Elevated type I interferon responses potentiate metabolic dysfunction, inflammation, and accelerated aging in mtDNA mutator mice

Abstract

Mitochondrial dysfunction is a key driver of inflammatory responses in human disease. However, it remains unclear whether alterations in mitochondria-innate immune cross-talk contribute to the pathobiology of mitochondrial disorders and aging. Using the polymerase gamma (POLG) mutator model of mitochondrial DNA instability, we report that aberrant activation of the type I interferon (IFN-I) innate immune axis potentiates immunometabolic dysfunction, reduces health span, and accelerates aging in mutator mice. Mechanistically, elevated IFN-I signaling suppresses activation of nuclear factor erythroid 2-related factor 2 (NRF2), which increases oxidative stress, enhances proinflammatory cytokine responses, and accelerates metabolic dysfunction. Ablation of IFN-I signaling attenuates hyperinflammatory phenotypes by restoring NRF2 activity and reducing aerobic glycolysis, which combine to lessen cardiovascular and myeloid dysfunction in aged mutator mice. These findings further advance our knowledge of how mitochondrial dysfunction shapes innate immune responses and provide a framework for understanding mitochondria-driven immunopathology in POLG-related disorders and aging.

Fig. 1. POLG mutator mice exhibit a hyperinflammatory phenotype to LPS challenge due to increased CD11b⁺ myeloid cells in the blood.

(A and B) Twelve-month-old WT (n = 8) and POLG (n = 7) mice were challenged with LPS [50 mg/kg by intraperitoneal (i.p.) injection]. Kaplan-Meyer survival analysis was performed (A). Plasma cytokine profiles were determined by multianalyte bead-based immunoassay on n ≥ 6 biological samples per group (B). Log-rank (Mantel-Cox) test was used to compare percent survival between different groups. (C and D) CD11b⁺Ly6C^hi inflammatory monocyte population in whole blood from 12-month-old WT and POLG mice was evaluated by flow cytometry. Pseudo-color plots are representative of four independent experiments (C), and quantification of the percentage of CD11b⁺Ly6C^hi cells is shown in (D). (E and F) CD11b⁺Ly6G⁺ blood neutrophil population in 12-month-old WT and POLG mice was determined by flow cytometry. Pseudo-color plots are representative of four independent experiments (E), and quantification of the percentage of CD11b⁺Ly6G⁺ cells is shown in (F). (G to I) CD11b⁺Ly6C^hiTNFα⁺ inflammatory monocyte population in unstimulated (C) or LPS-challenged (L) whole blood from 12-month-old WT and POLG mice was evaluated by flow cytometry. Histograms are representative of four independent experiments (G). Quantification of CD11b⁺Ly6C^hiTNFα⁺ mean fluorescent intensity (MFI) is shown in (H), and the percentage of CD11b⁺Ly6C^hiTNFα⁺ cells is shown in (I). (J to L) CD11b⁺Ly6G⁺TNFα⁺ neutrophil population in unstimulated or LPS-challenged whole blood from 12-month-old WT and POLG mice was evaluated by flow cytometry. Histograms are representative of four independent experiments (J). Quantification of CD11b⁺Ly6G⁺TNFα⁺ MFI is shown in (K), and the percentage of CD11b⁺Ly6G⁺TNFα⁺ neutrophils is shown in (L). Unless stated, statistical significance was determined using unpaired Student’s t test after Shapiro-Wilk normality test. *P < 0.05, **P < 0.01, and ***P < 0.001. Error bars represent SEM.

Fig. 2. POLG mutator macrophages exhibit enhanced IFN-I and proinflammatory responses after innate immune stimulation.

(A and B) Heatmaps of RNA-seq data displaying most up-regulated ISGs in POLG mutator PerMacs (A) and BMDMs (B) at baseline and after LPS challenge (200 ng/ml for 6 hours). Log₂ fold changes (Log₂ FC) are relative to WT controls. (C to F) qRT-PCR analysis of ISG and proinflammatory cytokine expression in WT and POLG PerMacs (C and E) and BMDM (D and F) after 4 or 6 hours of LPS stimulation. (G and H) Proinflammatory cytokine secretion in WT and POLG PerMacs (G) or BMDMs (H) after 4 hours of LPS stimulation (20 ng/ml). (I) Nitrite levels in WT and POLG BMDM after 17 hours of LPS (20 ng/ml) + IFNγ (50 ng/ml) treatment. (J) qRT-PCR analysis of ISGs and cytokine expression in WT and POLG BMDMs after 4 hours of ISD transfection (2 μg/ml). Statistical significance was determined using unpaired Student’s t tests. *P < 0.05, **P < 0.01, and ***P < 0.001. Error bars represent SEM.

Fig. 3. The cGAS-STING–IFN-I signaling axis regulates inflammatory monocyte expansion and elevated cytokine secretion in POLG mutator mice.

(A and B) Twelve-month-old WT, POLG, POLG cGAS^−/−, POLG Sting^−/−, and POLG Ifnar^−/− (n = 5 to 8 per group) mice were intraperitoneally injected with LPS (50 mg/kg). Kaplan-Meyer survival analysis was performed (A). Plasma was collected at indicated time points (n ≥ 6 at 2 hours and n = 4 at 6 hours) and subjected to multianalyte cytokine analysis (B). Statistical comparisons in (B) were made against LPS-injected POLG mice. Log-rank (Mantel-Cox) test was used to compare percent survival between different groups. (C and D) CD11b⁺Ly6C^hi inflammatory monocyte population in whole blood from 12-month-old mice was evaluated by flow cytometry. Pseudo-color plots are representative of four independent experiments (C), and quantification of the percentage of CD11b⁺Ly6C^hi cells is shown in (D). (E and F) CD11b⁺Ly6C^hiTNFα⁺ inflammatory monocyte population in unstimulated or LPS challenged whole blood from 12-month-old cohorts was evaluated by flow cytometry. Quantification of CD11b⁺Ly6C^hiTNFα⁺ MFI is shown in (E), and the percentage of CD11b⁺Ly6C^hiTNFα⁺ cells is shown in (F). Unless stated, statistical significance was determined using analysis of variance (ANOVA) and Tukey post hoc test. *P < 0.05, **P < 0.01, and ***P < 0.001. Error bars represent SEM.

Fig. 4. NRF2 suppression contributes to the hyperinflammatory phenotype of POLG mutator macrophages.

(A) Heatmaps of RNA-seq data displaying the most down-regulated NRF2 target genes in POLG mutator PerMacs and BMDMs after LPS challenge (200 ng/ml for 6 hours). Log₂ fold changes are relative to WT controls. (B) Representative confocal microscopy images of LPS-treated PerMacs stained with anti-NRF2 and anti-HSP60 antibodies and 4′,6-diamidino-2-phenylindole (DAPI). (C and D) Quantification of nuclear NRF2 staining intensity in WT and POLG PerMacs 4 hours (C) or 24 hours (D) after LPS stimulation. a.u., arbitrary units. (E and F) qRT-PCR analysis of NRF2 target (E) or proinflammatory cytokine (F) gene expression in WT and POLG PerMacs after LPS or LPS + KI696 treatment. (G) Proinflammatory cytokine secretion by WT and POLG PerMacs after LPS or LPS + KI696 treatment. (H) Protein expression in siCtrl or siKeap1-transfected WT and POLG BMDMs after LPS treatment (20 ng/ml for 6 hours). SE, short exposure; LE, long exposure. (I) TNFα secretion in siCtrl or siKeap1-transfected WT and POLG BMDMs after LPS treatment (20 ng/ml). Statistical significance was determined using unpaired Student’s t tests (C and D) or ANOVA and Tukey post hoc test (E to G and I). *P < 0.05, **P < 0.01, and ***P < 0.001. Error bars represent SEM.

Fig. 5. Elevated IFN-I signaling represses NRF2 activity and drives proinflammatory metabolic phenotypes in POLG mutator macrophages.

(A) qRT-PCR analysis of ISG expression in BMDMs after LPS challenge. (B) Representative confocal microscopy images of LPS-treated BMDMs stained with anti-NRF2 and anti-HSP60 antibodies and DAPI. (C and D) Quantification of nuclear NRF2 staining intensity in BMDMs 6 hours (C) or 24 hours (D) after LPS exposure. (E) qRT-PCR analysis of NRF2 target gene expression in BMDMs after LPS exposure. POLG BMDMs were normalized to WT BMDMs, and POLG Ifnar^−/− BMDMs were normalized to Ifnar^−/− BMDMs. (F) Protein expression of NRF2 targets and lactate dehydrogenase A (LDHA) in BMDMs after 6-hour LPS (200 ng/ml) exposure. (G) Proinflammatory cytokine secretion from WT, POLG, and POLG Ifnar^−/− BMDMs after LPS stimulation (20 ng/ml for 4 hours). (H) Protein expression in siCtrl or siNrf2-transfected WT, POLG, and POLG Ifnar^−/− BMDMs after LPS treatment (20 ng/ml for 6 hours). (I) Proinflammatory cytokines secretion in siCtrl or siNrf2-transfected WT, POLG, and POLG Ifnar^−/− BMDMs after LPS treatment (20 ng/ml for 24 hours). (J) Quantification of MFI of MitoSOX staining in LPS-treated BMDMs. (K) Seahorse ECAR analysis of WT, POLG, Ifnar^−/−, and POLG Ifnar^−/− BMDMs exposed to overnight LPS challenge (10 ng/ml). 2-DG, 2-deoxyglucose. (L) Relative LDH activity in BMDMs 24 and 48 hours after LPS exposure (normalized to WT). (M) Extracellular l-lactate level in culture media of resting (C) or LPS exposed (L) BMDMs. WT and POLG were normalized to WT resting BMDMs, and Ifnar^−/− and POLG Ifnar^−/− were normalized to Ifnar^−/− resting BMDMs. Statistical significance was determined using ANOVA and Tukey post hoc test. *P < 0.05, **P < 0.01, and ***P < 0.001. n.s., not significant. Error bars represent SEM.

Fig. 6. Ablation of IFN-I signaling lessens multiorgan pathology and extends life span in POLG mutator mice.

(A) Representative B-mode echocardiogram images of 9- to 10-month-old WT, POLG, Ifnar^−/−, and POLG Ifnar^−/− mouse hearts. (B) LVID;d and LVEF calculated from M-mode or B-mode images using Vevo LAB software. n = 5 to 8 animals per genotype. (C and D) Representative hematoxylin and eosin (H&E) staining of heart sections from WT, POLG, Ifnar^−/−, and POLG Ifnar^−/− mice (C) and quantification of cardiomyocyte width (D). Yellow arrows indicate infiltrating immune cells. Five myocytes per section and six animals per genotype were quantified in a blinded fashion (D). (E) Red blood cell (RBC) counts, hemoglobin (HGB) concentration, and hematocrit (HCT) were measured in WT, POLG, Ifnar^−/−, and POLG Ifnar^−/− mouse whole blood using the HM5 Hematology Analyzer. n = 6 animals per genotype. (F) Representative H&E staining showing white pulp and red pulp organization in WT, POLG, Ifnar^−/−, and POLG Ifnar^−/− mouse spleens. (G) Representative H&E-stained liver sections from WT, POLG, Ifnar^−/−, and POLG Ifnar^−/− cohorts. (H and I) Percent survival (H) and survival time (I) of POLG, POLG cGAS^−/−, POLG Sting^−/−, and POLG Ifnar^−/− mice. Log-rank (Mantel-Cox) test was used to compare percent survival between different groups. n = 10 animals per genotype. Unless stated, statistical significance was determined using ANOVA and Tukey post hoc test.*P < 0.05, **P < 0.01, and ***P < 0.001. n.s., not significant. Error bars represent SEM.

Fig. 7. Imbalances in IFN-I and NRFf2 signaling contribute to inflammatory and age-related phenotypes in POLG mutator mice.

mtDNA instability and mitochondrial dysfunction in POLG mutator mice lead to elevated IFN-I responses, which subsequently repress NRF2 activity and enhance aerobic glycolysis. Consequently, chronic IFN-I responses augment the expansion and inflammatory potential of CD11b⁺ myeloid cells and macrophages, while also contributing to cardiomyopathy and anemia in mutator mice. Genetic ablation of IFN-I signaling relieves the break on NRF2 and markedly improves health span by limiting myeloid reprogramming, inflammation, and tissue dysfunction in mtDNA mutator mice.