild-type parabionts at the indicated time points of K/BxN serum transfer arthritis. Data presented show the percentage of partner-derived PMNs (k) and monocytes/macrophages (l) within the blood circulation and the synovial tissue and are used to calculate the individual chimerism of tissue- and blood-derived cells. Mean ± s.e.m. For j, blood day 0 n = 6; synovial tissue day 0, n = 6; blood day 5, n = 8; synovial tissue day 5, n = 7. For k, blood day 0, n = 6; synovial tissue day 0, n = 5; blood day 5, n = 7; synovial tissue day 5, n = 8.
a, Gating strategy for CD45+CD11b+Ly6G−CD115+ classical Ly6Chigh and non-classical Ly6Clow monocytes of Cx3cr1creERR26-tdTomato mice. b, Gating strategy for DAPI− living, CD45+CD11b+Ly6G−F4/80+ macrophages of Cx3cr1creERR26-tdTomato mice. c, Evaluation of tdTomato expression in blood monocytes and synovial macrophages two days and four weeks after tamoxifen pulse. Mean ± s.e.m.; n = 4 per group. d, e, BFM images of knee joint synovial membranes of Cx3cr1creERtdTomato mice four weeks after systemic tamoxifen pulse (d) and five days after local injection of (Z)-4-hydroxytamoxifen (e) at day 0 and day 7 after the induction of K/BxN STA showing selective tdTomato (red) expression in synovial lining macrophages. The smaller graphs to the right in e show the absence of tdTomato expression in blood monocytes after local (Z)-4-hydroxytamoxifen injection. Scale bars, 25 µm. f, Gating strategy for DAPI− living, CD45+CD11b+Ly6G−F4/80+ macrophages of Cx3cr1creERR26-tdTomato mice four weeks after tamoxifen pulse, used to calculate the absolute numbers of tdTomato+ macrophages during steady state and K/BxN STA. g, Gating strategy after EdU labelling of proliferating macrophages (CD45+CD11b+Ly6G− F4/80+) of Cx3cr1creERR26-tdTomato mice. h, Quantification of total tdTomato+ and tdTomato− macrophages in paws of Cx3cr1creERR26-tdTomato mice 4 weeks upon tamoxifen pulse at day 0, 2 and 5 after induction of STA. Mean ± s.e.m. Day 0, n = 6; day 2, n = 5; day 5, n = 6.
a, Sorting strategy for bulk RNA sequencing analysis of synovial macrophages of Cx3cr1GFP mice. Macrophages were defined as CD45+, Ly6G−, CD11+ and F4/80+. GFP discriminated GFP+ lining macrophages and GFP− interstitial macrophages. b, Hierarchical clustering of z-score (left) and log2 counts (right) of selected genes of sorted GFP+ lining macrophages, GFP− interstitial macrophages and BMDMs generated from bulk RNA sequencing. c, Differential gene expression (mean fold change, log2(differentially expressed genes) (n = 3 per group) of tight-junction-associated genes comparing CX3CR1+ lining macrophages and BMDMs. Differential expression was performed with DESeq2. A Wald test was used to calculate two-sided P values; adjustment for multiple comparisons was performed with the Benjamini–Hochberg method. *P ≤ 0.05. d, Sorting strategy for synovial macrophages of Cx3cr1GFP mice for confirmatory quantitative analysis by PCR with reverse transcription (RT–PCR). Macrophages were defined as CD45+, Ly6G−, CD11+ and F4/80+. GFP discriminated GFP+ lining macrophages and GFP− interstitial macrophages. A dump channel using anti-CD31 and anti-E-cadherin was integrated to avoid endothelial cell or epithelial cell contaminations. e, Confirmatory quantitative RT–PCR analysis in synovial macrophage subsets determining expression of mRNAs encoding TJP1 (BMDM, n = 3; lining macrophage, n = 2), claudin 5 (n = 3 per group) and claudin 10 (n = 3 per group) in sorted GFP+ lining macrophages and in vitro cultured BMDMs, mean ± s.e.m.; two-tailed Student’s t-test, *P = 0.012. f, t-SNE profile of sorted synovial CD45+CD11b+Ly6G− mononuclear phagocytes of Cx3cr1creERR26-tdTomato mice analysed four weeks after tamoxifen pulse during steady-state conditions (top). After clustering, cell-cycle phase scoring based on canonical markers and regression was performed to determine clustering independent of cell cycle phase (middle and bottom). n = 7,362 cells. g, Gene ontology enrichment analysis of biological processes in cells of the proliferating Stmn1+ cluster of sorted CD45+CD11b+Ly6G− mononuclear phagocytes of a healthy tamoxifen-pulsed Cx3cr1creERR26-tdTomato mouse. The top 51 cluster marker genes determined with Seurat were used to perform a PANTHER overrepresentation test. The list of markers for the Stmn1+ cluster was compared to the reference list using Fisher’s exact test with false discovery rate correction. h, t-SNE profile of sorted synovial CD45+CD11b+Ly6G− mononuclear phagocytes of a healthy tamoxifen-pulsed Cx3cr1creERR26-tdTomato mouse after excluding Acp5+ osteoclast precursors revealing four remaining clusters (left). Single-cell trajectory analysis integrating cluster information (middle) and pseudotime (right) show a branch point of cellular differentiation into lining macrophages (red) or interstitial Retnla+ macrophages (dark blue) starting from proliferating MHCII+ macrophages (light blue). n = 7,028 cells. i, Differential gene expression analysis as a function of pseudotime in a branch-dependent manner showing a common gene signature of a pre-branch precursor cell population choosing two main cell fates: either Cx3cr1+ lining macrophage or interstitial Retnla+ macrophage. j, Gene expression changes of selected marker genes as a function of pseudotime reflecting the cellular differentiation into Retnla+ interstitial macrophages (solid line) and Cx3cr1+ lining macrophages (dashed line). n = 7,028 cells. k, BFM images of knee joints of Csf1rcreERR26-tdTomato mice (tdTomato, red) determining tdTomato expression in CD68+ (green) lining macrophages, MHCII+ interstitial macrophages (MHCII, white; top) and RELM-α+ interstitial macrophages (RELM-α, white; bottom) at indicated times after the start of tamoxifen treatment. Scale bars, 50 µm. l, m, Quantification of relative changes in tdTomato+ cells among CD68+ lining macrophages, RELM-α+ interstitial macrophages and MHCII+ interstitial macrophages in Csf1rcreERR26-tdTomato mice at indicated times after the start of tamoxifen treatment. n = 3 mice per group. Data are mean ± s.e.m. n, tdTomato (red) expression in CD68+ (green) macrophages in synovial tissue of the knee joint of RetnlacreR26-tdTomato mice. Scale bars, 250 µm (left), 25 µm (right). o, p, BFM images (o) and quantification of changes (p) in CD68+ (red) lining macrophages and MHCII+ (white) interstitial macrophages in LysMcreCD115DTR mice after 10 days of DT treatment, at the indicated time points after the beginning of DT treatment. Scale bars, 50 µm. n = 3 technical replicates. Data are mean ± s.e.m. q, Scheme of the postulated dynamic continuum of differentiating tissue-resident macrophages within the synovial tissue.
a, t-SNE scRNA-seq profiles of sorted synovial CD45+CD11b+Ly6G− mononuclear phagocytes of Cx3cr1creERR26-tdTomato mice analysed four weeks after tamoxifen pulse at the indicated time points after the induction of K/BxN STA, coloured by cluster assignment and annotated post hoc. Day 1, n = 4,640 cells; day 2, n = 2,722 cells; day 5, n = 3,237 cells. b, scRNA-seq-derived expression patterns of indicated genes within synovial mononuclear phagocytes at indicated time points after the induction of STA. Day 1, n = 4,640 cells; day 2, n = 2,722 cells; day 5, n = 3,237 cells. c, t-SNE plots of sorted CD45+CD11b+Ly6G− cells from arthritic hind paws at day 1 and day 5 after K/BxN serum transfer, showing the expression of Cx3cr1, Axl and Mfge8 within the cluster of lining macrophages. d, Comparison of available scRNA-seq datasets from monocytes of human synovial tissue derived from patients suffering from rheumatoid arthritis and osteoarthritis8 with scRNA-seq profiles of mouse CD45+CD11b+Ly6G− cells on day 5 after the induction of STA. Values represent the quotient of the numbers of all co-expressed marker genes of the 5 macrophage clusters at day 5 to the top 20 provided human marker genes of the 4 described subpopulations of human monocytes SC-M1, SC-M2, SC-M3 and SC-M4.
Extended Data Fig. 6 Expression patterns of tight-junction proteins and gap-junction proteins in tdTomato+ lining macrophages.
Expression of claudin 5, TJP1/ZO-1 and claudin 13 as well as that of connexin 43 (grey, filled arrowheads) in synovial lining tdTomato+ (red) macrophages of Cx3cr1creR26-tdTomato mice during steady state and on days 1, 2 and 7 after the induction of K/BxN STA. Scale bars, 5 µm.
Extended Data Fig. 7 Ultrastructural characterization of cell–cell contacts between lining macrophages.
a, Representative CLSM of macrophages (tdTomato, red) within the synovial membrane of knee joints of Cx3cr1creERR26-tdTomato mice, visualizing the tight-junction protein ZO-1/TJP1 (white). Phalloidin, green; DAPI, blue. Scale bars, 5 µm. b, Transmission electron microscopy (TEM) images of the synovial membrane of a healthy knee joint showing tight junctions (tj), adherens junctions (aj), desmosomes (ds) and interdigitations connecting synovial lining macrophages. c, TEM micrograph showing synovial lining macrophages (red) constituting a dense physical barrier segregating the synovial fluid from sublining interstitial tissue containing synovial fibroblasts (cyan), endothelial cells (purple) embedded into the extracellular matrix (beige). d, TEM micrograph demonstrating synovial macrophages (red) forming the uppermost cell layer covering the layer of synovial fibroblasts (cyan). e, f, TEM micrographs of an inflamed synovial membrane two days after induction of K/BxN STA, showing the disruption of the covering synovial macrophage (red) layer and a reorientation of synovial macrophages (red) and synovial fibroblasts (cyan) directed to the synovial cavity. g, A TEM micrograph of an inflamed synovial membrane two days after the induction of STA reveals the emergence of macrophages containing large amounts of vacuoles filled with phagocytosed material. h, i, Recruited monocytes and granulocytes as well as free DNA of neutrophil extracellular traps (blue) within the synovial cavity of knee joints two days after the induction of STA. Filled arrowheads point at an exemplary monocyte engulfing free DNA.
a, Histological sections of healthy (STA day 0, left) and inflamed (STA day 7, right) mouse knee joints of Cx3cr1creR26-tdTomato mice, showing the expression of TREM2 (green; filled arrowheads) in lining macrophages (tdTomato, red). Scale bars, 100 µm (top), 10 µm (bottom). b, c, Histological sections of synovial tissue of human knee joints isolated from patients diagnosed with osteoarthritis (OA) and rheumatoid arthritis (RA) determining expression of TREM2 (green; filled arrowheads) (b) and TJP1 (green; filled arrowheads) (c) in synovial macrophages (CD68, red). Scale bars, 100 µm (top), 10 µm (bottom). d, Flow-cytometric analysis of the composition and frequencies of MHCII+TREM2− and MHCII−TREM2+ mononuclear phagocytes in synovial tissue samples isolated from human knee joints of patients diagnosed with osteoarthritis and rheumatoid arthritis. e, Histology-based quantification of the density of the synovial macrophage lining (defined as percentage of CD68+TREM2+ macrophages among total lining cells) in synovial tissue sections of patients diagnosed with osteoarthritis (n = 4) and rheumatoid arthritis (n = 5), respectively. Data are mean ± s.e.m., two-tailed Student’s t-test.
a, To quantify lining density, tdTomato+ macrophages (red) were manually isolated from 3D reconstructions of optically cleared and LSFM-imaged Cx3cr1creR26-tdTomato knee joints (autofluorescence, grey; CD31, blue) using Imaris software. Isolated surfaces (yellow) were volume-rendered for tdTomato+ macrophages (red) and whole-area volume (green). Lining density was calculated from the ratio of whole-area volume to macrophage volume. An exemplary image of the same knee joint before and after isolation of lining macrophages is shown. Scale bars, 200 µm. b, Dynamic-contrast-enhanced magnetic resonance imaging (DCE-MRI) data analysis. The red line drawn in the sagittal T1-weighted image after administration of contrast agent marks the transverse plane used for T1-weighted DCE-MRI analysis. The DCE curve generated from the region of interest (synovial tissue) was normalized to the measurement time point after complete injection of contrast agent, and the time of measurements was converted to distinctive measurements. c, CLSM images of knee joints of Cx3cr1GFP mice injected with protein-G-purified and Alexa-Fluor-647-labelled K/BxN serum IgG (grey) at the indicated time points after IgG injection, determining the uptake of labelled IgG by macrophages (GFP, green; CD68, red) in the synovial tissue and the synovial lining (synovial cavity, sc). Scale bars, 10 µm. d, CLSM scan with higher magnification showing localization of labelled IgG (grey) inside the vacuoles of CD68+ (red) lining and interstitial synovial macrophages 24 h after injection. Scale bars, 10 µm. e, Clinical course of K/BxN STA in wild-type mice that were treated with an anti-GR1 antibody to deplete PMNs and inflammatory Ly6Chigh monocytes and a control antibody (LTF-2), one day before induction of STA. Mean ± s.e.m.; n = 5 per group. f, Histological CLSM analysis of lining morphology after anti-GR1 antibody-mediated neutrophil/monocyte depletion one day after induction of STA. Lining macrophages (tdTomato, red). Scale bars, 20 µm. g, Flow cytometry analysis of synovial macrophages and blood Ly6Chigh or Ly6Clow monocytes of Cx3cr1creiDTR mice and iDTR control mice one day and five days after two injections of DT (500 ng per mouse per day, i.p.). Mean ± s.e.m; For day 1: iDTR, n = 5; Cx3cr1creiDTR, n = 7; for day 5, n = 3 per group. Two-tailed Student’s t-test, ***P < 0.0001. h, Representative BFM images of the infiltration of PMNs (Ly6G, green) and neutrophil extracellular trap formation (filled arrowheads, DAPI, blue) within the synovial cavity of knee joints of Cx3cr1creiDTR (n = 3) and iDTR control (n = 3) mice 6 days after injection of DT and 24 h after induction of STA (CD68, red). Scale bars, 200 µm and for magnified view, 50 µm. i, Treatment scheme and clinical course of STA in Cx3cr1creiDTR and iDTR control mice that had received a unilateral local injection of DT (n = 7) and PBS (n = 7), respectively. P values calculated using two-tailed paired t-test, **P = 0.008. j, Clinical course of STA including AUC of the corresponding clinical index in C57BL/6 wild-type mice treated with C5C2 claudin peptidomimetics (3.5 µmol kg−1, i.v., n = 8) or scrambled C5C2 control peptide (C5C2scr; 3.5 µmol kg−1, i.v., n = 6) one day before and after the induction of STA. Data are mean ± s.e.m. Mann–Whitney U-test for clinical index with *P ≤ 0.05, and two-tailed Student’s t-test for AUC with **P = 0.0062. k, Normalized signal intensity curves of DCE-MRI of synovial tissue of knee joints over 90 measurements with intervals of 7 s at the indicated days after STA in C57BL/6 wild-type mice treated with C5C2 claudin peptidomimetics (3.5 µmol kg−1, i.v.) or vehicle one day before the induction of STA. Data are mean ± s.e.m. Day 0: vehicle, n = 10 knee joints; C5C2, n = 10 knee joints; day 1: vehicle, n = 9 knee joints; C5C2, n = 10 knee joints. P values for AUC were calculated using two-tailed Student’s t-test, *P = 0,0256. l, Flow cytometry of blood monocytes and neutrophils of LysMcreCD115DTR mice (n = 6) and CD115DTR control mice (n = 5) one day after two injections of DT (500 ng per mouse per day, i.p.). Mean ± s.e.m.; two-tailed Student’s t-test, *P = 0.0467, **P ≤ 0.0069, ***P = 0.0001.
Top, scheme of the postulated origin of resident synovial CX3CR1+ lining macrophages that constantly repopulate from proliferating tissue resident CX3CR1−MHCII+ interstitial macrophages. Bottom, tight-junction-forming resident lining macrophages form a protective barrier for joint structures that disintegrates during arthritis, enabling infiltration of inflammatory myeloid cells.
Marker genes of clusters of scRNA-seq data of sorted synovial CD45+CD11b+Ly6G- mononuclear phagocytes during steady state.
Marker genes of clusters of scRNA-seq data of sorted synovial CD45+CD11b+Ly6G- mononuclear phagocytes at day 5 post K/BxN serum transfer.
Supplementary experimental procedures and key resources.
Spatial arrangement of tdTomato+ macrophages at the synovial membrane. Optical sections of a light-sheet fluorescence microscopy scanned Cx3cr1creR26-tdTomato murine knee joint (Autofluorescence, grey; CD31, blue) show the dense and continuous localization of tdTomato+ macrophages (red) along the synovial cavity in all three dimensions.
High resolution scan of the synovial lining architecture. Processed confocal laser scanning microscopy scan of the synovial membrane of the knee joint in Cx3cr1creR26-tdTomato mice (tdTomato, red; Phalloidin, green; DAPI, blue) showing the dense spatial arrangement of lining macrophages (tdTomato, red) with high resolution.
Localization of tdTomato+ macrophages (MΦs) at the synovial membrane. 3D reconstruction of an optically cleared and light-sheet fluorescence microscopy scanned Cx3cr1creR26-tdTomato knee joint shows the general tissue morphology (autofluorescence, grey), vascularization (CD31, blue), and distribution of tdTomato+ macrophages (red) in the tissue.
Physical barrier of tdTomato+ macrophages (MΦs) between the synovial capillary network and intraarticular space. Manually isolated tdTomato+ lining macrophages (red) from a light-sheet fluorescence microscopy scanned knee joint of a Cx3cr1creR26-tdTomato mouse form a barrier between the dense capillary network (CD31, blue) within the sublining synovial tissue and the synovial cavity.
Morphology of the membrane-forming lining macrophages (MΦs) at D0 of STA. 3D reconstruction of a light-sheet fluorescence microscopy scanned Cx3cr1creR26-tdTomato knee joint at steady state showing a healthy morphology of the synovial membrane. TdTomato+ lining macrophages (red) form a dense barrier along the synovial cavity and neutrophil granulocytes (Ly6G, green) are exclusively located in the bone marrow (autofluorescence, grey).
Morphology of the membrane-forming lining macrophages (MΦs) at D2 of STA. 3D reconstruction of a light-sheet fluorescence microscopy scanned Cx3cr1creR26-tdTomato knee joint at day 2 (D2) after K/BxN serum transfer showing a massive reduction in the density of tdTomato+ lining macrophages (red) in the synovial membrane (autofluorescence, grey). Additionally, neutrophils (Ly6G, green) sporadically infiltrate the synovial fat pad.
Morphology of the membrane-forming lining macrophages (MΦs) at D7 of STA. 3D reconstruction of a light-sheet fluorescence microscopy scanned Cx3cr1creR26-tdTomato knee joint at day 7 (D7) after K/BxN serum transfer (autofluorescence, grey). Neutrophils (Ly6G, green) and macrophages (tdTomato, red) infiltrate the synovial fat pad and the synovial cavity.
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Culemann, S., Grüneboom, A., Nicolás-Ávila, J.Á. et al. Locally renewing resident synovial macrophages provide a protective barrier for the joint. Nature 572, 670–675 (2019). https://doi.org/10.1038/s41586-019-1471-1
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