Synovial macrophages: from ordinary eaters to extraordinary multitaskers
Authors: Nicole Hannemann, Florence Apparailly, Gabriel Courties
Journal: Trends in Immunology (2021)

Like other tissues, joints contain resident macrophages, and their diversity is only beginning to be characterized. Based on the highlights of recent studies, we discuss where current challenges lie and propose new avenues for future research in the osteoarticular field. Like other tissues, joints contain resident macrophages, and their diversity is only beginning to be characterized. Based on the highlights of recent studies, we discuss where current challenges lie and propose new avenues for future research in the osteoarticular field. In adult mammals, macrophages are found in all tissues and organs of the body. Tissue macrophages are specialized phagocytes that have a variety of supportive functions in tissue development, homeostasis, and immunity. They are extremely heterogeneous in terms of their origin, phenotype, and functions, and have a genetic program that includes a conserved core signature unique to each tissue niche [1.The Immunological Genome Consortium et al Gene-expression profiles and transcriptional regulatory pathways that underlie the identity and diversity of mouse tissue macrophages.Nat. Immunol. 2012; 13: 1118-1128Crossref PubMed Scopus (1193) Google Scholar]. Recent reports have revealed the diversity of macrophages within human and mouse joint tissues [2.Culemann S. et al.Locally renewing resident synovial macrophages provide a protective barrier for the joint.Nature. 2019; 572: 670-675Crossref PubMed Scopus (122) Google Scholar, 3.Alivernini S. et al.Distinct synovial tissue macrophage subsets regulate inflammation and remission in rheumatoid arthritis.Nat. Med. 2020; 26: 1295-1306Crossref PubMed Scopus (55) Google Scholar, 4.Zhang F. et al.Defining inflammatory cell states in rheumatoid arthritis joint synovial tissues by integrating single-cell transcriptomics and mass cytometry.Nat. Immunol. 2019; 20: 928-942Crossref PubMed Scopus (218) Google Scholar, 5.Kuo D. et al.HBEGF+ macrophages in rheumatoid arthritis induce fibroblast invasiveness.Sci. Transl. Med. 2019; 11eaau8587Crossref PubMed Scopus (42) Google Scholar]. Emerging evidence indicates that tissue-resident macrophages and their neighbors constitute complex microenvironments known as niches [6.Guilliams M. et al.Establishment and maintenance of the macrophage niche.Immunity. 2020; 52: 434-451Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar]. Similarly to a forest ecosystem, a tissue niche functions as a unit in which all its components sense and interact with each other to maintain tissue homeostasis. Synovial tissue can also be viewed as a biological niche in which macrophages occupy a defined place that supports their functions. The architecture of bony vertebrate synovial joints comprises a complex environment that bridges two articulating bones. Synovial joints contain synovial tissue that is divided into two well-defined areas: (i) the lining layer, which is formed by fibroblasts and macrophages that build an epithelial-like barrier flanking the intra-articular fluid, bone, or cartilage, as well as ligaments and tendons; and (ii) a sublining layer – a loose connective tissue formed by fibroblasts, lymphatic vessels, small venules, and also macrophages. Recent discoveries of heterogeneity among synovial cells in general, and among macrophages in particular, match this historical dual synovial architecture. Indeed, human and mouse studies have identified distinct macrophage clusters in specific areas of the synovial membrane (Table 1), thus reinforcing the idea that the lining and sublining layers are truly distinct niches within the synovial tissue that not only differ in terms of structure and density but also in terms of cell composition and identity [2.Culemann S. et al.Locally renewing resident synovial macrophages provide a protective barrier for the joint.Nature. 2019; 572: 670-675Crossref PubMed Scopus (122) Google Scholar, 3.Alivernini S. et al.Distinct synovial tissue macrophage subsets regulate inflammation and remission in rheumatoid arthritis.Nat. Med. 2020; 26: 1295-1306Crossref PubMed Scopus (55) Google Scholar, 4.Zhang F. et al.Defining inflammatory cell states in rheumatoid arthritis joint synovial tissues by integrating single-cell transcriptomics and mass cytometry.Nat. Immunol. 2019; 20: 928-942Crossref PubMed Scopus (218) Google Scholar, 5.Kuo D. et al.HBEGF+ macrophages in rheumatoid arthritis induce fibroblast invasiveness.Sci. Transl. Med. 2019; 11eaau8587Crossref PubMed Scopus (42) Google Scholar].Table 1Macrophage clusters identified in human and mouse synovial tissuesaThis classification is based on human macrophage phenotypic markers [3–5] identified in [3], cluster-defining genes, their subanatomical locations, gene ontology (GO)-based putative functions, and their respective abundance in health and disease. The right column presents their mouse counterparts when available [2].Phenotypic marker [3.Alivernini S. et al.Distinct synovial tissue macrophage subsets regulate inflammation and remission in rheumatoid arthritis.Nat. Med. 2020; 26: 1295-1306Crossref PubMed Scopus (55) Google Scholar]Subset [3.Alivernini S. et al.Distinct synovial tissue macrophage subsets regulate inflammation and remission in rheumatoid arthritis.Nat. Med. 2020; 26: 1295-1306Crossref PubMed Scopus (55) Google Scholar]Cluster [3.Alivernini S. et al.Distinct synovial tissue macrophage subsets regulate inflammation and remission in rheumatoid arthritis.Nat. Med. 2020; 26: 1295-1306Crossref PubMed Scopus (55) Google Scholar]Putative functions/signature(GO-based)AnatomicdistributionConditionOverlappingclustersMouse counterparts [2.Culemann S. et al.Locally renewing resident synovial macrophages provide a protective barrier for the joint.Nature. 2019; 572: 670-675Crossref PubMed Scopus (122) Google Scholar]MerTK+CD206+TREM2TIMD4PhagocytosisEfferocytosisHost defenseImmune regulationMatrix remodelingLiningnicheHealth and remissionNUPR1 [4]C1QA [4.Zhang F. et al.Defining inflammatory cell states in rheumatoid arthritis joint synovial tissues by integrating single-cell transcriptomics and mass cytometry.Nat. Immunol. 2019; 20: 928-942Crossref PubMed Scopus (218) Google Scholar]Cluster 2 [5.Kuo D. et al.HBEGF+ macrophages in rheumatoid arthritis induce fibroblast invasiveness.Sci. Transl. Med. 2019; 11eaau8587Crossref PubMed Scopus (42) Google Scholar]CX3CR1TREM2FOLR2LYVE1RELM-αICAMCytokines/chemokinesCX3CR1ID2Self-maintenanceMerTK−CD206−HLAISG15InterferonProinflammatorySubliningnicheActive arthritisIFN [4.Zhang F. et al.Defining inflammatory cell states in rheumatoid arthritis joint synovial tissues by integrating single-cell transcriptomics and mass cytometry.Nat. Immunol. 2019; 20: 928-942Crossref PubMed Scopus (218) Google Scholar]Cluster 4 [5.Kuo D. et al.HBEGF+ macrophages in rheumatoid arthritis induce fibroblast invasiveness.Sci. Transl. Med. 2019; 11eaau8587Crossref PubMed Scopus (42) Google Scholar]CCR2/ARG1CLEC10AAntigen presentationIL1B [4.Zhang F. et al.Defining inflammatory cell states in rheumatoid arthritis joint synovial tissues by integrating single-cell transcriptomics and mass cytometry.Nat. Immunol. 2019; 20: 928-942Crossref PubMed Scopus (218) Google Scholar]Cluster 1 [5.Kuo D. et al.HBEGF+ macrophages in rheumatoid arthritis induce fibroblast invasiveness.Sci. Transl. Med. 2019; 11eaau8587Crossref PubMed Scopus (42) Google Scholar]CCR2/IL1BCD48S100A12Leukocyte recruitmentProinflammatorySPP1Bone resorptionCluster 4 [5.Kuo D. et al.HBEGF+ macrophages in rheumatoid arthritis induce fibroblast invasiveness.Sci. Transl. Med. 2019; 11eaau8587Crossref PubMed Scopus (42) Google Scholar]CCR2/ARG1a This classification is based on human macrophage phenotypic markers [3.Alivernini S. et al.Distinct synovial tissue macrophage subsets regulate inflammation and remission in rheumatoid arthritis.Nat. Med. 2020; 26: 1295-1306Crossref PubMed Scopus (55) Google Scholar, 4.Zhang F. et al.Defining inflammatory cell states in rheumatoid arthritis joint synovial tissues by integrating single-cell transcriptomics and mass cytometry.Nat. Immunol. 2019; 20: 928-942Crossref PubMed Scopus (218) Google Scholar, 5.Kuo D. et al.HBEGF+ macrophages in rheumatoid arthritis induce fibroblast invasiveness.Sci. Transl. Med. 2019; 11eaau8587Crossref PubMed Scopus (42) Google Scholar] identified in [3.Alivernini S. et al.Distinct synovial tissue macrophage subsets regulate inflammation and remission in rheumatoid arthritis.Nat. Med. 2020; 26: 1295-1306Crossref PubMed Scopus (55) Google Scholar], cluster-defining genes, their subanatomical locations, gene ontology (GO)-based putative functions, and their respective abundance in health and disease. The right column presents their mouse counterparts when available [2.Culemann S. et al.Locally renewing resident synovial macrophages provide a protective barrier for the joint.Nature. 2019; 572: 670-675Crossref PubMed Scopus (122) Google Scholar]. Open table in a new tab Over the past few years elegant single-cell RNA sequencing (scRNA-seq) and fate-mapping approaches have revealed the existence of several synovial macrophage subsets in healthy joints [2.Culemann S. et al.Locally renewing resident synovial macrophages provide a protective barrier for the joint.Nature. 2019; 572: 670-675Crossref PubMed Scopus (122) Google Scholar, 3.Alivernini S. et al.Distinct synovial tissue macrophage subsets regulate inflammation and remission in rheumatoid arthritis.Nat. Med. 2020; 26: 1295-1306Crossref PubMed Scopus (55) Google Scholar, 4.Zhang F. et al.Defining inflammatory cell states in rheumatoid arthritis joint synovial tissues by integrating single-cell transcriptomics and mass cytometry.Nat. Immunol. 2019; 20: 928-942Crossref PubMed Scopus (218) Google Scholar]. Mouse studies have shown that the two synovial layers exhibit uneven macrophage heterogeneity, with a single homogeneous population in the lining layer and up to four different subsets in the sublining layer of healthy tissues [2.Culemann S. et al.Locally renewing resident synovial macrophages provide a protective barrier for the joint.Nature. 2019; 572: 670-675Crossref PubMed Scopus (122) Google Scholar]. Four subsets with similar profiles were found in human synovium [3.Alivernini S. et al.Distinct synovial tissue macrophage subsets regulate inflammation and remission in rheumatoid arthritis.Nat. Med. 2020; 26: 1295-1306Crossref PubMed Scopus (55) Google Scholar, 4.Zhang F. et al.Defining inflammatory cell states in rheumatoid arthritis joint synovial tissues by integrating single-cell transcriptomics and mass cytometry.Nat. Immunol. 2019; 20: 928-942Crossref PubMed Scopus (218) Google Scholar, 5.Kuo D. et al.HBEGF+ macrophages in rheumatoid arthritis induce fibroblast invasiveness.Sci. Transl. Med. 2019; 11eaau8587Crossref PubMed Scopus (42) Google Scholar], highlighting the conserved features of these subsets among these species [7.Hannemann N. et al.New insights into macrophage heterogeneity in rheumatoid arthritis.Joint Bone Spine. 2021; 88: 105091Crossref PubMed Scopus (2) Google Scholar] (Table 1). Beyond their transcriptional signatures, fate-mapping strategies in adult healthy mice – including lineage-tracing and parabiosis experiments combined with flow cytometry proliferation assays and immunofluorescence stainings – suggest that proliferating sublining macrophages contribute to the renewal of lining macrophages [2.Culemann S. et al.Locally renewing resident synovial macrophages provide a protective barrier for the joint.Nature. 2019; 572: 670-675Crossref PubMed Scopus (122) Google Scholar]. Although no contribution from circulating monocytes to tissue-resident macrophages was evidenced from parabiosis experiments, an earlier study using bone marrow transplantation showed that a sublining subset was replaced by macrophages derived from blood monocytes [8.Misharin A.V. et al.Non-classical Ly6C− monocytes drive the development of inflammatory arthritis in mice.Cell Rep. 2014; 9: 591-604Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar]. The clusters identified from scRNA-seq screens, their ontogenic origins, and developmental lineage relationships therefore remain to be explored. In humans, gene ontology-based analyses indicate that lining MerTK+ macrophages include clusters that exhibit homeostatic functions such as efferocytosis, matrix remodeling, and antimicrobial activities [3.Alivernini S. et al.Distinct synovial tissue macrophage subsets regulate inflammation and remission in rheumatoid arthritis.Nat. Med. 2020; 26: 1295-1306Crossref PubMed Scopus (55) Google Scholar]. Another cell type rooted in the synovial niche is the fibroblast. Fibroblasts are the source of several extracellular matrix proteins including collagens, fibronectin, hyaluronic acid, and glycosaminoglycans that provide the scaffolding components for structuring and maintaining the niche ecosystem. Like macrophages, they exhibit a high degree of heterogeneity according to their anatomic distribution in the lining and sublining synovial niches of human and mouse synovial tissues [4.Zhang F. et al.Defining inflammatory cell states in rheumatoid arthritis joint synovial tissues by integrating single-cell transcriptomics and mass cytometry.Nat. Immunol. 2019; 20: 928-942Crossref PubMed Scopus (218) Google Scholar,9.Croft A.P. et al.Distinct fibroblast subsets drive inflammation and damage in arthritis.Nature. 2019; 570: 246-251Crossref PubMed Scopus (182) Google Scholar,10.Mizoguchi F. et al.Functionally distinct disease-associated fibroblast subsets in rheumatoid arthritis.Nat. Commun. 2018; 9: 789Crossref PubMed Scopus (158) Google Scholar]. Overall, based on transcriptomic analyses, cells from the sublining niche seem to be more diverse than those in the lining niche. In the context of joint inflammation, the ecosystem and the network of synovial niches change substantially. The thin lining layer rapidly loses its barrier integrity, followed by hyperplasia, and the sublining area undergoes extensive remodeling concomitantly with massive infiltration by blood leukocyte invaders, including monocytes and lymphocytes. In vitro stimulation assays on lining and sublining macrophages isolated from rheumatoid arthritis (RA) synovial biopsies showed comparable production of anti-inflammatory and tissue-remodeling cytokines in response to lipopolysaccharide [3.Alivernini S. et al.Distinct synovial tissue macrophage subsets regulate inflammation and remission in rheumatoid arthritis.Nat. Med. 2020; 26: 1295-1306Crossref PubMed Scopus (55) Google Scholar]. However, activated sublining macrophages produce more proinflammatory cytokines than lining macrophages, whereas release of the inflammation-resolving lipid mediator resolvin D1 is restricted to lining macrophages [3.Alivernini S. et al.Distinct synovial tissue macrophage subsets regulate inflammation and remission in rheumatoid arthritis.Nat. Med. 2020; 26: 1295-1306Crossref PubMed Scopus (55) Google Scholar]. Several lines of evidence suggest that there is dynamic interplay between fibroblasts and macrophages in the synovial environment. In vitro coculture experiments using isolated cells from biopsies of RA patients indicated that macrophages influence fibroblast transcriptional profiles. Indeed, lining macrophages can license fibroblast repair responses by increasing the expression of transforming growth factor β and collagen genes. By contrast, sublining macrophages trigger the emergence of a pro-destructive fibroblast subset enriched for the expression of genes encoding proinflammatory mediators such as interleukin (IL)-6, as well as mediators of cartilage degradation and bone erosion such as receptor activator of nuclear factor κB ligand (RANKL) and matrix metalloproteinases (MMPs) [3.Alivernini S. et al.Distinct synovial tissue macrophage subsets regulate inflammation and remission in rheumatoid arthritis.Nat. Med. 2020; 26: 1295-1306Crossref PubMed Scopus (55) Google Scholar]. In turn, fibroblasts provide a set of niche factors that rewire macrophages towards proinflammatory responses, thereby leading to activation and enhanced recruitment of immune cells [5.Kuo D. et al.HBEGF+ macrophages in rheumatoid arthritis induce fibroblast invasiveness.Sci. Transl. Med. 2019; 11eaau8587Crossref PubMed Scopus (42) Google Scholar,9.Croft A.P. et al.Distinct fibroblast subsets drive inflammation and damage in arthritis.Nature. 2019; 570: 246-251Crossref PubMed Scopus (182) Google Scholar]. As such, the epidermal growth factor (EGF)–EGF receptor (R) axis contributes to the dialog between proinflammatory macrophages and fibroblasts, and promotes fibroblast invasiveness in a Matrigel invasion assay [5.Kuo D. et al.HBEGF+ macrophages in rheumatoid arthritis induce fibroblast invasiveness.Sci. Transl. Med. 2019; 11eaau8587Crossref PubMed Scopus (42) Google Scholar,11.Zhou X. et al.Circuit design features of a stable two-cell system.Cell. 2018; 172: 744-757Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar]. Although these pioneering studies have identified the cellular sources of several cytokines, chemokines, and maintenance factors, together with signaling interplay between macrophages and fibroblasts, most synovial niche-specific cell–cell circuits that provide joint integrity and functions remain to be discovered. The current paradigm is that tissue macrophages are established before birth and self-maintain throughout mammalian adulthood. In some organs, mouse macrophages can be of mixed origin, and bone marrow-derived monocytes contribute to the macrophage pool [12.Ginhoux F. Guilliams M. Tissue-resident macrophage ontogeny and homeostasis.Immunity. 2016; 44: 439-449Abstract Full Text Full Text PDF PubMed Scopus (654) Google Scholar]. So far, most mouse synovial macrophages are considered to be of embryonic origin, although initial work using bone marrow transplantation suggests that a discrete subset might originate from blood monocytes [8.Misharin A.V. et al.Non-classical Ly6C− monocytes drive the development of inflammatory arthritis in mice.Cell Rep. 2014; 9: 591-604Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar]. The recently discovered diversity of synovial macrophages questions the origins of macrophages in both the lining and sublining niches, particularly those from the sublining area. Indeed, in contrast to their embryonic counterparts, the residence time of adult monocyte-derived macrophages within a tissue is generally considered to be short. If a pool of macrophages can be acquired during adulthood, how do those new immigrants successfully adapt to and integrate with their new community? Can they completely overcome the functions of the embryonic pool? How and to what extent is the synovial niche permissive? Answering these questions will be crucial for understanding how niche remodeling occurs upon infection or trauma, and how it can affect, irreversibly or not, the health span and longevity of joints. Beyond the gene expression profiles of macrophages, the functional properties of macrophage subsets in the tissue remain to be identified. Of note, lung interstitial tissue can harbor two distinct blood-derived macrophage populations that reside close to either nerve fibers or blood vessels; these subsets have been shown to have nonoverlapping functions in contributing to tissue homeostasis [13.Chakarov S. et al.Two distinct interstitial macrophage populations coexist across tissues in specific subtissular niches.Science. 2019; 363eaau0964Crossref PubMed Scopus (203) Google Scholar]. Because the synovial tissue is highly innervated and vascularized, a topic of investigation is whether synovial macrophage heterogeneity can also be associated with defined subanatomical localizations. These studies also challenge the current dualistic view that circulating monocytes are the 'bad guys' relative to embryonic macrophages. Given that scRNA-seq approaches can help us to dig deeply into macrophage heterogeneity, understanding how these gene-enriched clusters relate to macrophage quiescent or activated states is paramount. Indeed, we need to bear in mind that macrophages were initially shown to exhibit remarkable plasticity. One can also compare the available datasets of murine macrophages from multiple territories at steady-state, as well as during distinct inflammatory settings, and be amazed by the extent of the similarity of some clusters. Beyond the list of enriched gene expression that defines a specific cluster, it will be interesting to integrate the epigenetic influences, transcriptional regulatory networks, and synovial niche-specific signals that drive joint macrophages into distinct clusters; this may allow a better understanding of the underlying mechanisms licensing their unique functions. In addition to 'omic' methods, investigating downstream activation pathways at the protein level might enable translation of these findings to the functional level. With multiplexed immunofluorescence and imaging mass cytometry, we can now examine in unprecedented detail the complex network of any tissue microenvironment. These technical imaging advances can allow the composition of synovial niches to be mapped under physiological conditions and for various joint disorders, including RA, ankylosis spondylarthritis, and osteoarthritis. Side-by-side comparisons may provide answers to why distinct joint microenvironments are associated with specific joint disorders, and subsequently inform different clinical outcomes of treatments. Ultimately, they might serve to optimize synovial biopsy-based precision and personalized medical strategies. For all these lingering questions, the osteoarticular and immunological research community needs to further investigate the respective contributions of cell-intrinsic versus -extrinsic factors in the fate, heterogeneity, and plasticity of macrophage subsets. As in any sociological integration process, successful integration of the new immigrant macrophages into the tissue niche requires the establishment of multidirectional communication systems between all actors involved, starting with the macrophages and their niche neighbors. This work was supported in part by INSERM, the University of Montpellier, the French National Research Agency (ANR-18-CE14-0042-01), and the French Society for Rheumatology (SFR 4939). The authors declare no conflicts of interest.