Fig 2: EIF3C is a potential target for CFH to play a role in inhibiting FLSs and monocyte function. Monocytes and FLS from RA patients were selected respectively, and cells from each patient were divided into two groups. One group was left untreated, and one group was treated with 5 μg/ml CFH for 6 h. A Volcano plot indicated up-regulated (red dots) and down-regulated (blue dots) genes (P value < 0.05 and |log2FC|> 0.5) by RNA sequencing in CFH-treated versus untreated RA monocytes. EIF3C is indicated. B Volcano plot indicated up-regulated (red dots) and down-regulated (blue dots) genes (P value < 0.05 and |log2FC|> 0.5) by RNA sequencing in CFH-treated versus untreated RA monocytes. EIF3C and EIF3CL are indicated. C We selected the genes upregulated in the CFH-treated monocytes and the genes upregulated in the CFH-treated FLS identified by RNA-sequencing and determined the intersecting proteins. We obtained one gene, namely EIF3C. DEGs: Differentially Expressed Genes. D The boxplot shows the relative expression of EIF3C and EIF3CL. E CFH upregulated the protein expression of EIF3C by using Western blot. F CFH combined with TNF-α upregulated protein expression of EIF3C in both RA FLSs and monocytes by using Western blot. Data are expressed as mean ± SEM (n = 6). *p < 0.05

Fig 3: CFH attenuates TNF-α-induced pyroptosis and inflammatory cytokines release of RA monocytes. Purified CD14+ monocytes were pretreated with TNF-α(50 ng/ml), TNF-α(50 ng/ml) + CFH (5 μg/ml), or CFH (5 μg/ml) for 24 h (n = 6–9). A The expression of secreted CFH in the culture supernatant of RA patients and healthy control stimulated with TNF-α, IL-1β, and IL-6 detected by ELISA. B The expression of secreted cytokines (IL-1β, IL-6) in the culture supernatant of RA monocytes in each group was detected by ELISA. C Flow cytometric analysis of cells stained with annexin V/7-AAD to determine cell death and percentage of 7-AAD-positive cells. D The protein expression of c-caspase3, GSDME-N, and GSDME-F was measured by using Western blot. E Representative phase-contrast microscopy images of monocytes treated as indicated. Arrows indicate pyroptotic cell bubbles. Data are expressed as mean ± SEM. *p < 0.05; **p < 0.01; NC: negative control; T: TNF-α; T + H: TNF-α + CFH; H: CFH; IL-6: interleukin-6; IL-1β: interleukin-1β; c-caspase3: cleaved caspase-3; GSDME-N: gasdermin E N-terminal; GSDME-F: gasdermin E full length

Fig 4: CFH inhibits the migration, invasion, and TNF-α-induced expression of inflammatory mediators of RA FLSs. The FLSs were pretreated with TNF-α（50 ng/ml), TNF-α（50 ng/ml) + CFH (5 μg/ml), or CFH (5 μg/ml) for 24 h (n = 5–10). A The expression of secreted CFH in the culture supernatant of RA-FLS and OA-FLS detected by ELISA. B-C The effect of CFH on cell migration was detected using wound healing assay. The scratching area was photographed at 0 h and 24 h. The scratch assay was presented as the percentage by which the original scratch area decreased at 24 h. Representative images (original magnification, × 200) are shown. D-E FLS invasion ability was measured by Transwell assay, and the invaded cells were photographed. The relative invasion rate was calculated by counting mean invaded cells from 5 randomly selected fields and then normalized to that in the NC group. Representative images (original magnification, × 200) are shown. F–G The expression of secreted cytokines (IL-6, IL-8) and MMPs (MMP-1, MMP-3) in culture supernatant of RA-FLS and OA-FLS in each group were detected by ELISA. Data are expressed as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. MMP-1: matrix metalloproteinase-1; MMP-3: matrix metalloproteinase-3

Fig 5: CFH is elevated in the serum and synovial fluid from RA. A CFH in serum from RA patients and healthy control detected by ELISA. (RA: n = 52; HC: n = 28). B CFH in synovial fluid from RA and OA patients detected by ELISA. (RA: n = 8; OA: n = 4). C Correlation analysis of CFH expression in serum of RA patients with ESR, DAS28, and hs-CRP respectively. Data are expressed as mean ± SEM. ***p < 0.001; RA: rheumatoid arthritis; OA: osteoarthritis; HC: healthy control; hs-CRP: high sensitivity C-reactive protein; ESR: erythrocyte sedimentation rate; DAS28: disease activity score 28