Ontogeny of Synovial Macrophages and the Roles of Synovial Macrophages From Different Origins in Arthritis

Abstract

The ontogeny of macrophages in most organ/tissues in human body has been proven. Due to the limited number and inaccessibility of synovial macrophages (SM), the origin of SM has not been fully illuminated. The objective of this study was designed to investigate the ontogeny of SM and to evaluate the role of SM from different origins in arthritis. Two origins of SM, embryonic SM (ESM) and bone marrow SM (BMSM) were identified in Cx3cr1-EGFP mice, CCR2^-/- mice and bone marrow (BM) chimera model by using a stringent sorting strategy. The cellular features, including dynamic total cell number, in situ proliferation, phagocytosis and expressions of pro-inflammatory and anti-inflammatory genes, of ESM and BMSM were compared. In addition, ESM and BMSM showed different expression patterns in Rheumatoid Arthritis (RA) patients' synovium and during the developmental process of collagen-induced arthritis (CIA) mice. Taken together, these results demonstrated that the SM at least has two origins, ESM and BMSM. The different cellular property and dynamic expression patterns in RA patients/CIA mice highlight the notion that ESM and BMSM might play different role in arthritis.

Keywords: BMSM; CIA; ESM; RA; SM; ontogeny.

Figure 1

The origin of SM at embryonic stage. (A) The development of mice joint synovium during embryonic stage; (B) Cx3cr1+/GFP cells localize around joint at E12.5; (C,D) Resident macrophage marker F4/80 appears at joint synovium as early as E12.5; (E) The number of F4/80⁺ ESM gradually increase with embryonic development. Each bar in the figure represents mean ± SEM of triplicates. 10 mouse embryos were used for each FACS experiment.

Figure 2

The ontology of SM at perinatal to adult stage. (A) F4/80⁻CD11b⁺ BMSM appear in joint synovium at E20.5; (B) The expression of Ly6C and MerTK in F4/80⁻CD11b⁺ BMSM; (C) ESM gradually increase from neonatal to adult stage and BMSM show the opposite expression pattern. F4/80⁺CD11b⁺ SM appear at neonatal stage and progressively increase up to adult; (D) To further divide F4/80⁺CD11b⁺ SM by Ly6C and MHC II in WT mice and CCR2-/- mice. (E) The CX3CR1 expression in three subgroups of F4/80⁺CD11b⁺ SM. Each bar in the figure represents mean ± SEM of triplicates. For embryonic stage, 8–10 mouse embryos were used for each FACs experiment. For neonatal stage, 6 cubs were used for each FACS experiment. **P < 0.01.

Figure 3

Identification of SM from different origins. (A) The expression of the macrophage-specific markers CD64, CD14, and CX3CR1 in ESM and BMSM; (B) The comparison of phagocytic ability of ESM and BMSM; (C) Expression of IL-1β, TNF-α, IL-4, and IL-10 in ESM and BMSM. Each bar in the figure represents mean ± SEM of triplicates. *P < 0.05, **P < 0.01.

Figure 4

The proliferative ability of SM. (A) in situ proliferation of the ESM at prenatal and postnatal stages; (B) PCNA staining in joint synovium in situ at postnatal stage; (C) A chimera approach by joining congenic wild-type CD45.1⁺ and CD45.2⁺ mice to assess the contribution of non-host cells in synovium. Each bar in the figure represents mean ± SEM of triplicates. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 5

The role of SM from different origins in CIA mice. (A,B) H&E staining and Masson staining of synovium and joint of control and CIA mice; (C) The dynamic expression pattern of SM in the developmental process of CIA model; (D) The expressionfo M1(pro-inflammatory)/M2(anti-inflammatory) markers of ESM and BMSM at different developmental stage of CIA model. Each bar in the figure represents mean ± SEM of triplicates. 5 mice were used for each FACS experiment. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 6

The role of SM from different origins in RA patients' synovium. (A) H&E staining and CD11b immunofluorescence of synovium from OA and RA patients; (B) EMR1(F4/80) and CD11b immunofluorescence in RA synovium; (C) The in vitro culture of isolated EMR1(F4/80)⁺CD11b⁺ SM from RA synoivum by using a similar sorting strategy in mice; (D) The ratio of ESM and BSM in OA and RA synovium; (E) The expression of M1(pro-inflammatory)/M2(anti-inflammatory) markers of ESM and BMSM in RA synovium. Each bar in the figure represents mean ± SEM of six experiments. *P < 0.05.