Fig. 3: C5 acts through SLC15A4 to selectively inhibit TLR7/8-induced IRF5 responses in cells from healthy donors and SLE patients.

a PBMCs or b isolated CD14⁺ monocytes from healthy donors were pre-treated for 24 h with C5 (10 µM) and stimulated for 16 h with R848 (5 µg/ml). Supernatants were analyzed by ELISA as indicated. Data show mean ± SD, n = 8 donors. c Representative confocal microscopy images of PBMCs pre-treated for 24 h with C5 (10 µM) and stimulated for 3 h with R848 (5 µg/ml) stained for IRF5 and NF-κB p65. d PBMCs from healthy donors pre-treated for 24 h with DMSO or C5 (10 µM) and stimulated with R848 (5 µg/ml) for 3 h as indicated were analyzed for CD19⁺ B cell-specific IRF5 and NF-κB p65 nuclear translocation. n = 7 donors. e PBMCs from SLE patients were pre-treated for 24 h with C5 (10 µM) and stimulated for 16 h with R848 (5 µg/ml). Supernatants were analyzed by ELISA as indicated. Data show mean ± SD, n = 9 donors. f PBMCs of SLE patients pre-treated for 24 h with DMSO or C5 (10 µM) and stimulated with R848 (5 µg/ml) for 3 h as indicated were analyzed for CD19⁺ B cell-specific IRF5 and NF-κB p65 nuclear translocation. n = 8 donors. g, h Flow cytometry analysis (g) or live cell fluorescence microscopy (h) of HEK293T cells stably expressing the indicated constructs after 24 h treatment with DMSO or 10 μM C5. g Mean of integrated GFP signals from two independent experiments shown. See Supplementary Note 2 for gating strategy. i Immunoblot of control sgRen or sgSLC15A4 CAL-1 cells stably reconstituted with indicated constructs. Cells were pre-treated with C5 (10 μM) for 24 h before R848 (5 μg/ml) treatment for the indicated time. j Supernatants from indicated CAL-1 cell lines pre-treated with C5 for 24 h before R848 (5 μg/ml, 24 h) stimulation were analyzed for IL-6 production by ELISA. Data show mean of three stimulation replicates from one experiment representative of two independent experiments. (a, b, d, f) Significance was assessed by paired two-sided t-test. (c, h) Scale bars, 10 μm. (d, f) Box plots defined as in Fig. 2a. In (h–j) data representative of at least two independent experiments.