Gpx4 Regulates Invariant NKT Cell Homeostasis and Function by Preventing Lipid Peroxidation and Ferroptosis

Abstract

Invariant NKT (iNKT) cells are a group of innate-like T cells that plays important roles in immune homeostasis and activation. We found that iNKT cells, compared with CD4+ T cells, have significantly higher levels of lipid peroxidation in both mice and humans. Proteomic analysis also demonstrated that iNKT cells express higher levels of phospholipid hydroperoxidase glutathione peroxidase 4 (Gpx4), a major antioxidant enzyme that reduces lipid peroxidation and prevents ferroptosis. T cell-specific deletion of Gpx4 reduces iNKT cell population, most prominently the IFN-γ-producing NKT1 subset. RNA-sequencing analysis revealed that IFN-γ signaling, cell cycle regulation, and mitochondrial function are perturbed by Gpx4 deletion in iNKT cells. Consistently, we detected impaired cytokine production, elevated cell proliferation and cell death, and accumulation of lipid peroxides and mitochondrial reactive oxygen species in Gpx4 knockout iNKT cells. Ferroptosis inhibitors, iron chelators, vitamin E, and vitamin K2 can prevent ferroptosis induced by Gpx4 deficiency in iNKT cells and ameliorate the impaired function of iNKT cells due to Gpx4 inhibition. Last, vitamin E rescues iNKT cell population in Gpx4 knockout mice. Altogether, our findings reveal the critical role of Gpx4 in regulating iNKT cell homeostasis and function, through controlling lipid peroxidation and ferroptosis.

Figure 1.. iNKT cells have distinct redox regulation and higher lipid peroxidation.

(a) Ingenuity Pathway Analysis (IPA) of the canonicals pathways enriched in the differentially expressed proteins between NKT1 and naïve CD4⁺ T cells. (b) Heatmap of the differentially expressed proteins associated with oxidative stress and ferroptosis pathways (columns represent biological replicates of each cell type). (c) Histogram overlays and mean fluorescence intensities (MFI) of oxidized C11-BODIPY staining signal reporting lipid ROS in CD4⁺ T and iNKT from the spleens of C57BL/6J mice or the PBMC from healthy donors, considering the difference in cell size as detailed in Supplemental Fig. 2. The data on mice shown in c is representative of four independent experiments, while human PBMC data depict representative histograms and individual responses from 18 donors. **p < 0.01, ****p < 0.0001, paired Student’s t test.

Figure 2.. Gpx4 is required for iNKT homeostasis.

(a) Left panel: representative flow cytometry plots of iNKT cells from thymus, spleen, and liver of WT and littermate Gpx4-cKO mice. Percentages show iNKT cells among live, CD19⁻CD8a⁻ splenocytes; Right panel: The percentage among total live cells and absolute cell numbers of iNKT cells. (b) Left panel: representative flow cytometry plots of NKT1 (Tbet⁺ RORγt⁻), NKT2 (Tbet⁻ RORγt⁻), and NKT17 (Tbet⁻ RORγt⁺) cells among total iNKT cells in WT and littermate Gpx4-cKO mice. Right panel: The percentages among total iNKT cells and absolute cell numbers of all subsets. (c) Total iNKT cell percentages among total live cells and cell numbers in Bcl-XL-Tg⁺; Gpx4-flox/flox; CD4cre⁻ and littermate Bcl-XL-Tg⁺; Gpx4-flox/flox; CD4cre⁺ mice. (d) iNKT cell subsets among total iNKT cells and subset cell number in Bcl-XL-Tg⁺; Gpx4-flox/flox; CD4cre⁻, and littermate Bcl-XL-Tg⁺; Gpx4-flox/flox; CD4cre⁺ mice. Live/death blue stain was used for cell viability. The data shown in a and b are from one representative experiment out of three independent experiments, and the data shown in c and d are pooled from three independent experiments. ns: non-significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, unpaired Student’s t test.

Figure 3.. Lipid peroxidation promotes ferroptosis in Gpx4 deficient iNKT cells

(a) Cell viability of WT and Gpx4-cKO iNKT cells from littermate mice measured immediately ex vivo (0 h) or after 4 h culture at 37°C. (b) Normalized cell viability of WT and Gpx4-cKO iNKT cells from littermate mice cultured for 4 h in the presence of various compounds. (c) Cytosolic ROS level assessed by CM-H₂DCFA and (d) lipid ROS by C11-BODIPY in iNKT cells. (c, d) Intensities normalized to MFI signals in WT cells. (e) WT iNKT cells cultured for 4 h as in (b) with or without RSL3 (10 μM), by itself or in combination with other compounds as indicated, before lipid ROS was assessed by C11-BODIPY staining. (f) Normalized cell viability of WT and Gpx4-cKO iNKT cells from littermate mice cultured for 4 h with or without MK-4 or αToc. For cell viability, live/death blue was used. The data shown is from one representative experiment out of three independent experiments. ns: non-significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, unpaired Student’s t test.

Figure 4.. iNKT cell function is regulated by Gpx4.

(a) Splenocytes from WT and littermate Gpx4-cKO mice were cultured in the presence of αGC (250 ng/ml) and BFA (2.5 μg/ml) for 4 h at 37°C and intracellular cytokines in live iNKT cells assessed by flow cytometry. (b, c) WT splenocytes were cultured for 4 h with αGC (250 ng/ml) and BFA (2.5 ug/ml) in the absence or presence of RSL3 (10 μM), by itself or in combination with other compounds as indicated. Cell viability (b) and relative cytokine productions (c) from total live INKT cells relative to αGC stimulated control were shown. For cell viability, live/death blue was used. The data shown in a are pooled from four independent experiments, and the data shown in b and c are from one representative experiment out of three independent experiments. ns: non-significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, unpaired Student’s t test.

Figure 5.. Gpx4 deficiency leads to abnormal proliferation and accumulation of mitochondrial ROS in iNKT.

(a) Ingenuity Pathway Analysis (IPA) of canonical pathways enriched in the differentially expressed genes comparing Gpx4-cKO to WT iNKT cells. (b) Proliferation of iNKT subsets from thymus, spleen, and liver was assessed by Ki-67 expression. Measurement of mitochondrial mass using MitoTracker Green (MTG) (c), mitochondrial ROS using MitoSOX (d) and MitoTracker orange CM-H₂TMRos (MTO) (e) in WT and Gpx4-cKO iNKT cells from littermate mice, as well as normalization by MFI (MTG) were shown (d, e). Live/death blue was used for viability. The data shown in b and c are pooled from four and three independent experiments, respectively. The data shown in d, e and f are from one representative experiment out of three independent experiments. ns: non-significant, *p < 0.05, ***p < 0.001, ****p < 0.0001, unpaired Student’s t test.

Figure 6.. Vitamin E rescues iNKT homeostasis in Gpx4-cKO mice

(a) Experimental scheme to study the effects of αToc/Vit E supplementation from perinatal day 6 in WT and littermate Gpx4-cKO mice. (b) Left panel: Representative flow cytometry plots of splenic iNKT cells from WT and littermate Gpx4-cKO mice, treated with vehicle (Corn oil) or αToc (50 mg/kg). Right panel: The percentages among total live cells and absolute cell numbers of iNKT. (c) Left panel: Representative flow cytometry plots of NKT1, NKT2, and NKT17 cells among total iNKT cells in WT and littermate Gpx4-cKO mice. Right panel: The absolute cell numbers of iNKT subsets. (d) Proliferation of splenic iNKT subsets in different experimental groups as assessed by Ki-67 expression. Live/death blue stain was used for viability. The data shown is each from one representative experiment out of three independent experiments. ns: non-significant, **p < 0.01, ***p < 0.001, ****p < 0.0001, unpaired Student’s t test.