Fig. 5: Blocking of TRPM8 induces lactate-mediated activation of IL-10 pathway.
From: Dietary targeting of TRPM8 rewires macrophage immunometabolism reducing colitis severity
Luteolin, via TRPM8, increases lactate levels necessary for IL-10 pathway activation. A Relative lactate levels measured by GC-MS metabolomic analysis of pro-inflammatory BMDMs treated or not with Luteolin (10 μM). The analysis was performed after 3 or 6 h of polarization with LPS + IFN-γ. Data are presented as peak area normalized on μg of protein of n = 3 independent experiments, each performed in technical triplicate. Error bars represent ±SEM. P value was determined using unpaired Student’s t test. *p < 0.05. B Relative lactate levels measured by GC-MS metabolomic analysis of WT and Trpm8−/− BMDMs. The analysis was performed in basal conditions (t0) and after 3 or 6 h of polarization with LPS + IFN-γ. Data are presented as peak area normalized on μg of protein of n = 3 independent experiments, each performed in technical triplicate. Error bars represent ± SEM. P value was determined using unpaired Student’s t test. *p < 0.05; **p < 0.01. IL-10 (C) and IL-6, TNF-α, and IL-1β (D) mRNA relative expression measured in pro-inflammatory WT BMDMs treated or not with luteolin (10 μM) and LDH-a inhibitor GSK2837808A (10 μM). The analysis was performed after 0, 1, 3, or 6 h of polarization with LPS + IFN-γ. Data are expressed as relative to basal levels (0 h timepoint) and are presented as the average of n = 5 independent experiments. Error bars represent ±SEM. P value was determined using multiple paired Student’s t test. *p < 0.05; **p < 0.01; ***p < 0.001 Pro-inflammatory+Lut10μM vs Pro-inflammatory; #p < 0.05 Pro-inflammatory+LDHAi+Lut10μM vs Pro-inflammatory+LDHAi. E IL-6, IL-1β, and TNF-α levels measured in the supernatant of IL-10fl/fl WT and IL-10fl/fl LysMCre BMDMs treated or not with luteolin (10 μM), after 3, 6, or 12 h of polarization with LPS + IFN-γ. Data are presented as the average of n = 5 independent experiments. Error bars represent ±SEM. P value was determined using multiple paired Student’s t test. *p < 0.05; ** p < 0.01 vs IL-10fl/fl WT pro-inflammatory. F ECAR kinetics (left) and glycolytic parameters (right) of naïve and pro-inflammatory IL-10fl/fl WT and IL-10fl/fl LysMCre BMDMs treated or not with Luteolin (10 μM). Analysis was performed 18 h after polarization with LPS + IFN-γ. Data are presented as the average of n = 3 independent experiments. Error bars represent ±SEM. P value was determined using one-way ANOVA followed by Tukey’s multiple comparisons test. n.s. non-significant, *p < 0.05; **p < 0.01; ****p < 0.0001.
