Glucocorticoid receptor modulates myeloid-derived suppressor cell function via mitochondrial metabolism in immune thrombocytopenia

Abstract

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature cells and natural inhibitors of adaptive immunity. Intracellular metabolic changes in MDSCs exert a direct immunological influence on their suppressive activity. Our previous study demonstrated that high-dose dexamethasone (HD-DXM) corrected the functional impairment of MDSCs in immune thrombocytopenia (ITP); however, the MDSC population was not restored in nonresponders, and the mechanism remained unclear. In this study, altered mitochondrial physiology and reduced mitochondrial gene transcription were detected in MDSCs from HD-DXM nonresponders, accompanied by decreased levels of carnitine palmitoyltransferase-1 (CPT-1), a rate-limiting enzyme in fatty acid oxidation (FAO). Blockade of FAO with a CPT-1 inhibitor abolished the immunosuppressive function of MDSCs in HD-DXM responders. We also report that MDSCs from ITP patients had lower expression of the glucocorticoid receptor (GR), which can translocate into mitochondria to regulate the transcription of mitochondrial DNA (mtDNA) as well as the level of oxidative phosphorylation. It was confirmed that the expression of CPT-1 and mtDNA-encoded genes was downregulated in GR-siRNA-treated murine MDSCs. Finally, by establishing murine models of active and passive ITP via adoptive transfer of DXM-modulated MDSCs, we confirmed that GR-silenced MDSCs failed to alleviate thrombocytopenia in mice with ITP. In conclusion, our study indicated that impaired aerobic metabolism in MDSCs participates in the pathogenesis of glucocorticoid resistance in ITP and that intact control of MDSC metabolism by GR contributes to the homeostatic regulation of immunosuppressive cell function.

Keywords: HD-DXM; glucocorticoid receptor; immune thrombocytopenia; myeloid-derived suppressor cell.

Fig. 1

Mitochondrial activity level and OCR in MDSCs from ITP patients and controls. A Immunofluorescence of mitochondria in MDSCs from ITP patients and controls. Representative merged images of CD33/MitoTracker Red double-positive cells. The mitochondrial activity (mean fluorescence intensity; MFI = IntDen/Area) in MDSCs from the (i) control, (ii) remission and (iii) refractory groups (n = 11 for each group). Original magnification: 630×. Scale bars, 10 μm. B Representative respiration curves of MDSCs in the refractory group showed an overall lower respiration level in this group than in the remission group or normal control group. Mitochondrial respiratory parameters (basal respiration rate, ATP production, maximal respiration rate) in the refractory group, remission group, and normal control group (n = 5 for each group). Differences between the refractory group and remission group or between the refractory group and control group were determined using analysis of variance. *P < 0.05; **P < 0.01

Fig. 2

CPT-1 inhibitor treatment impaired the suppressive function of MDSCs against CD4⁺ T cells. A, B The expression of CPT-1 was significantly decreased in MDSCs from the refractory group compared to those from the remission group and control group at both the mRNA level (n = 10 for each group) and the protein level (n = 5 for each group). Differences between groups were determined using analysis of variance or the Kruskal–Wallis test. C CD4⁺ T-cell proliferation was inhibited by MDSCs. The peaks indicate cell division processes. D The division index of CD4⁺ T cells cocultured with MDSCs from five nonresponders and five responders at MDSC:CD4⁺ T cell ratios of 1:2 and 1:4. Differences between two independent groups were compared using an independent t test. Differences between the etomoxir treatment and nontreatment groups were determined by a paired samples t test. *P < 0.05; **P < 0.01

Fig. 3

GR mRNA expression and binding of GR to D-loop regions of mitochondrial DNA in MDSCs. A, B MDSCs in the refractory group had significantly lower levels of GR expression than those in the remission group or control group at both the mRNA level (n = 10 for each group) and the protein level (n = 5 for each group). Differences between the refractory group and remission group or between the refractory group and control group were determined using the Kruskal–Wallis test. C The human mitochondrial genome. Primer locations are indicated by the arrows. D MDSCs were subjected to a ChIP assay using antibodies specific for GR or nonspecific IgG as the control. The relative quantity of GR binding to D-loop regions of mtDNA was significantly decreased in the refractory group (n = 5) compared to the remission group (n = 5) or normal control group (n = 5), as assessed by qPCR. Differences between groups were determined using analysis of variance. *P < 0.05; **P < 0.01

Fig. 4

Expression of 13 mtDNA-encoded genes in MDSCs from ITP patients and controls. The expression of ND1, ND-2, ND6, Cyt-b, COX-1, COX-3 and ATP-8 in MDSCs from the refractory group (n = 10) was significantly decreased, and the expression of ND3, COX-2 and ATP-6 tended to be lower in the refractory group than in the remission group (n = 10) or normal control group (n = 10). Differences between the refractory group and remission group or between the refractory group and control group were determined using analysis of variance or the Kruskal–Wallis test. *P < 0.05; **P < 0.01

Fig. 5

Metabolomic analysis of MDSCs in ITP patients. A Heatmap of metabolites present in different quantities in MDSCs. B The generation of aerobic metabolites (via the tricarboxylic acid cycle and oxidative phosphorylation) in MDSCs was higher in the remission group (n = 3) than in the refractory group (n = 3). *P < 0.05; **P < 0.01

Fig. 6

GR-siRNA treatment offset the rescue effect of MDSC treatment in the murine model of passive ITP. A GR-siRNA treatment decreased GR gene expression in MDSCs of WT mice. Differences between the two groups were compared using a paired samples t test (n = 6). B The timelines for MDSC treatment. (C–H) Change in the platelet count over time in Group A (C) and Group B (F). Thrombocytopenia occurred and the platelet count fell below 300 × 10⁹/L in all mice in all three groups on Day 1. D, G Mice receiving NC-siRNA-treated MDSCs showed significantly higher platelet counts than mice receiving GR-siRNA-treated MDSCs or PBS five days after immunization. E, H The platelet counts in mice receiving NC-siRNA-treated MDSCs also tended to be higher than those in mice in the other two groups seven days after immunization. Differences among the groups were determined using analysis of variance (n = 6). I Decreased transcription levels of mtDNA in murine GR-siRNA-treated MDSCs. Differences between the NC-siRNA treatment group (n = 5) and GR-siRNA treatment group (n = 5) were determined using a paired samples t test or the Wilcoxon signed rank test. J Compared to NC-siRNA treatment, GR-siRNA treatment decreased CPT-1 expression in murine MDSCs, as determined using a paired samples t test (n = 6). *P < 0.05; **P < 0.01

Fig. 7

GR-siRNA treatment offset the rescue effect of MDSC treatment and the rebalancing of T-cell subsets in the murine model of active ITP. A The timelines for MDSC treatment. B Change in the platelet count over time in mice with active ITP; mice receiving NC-siRNA-treated MDSCs showed significantly higher platelet counts than mice receiving GR-siRNA-treated MDSCs or PBS 21 and 28 days after irradiation. C–H The percentages of CD8⁺ T cells (C) and Th1 cells among CD4⁺ T cells (E) were significantly decreased and the percentages of Tregs (D) and Th2 cells (F) among CD4⁺ T cells were significantly increased in mice receiving NC-siRNA-treated MDSCs group. No significant differences were found between the percentages of Th17 (G) and Th22 (H) cells among splenic CD4⁺ T cells. Differences among groups were determined using analysis of variance (n = 5). *P < 0.05; **P < 0.01