Fig. 4: ROS are the upstream effector that activate TRPML1 channels to switch macrophages from a pro-inflammatory to an anti-inflammatory state.

a A heat map representation of the relative mRNA expressions of M1 and M2 markers in LPS-stimulated BMDMs treated with either of ML-SA5 (0.1 μM) or control by RNA-seq analysis. b Relative IL1B mRNA expression in WT or MCOLN1-null BMDMs under the treatment of LPS (100 ng/mL), LPS + NAC (3.5 mM), LPS + 2-2’ Bipyridyl (150 μM), or LPS + BAPTA-AM (20 μM). n = 4–5. c Relative mRNA levels of CXCL11, CXCL12, CD74 and CCL22 in BMDMs under LPS (100 ng/mL), LPS + NAC (3.5 mM), LPS + 2-2’ Bipyridyl (150 μM), or LPS + BAPTA-AM (20 μM) treatment. n = 4–7. All treatments were for 6 h. d, e Pro IL-1β protein levels (d) and secreted IL-1β levels (e) in WT or MCOLN1-null BMDMs under LPS (100 ng/mL) or LPS + NAC (3.5 mM) conditions. ATP (4 mM for 1 h) was added to facilitate the secretion of IL-1β in all groups. The line inside the box denotes the median value, while the top and bottom of the box contain the 25th to 75th percentiles of the dataset. The whiskers indicate the minimum and maximum values. n = 3–5. f Representative images of Perls’ Prussian blue staining for WT BMDMs under the treatment of control, LPS (100 ng/mL), or LPS + NAC (3.5 mM). Arrows denote accumulated Fe³⁺. Scale bar = 50 μm. The average number of Perls’ positive cells per region of interest in BMDMs under the treatments of control, LPS, or LPS + NAC. n = 5. g Representative images of lysosomal Fe²⁺, stained by the HMRhoNox-M probe, in WT and MCOLN1-null BMDMs under the treatment of control, LPS (100 ng/mL), or LPS + NAC (3.5 mM). Scale bar = 5 μm. Relative fluorescence intensity of HMRhoNox-M was normalized to the control condition in WT or MCOLN1-null BMDMs group. n = 25. Both WT and MCOLN1-null BMDMs were preloaded with 100 μM ferric ammonium citrate (FAC) for 2 h. Means ± SEMs were shown in b–d, f, g. Significant differences were evaluated using one-way ANOVA followed by Tukey’s test.