SIRT3 is essential for host defense against mycobacterial infection in vivo and in vitro. (a and b) WT BMDMs were infected with Mabc-R (MOI = 3) at the indicated times. Actin protein levels were evaluated by immunoblotting as an internal control. (b) Quantification of results on (a). (c) Western blot analysis of the lung tissues from SIRT3 WT and KO mice left uninfected or infected intranasally with Mabc-R (1 × 10⁷ CFU) for 3 days. Quantifiation of results on top. (d) SIRT3 WT and KO mice were infected intranasally with various CFUs of Mabc-R (1 × 10⁷ CFU) or Mabc-S (1 × 10⁷ CFU) and monitored at 5 or 7 days post-infection (dpi). Data are shown as log pulmonary CFU. (e) Lung histopathology by H&E staining of SIRT3 WT and KO mice infected with Mabc-R for 5 days. Right, Quantification of results on left. Scale bar, 5 mm. (f) Intracellular survival of Mabc-S assessed by a CFU assay. SIRT3 WT and KO BMDMs were infected with Mabc-S (MOI = 1, for left; MOI = 3, for right) for 4 h, and then lysed to determine intracellular bacterial loads at 0 and 3 dpi. *P < 0.05, **P < 0.01, ***P < 0.001. Non-parametric test (b and d); Student’s t-test (c below and e right); One-way ANOVA (f). Data represent three independent experiments (a, c top, and e left), and values represent means (± SEM) from three or four independent experiments performed in triplicate (b, c bottom, e right, and f).

SIRT3 is required to control pathological inflammation and mitochondrial damage during Mabc-R infection. (a and b) SIRT3 WT and KO mice (n = 8 each group) were infected intranasally with Mabc-R (1 × 10⁷ CFU) and monitored at 1 and 3 dpi. (a) Lung tissues were subjected to quantitative real-time PCR analysis for the measurement of mRNA expression of various cytokines/chemokines. (b) The supernatants from lung lysates were subjected to ELISA analysis of TNF (at 3 dpi). (c) SIRT3 WT and KO mice (n = 3 each group) were infected intranasally with Mabc-R (1 × 10⁷ CFU) and monitored at 5 dpi. The lung tissues were harvested and then subjected to TEM analysis (left). Mitochondria with complete cristae are shown in a; swollen mitochondria with vacuolation in the cristae are shown in b. Right, Quantitative analysis of at least 8 EM images in the lung tissues from SIRT3 WT and KO mice infected intranasally with Mabc-R (1 × 10⁷ CFU; n = 3 each group). The ratio of damaged mitochondria in total mitochondria was calculated quantitatively. Scale bars, 500 nm. *P < 0.05, **P < 0.01, ***P < 0.001, n.s., not significant compared with SIRT3 WT conditions (a). Non-parametric test (a, b, and c right). Data represent three independent experiments (c left), and values represent means (± SEM) from three or four independent experiments performed in triplicate (a and b).

SIRT3 is essential for the amelioration of proinflammatory cytokine expression and controlling mitochondrial ROS production in BMDMs during Mabc-R infection. (a) BMDMs from SIRT3 WT and KO mice were infected with Mabc-R (MOI = 3) and incubated for 3, 6, or 18 h. (b and c) BMDMs (b) and PMs (c) were prepared from SIRT3 WT and KO mice, and were infected with Mabc-R (MOI = 3) in the presence or absence of 3-TYP (50 µM) for 3 h and quantitative real-time PCR analysis for Tnf, Il6, and Cxcl2 was performed. (d) SIRT3 WT and KO BMDMs were infected with Mabc-R (MOI = 3) for 2 h and subjected to MitoSOX Red staining (representative images are shown in the left panel; quantitative analysis is shown in the right panel). Scale bar, 50 μm. *P < 0.05, **P < 0.01, ***P < 0.001. U, uninfected. One-way ANOVA (a-d). Data are representative of three independent experiments (d left), and values represent means (± SEM) from three or four independent experiments performed in triplicate (a-c, d right).

SIRT3 is required for mitochondrial OXPHOS function and attenuation of cell death during Mabc-R infection. (a and b) SIRT3 WT and KO mice (n = 8 each group) were infected intranasally with Mabc-R (1 × 10⁷ CFU) and monitored at 1 and 3 dpi. Lung tissues were collected and subjected to (a) Western blot analysis for measurement of OXPHOS protein expression (Left, representative images; right, quantitative analysis), and (b) qRT-PCR analysis. (c) Oxygen consumption rate (OCR) analysis of SIRT3 WT and KO BMDMs untreated or treated with Mabc-R for 18 h. (d) Quantitative analysis of basal respiration, spare respiratory capacity (SRC), ATP production, and maximal respiration analysis from (c). (e) PI staining after infection (Left, representative images; right, quantitative analysis). Scale bar, 300 μm. *P < 0.05, **P < 0.01, ***P < 0.001. n.s., not significant. Paired t-test (a right); non-parametric test (b and e right); One-way ANOVA (d). Data are representative of three independent experiments (a left, c, and e left), and values represent means (± SEM) from three or four independent experiments performed in triplicate (a right, b, d, and e right).

Administration of MIT-001 in mice led to a protective effect against Mabc-R infection. (a) WT BMDMs were infected with Mabc-R (MOI = 5) for 2 h in the presence or absence of MIT-001 (20 µM) and subjected to MitoSOX Red staining (Left, representative images; right, quantitative analysis). Scale bar, 50 μm. (b-e) WT mice (n = 5 each group) were left uninfected or infected intranasally with Mabc-R (1 × 10⁷ CFU), prior to treatment with or without MIT-001 (30 mg/kg) and monitored at 10 days post-infection (dpi). Lung tissues were subjected to a (b) pulmonary CFU assay, (c) qRT-PCR analysis for cytokines/chemokines, (d) TEM analysis. Mitochondria with complete cristae are shown in a; swollen mitochondria with vacuolation in the cristae are shown in b. Right, Quantitative analysis of at least 8 EM images in the lung tissues from each group of mice infected intranasally with Mabc-R (1 × 10⁷ CFU; n = 3 each group). The ratio of damaged mitochondria in total mitochondria was calculated quantitatively. Scale bars, 200 nm. (e) qRT-PCR analysis for Sirt3 mRNA expression. *P < 0.05, **P < 0.01, ***P < 0.001, n.s., not significant. One-way ANOVA (a right) or non-parametric test (b, c, d right and e). Data represent three independent experiments (a left and d), and values represent means (± SEM) from three or four independent experiments performed in triplicate (a right, b, c, and e).

SIRT3 activation by RSV enhances antimicrobial responses and ameliorates mitochondrial oxidative stress during Mabc-R infection. (a and b) SIRT3 WT and KO mice (n = 4 each group) were infected with Mabc-R (1 × 10⁶ CFU), followed by treatment with or without RSV (50 mg/kg), and monitored at 7 dpi. (a) Data are shown as log pulmonary CFUs. (b) The lung tissues of the mice were subjected to qRT-PCR analysis. (c) MitoSOX Red staining for Mabc-infected WT BMDMs in the presence or absence of RSV (20 µM). Left, representative images; right, quantitative analysis. Scale bar, 50 μm. *P < 0.05, **P < 0.01, ***P < 0.001. Non-parametric test (a and b; c right); Data represent three independent experiments (c left), and values represent means (± SEM) from three or four independent experiments performed in triplicate (a, b, and c right).

Efficacy of the RSV in Mabc-R infected ZF. (a) The panels show representative dissemination of Mabc-R-mWasabi in ZF. The fluorescent bacterial dissemination and bacterial burden after treatment with different doses of RSV (1, 5, or 10 µM) were assessed. Statistical significance was determined by ANOVA using Tukey?s multiple comparison test. **P < 0.01, ***P < 0.001. Scale bar, 0.5 mm. (b) Survival curve of embryos infected with Mabc-R. The embryos were infected with Mabc-R, followed by treatment with RSV or solvent control (S. C.; DMSO, 1%). As a positive control, ZF without infection was used. RSV, Resveratrol; UNT, untreated. *P < 0.05.