Fig 2: Blockade of B1R attenuates ZIA and K/BxN serum transfer arthritis in mice.(A) B1R antagonist SSR240612 significantly reduced ZIA in WT mice compared with vehicle-treated mice. n = number of mice per group: PBS, n = 5; zymosan, n = 10; zymosan + SSR, n = 10. (B) Knee homogenates from SSR240612-treated mice showed significantly lower IL-1β and IL-6 in comparison with vehicle-treated mice. n = number of mouse knees per group: PBS, n = 3; zymosan, n = 5; zymosan + SSR, n = 5. (C) Immunofluorescence of knees from SSR240612-treated mice shows a marked decrease in macrophage marker F4/80 (green). Representative sections from each group are shown. Original magnification, ×200. (D) Vehicle-treated WT mice (n = 9–13) had profound serum transfer arthritis as measured by joint circumference compared with SSR240612-treated WT mice (n = 10). There was no difference in arthritis between SSR240612-treated Cd13–/– mice (n = 6) and vehicle-treated Cd13–/– mice (n = 10) except for day 8. (E) Significant decrease in MCP-1/CCL2 and IL-6 in ankle homogenates from SSR240612-treated WT (n = 9) compared with vehicle-treated WT mice (n = 13) is shown. In contrast, significant increase in the cytokines was observed in the SSR240612-treated Cd13–/– mice (n = 8) compared with vehicle-treated Cd13–/– mice (n = 6). (F) SSR240612-treated WT and Cd13–/– mouse cryosections showed a marked decrease in F4/80 staining compared with sections from vehicle-treated mice. Red staining represents F4/80 for MNs/macrophages, while blue (DAPI) stains nucleus. Original magnification, ×200. Results are expressed as mean ± SD. *P < 0.05. Significance was determined by Kruskal-Wallis test (A and E), 1-way ANOVA (B and F), and 2-way ANOVA (D).

Fig 3: B1R is expressed in RA FLSs and ST.(A and B) IL-1β and TNF-α induced B1R mRNA and protein expression in RA FLSs in a time-dependent manner as determined by quantitative PCR and Western blotting. n = 3 RA FLSs used in each group. (C) B1R is highly expressed in RA in vivo. Immunofluorescence staining was performed targeting B1R (green), myeloid cell marker CD68 (red), fibroblast marker CD55 (red), EC marker vWF (red), and nuclear staining DAPI (blue). Representative pictures from 3 RA patients. Quantification was done by counting of B1R-positive cells in 5 fields per patient. Original magnification, ×200 and ×400. (D) Analysis of single-cell RNA-Seq data from Zhang et al. (71). showed that BDKRB1 is predominantly expressed on FLSs, while ANPEP and its sheddase MMP14 are expressed on FLSs and MNs. (E) All FLS (blue boxes) subsets coexpress ANPEP, MMP14, and BDKRB1, while the 2 MN clusters (red boxes) have high levels of ANPEP and MMP14 but low expression of BKDRB1. Results are shown as mean ± SD. *P < 0.05. Significance was determined by Kruskal-Wallis test or 1-way ANOVA (A).

Fig 4: Cd13-knockout (Cd13–/–) mice develop less inflammation compared with WT mice when injected with TNF-α.TNF-α was injected into Cd13–/– and C57BL/6J WT mouse knee joints. PBS served as a negative control. (A) Cd13–/– mice developed significantly less knee swelling when TNF-α was injected into their knees compared with WT mice. n = number of knee joints per group: WT-PBS, n = 16; WT–TNF-α, n = 18; Cd13–/–-PBS, n = 16; Cd13–/––TNF-α, n = 10. (B) sCD13 was significantly elevated in TNF-α–injected knees (n = 8) in WT mice compared with PBS-injected mice (n = 7). (C) Knee homogenates from TNF-α–treated WT mice (n = 9) showed significantly higher MCP-1/CCL2 and IL-1β compared with TNF-α–treated Cd13–/– mice (n = 11). n = number of mouse knees per group. (D) TNF-α–treated WT mouse cryosections showed a marked increase in F4/80 staining compared with sections from PBS-treated mice, and this was not observed in the TNF-α–treated Cd13–/– mice. Red staining represents F4/80 for MNs/macrophages, while blue (DAPI) stains nucleus. Original magnification, ×200. Results are expressed as mean ± SD. *P < 0.05. Significance was determined by Kruskal-Wallis test (A and C), unpaired, 2-tailed Student’s t test (B), and 1-way ANOVA (C and D).