Antimicrobial mitochondrial reactive oxygen species induction by lung epithelial immunometabolic modulation

Pneumonia is a worldwide threat, making discovery of novel means to combat lower respiratory tract infection an urgent need. Manipulating the lungs\u27 intrinsic host defenses by therapeutic delivery of certain pathogen-associated molecular patterns protects mice against pneumonia in a reactive oxygen species (ROS)-dependent manner. Here we show that antimicrobial ROS are induced from lung epithelial cells by interactions of CpG oligodeoxynucleotides (ODN) with mitochondrial voltage-dependent anion channel 1 (VDAC1). The ODN-VDAC1 interaction alters cellular ATP/ADP/AMP localization, increases delivery of electrons to the electron transport chain (ETC), increases mitochondrial membrane potential (ΔΨm), differentially modulates ETC complex activities and consequently results in leak of electrons from ETC complex III and superoxide formation. The ODN-induced mitochondrial ROS yield protective antibacterial effects. Together, these studies identify a therapeutic metabolic manipulation strategy to broadly protect against pneumonia without reliance on antibiotics

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Pneumonia is a worldwide threat, making discovery of novel means to combat lower respiratory tract infection an urgent need. Manipulating the lungs’ intrinsic host defenses by therapeutic delivery of certain pathogen-associated molecular patterns protects mice against pneumonia in a reactive oxygen species (ROS)-dependent manner. Here we show that antimicrobial ROS are induced from lung epithelial cells by interactions of CpG oligodeoxynucleotides (ODN) with mitochondrial voltage-dependent anion channel 1 (VDAC1). The ODN-VDAC1 interaction alters cellular ATP/ADP/AMP localization, increases delivery of electrons to the electron transport chain (ETC), increases mitochondrial membrane potential (ΔΨm), differentially modulates ETC complex activities and consequently results in leak of electrons from ETC complex III and superoxide formation. The ODN-induced mitochondrial ROS yield protective antibacterial effects. Together, these studies identify a therapeutic metabolic manipulation strategy to broadly protect against pneumonia without reliance on antibiotics.</div

VBIT-4 increases epithelial mtROS generation and Δ_Ψm.

MitoSOX fluorescence (A) and JC-1 ratio (B) of HBEC3-KT cells treated for 100 min with PBS, ODN or VDAC1 inhibitor VBIT-4. * p (EPS)</p

Uncut immunoblots for mitochondria mass analysis.

HBEC3-KT cells were exposed to ODN for 0, 50, or 100 min. Shown are the uncut immunoblots for the bands shown in Fig 2B. These include (A) SDHB, succinate dehydrogenase subunit B; (B) COX4, cytochrome c oxidase subunit IV; (C) ATP5A, ATP synthase alpha-subunit; (D) CS, citrate synthase; (E) VDAC1, voltage dependent anion channel 1; and (F) β-Actin, used as a loading control. (EPS)</p

Effect of TCA cycle metabolites on ODN-induced mtROS generation.

mtROS dose response to ODN in HBEC3-KT cells supplemented with the TCA metabolites or metabolite analogues (A) citrate, (B) pyruvate, (C) α-ketoglutarate, (D) dimethyl succinate, (E) dimethyl malonate, (F) dimethyl fumurate or (G) oxaloacetate. * p≤0.003 vs. 0 μM ODN treated with no metabolite pretreatment by one-way ANOVA using Holm-Sidak method. † p≤0.05 vs. same ODN dose with no metabolite pretreatment by one-way ANOVA using Holm-Sidak method. ‡ p (EPS)</p

ODN treatment increases electron delivery to complex II.

(A) Schematic overview of immunometabolic modulation by ODN. (B) Acetyl-CoA levels in ODN-treated HBEC3-KT cells. (C) Fatty acid oxidation after ODN treatment of different intervals. (D) ODN-induced fatty acid oxidation in AMPK-deficient cells. (E) mtROS production following treatment with ODN and/or β-oxidation inhibitor etomoxir. (F) Oxygen consumption following the indicated treatments, shown as mean ± SEM. (G) HBEC3-KT cell complex II activity following treatment with the indicated agents.ODN-induced mtROS production in cells with knockdowns of gene CPT1A (H) and the genes for electron shuttles GPD2 (I) or ETFDH (J). (K) Ratio of reduced:oxidized CoQ in mitochondria isolated from HBEC3-KT cells treated with PBS or ODN. * p <0.01 vs 0 min; † p <0.001 vs. (syngeneic) PBS treated; ǂ p < 0.05 vs (syngeneic) PBS treated. # p = 0.008 vs PBS-treated by one-way ANOVA using Dunnett’s test for multiple comparisons. RPPA, reverse phase protein array; AMPK, AMP-activating protein kinase; ACC, acetyl-CoA carboxylase; AdV, adenovirus; OCR, oxygen consumption rate; Scr, scrambled shRNA control; CPT1A, carnitine palmitoyltransferase 1A; GPD2, glycerol-3-phosphate dehydrogenase 2; ETFDH, electron transfer flavoprotein-ubiquinone dehydrogenase.</p

Alternate electron sources that contribute to mtROS generation.

(A) ODN-induced mtROS generation in the presence of absence of glycolysis inhibitor UK5099, β-oxidation inhibitor etomoxir, or glutaminolysis inhibitor BPTES. mtROS dose response to ODN in the presence or absence of (B) BPTES, (C) etomoxir, or (D) UK5099. (E) ODN-induced mtROS in the presence of single or combined inhibitors. (F) mtROS dose response to ODN in the presence or absence of (2-DG), a D-glucose analogue. (G) Mitochondrial oxygen consumption in oligomycin-inhibited HBEC3-KT cells following treatment with ODN in the presence of the indicated inhibitors measured using a Seahorse XFe96 Flux Analyzer, shown as mean ± SEM. 2-DG, 2-deoxy-D-glucose; OCR, oxygen consumption rate. * p≤0.04 vs. no ODN with same pretreatment by one-way ANOVA using Holm-Sidak method. † p≤0.002 vs. same ODN dose with no inhibitor pretreatment by one-way ANOVA using Holm-Sidak method. ‡ p≤0.008 vs. same pretreatment without ODN by one-way ANOVA using Holm-Sidak method. § p (EPS)</p

Induction of epithelial mtROS by CpG ODN.

(A) mtROS production from HBEC3-KT cells after treatment with pathogen associated molecular patterns. (B) mtROS production from HBEC3-KT cells after treatment with the indicated ODNs. mtROS production after treatment with ODN from mouse lung epithelial cell lines (C) and primary human (D) and primary mouse (E) lung epithelial cells. (F) Representative fluorescence images primary tracheal epithelial cells harvested from mt-roGFP mice treated with PBS or ODN. Images shown as gradient of color intensity from the reduced (blue) form to the oxidized (green) form of roGFP. Scale bar, 50 μm. (G) Ratio of the fluorescence intensity of the oxidized:reduced roGFP from F, quantified at 488 nm and 405 nm, respectively. (H) Oxygen consumption following the indicated treatment by Seahorse XFe96 Flux Analyzer, shown as mean ± SEM. (I) ODN-induced mtROS production from HBEC3-KT cells in the presence of the indicated inhibitors. Mitochondrial membrane potential ΔΨm measurement in HBEC3-KT cells after ODN treatment assessed by JC-1 (J), MitoTracker (K) and TMRM (L). * p<0.001 vs. PBS by one-way ANOVA using Holm-Sidak method, except A which use Tukey method due to failed normality testing; † p<0.001 vs PBS by two-way Student’s t test; ‡ p <0.008 vs PBS treated by one-way ANOVA using Dunnett’s test for multiple comparisons. ODN, oligodeoxynucleotide; ISD, immune stimulating DNA; mTEC, primary mouse tracheal epithelial cells; NHBE, primary normal human bronchial epithelial cells; GFP, green fluorescent protein; OCR, oxygen consumption rate; TMRM, tetramethylrhodamine.</p

Correlation between ODN and VDAC1 pixel intensity by linear regression.

Pixel intensity values for FITC-labeled ODN and Alexa Fluor 555-labeled anti-VDAC1 antibody were plotted against each other and a simple linear regression model was fit to the data. Pearson’s correlation coefficients were calculated to determine whether VDAC1 pixel intensity tended to accumulate with ODN1 pixel intensity. FITC, Fluorescein isothiocyanate; VDAC1, voltage dependent anion channel 1. (EPS)</p

Inhibition of ODN-induced increase of mtROS and membrane potential ΔΨm by TTFA and FCCP treatment.

HBEC3-KT cells were pre-treated (or not) with either TTFA or FCCP or both, then treated for 100 min with the indicated concentration of ODN. Shown are mitoSOX fluorescence with (A) TTFA, (B) FCCP and (C) TTFA-FCCP. (D) MitoTracker fluorescence, or (E) JC-1 ratio of aggregates:monomers with TTFA-FCCP. (F) Bacterial burden of HBEC3-KT cells treated with the indicated ligands with or without TTFA or FCCP or both. * p≤0.001 vs PBS treated without inhibitor by Kruskal-Wallis one-way ANOVA using Tukey test. † p (EPS)</p