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. 2023 Feb 2;15(5):1161–1179. doi: 10.1016/j.jcmgh.2023.01.010

Figure 9.

Figure 9

STING agonist modulates T cell metabolism to upregulate IL-10 production in Th1 cells. (A–C) The real-time levels of (A) ECAR and (B) OCR in splenic naïve CD4+ T cells were measured before and after treatment of DMXAA (1 μg/mL) using an extracellular flux Seahorse analyzer (n = 4/group). (C) The ratio of OCR/ECAR at the last time point. (D–F) The real-time levels of (D) ECAR and(E) OCR in splenic activated CD4+ T cells were measured before and after treatment of DMXAA (1 μg/mL) using an extracellular flux Seahorse analyzer (n = 4/ group). (F) The ratio of OCR/ECAR at the last time point. (G-J) The parameters of mitochondrial respiration in control Th1 and DMXAA-pretreated Th1 cells were measured by a Mito Stress Test Kit (n = 8/group). (G) The OCR profile. (H) Basal respiration. (I) Adenosine triphosphate (ATP)–related respiration. (J) Maximal respiration. (K, L) Representative flow cytometry plots and dot plots of mitochondrial mass in control Th1 and DMXAA-pretreated Th1 cells. (M, N) Representative flow cytometry plots and dot plots of mitochondrial membrane potential in control Th1 and DMXAA-pretreated Th1 cells. (O) Mouse splenic CD4+ T cells were activated with anti-CD3 (5 μg/mL) and anti-CD28 (2 μg/mL) antibodies in the presence or absence of DMXAA (1 μg/mL) under Th1 conditions for 6 hours, and the phosphorylated STAT3 (pSTAT3) site Ser727 and total STAT3 were determined by Western blot. (P) A diagram of different metabolic pathways contributing to mitochondrial oxidation. (Q–R) Mouse splenic CD4+ T cells were activated with anti-CD3 (5 μg/mL) and anti-CD28 (2 μg/mL) antibodies in the presence or absence of DMXAA (1 μg/mL) with or without UK5099 (30 μM)/DON (1 μM)/etomoxir (50 μM) under Th1 conditions for 5 days. (Q) Representative flow cytometry plots of IL-10+ CD4+ T cells. (R) Dot plots of IL-10+ CD4+ T cells. Data were shown as mean ± SEM. One representative of 2 (A-J) or 3 (K-O and Q-R) independent experiments. (C, F, H, I, J, L, N) Unpaired Student’s t test; (R) 1-way analysis of variance with Dunnett's multiple comparisons test. ∗∗P < .01, ∗∗∗P < .001.