Fig. 7.

The FBG domains of FIB-G and FIC-1 exhibit pro-inflammatory effects in vitro and in vivo. a–c Primary human macrophages were stimulated with different concentrations of FBG-C, FIB-G, FIC-1, and ALP-4, or were left unstimulated (−) for 24 h. Cytokine levels were measured by ELISA. Data shown as mean ± SEM from at least three independent donors. One-way ANOVA vs. non-stimulated, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. d Primary human macrophages were pre-incubated for 6 h with 3 µM TAK 242 prior to stimulation with FBG-C, FIB-G, FIC-1, and ALP-4 (1 µM), or no stimulation (−) for 24 h. Cytokine synthesis was measured by ELISA. Data shown as mean ± SEM from at least three independent donors. Paired t-test vs. non-treated, *p < 0.05, **p < 0.01, ***p < 0.001. e 96-well plates were coated with 1 µg ml⁻¹ of FBG-C, FBG-X, FIB-G, FIC-1, and ALP-4, or PBS, and incubated with increasing doses of TLR4. Curves were fitted in GraphPad Prism using one-binding site hyperbola equation. Data are shown as mean ± SEM from three independent experiments. f, g Synovial inflammation was assessed 3 days post injection of each protein (1 µg) or PBS alone into the knees of DBA-1 mice. The histological score was calculated as the mean of seven sections from each knee joint per mouse. n = 5 mice per group except for FIC-1 (n = 4) (f). Mann–Whitney non-parametric test vs. PBS, *p < 0.05, **p < 0.01. Images show representative sections stained by haematoxylin and eosin (left panels) or safranin-O (right panels) (g). Mice injected with FBG-C, FIB-G, and FIC-1 exhibit cell infiltration into a thickened synovial lining layer, cellular invasion into the subchondral bone (arrows indicate bone erosion) and loss of articular cartilage proteoglycan (cp), pathological features not observed in mice injected with FBG-C mut or ALP-4.Scale bar left panels: 100 μM, right panels: 50 μM