Inhibition of the mitochondrial pyruvate carrier simultaneously mitigates hyperinflammation and hyperglycemia in COVID-19

Abstract

The relationship between diabetes and coronavirus disease 2019 (COVID-19) is bidirectional: Although individuals with diabetes and high blood glucose (hyperglycemia) are predisposed to severe COVID-19, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection can also cause hyperglycemia and exacerbate underlying metabolic syndrome. Therefore, interventions capable of breaking the network of SARS-CoV-2 infection, hyperglycemia, and hyperinflammation, all factors that drive COVID-19 pathophysiology, are urgently needed. Here, we show that genetic ablation or pharmacological inhibition of mitochondrial pyruvate carrier (MPC) attenuates severe disease after influenza or SARS-CoV-2 pneumonia. MPC inhibition using a second-generation insulin sensitizer, MSDC-0602K (MSDC), dampened pulmonary inflammation and promoted lung recovery while concurrently reducing blood glucose levels and hyperlipidemia after viral pneumonia in obese mice. Mechanistically, MPC inhibition enhanced mitochondrial fitness and destabilized hypoxia-inducible factor-1α, leading to dampened virus-induced inflammatory responses in both murine and human lung macrophages. We further showed that MSDC enhanced responses to nirmatrelvir (the antiviral component of Paxlovid) to provide high levels of protection against severe host disease development after SARS-CoV-2 infection and suppressed cellular inflammation in human COVID-19 lung autopsies, demonstrating its translational potential for treating severe COVID-19. Collectively, we uncover a metabolic pathway that simultaneously modulates pulmonary inflammation, tissue recovery, and host metabolic health, presenting a synergistic therapeutic strategy to treat severe COVID-19, particularly in patients with underlying metabolic disease.

Fig. 1.. Disruption of MPC-mediated oxidative pyruvate metabolism mitigates IAV- or SARS-CoV-2-induced pathogenesis.

(A) Diagram of pyruvate metabolic pathways. (B to D) MPC2^fl/fl and MPC2^ΔLyz2 mice were infected with sub-lethal (B and D) and lethal (C) doses of IAV, respectively. Host morbidity (B) and mortality (C) were monitored. (D) BAL cytokine levels at 4 d.p.i. (n = 9). (E) Schematic diagram for viral infected C57BL/6 WT mice with vehicle or MSDC-0602 k (MSDC) treatment. (F to I) Mice were infected with sub-lethal (F, H, and I) and lethal (G) doses of IAV. Host morbidity (F) and mortality (G) were monitored. (H) RNA-seq analysis of lungs at 4 d.p.i. (n = 3). GSEA of inflammatory response gene set shown. (I) H&E staining of lung section (n = 4–5) at 14 d.p.i. Scale bar, 200 μm (I). (J to N) Mice were infected with SARS-CoV-2 MA10 virus. (J) Schematic diagram for viral infected C57BL/6 WT mice with vehicle or MSDC treatment. (K) Host morbidity was monitored. (L) H&E staining of lung section (n = 5) at 8 d.p.i. Scale bar, 200 μm. (M) BAL viral titers were measured at the indicated time points (n = 5). n.d., not detected. (N) BAL cytokine levels at 8 d.p.i. (n = 9–10). (O) MPC2^fl/fl and MPC2^ΔLyz2 mice were infected with SARS-CoV-2 MA10. Host morbidity was monitored. Representative (I and L) or pooled data (B, C, D, F, G, K, N and O) from at least two independent experiments. Data are presented as means ± SEM. *, p < 0.05; **, p < 0.01; ***, p < 0.001. The p value was determined by multiple t-tests (B, F, K and O), Logrank test (C and G) or a two-tailed Student’s t-test (D, M and N).

Fig. 2.. Murine and human lung macrophages are prominent target of MPC inhibition by MSDC.

(A to E) scRNA-seq analysis of lungs from IAV-infected C57BL/6 WT mice with vehicle or MSDC treatment at 4 d.p.i. Lung cells were pooled from three individual mice from each group. (A) UMAP plot visualization of lung cells from vehicle- or MSDC-treated mice. (B) The relative contributions of indicated clusters by each group. (C) Dot plot showing enrichment of Gene Ontology biological processes pathways enriched in MSDC-treated lungs. The color of the dots represents the adjusted P value. Dot size represents the enrichment score. (D) UMAP showing clusters of monocytes and macrophages from (A) in vehicle- or MSDC- treated lung cells (upper panel). The percentages of each cluster in each studied subject was shown on the lower panel. (E) Volcano plot showing the differentially expressed genes in AMs (cluster 2, 4, 6 and 7) of vehicle (blue) and MSDC (red) treated mice. (F) The mRNA levels of Tnf, Il1b and Ccl2 in AMs, CD11b⁺ macrophages (Mac) and monocytes (Mono) sorted from lungs at 4 d.p.i. (G) RNA-seq analysis of mouse AMs stimulated with or without Poly IC in the presence of vehicle or MSDC overnight in vitro. Heatmap of K-means clustering of differentially expressed genes and KEGG enrichment analysis. (H) The mRNA levels of TNF, IL1B and CCL2 in human AMs, monocyte-derived macrophages (MdM), and monocytes (Mono) stimulated with or without Poly IC in the presence of vehicle or MSDC overnight in vitro. Data are presented as means ± SEM. *, p < 0.05; **, p < 0.01; ***, p < 0.001. The p value was determined by multiple t tests (F) and one-way ANOVA (H).

Fig. 3.. MPC inhibition dampens HIF-1α levels in lung macrophages.

(A to C) Immunoblot analysis of indicated total or phosphorylated protein levels in mouse AMs (A) and BMDM (C) stimulated with or without Poly IC in the presence of vehicle or MSDC in vitro. (B) Quantification of HIF-1α levels in (A) of three experiments were shown. (D) Flow cytometry analysis of HIF-1α levels in AMs from naïve or IAV-infected wt mice treated with vehicle or MSDC at 4 d.p.i. (n = 5). (E) GSEA of hypoxia gene set shown for Poly IC stimulated AMs with vehicle or MSDC treatment. (F) Immunoblot analysis of HIF-1α in human AMs stimulated with or without Poly IC in the presence of vehicle or MSDC overnight in vitro (left). Quantitation on the right (n = 3 donors). (G) Ccl2 and Tnf levels in mouse AMs under indicated conditions overnight in vitro. Representative immunoblots (A, C and F) were from three independent experiments. Data are presented as means ± SEM. *, p < 0.05. The p value was determined by a two-tailed Student’s t-test (B, D, and F) and one-way ANOVA (G).

Fig. 4.. MPC inhibition promotes mitochondrial fitness and diminishes HIF-1α stabilizing metabolites in lung macrophages.

(A and B) AMs were stimulated with or without Poly IC in the presence of vehicle or MSDC overnight in vitro. (A) OCR of AMs and quantification of respiratory reverse. (B) Flow cytometry showing mitochondrial mass by Mitotracker green versus Mitotracker deep red in AMs on the left and quantification on the right. (C and D) OCR (C) or mitochondrial mass (D) in AMs from naïve or IAV-infected wt mice treated with vehicle or MSDC at 4 d.p.i. (n = 5). (E) Heatmap showing TCA cycle metabolites measured in AMs (n = 3). (F) Succinate to Ketoglutarate (α-KG) ratios in (E). (G) Acetyl-CoA concentrations in BMDM and AMs treated with vehicle or MSDC overnight in vitro. (H) BMDM and AMs were stimulated with Poly IC in the presence of vehicle or MSDC overnight in vitro. MG132 were added 4 h before cell harvesting. Coimmunoprecipitation (IP) was performed with anti-HIF-1α antibodies followed by immunoblot analysis of HIF-1α and HIF-1α acetylation (Ace-K, with anti-ace-lysine antibodies) levels. Quantification of HIF-1α acetylation was shown. (I and J) AMs were stimulated with or without Poly IC in the presence of vehicle, MSDC, dimethyl succinate (DMS), or sodium acetate (SDA) overnight in vitro. (I) HIF-1α protein levels in AMs. (J) Tnf and Ccl2 gene expression in AMs. Representative immunoblots (H and I) were from three independent experiments. Data are presented as means ± SEM. *, p < 0.05; **, p < 0.01; ***, p < 0.001. The p value was determined by a two-tailed Student’s t-test (A, D, F, G, and H) and one-way ANOVA (B and J).

Fig. 5.. MSDC treatment simultaneously promotes host metabolic health, dampens pulmonary inflammation and enhances tissue recovery.

(A) Schematic diagram for treatment of IAV-infected DIO mice. (B to E) DIO mice were infected with sub-lethal (B to D) or lethal (E) doses of IAV and treated with vehicle or MSDC. (B) Blood glucose concentration measured by an i.p. glucose tolerance test at 5 d.p.i. (n = 9–10). (C) Total cholesterol concentrations in the blood at 5 d.p.i. (n = 4–5). (D) BAL cytokine levels at 5 d.p.i. (n = 13–14). (E) Host mortality was monitored. (F to K) DIO mice were infected with SARS-CoV-2 MA10 virus and treated with vehicle or MSDC. (F) Schematic diagram. (G) BAL cytokine levels at 5 d.p.i. (n = 5). (H) Blood glucose and total cholesterol levels were measured at 5 d.p.i. (n = 5). (I) H&E staining of lung section (n = 5) and quantification of pathological lesions at 5 d.p.i. Scale bar, 200 μm. HM, hyaline membranes. (J) Fluorescence microscopy images of PDPN, proSP-C and DAPI staining in fixed lung tissues at 5 d.p.i (n = 5). Scale bar, 50 μm. Quantification of proSP-C⁺ cell number was performed using at least 10 random fields (10x) of alveolar space per mouse lung. (K) Host mortality was monitored. Representative (I and J) or pooled data (B, D, E and K) from at least two independent experiments. Data are presented as means ± SEM. *, p < 0.05; **, p < 0.01. The p value was determined by Logrank test (E and K), one-way ANOVA (B), or a two-tailed Student’s t-test (C, D and G to J).

Fig. 6.. MSDC diminishes COVID-19-associated lung inflammation and exhibits synergy with anti-viral therapy.

(A) Human AMs from BAL of non-infectious donors were infected with or without SARS-CoV-2 in the presence of vehicle or MSDC (n = 3 donors) for 48 h in vitro. Nanostring analysis of inflammatory genes in AMs. (B) Cytokine expression in lung cells from COVID-19 patient autopsies following vehicle or MSDC treatment overnight ex vivo (n = 7 subjects). (C and D) DIO mice were infected with SARS-CoV-2 MA10 virus and treated with vehicle, Nirmatrelvir, or Nirmatrelvir plus MSDC. (C) Host mortality was monitored and survival rate is shown. (D) H&E staining of lung section (n = 5) and quantification of pathological lesions at 21 d.p.i. Scale bar, 200 μm. HM, hyaline membranes. Representative (D) or pooled data (B and C) from at least two independent experiments. Data are presented as means ± SEM. *, p < 0.05; **, p < 0.01; ***, p < 0.001. The p value was determined by paired t test (B), Logrank test (C), and a two-tailed Student’s t-test (D).