Antimicrobial mitochondrial reactive oxygen species induction by lung epithelial metabolic reprogramming

Pneumonia is a worldwide threat, making discovery of novel means to combat lower respiratory tract infections an urgent need. We have previously shown that manipulating the lungs’ intrinsic host defenses by therapeutic delivery of a unique dyad of 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 (ODNs) with mitochondrial voltage-dependent anion channel 1 (VDAC1) without dependence on Toll-like receptor 9 (TLR9). The ODN-VDAC1 interaction alters cellular ATP/ADP/AMP localization, increases delivery of electrons to the electron transport chain (ETC), enhances mitochondrial membrane potential (ΔΨm), and differentially modulates ETC complex activities. These combined effects promote leak of electrons from ETC complex III, resulting in superoxide formation. The ODN-induced mitochondrial ROS yield protective antibacterial effects. Together, these studies identify a therapeutic metabolic manipulation strategy that has the potential to broadly protect patients against pneumonia during periods of peak vulnerability without reliance on currently available antibiotics.

electron transport chain (ETC), enhances mitochondrial membrane potential (ΔΨm), and 24 differentially modulates ETC complex activities. These combined effects promote leak of 25 electrons from ETC complex III, resulting in superoxide formation. The ODN-induced 26 mitochondrial ROS yield protective antibacterial effects. Together, these studies identify a 27 therapeutic metabolic manipulation strategy that has the potential to broadly protect patients 28 against pneumonia during periods of peak vulnerability without reliance on currently available 29 antibiotics. CpG ODN blocks mitochondrial nucleotide transition 121 As TLR9 is the established intracellular sensor for CpG ODNs (24), we examined whether TLR9 122 activation regulates ODN-induced mtROS generation. To our surprise, ODN treatment still fully 123 induced mtROS production in primary mouse lung epithelial cells isolated from Tlr9 knockout 124 mice or from mice lacking downstream TLR signaling molecules MyD88 or TRAF6 ( Figure S5), 125 indicating that ODN-induced mtROS generation does not require TLR9 signaling. 126 Seeking to identify a TLR9-independent mechanism by which ODN alters mitochondrial 127 energy metabolism, we investigated whether ODNs could directly stimulate mtROS production 128 in isolated mitochondria. Remarkably, we found that direct ODN treatment of mitochondria 129 isolated from HBEC3-KT cells recapitulates the inducible mtROS generation and increased 130 ΔΨm observed in whole cells ( Figure 3A-B). Alternately, to examine whether ODN interacts with 131 mitochondria in intact cells, whole cells were treated with fluorescently-labeled ODN, then the 132 mitochondria were isolated and assessed for fluorescence intensity. As in Figure 3C, 133 mitochondria from cells treated with labeled ODN displayed significant fluorescence, supporting 134 a hypothesis that ODN can directly interact with mitochondria to stimulate mtROS production. 135 To identify potential mitochondrial ODN binding partners, HBEC3-KT cells were treated with 136 biotin-labeled ODN, then the streptavidin precipitants from mitochondria lysates were resolved 137 on an SDS PAGE gel. The silver stain in Figure 3D demonstrates bands present only after ODN 138 treatment. The boxed area was excised and liquid chromatography-mass spectrometry 139 proteomic analysis generated a list of candidate targets.  are ETC complex II inhibitors while α-ketoglutarate inhibits glutaminolysis (41-43).

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In the ETC, CoQH2 is generated when CoQ accepts electrons from FADH2. The

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CoQH2:CoQ ratio has been described as an indicator of ETC efficiency, with an increased ratio 198 associated with increased mtROS production (44). As in Figure 4O, ODN treatment caused an 199 increase in CoQH2:CoQ ratio. Although augmented β-oxidation is required for maximal  induced mtROS production, inducible mtROS production can also be partially attenuated by 201 inhibiting glycolysis and/or glutaminolysis ( Figure S12). Future work will explore the 202 contributions of these pathways.

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Together, these results indicated that ODN-induced and AMPK-regulated metabolic 204 reprogramming enhances electron delivery to ETC, increases complex II activity and eventually 205 drives mtROS induction. These findings were schematically summarized in Figure 4P.  208 It is intriguing that ODN simultaneously increases complex II activity and decreases complex III 209 activity. We hypothesized that these changes in ETC complex activity might provide insights into 210 the site(s) of mtROS generation. A series of ETC complex inhibitors were used to determine the 211 roles of ETC complexes in ODN-induced mtROS generation.  Figure S13). Similarly, while TCA cycle input can support 217 reverse election transport from complex II to complex I in the setting of high ΔΨm (48), we found 218 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made

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These findings, along with the modest ODN impact on complex I activity (Figure 2A, Figure S2), 220 indicated that complex I is not a major site of ODN-induced mtROS production. In contrast, 221 while inhibitors of complex II activity reduced ODN-induced mtROS, inhibitors of complex III 222 enhanced ODN-induced mtROS dramatically ( Figure S13). We thus concluded that complex III 223 is the main ODN-induced mtROS generating site following forward electron transfer.

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Additionally, while complex IV inhibition had no impact on mtROS induction, the complex V 225 inhibitor oligomycin decreased ODN-induced mtROS production. As oligomycin treatment 226 collapses mitochondrial membrane potential ΔΨm, this result suggested that ODN-induced ΔΨm 227 increases may be required for mtROS formation. 228 We compared the impacts of ODN and complex III inhibitor antimycin on complex III 229 activity and mtROS induction. Although treatment with antimycin or ODN resulted in similar 230 inhibition of complex III enzymatic activity and initial mtROS production ( Figure 5A CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 21, 2023. ; https://doi.org/10.1101/2023.01.19.524841 doi: bioRxiv preprint oxidation of cytochrome bH is ΔΨm-dependent, whereas FCCP did not alter the oxidation of 244 cytochrome bH in antimycin-treated mitochondria ( Figure 5L). Congruently, in isolated 245 mitochondria, FCCP reversed ODN-impaired complex III electron transfer activity but had no 246 such effect on antimycin treated mitochondria ( Figure 5M). The generation of ODN-induced 247 mtROS at complex III is graphically displayed in Figure 5N. Under homeostatic conditions, 248 complex III quickly transfers CoQH2-carried electrons to cytochrome c1, which, in turn, transfers  Thus, while dissecting the process of ODN-induced antimicrobial mtROS formation, we 257 identified that mtROS induction requires both AMPK-directed metabolic reprograming to 258 augment electron delivery to ETC complex II ( Figure 4P) and increased ΔΨm to retard electron 259 transfer at complex III ( Figure 5N).  (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 21, 2023. ; https://doi.org/10.1101/2023.01.19.524841 doi: bioRxiv preprint reduced:oxidized ratio of ODN-treated cells ( Figure 6A-B). As shown in Figure 6C and Figure   269 S15, the bacterial killing induced by Pam2 and ODN was obviated in HBEC3-KT cells when the 270 cells were pretreated with TTFA-FCCP. Congruently, wild type mice with impaired lung epithelial 271 mtROS generation due to aerosolized TTFA-FCCP pretreatment prior to treatment with Pam2 272 and ODN were less protected against P. aeruginosa pneumonia than were mice who received 273 sham aerosol pretreatment prior to receiving Pam2-ODN ( Figure 6D). Notably, both the Pam2-  Figure 6F). Strikingly, when delivered by aerosol with Pam2, erastin yielded a similar 281 survival advantage to ODN following P. aeruginosa pneumonia challenge in mice ( Figure 6G).

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Even when delivered without Pam2, both ODN and erastin induced significant reductions in the 283 lung bacterial burden ( Figure 6H).

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In summary, we found that antimicrobial mtROS are generated from lung epithelial cells 285 following ODN treatment. ODN interacts with VDAC1 to alter mitochondrial nucleotide transport, 286 driving AMPK-ACC-CPT1-mediated electron delivery to ETC complex II and increasing ΔΨm to 287 promote superoxide production at complex III.  (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made Here, we report previously unknown, TLR9-independent induction of immunometabolic 294 reprogramming by ODN that results in generation of pneumonia-protective mtROS.

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ODN-induced mtROS formation is fundamentally a manifestation of altered energy 296 metabolism. ODN interacts with VDAC1 and ANT1 localized in the mitochondrial membranes.

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The VDAC1-ANT1-mCK complex regulates the exchange of metabolites between the 298 mitochondria and cytosol (25, 26). We show that VDAC1 binding ODN perturbs cellular 299 nucleotide distribution, activating the AMPK-ACC pathway, and promoting fatty acid β-oxidation.   (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 21, 2023. ; https://doi.org/10.1101/2023.01.19.524841 doi: bioRxiv preprint Although ROS production is often regarded as an untoward cellular event that 318 contributes to degenerative diseases (64, 65), there is robust evidence that controlled mtROS 319 generation contributes to critical signaling events in a wide range of physiologic processes that  Here, we demonstrate that complex III-dependent mtROS induction is required for maximally  Wild type C57BL/6J mice were purchased from The Jackson Laboratory (Bar Harbor, ME).

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Prkaa1 fl and Prkaa2 fl mice were purchased from Jackson. TLR9 -/mice were provided by Dr.

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Shizuo Akira (77). CMV mt-roGFP mice were generated by Dr. D James Surmeier and kindly 364 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 21, 2023.    BioTek Synergy2 plate reader for 3 h immediately after ODN or PBS addition.  To scavenge mtROS, HBEC3-KT cells were exposed to 100 nM MitoTEMPO or 10 µM MitoQ 412 for 1 h prior to fluorescent mtROS detector incubation and ODN or PBS treatment.

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. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made To disrupt mtROS production, HBEC3-KT cells were exposed to compounds that inhibit  To inhibit mtROS generation in vivo, mice were exposed to TTFA (200 mM) and FCCP (800  (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 21, 2023. ; https://doi.org/10.1101/2023.01.19.524841 doi: bioRxiv preprint permeabilized with 0.1% Triton X-100, and blocked with 2% goat serum in 1× PBS. Cells or lung

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Oxygen consumption rate (OCR) was measured using a Seahorse XFe96 extracellular flux 464 analyzer (Agilent, Santa Clara, CA). HBEC3-KT cells (1.5×10 4 per well) were seeded into a 465 XFe96 microplate and grew overnight in complete media. 1 h before the assay, the media was 466 changed to Seahorse XF assay media (Agilent) and incubated in a non-CO2 incubator at 37 °C.

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The microplate was loaded onto the analyzer and basal respiration in these cells were recorded     The electron transport chain enzymes were assayed at 30° C using a temperature-controlled (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 21, 2023. ; https://doi.org/10.1101/2023.01.19.524841 doi: bioRxiv preprint Complex III activity assay kit (BioVision). Complex V activity was measured using a 533 Quantichrom ATPase assay kit (Bioassay Systems).    The method of cytochrome bH measurement was adapted from that described by Quinlan et al.

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Absorbance change in Cytochrome bH was measured after ODN or antimycin A addition using a 572 NanoDrop One UV-Vis spectrophotometer (ThermoFisher Scientific). The Cytochrome bH signal 573 was recorded by spectrum scanning at the wavelength pair 566-575 nm at 37 °C with stirring 574 and normalized based on the assumption that reductions of bH are 0% with no added substrate 575 and 100% with saturating substrates plus 2 µM DTT. In parallel with cytochrome bH 576 measurement, mtROS generation was detected in isolated mitochondria (0.5 mg/ml) treated by 577 ODN or antimycin A using mitoSOX red as above described. To determine the effect of the 578 mitochondria membrane potential on Cytochrome bH redox state and mtROS generation in 579 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 21, 2023. ; https://doi.org/10.1101/2023.01.19.524841 doi: bioRxiv preprint isolated mitochondria, bH %red and mtROS were analyzed after addition of 500 nM FCCP in the 580 assay buffer along with ODN or antimycin A treatment.  (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made

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. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made    (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made   (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made