Fig. 2: TRPM8 channel activity controls macrophage response to LPS.
From: Dietary targeting of TRPM8 rewires macrophage immunometabolism reducing colitis severity
Over-expression of TRPM8 in pro-inflammatory macrophages is essential for LPS-induced intracellular Ca2+ increase. A Representative image of TRPM8 (orange), and 4′,6-diamidino-2-phenylindole (DAPI) (blue) immunofluorescence staining in naïve and pro-inflammatory WT BMDMs treated or not with luteolin (10 μM). Analysis was performed 18 h after polarization with LPS + IFN-γ. B Relative changes in the fluorescence of Fluo-4AM-loaded naïve BMDMs, reflecting changes in intracellular Ca2+ concentration [Ca2+]i after icilin (40 μM) perfusion for 1 min. Data are presented as the average of n = 3 independent experiments. Error bars represent ±SEM (n = 58 and n = 60 for Vehicle and Luteolin 10 μM, respectively). C Representative fluorescence images from indicated time points in the experiments shown in (B). D Statistical representation of total peak area of Fluo-4 fluorescence after icilin perfusion from (B). Error bars represent ±SEM. P value was determined using unpaired Student’s ttest. *p < 0.05. E Relative changes in the fluorescence of Fluo-4AM-loaded naïve WT and Trpm8−/− BMDMs, reflecting changes in intracellular Ca2+ concentration [Ca2+]i after LPS (100 ng/mL) perfusion for 30 s. Data are presented as the average of n = 3 independent experiments. Error bars represent ±SEM (n = 86 and n = 112 for WT and Trpm8−/−, respectively). F Quantification of basal Ca2+ levels in Fluo-4AM-loaded naïve WT and Trpm8−/− BMDMs. Error bars represent ±SEM. P value was determined using unpaired Student’s ttest. ****p < 0.0001. G Representative fluorescence images from indicated time points in the experiments shown in (E). H Statistical representation of total peak area of Fluo-4 fluorescence after LPS perfusion from panel E. Error bars represent ±SEM. P value was determined using unpaired Student’s t-test. ****p < 0.0001. I Relative changes in the fluorescence of Fluo-4AM-loaded naïve BMDMs, reflecting changes in intracellular Ca2+ concentration [Ca2+]i BMDMs after LPS (100 ng/mL) perfusion for 30 s. Data are presented as the average of n = 3 independent experiments. Error bars represent ± SEM (n = 108 and n = 113 for Vehicle and Luteolin 10 μM, respectively). J Representative fluorescence images from indicated time points in the experiments shown in (I). K Statistical representation of total peak area of Fluo-4 fluorescence after LPS perfusion from panel I. Error bars represent ±SEM. P value was determined using unpaired Student’s t test. ****p < 0.0001.
