Citations

Airway inflammation and pro-inflammatory cytokines such as tumor necrosis factor alpha (TNFα) underlie the pathophysiology of respiratory diseases, including asthma. Previously, we showed that TNFα selectively activates the inositol-requiring enzyme 1α (IRE1α)/X-box binding protein 1 spliced (XBP1s) ER stress pathway in human airway smooth muscle (hASM) cells. The ER stress pathway is activated by the accumulation of unfolded proteins in the ER. Accordingly, chemical chaperones such as 4-phenylbutyrate (4-PBA) may ameliorate ER stress activation. In the present study, we hypothesize that 4-PBA mitigates TNFα induced ER stress in hASM cells. hASM cells were isolated from bronchiolar tissue obtained from 5 donors with no history of current smoking or respiratory diseases. hASM cells were serum-deprived for 48 h, and phenotype confirmed by expression of smooth muscle actin and fusiform morphology. hASM cells from the same donor were then separated into 4 12-h treatment groups: 1) TNFα (20 ng/mL), 2) TNFα + 4-PBA (30 min pretreatment), and 3) untreated control. Protein expression and phosphorylation of IRE1α and XBP1s were determined by Western blot. Gene expression of XBP1s was analyzed by PCR. We found that TNFα induced IRE1α phosphorylation and XBP1 splicing, which were significantly reduced by pretreatment with 4-PBA. These results support our hypothesis and suggest that, in the presence of chemical chaperone 4-PBA, TNFα induced ER stress is significantly decreased. 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.

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.

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.

Previously, we reported that airway inflammation mediated by pro‐inflammatory cytokines such as TNFα induces hypercontractility and increased ATP consumption in human airway smooth muscle (hASM). We also found that TNFα induces an increase in maximum O 2 consumption rate as well as mitochondrial biogenesis and an increase in mitochondrial volume density. However, when normalized to mitochondrial volume, maximum O 2 consumption rate per mitochondrion is reduced in hASM after TNFα exposure. Unfortunately, standard respirometry (e.g., Seahorse) measures only the averaged maximum O 2 consumption rate of a large number of cells. In the present study, we used a quantitative histochemical technique to determine the maximum velocity of the succinate dehydrogenase reaction (SDH max ) together with mitochondrial volume density in individual hASM cells. SDH is a key enzyme in the tricarboxylic acid (TCA) cycle as well as complex II in the electron transport chain (ETC), and SDH max reflects the maximum respiratory capacity of individual mitochondrion. We hypothesized that TNFα decreases SDH max per mitochondrion in hASM cells. hASM cells were dissociated from bronchial biopsies obtained during thoracic surgeries in patients with no history of asthma or chronic obstructive pulmonary disease. The hASM cells were treated with TNFα (20 ng/ml) or vehicle (DMSO) for 24 h, and SDH max was measured using a solution containing 80 mM succinate (maximum substrate concentration) and 1.5 mM nitro blue tetrazolium (NBT) as the reaction indicator. As the SDH reaction proceeded, the hASM cells were imaged in 3D (Z optical slice of 0.5 μm) using an oil‐immersion ×60/1.4 NA objective on a Nikon Eclipse A1 laser scanning confocal system. The change in optical density (OD), reflecting the accumulation of NBT diformazan reaction product in the hASM cell, was measured every 15 s over a 10 min period. Based on the Beer‐Lambert equation, the slope of the change in OD was used to determine SDH max expressed as mM fumarate/L tissue/min. To determine mitochondrial volume density, the same hASM cells were also loaded with MitoTracker Green (500 nM) and imaged in 3D. We found that mitochondria were more fragmented after TNFα exposure although mitochondrial volume density increased. When SDH max was normalized to mitochondrial volume, we found that SDH max per mitochondrion decreased in hASM cells, consistent with the reduction in maximum O 2 consumption per mitochondrion that we previously observed. Thus, the increased energetic demand induced by inflammation (TNFα) are met by mitochondrial fragmentation leading to biogenesis and an increase in mitochondrial volume density rather than an increase in mitochondrial respiratory capacity.

Inflammation triggers a homeostatic response that is mediated by pro‐inflammatory cytokines such as TNFα. We hypothesize that in a motor neuron‐like cell line, NSC‐34, TNFα decreases mitochondrial function, specifically succinate dehydrogenase (SDH) activity, which is a key enzyme in the tricarboxylic acid (TCA) cycle as well as complex II in the electron transport chain (ETC). Experiments were performed on cultured NSC‐34 cells that were differentiated by serum depletion until neurite structures (>50 μm) were observed. The differentiated cells were treated with TNFα (20 ng/ml) for 24 h. To measure the maximum velocity of the SDH reaction (SDH max ), cells were incubated in a solution containing 80 mM succinate and 1.5 mM nitro blue tetrazolium (NBT), the reaction indicator. As the SDH reaction proceeded, images were acquired every 15 s using a 40× objective on an Olympus IX71 inverted microscope. Linearity of the SDH reaction was confirmed across the 8‐min period, and SDH max was determined and expressed as mM fumarate/L tissue/min. To determine mitochondrial volume density, the cells were loaded with MitoTracker Green and imaged in 3D using a confocal microscope. The cell volume occupied by labeled mitochondria was determined from 3D reconstructions of 1 mm optical slices. In NSC‐34 cells exposed to TNFα, we found that mitochondrial volume density increased, and mitochondria were fragmented as compared to untreated controls. We also found that TNFα exposure decreased SDH max when normalized for mitochondrial volume density. In other cell types, we found that TNFα‐induces an increase in PGC1a and Drp1 expression and a decrease in Mfn2 expression, all of which affect mitochondrial structure. In related studies, we also found that TNFα exposure decreases O 2 consumption per mitochondrion in NSC‐34 cells. We conclude that by reducing the redox potential (SDH max ) of mitochondria, O 2 consumption per mitochondrion is reduced, thereby damping ROS formation. The impact of this homeostatic response is offset by mitochondrial biogenesis and an increase in mitochondrial volume density, thereby maintaining overall cellular O 2 consumption and ATP production but at reduced cost of ROS formation. Support or Funding Information Supported by NIH grants AG44615 and HL105355.

Neuroinflammation triggers a homeostatic response that is mediated by pro‐inflammatory cytokines such as TNFα. We hypothesize that in a motor neuron‐like cell line, NSC‐34, TNFα decreases mitochondrial O 2 consumption. NSC‐34 cells were differentiated by serum depletion (~24 h) until neurite structures (>50 μm) were observed. The cells were then exposed to TNFα (20 ng/ml) for 24 h. To determine mitochondrial volume density, the cells were loaded with MitoTracker Green and imaged in 3D using a Nikon A1R confocal microscope (60x oil immersion 1.4 NA). After treatment with TNFα, mitochondria appeared fragmented but mitochondrial volume density increased by ~50%, as compared to control untreated cells. A Agilent Seahorse XF Analyzer was used to determine the O 2 consumption of treated and untreated cells during a stress test that determines basal respiration, ATP production and maximum respiration. O 2 consumption was normalized to mitochondrial volume density in both treated and untreated groups. After TNFα exposure, basal respiration, ATP production and maximum respiration decreased per mitochondrion. However, overall O 2 consumption of the cells was unchanged after TNFα exposure due to an offsetting increase in mitochondrial volume density. Using MitoSox, we also measured reactive oxygen species (ROS) formation in treated and untreated NSC‐34 cells. ROS is a byproduct of mitochondrial O 2 consumption, and can cause cellular damage. After TNFα exposure, ROS formation increased compared to untreated cells. We conclude that mitochondrial biogenesis and an increase in mitochondrial volume density represent a homeostatic mechanism to reduce O 2 consumption the per mitochondrion, thereby damping ROS formation while maintaining overall cellular O 2 consumption and ATP production. Support or Funding Information Supported by NIH grant AG44615 and HL105355.