Mitochondrial DNA damage and subsequent activation of Z-DNA binding protein 1 links oxidative stress to inflammation in epithelial cells

This report identifies mitochondrial DNA (mtDNA) as a target and active mediator that links low-level oxidative stress to inflammatory response in pulmonary epithelial cells. Extrusion of mtDNA into the bronchoalveolar lavage fluid occurs as an early event in mice subjected to cigarette smoke injury, concomitantly with the depletion of mtDNA in the lung tissue. In cultured lung epithelial cells, prolonged, low-level oxidative stress damages the mtDNA, without any detectable damage to the nuclear DNA. In turn, cellular depletion of the mtDNA occurs, together with a transient remodeling of cellular bioenergetics and morphology - all without any detectable impairment in overall cell viability. Damaged mtDNA first enters the cytoplasm, where it binds to Z-DNA binding protein 1 (ZBP1) and triggers inflammation via the TANK-binding kinase 1 /interferon regulatory factor 3 signaling pathway. Fragments of the mtDNA are subsequently released into the extracellular space via exosomes. MtDNA-containing exosomes are capable of inducing an inflammatory response in naïve (non-oxidatively stressed) epithelial cells. In vivo, administration of isolated mtDNA into the in lungs of naïve mice induces the production of pro-inflammatory mediators, without histopathologic evidence of tissue injury. We propose that mtDNA-specific damage, and subsequent activation of the ZBP1 pathway, is a mechanism that links prolonged, low-level oxidative stress to autocrine and paracrine inflammation during the early stages of inflammatory lung disease.

Lactate dehydrogenase release was used as a cytotoxicity assay to measure permeability of the plasma membrane. Briefly, 30 µl of supernatant was saved from BEAS 2B cells treated with GOx and mixed with 100 µl of freshly prepared LDH assay reagent to reach final concentrations of 85 mM lactic acid, 1040 mM nicotinamide adenine dinucleotide (NAD + ), 224 mM Nmethylphenazonium methyl sulfate (PMS), 528 mM 2-(4-Iodophenyl)-3-(4-nitrophenyl)-5phenyl-2H-tetrazolium chloride (INT) and 200 mM Tris (pH 8.2). The changes in absorbance were read kinetically at 492 nm for 15 min (kinetic LDH assay) on a monochromator-based reader (Powerwave HT, Biotek) at 37 ºC. LDH activity values are shown as V max for the kinetic assays in mOD/min. Citrate synthase activity, an indicator of the mitochondrial volume, was measured with a Citrate Synthase Assay Kit (Sigma-Aldrich, CS0720). Briefly, total cell extract was prepared using NP-40 lysis buffer (100 mM NaCl, 50 mM Tris-Cl pH 8.0, 1 % NP-40). 10 µg of cell extract was used for determination of citrate synthase specific activity by measuring amount of 5-thio-2-nitrobenoic acid (TNB, yellow product) from reaction of DTNB with thiol-containing acetyl coenzyme A produced from acetyl coenzyme A and oxaloacetate by citrate synthase.
Mitochondrial membrane potential was measured using the TMRE-Mitochondrial Membrane Potential Assay kit (Abcam, ab113852).
MitoSOX Red staining, a mitochondrial superoxide indicator (Invitrogen M36008) was used to detect mitochondrial ROS production. Briefly, 40,000/well of BEAS 2B cells were seeded in Lab-Tek II chamber slides (Nalgen Nunc International) and incubated at 37 o C, 5% CO 2 3 humidified incubator overnight. The cells were loaded with 5 µM of MitoSOX Red dye (Invitrogen M36008) for 10 min. For analysis, cells were washed three times with PBS and dye's specific fluorescence was visualized using Nikon eclipse 80i inverted microscope with Photometric CoolSNAP HQ2 camera and NIS-Elements BR 3.10 software.
Nanoparticle tracking analysis (NTA). NTA measurements were performed for size and concentration of nanoparticles using a NanoSight NS300 instrument (NanoSight, Malvern Instruments, USA), following the manufacturer's instructions. Medium of BEAS 2B cells were diluted to reach a particle concentration suitable for analysis with NTA (10 6 to 10 9 particles/ml).
Two different dilutions for each sample were prepared and analyzed each one three times. The samples were injected into the NS300 unit (approximately 300 µl) with a 1 ml sterile syringe.
The capturing settings (shutter and gain) and analyzing settings were manually set according to the protocol suggested in the technical note and then optimized for specific nanoparticles. The NanoSight NS300 recorded 60 second sample videos which were than analyzed with the Nanoparticle Tracking Analysis (NTA) 2.0 Analytical software. Size/concentration PDF reports and videos were obtained for each set of data (5 fields recorded for each sample). The average of all 6 reads per sample are shown.
Mitochondrial DNA size analysis in exosomal fraction was conducted with the 2100 expert Bioanalyzer (Agilent) using the Agilent 1200 kit. Briefly, 10 6 cells/well were seeded in 6 well plates. Next day cells were treated with GOx and at 24 h post-treatment total DNA from the exosomal fraction of the supernatant (1 ml) was extracted using DNasey Blood and Tissue Kit