Glutaminase 1 Inhibition Reduces Glycolysis and Ameliorates Lupus-like Disease in MRL/lpr Mice and Experimental Autoimmune Encephalomyelitis

Abstract

Objective: Glutaminase 1 (Gls1) is the first enzyme in glutaminolysis. The selective Gls1 inhibitor bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide (BPTES) suppresses Th17 development and ameliorates experimental autoimmune encephalomyelitis (EAE). The present study was undertaken to investigate whether inhibition of glutaminolysis is beneficial for the treatment of systemic lupus erythematosus (SLE), and the involved mechanisms.

Methods: MRL/lpr mice were treated with BPTES or vehicle control, and disease activity was examined. Then naive CD4+ T cells from patients with SLE were cultured under Th17-polarizing conditions with BPTES or vehicle. Furthermore, using newly generated Gls1 conditional-knockout mice, in vitro Th17 differentiation was examined, and EAE was induced in the mice. Glutaminolysis and glycolysis were measured with an extracellular flux analyzer. The expression of hypoxia-inducible factor 1α (HIF-1α) was examined by Western blotting.

Results: Treatment of MRL/lpr mice with BPTES improved autoimmune pathology in a Th17-dependent manner. T cells from patients with SLE treated with BPTES displayed decreased Th17 differentiation (P < 0.05). Using the conditional-knockout mice, we demonstrated that both in vitro Th17 differentiation (P < 0.05) and the development of EAE were dependent on Gls1. Gls1 inhibition reduced glycolysis and the expression of HIF-1α protein, which induces glycolysis.

Conclusion: We demonstrated that inhibition of glutaminolysis represents a potential new treatment strategy for patients with SLE and Th17-related autoimmune diseases. Mechanistically, we have shown that inhibition of glutaminolysis affects the glycolysis pathway by reducing HIF-1α protein in Th17 cells.

Figure 1.

BPTES treatment ameliorates lupus disease. A-D, MRL/lpr mice were treated with DMSO or BPTES twice a week intraperitoneally. All mice were euthanized at 16 weeks of age. A, Ratio of urinary albumin (Alb) and creatinine (Cre) quantified by ELISA. Cumulative results of 12–13 mice per group were shown. B and C, PAS-stained kidney section and disease scores were shown. Representative histopathologic images (B) and quantitative cumulative data (C) were shown. Scale bars, 50 μm; n = 9. D, Kidney infiltrated CD45⁺ cells (ZA^- CD45⁺), T cells (ZA^- CD45⁺ Th1.2⁺), and IL-17A producing T cells (ZA^- CD45⁺ Th1.2⁺ IL-17A⁺) were evaluated by flow cytometry. Cumulative results of 9 mice per group were shown. E, Naïve CD4⁺ T cells from patients with SLE (n = 6) and healthy donors (HD; n = 6) were cultured under Th17-polarizing conditions in the presence of DMSO or BPTES for 7 days. Percentage of IL-17A positive cells (ZA^- IL-17A⁺) was measured by flow cytometry (ZA^- IL-17A⁺). Cumulative data were shown. The data represents the mean ± SEM; *P < 0.05, **P < 0.01, ns, not significant.

Figure 2.

Gls1 is requisite for Th17 differentiation and promotes glycolysis in MRL/lpr mice. A-F, Naïve CD4⁺ T cells from MRL/lpr mice were cultured under Th17 polarizing condition. A, Cells were cultured in media containing the indicated doses of glutamine (Gln) (0–2.0 mM) for 3 days. Representative flow plots are shown. B, Cumulative data are shown; n = 4. C, Oxygen consumption rate (OCR) was measured by extracellular flux analyzer. Cumulative data of calculated glutaminolysis on day 2 were shown; n = 4. D and E, Cells were cultured in the presence of increasing concentration of BPTES (0–10 μΜ) for 3 days. Percentage of IL-17A positive cells (ZA^- IL-17A⁺) was measured by flow cytometry. Representative data (D) are shown. Cumulative data (E) were shown; n = 3. F, Gls1-shRNA or control-shRNA containing lentiviral particles were infected on day 1. Percentage of IL-17A positive cells (ZA^- IL-17A⁺) was measured by flow cytometry on day 4. Cumulative data were shown; n = 4. The data represents the mean ± SEM; *P < 0.05, **P < 0.01.

Figure 3.

Gls1 inhibition reduces glycolysis. A-D, Naïve CD4⁺ T cells from MRL/lpr (A and B) or B6 (C and D) mice were cultured under Th17 polarizing condition with DMSO or 10 mM BPTES. Glycolysis (A and C) and glycolytic capacity (B and D) were assessed by measuring extracellular acidification rate (ECAR) on day 2, 3, and 5. Cumulative data of extracellular acidification rate (ECAR) were shown; n = 3. The data represents the mean ± SEM; *P < 0.05, **P < 0.01.

Figure 4.

Gls1 deficiency reduces Th17 cell differentiation and glycolysis. A-F, Naïve CD4 T cells from Gls1^+/+Il17Cre mice and Gls1^fl/flIl17Cre mice were cultured under Th17 polarizing conditions. A, Oxygen consumption rate (OCR) was measured by extracellular flux analyzer. Cumulative data of calculated glutaminolysis on day 2 are shown; n = 4. B, Percentage of IL-17A positive cells was measured by flow cytometry on day 5. Representative data are shown. C, Cumulative data were shown; n = 4. D and E, Empty vector (Empty), or Gls1 overexpression (Gls1) plasmids were transfected on day 1. Percentage of IL-17A positive cells was measured by flow cytometry on day 5. Representative data (D) and cumulative data (E) were shown; n = 3. F, Glycolysis and glycolytic capacity were assessed by measuring ECAR on day 5. Cumulative data were shown; n = 4. The data represents the mean ± SEM; *P < 0.05, **P < 0.01.

Figure 5.

Gls1 deficiency ameliorates EAE disease activity. A-D, EAE was induced in Gls1^+/+Il17Cre mice and Gls1^fl/flIl17Cre mice. The clinical scores (A) are shown. Cumulative results of 10 mice per group are shown. B and C, Spinal cords were harvested at day 14 and stained with H&E to assess inflammation. Representative histopathologic images (B) and quantitative cumulative data (C) are shown. Scale bars, 500 μm or 100 μm (magnified panels); n = 9 (Gls1^+/+Il17Cre mice). n = 11 (Gls1^fl/flIl17Cre mice). D, Absolute cell numbers of spinal-cord infiltrated CD4⁺ T cells (ZA^- Th1.2⁺ CD4⁺) and IL-17A producing CD4⁺ T cells (ZA^- Th1.2⁺ CD4⁺IL-17A⁺) from recipient mice were evaluated by flow cytometry on day 14. Cumulative data are shown; n=5. The data represents the mean ± SEM; *P < 0.05, **P < 0.01.

Figure 6.

Gls1 inhibition reduces Hif1α expression and Th17 differentiation. A, Naïve CD4⁺ T cells from MRL/lpr mice were cultured under Th17-polarizing conditions. Gls1-shRNA or control-shRNA containing lentiviral particles were infected on day 1 and cells were harvested on day 4. Indicated protein expression was assessed by western blotting. Representative blots are shown. Data are representative of 3 experiments. B and C, Naïve CD4⁺ T cells from Gls1^+/+Il17Cre mice and Gls1^fl/flIl17Cre mice were cultured under Th17-polarizing conditions. Cells were cultured in the presence of VH298 (0, 30 and 100 μΜ) for 5 days. Percentage of IL-17A positive cells was measured by flow cytometry on day 5. Representative data (B) and cumulative data (C) were shown; n=4. D, Schematic representation of mechanism whereby Gls inhibition reduces glycolysis in Th17 cells. Gls inhibition reduces the expression of HIF1α, which is a canonical glycolysis master regulator. Reduction of Hif1α diminishes glycolysis and Th17 differentiation. The data represents the mean ± SEM; *P < 0.05, **P < 0.01.