Fig. 5: Interaction between E4 and macrophages.

A Removal of Fc N-glycan abolishes the protective effect of E4 on CAIA. Arthritogenic antibody cocktail (3 mg M2139 + 3 mg ACC1) together with 3 mg of either E4, E4m, or E4-EndoS (Fc N-glycan cleaved) were injected to B10.Cia9i mice (n = 4 for M2139 + ACC1 and 5 for others) on day 0, followed by intraperitoneal injection of LPS on day 3. Arthritis scores are assessed using Mann–Whitney test (two-tailed) and presented as mean ± SD. B E4 does not protect FCGR2B KO mice from CAIA. Indicated antibodies were injected to FCGR2B KO mice (n = 6) on day 0 and LPS on day 5. Data were analyzed by Mann–Whitney test (two-tailed) and presented as mean ± SD. C, D E4 and L2 bind to LPS-stimulated macrophages. Mouse bone marrow-derived macrophages (BMDMs) were differentiated with M-CSF (20 ng/ml) for 6 days followed by overnight stimulation with LPS (100 ng/ml) or PBS (n = 3 biologically independent mice). Biotinylated antibodies (20 μg/ml) were added on day 7 for 1 h before flow cytometry. CD11b⁺F4/80⁺CD38⁺ macrophages bound by antibodies were gated by streptavidin⁺ population. Data are analyzed using one-way ANOVA and presented as mean ± SD. E E4 does not bind to FCGR2B KO macrophages. BMDMs from wildtype and FCGR2B knockout mice were similarly differentiated and stimulated with LPS. Macrophages were labeled with F4/80-specific antibody, and the binding of E4/E4m/L2 to macrophages was visualized using goat anti-mouse IgG-AF488. Confocal microscopy images are presented as single channels and merges, magnification ×20. Scale bars represent 100 µm. F, G E4 attenuates osteoclastogenesis. BMDMs were differentiated for 7 days, followed by 7 days of culture with 50 ng/ml RANKL and 5 μg/ml indicated antibodies before IF staining (n = 5 mice for RANKL group and 3 for others). Osteoclasts were visualized by phalloidin (green) and Hoechst 33342 (blue), confocal microscopy images were presented as merges, scale bars represent 100 µm, and the number of osteoclasts per field was counted. Data are analyzed using one-way ANOVA and presented as mean ± SEM.