Fig. 4

Repeated exposure to CKLF1-induced metabolic abnormalities and immune tolerance in microglia. A Timeline of repeated exposure of microglia to CKLF1. Microglia were stimulated by CKLF1 or vehicle for 24 h. After being washed, the cells were further cultured in medium without C27 for three days and then restimulated with CKLF1 or vehicle for 24 h, resulting in three experimental groups: Veh, acute, and chronic stimulation. B The level of IL-6 in microglia was determined by western blot (n = 3 per group). F (2, 6) = 17.97. C Relative mRNA levels of inflammatory cytokines in microglia (n = 6 per group). IL-1β: F (2, 15) = 9.64; IL-6: F (2, 15) = 18.18; TNF-α: F (2, 15) = 10.15. D Repeated exposure to C27 failed to induce phagocytosis in microglia. After microglia were induced with CKLF1, the amount of zymosan phagocytosed by cells in each group was detected by imaging and flow cytometry (n = 3 per group). 1 bead: F (2, 6) = 67.6; 2 beads: F (2, 6) = 78.46; 3 beads: F (2, 6) = 91.19; 4 beads: F (2, 6) = 108.5. E, F Repeated exposure to C27 led to metabolic abnormalities in microglia. The cell energy metabolism analyzer can detect real-time changes in extracellular acidification and quantitatively determine the basic glycolytic capacity, maximum glycolytic capacity and glycolytic reserve capacity to measure the ECAR (n = 6 per group). Basal glycolysis: F (2, 15) = 7.993; Max glycolysis: F (2, 15) = 9.294; Reversed glycolysis: F (2, 15) = 20.16. G Statistical analysis of the relative mRNA expression of glycolytic genes (n = 5–6 per group). HK: F (2, 12) = 17.34; GLUT1: F (2, 15) = 16.16; PFKFB3: F (2, 12) = 11.38; PKM2: F (2, 12) = 7.392. The data are presented as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 vs. vehicle group or acute group