Delmotte, Philippe

The impact of airway inflammation is mediated by pro-inflammatory cytokines such as tumor necrosis factor alpha (TNFα), which underlies several respiratory diseases, including COVID-19, asthma, chronic bronchitis, and chronic obstructive pulmonary disease (COPD). In human airway smooth muscle (hASM) cells, exposure to TNFα leads to an accumulation of unfolded proteins in the ER, which triggers a homeostatic ER stress response activating a signaling cascade to restore normal function. Previously, we showed that TNFα selectively activates the inositol-requiring enzyme 1α (IRE1α)/X-box binding protein 1 (XBP1) ER stress pathway in hASM cells. Most likely, the protein unfolding in hASM cells caused by TNFα results from an increase in reactive oxygen species (ROS) formation. We hypothesize that ER stress induced by TNFα is mitigated by the ROS scavenger TEMPOL. Primary hASM cells were isolated from bronchiolar tissue samples obtained from 5 patients (both sexes included) who had no history of respiratory diseases. hASM cells were serum deprived for 48 h before treatment with 1) TNFα (20 ng/ml) for 12 h; 2) TEMPOL (500 μM) for 30 min followed by TNFα (20 ng/ml) for 12 h; and 3) untreated control for 12 h. At the end of the treatment period, hASM cells were processed for determination of protein expression through Western blotting. Activation of the ER stress response was assessed by phosphorylation of IRE1α and splicing of XBP1. Confirming our previous results, we found that 12 h TNFα exposure induced an ER stress response in hASM cells, evidence by phosphorylation of IRE1α and XBP1 splicing. Treating hASM cells with TEMPOL before exposure to TNFα mitigated the ER stress response. These results support our hypothesis and indicate that ROS scavenging prevents TNFα induced protein unfolding and triggering of the IRE1α/XBP1 ER stress pathway. R01HL 157984 (GCS) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Proinflammatory cytokines such as TNFα mediate airway inflammation. Previously, we showed that TNFα increases mitochondrial biogenesis in human ASM (hASM) cells, which is associated with increased PGC1α expression. We hypothesized that TNFα induces CREB and ATF1 phosphorylation (pCREBS133 and pATF1S63), which transcriptionally co-activate PGC1α expression. Primary hASM cells were dissociated from bronchiolar tissue obtained from patients undergoing lung resection, cultured (one–three passages), and then differentiated by serum deprivation (48 h). hASM cells from the same patient were divided into two groups: TNFα (20 ng/mL) treated for 6 h and untreated controls. Mitochondria were labeled using MitoTracker green and imaged using 3D confocal microscopy to determine mitochondrial volume density. Mitochondrial biogenesis was assessed based on relative mitochondrial DNA (mtDNA) copy number determined by quantitative real-time PCR (qPCR). Gene and/or protein expression of pCREBS133, pATF1S63, PCG1α, and downstream signaling molecules (NRFs, TFAM) that regulate transcription and replication of the mitochondrial genome, were determined by qPCR and/or Western blot. TNFα increased mitochondrial volume density and mitochondrial biogenesis in hASM cells, which was associated with an increase in pCREBS133, pATF1S63 and PCG1α expression, with downstream transcriptional activation of NRF1, NRF2, and TFAM. We conclude that TNFα increases mitochondrial volume density in hASM cells via a pCREBS133/pATF1S63/PCG1α-mediated pathway.

Cellular mitochondrial function can be assessed using high resolution respirometry that measures O 2 consumption rate during various conditions that systematically alter the tricarboxylic acid (TCA) cycle or the electron transport chain (ETC). However, current high resolution respirometry does not measure O 2 consumption rate at the single cell level, but actually measures average mitochondrial function across a number of cells (either isolated or in tissues). Thus, respirometry assumes physiological homogeneity across cells. However, in many tissues, mitochondrial function varies across cells and this heterogeneity is physiologically important. Therefore, a direct measurement of cellular mitochondrial function will provide valuable novel information and physiological insight. In the present study, we used a quantitative histochemical technique to measure the activity of succinate dehydrogenase (SDH), a key enzyme located in the inner mitochondrial membrane, and the only enzyme to participate in both the TCA cycle and the ETC as Complex II. SDH mediates the oxidation of succinate to fumarate in the TCA cycle, which is coupled to the reduction of ubiquinone to ubiquinol in the ETC. In this study we determined the maximum velocity of the SDH reaction (SDH max ) in isolated human airway smooth muscle (hASM) cells using 1‐methoxyphenazine methosulphate (mPMS), as an exogenous electron carrier, and azide to inhibit cytochrome oxidase. To measure SDH max , the cells were exposed to a solution containing 80 mM succinate and 1.5 mM nitroblue tetrazolium (NBT) as the reaction indicator. hASM cells were imaged in 3D (Z optical slice of 0.5 μm) using a Nikon Eclipse A1 laser scanning confocal system with a ×60/1.4 NA oil‐immersion lens. In the quantitative histochemical procedure, changes in cell optical density (OD) due to the progressive reduction of NBT to its diformazan (peak absorbance wavelength of 570 nm) were measured every 15 s over a 10 min period. Linearity of the SDH reaction was confirmed across the 10 min period, and SDH max was expressed as mM fumarate/liter of tissue/min. Validation of this technique included specific ETC inhibitors including oligomycin (ATP synthase inhibitor), FCCP (proton ionophore), antimycin A (Complex III inhibitor) and rotenone (Complex I inhibitor), similar to those used in high resolution respirometry. We observed that FCCP‐mediated disruption of the mitochondrial proton gradient does not affect SDH max , while SDH max is decreased by rotenone and antimycin A. In addition, we used MitoTracker Green to label and image mitochondria in hASM cells and determined mitochondrial volume density. The SDH max was then normalized to mitochondrial content. Our results confirm that this quantitative technique is rigorous and reproducible, and that measurements of cellular SDH max can serve as a reliable surrogate for the measurement of maximum mitochondrial respiration in single cells.

Tumor necrosis factor α (TNFα) contributes to the pathophysiology of several inflammatory airway diseases such as asthma, chronic obstructive pulmonary disease (COPD), chronic bronchitis, and COVID‐19. Previously, we showed that acute (24‐h) exposure to TNFα promotes mitochondrial fragmentation in human airway smooth muscle (hASM) cells, which is associated with an increased level of cytosolic GTPase dynamin‐related protein 1 (DRP1), a major protein involved in mitochondrial fragmentation. Phosphorylation of DRP1 at Ser616 (S616) promotes its translocation and binding to the outer mitochondrial membrane and induces mitochondrial fragmentation; however, phosphorylation of DRP1 at the Ser637 (S637) residue reverses the total process and diminishes fragmentation. The balance between S616 and S637 phosphorylation of DRP1 regulates mitochondrial dynamics. In hASM cells, exposure to TNFα triggers protein unfolding and selectively activates the endoplasmic reticulum (ER) stress pathway involving the phosphorylation of inositol‐requiring enzyme 1α (pIRE1α) and subsequent splicing of X‐box binding protein 1 (XBP1s) that acts as a potent transcriptional activator for several downstream genes. We hypothesized that TNFα mediated activation of the pIRE1α/ XBP1 ER stress pathway transcriptionally activates genes that code for proteins involved in DRP1 phosphorylation at S616 leading to mitochondrial fragmentation. hASM cells were dissociated from bronchial biopsies from patients without a history of respiratory diseases and exposed to TNFα (20 ng/ ml for 6 h). Cells were also treated with Mdivi1 (50 μM for 6 h), a small‐molecule inhibitor of mitochondrial fragmentation that targets the GTPase activity of DRP1. Bioinformatic analysis revealed the presence of putative binding sites for XBP1 on the promoter region of genes that translate into kinases (cyclin dependent kinases CDK1 and CDK5, cyclin B1), which are reported to phosphorylate DRP1 at S616. Western blot and/or immunocytochemistry were employed to quantify the expression and phosphorylation status of IRE1α, DRP1, XBP1 splicing, CDKs and cyclin B1. Mitochondrial morphology was assessed by 3‐D confocal microscopy using MitoTracker green to label mitochondria. Chromatin immunoprecipitation (ChIP) and quantitative real time PCR was done to validate XBP1 target genes. Spliced XBP1 transcriptionally activates expression of CDK1, CDK5 and cyclin B1 and subsequent phosphorylation at S616 of DRP1 without significant alteration in S637 phosphorylation. As a result, TNFα induces an increase in S616/S637 phosphorylation ratio that promotes the translocation of DRP1 from cytosol to mitochondria and mediates mitochondrial fragmentation. Inhibition of DRP1 GTPase activity by Mdivi1 ameliorates DRP1 phosphorylation at S616 and significantly reduces mitochondrial fragmentation. These results reveal the mechanisms that underlie TNFα induced ER stress and downstream mitochondrial fragmentation.