Homocysteine activates T cells by enhancing endoplasmic reticulum-mitochondria coupling and increasing mitochondrial respiration

Abstract

Hyperhomocysteinemia (HHcy) accelerates atherosclerosis by increasing proliferation and stimulating cytokine secretion in T cells. However, whether homocysteine (Hcy)-mediated T cell activation is associated with metabolic reprogramming is unclear. Here, our in vivo and in vitro studies showed that Hcy-stimulated splenic T-cell activation in mice was accompanied by increased levels of mitochondrial reactive oxygen species (ROS) and calcium, mitochondrial mass and respiration. Inhibiting mitochondrial ROS production and calcium signals or blocking mitochondrial respiration largely blunted Hcy-induced T-cell interferon γ (IFN-γ) secretion and proliferation. Hcy also enhanced endoplasmic reticulum (ER) stress in T cells, and inhibition of ER stress with 4-phenylbutyric acid blocked Hcy-induced T-cell activation. Mechanistically, Hcy increased ER-mitochondria coupling, and uncoupling ER-mitochondria by the microtubule inhibitor nocodazole attenuated Hcy-stimulated mitochondrial reprogramming, IFN-γ secretion and proliferation in T cells, suggesting that juxtaposition of ER and mitochondria is required for Hcy-promoted mitochondrial function and T-cell activation. In conclusion, Hcy promotes T-cell activation by increasing ER-mitochondria coupling and regulating metabolic reprogramming.

Keywords: T cell; endoplasmic reticulum stress; homocysteine; mitochondria.

Figure 1

Reprogramming of mitochondrial metabolism in T cells from HHcy mice. Flow cytometry of splenic T cells from mice fed with or without Hcy and stained with MitoSOX Red (A) or Rhod-2 (B). Traces of OCR of splenic T cells from control or HHcy mice (C) or stimulated with anti-CD3 antibody for additional 24 h (D) as measured by the XF24 metabolic analyzer, with additions of mitochondrial effectors at time points indicated (left), and quantification of the basal OCR, max capacity OCR, and ATP-linked OCR (right). ECAR of splenic T cells from control or HHcy mice (E) or stimulated with anti-CD3 antibody for additional 24 h (F) as measured by the XF24 metabolic analyzer, and quantification of the basal and maximal ECAR. Flow cytometry of T cells stained with rhodamine 123 (G). (H) Confocol images of T cells loaded with MitoTracker Green. Results are mean ± SEM of six mice per group. *, P < 0.05 vs. control

Figure 2

Altered T-cell mitochondrial metabolism in response to Hcy stimulation. T cells were incubated with or without Hcy (50 μmol/L) in the presence of anti-CD3 antibody for 24 h. (A) Flow cytometry of T cells stained with MitoSOX Red (left), and quantification (right). (B) Confocol images of T cells loaded with Rho-2. (C) Traces of OCR of T cells as measured by the XF24 metabolic analyzer, with additions of mitochondrial effectors at time points indicated (left), and quantification of the basal OCR, max capacity OCR, and ATP-linked OCR (right). (D) Traces of ECAR of T cells as measured by the XF24 metabolic analyzer, and quantification of the basal and maximal ECAR (right). Flow cytometry of T cells stained with rhodamine 123 (E) and MitoTracker Green (F). Data are mean ± SEM from 3 independent experiments. *, P < 0.05 vs. control

Figure 3

Mitochondrial metabolism mediates Hcy-induced T cell activation. (A) Western blot of phosphorylated Akt in T cells. (B) ELISA analysis of T-cell IFN-γ secretion in response to Hcy (50 μmol/L, 24 h) stimulation in the presence of mitochondrial ROS scavengers APDC and SS31; (C) T cell proliferation by CCK8 assay. T-cell IFN-γ secretion (D) or proliferation (E) in response to Hcy stimulation with or without the mitochondrial Ca²⁺ uniporter inhibitors Ru360 (10 μmol/L), RuRed (10 μmol/L) or IP3 receptor inhibitor Xestospongin C (Xes, 2 μmol/L). IFN-γ secretion (F) or cell proliferation (G) in response to Hcy with or without mitochondrial OXPHOS inhibitor rotenone (1 μmol/L). Data are mean ± SEM from 3 independent experiments. *, P < 0.05 vs. control. #, P < 0.05 vs. Hcy

Figure 4

Hcy enhances T cell ER stress. T cells were incubated with or without Hcy (50 μmol/L) for 4 h with anti-CD3 antibody. Western blot analysis of phosphorylated eIF2α (A), phosphorylated PERK (B), and protein levels of IRE1α and spliced (S) and unspliced (U) XBP1 (C) in T cells with or without Hcy stimulation. β-Actin as a protein loading control. (D) CCK8 assay of cell proliferation with Hcy stimulation with or without ER stress inhibitor PBA or (F) ER stress stimulator DTT. ELISA of IFN-γ with Hcy stimulation with or without PBA (E) or DTT (G). Data are mean ± SEM from 3 independent experiments. *: P < 0.05 vs. Control. #, P < 0.05 vs. Hcy

Figure 5

Hcy reprograms mitochondrial metabolism and activates T cells through extending ER-mitochondria interaction. Western blot analysis of MFN2 (A), GPX7 (B), ERP44 (C), and VDAC (D). (E) Structured illumination microscopy of ER-mitochondria colocalization (left) and quantification of the Manders’ coefficiency (right) of T cells with or without Hcy treatment for 4 h and nocodazole pretreated for one hour. (F), (G) and (H) MFI in T cells loaded with rhodamine 123, Rhod-2, and MitoSOX Red respectively, and quantified by FACS after Hcy stimulation for 24 h with or without nocodazole pretreatment. (I) IFN-γ secretion and (J) cell proliferation in response to Hcy with or without nocodazole pretreatment. (K) Proposed model of T-cell activation in response to Hcy stimulation. Data are mean ± SEM from 3 independent experiments. *, P < 0.05 vs. Control. #, P < 0.05 vs. Hcy