Delmotte, Philippe

Airway inflammation is a key aspect of asthma and is associated with airway smooth muscle (ASM) hyperreactivity as well as airway remodeling. Proinflammatory cytokines, such as TNFα mediate this airway inflammatory response. Previously, we found that TNFα induces an increase airway smooth muscle (ASM) force and ATP hydrolysis. In response, TNFα may also induce an increase in ATP production in ASM. This could be achieved by either an increase in mitochondrial function and/or mitochondrial biogenesis. We previously found that TNFα decreases mitochondrial Ca 2+ influx. Since dehydrogenase enzymes in the TCA cycle are Ca 2+ dependent, this would tend to reduce mitochondrial function in response to agonist stimulation. We also found that TNFα increases mitochondrial fragmentation, an initial step in mitochondrial biogenesis. We hypothesized that TNFα increases mitochondrial biogenesis and decreases mitochondrial function in human ASM. Human ASM cells were isolated from lung specimens incidental to patient surgery. The patients were normal as evaluated by a clinical pathologist (no history of asthma or COPD). hASM cells were exposed to TNFα (20 ng/ml) for 24 h. To examine mitochondrial biogenesis, protein expression of porin (an indicator of mitochondrial density) and PGC1α (a regulator of mitochondria biogenesis) were determined by Western blot analysis. In addition, the relative number of copies of human mitochondrial DNA (mtDNA) was determined by PCR and normalized to nuclear DNA. Mitochondrial function was assessed by measuring O 2 Consumption Rate (OCR – using a Seahorse Bioscience XF24 Analyzer), succinate dehydrogenase (SDH) activity (using a quantitative histochemical technique) and mitochondrial membrane potential – (DY m – using TMRM and confocal microscopy). OCR and SDH activity measurements were normalized to mitochondrial volume density. Results were analyzed by one‐way ANOVA with post hoc analysis when justified. We found that TNFα increases mitochondrial biogenesis as indicated by an increase in porin and PGC1α protein expression in hASM and an increase in mtDNA copy numbers. We also found that TNFα decreases mitochondrial membrane potential in hASM cells, and also decreases OCR and SDH activity when normalized for the increase in mitochondrial volume density induced by TNFα. In a normal homeostatic response, an increase in hASM force and ATP hydrolysis (energy demand) would be met by increased mitochondrial ATP production. Our results indicate that TNFα disrupts this normal homeostatic response by reducing mitochondrial Ca 2+ influx and thereby reducing mitochondrial TCA activity. However, TNFα increase the number of mitochondria to compensate the decrease in mitochondrial TCA activity. Support or Funding Information Supported by NIH grant HL126451 This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Airway inflammation is a key trigger for asthma, and pro‐inflammatory cytokines such as TNFα mediate the airway hyperreactivity and remodeling characteristic of asthma. In various diseases, inflammation leads to an increase in mitochondrial fission, associated with decreased expression of mitochondrial fusion proteins, such as Mfn2. Mitochondrial fission is generally coupled with mitochondrial biogenesis and cell proliferation. Some studies in other cell types suggest that mitochondrial fission may be a result of the accumulation of unfolded or misfolded proteins i.e., endoplasmic reticulum (ER) stress. ER stress activates three intracellular pathways to induce a homeostatic response to restore normal ER function. The IRE1α pathway is activated by autophosphorylation and splices XBP1 mRNA. Previous studies have shown that the XBP1 has downstream effects on mitochondrial proteins. In this study we hypothesized that TNFα induces activation of the IRE1α pathway which leads to a decrease in Mfn2 expression. hASM cells were isolated from lung specimen incidental to patient surgery. The patients were normal as evaluated by the clinical pathologist (no history of asthma or COPD). hASM cells were stimulated with TNFα (20 ng/ml) for 1, 3, 6, 12 or 24 h. Expression of total and phosphorylated IRE1α, Mfn2 and cleaved and uncleaved XBP1 were evaluated by Western blot. RNAscope® was used to measure Mfn2 mRNA expression in hASM cells transfected with non‐phosphorylatable IREα ( Delta P‐IRE1α; GFP tagged) compared to non‐transfected cells. Results were analyzed by 2‐way ANOVA with Bonferroni analysis when justified. We found that exposure of hASM cells to TNFα activated the IRE1α/XBP1 pathway and decreased Mfn2 expression in a time dependent fashion. In addition we found that TNFα exposure in cells expressing the inactive Delta P‐IRE1α did not lead to a significant decrease in Mfn2 expression. Our results suggest that the IRE1α/XBP1 pathway may be involved in the regulation of mitochondrial fission, mitochondrial biogenesis and cell proliferation. Support or Funding Information Supported by NIH grant HL126451 (GCS) This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Airway inflammation is a key feature of asthma, with inflammatory cytokines (e.g., TNFα and IL‐13) triggering hyperreactivity (enhance contractile state) and proliferation and remodeling (enhanced synthetic state) in airway smooth muscle (ASM). We hypothesize that TNFα and IL‐13 induce sarcoplasmic reticulum/endoplasmic reticulum (SR/ER) stress, thereby altering expression of mitochondrial fission/fusion proteins (dynamin‐related protein ‐ Drp1 and mitofusin ‐ Mfn2) with downstream impact on ASM Ca 2+ regulation and contractility as well as proliferation. Human ASM cells were isolated from lung specimens obtained incidental to patient surgery. We found that after 24 h exposing ASM cells to TNFα and IL‐13: 1) expression of SR/ER stress protein markers (PERK, calnexin and XBP1) increased; 2) Drp1 expression increased Mfn2 expression decreased; 3) mitochondrial fragmentation increased; 4) [Ca 2+ ] cyt and [Ca 2+ ] mito responses to ACh stimulation were uncoupled; 5) net [Ca 2+ ] cyt increased; 6) contractility increased; and 7) cell proliferation increased. Together these results suggest that inflammation induces SR/ER stress that leads to changes in Mfn2 and Drp1 expression, which alter mitochondrial dynamics and Ca 2+ regulation resulting in ASM hyperreactivity and remodeling. Grant Funding Source : Supported by NIH grant HL74309 (GCS)

During agonist stimulation of airway smooth muscle (ASM), agonists such as ACh induce a transient increase in cytosolic Ca2+ concentration ([Ca2+]cyt), which leads to a contractile response [excitation-contraction (E-C) coupling]. Previously, the sensitivity of the contractile response of ASM to elevated [Ca2+]cyt (Ca2+ sensitivity) was assessed as the ratio of maximum force to maximum [Ca2+]cyt. However, this static assessment of Ca2+ sensitivity overlooks the dynamic nature of E-C coupling in ASM. In this study, we simultaneously measured [Ca2+]cyt and isometric force responses to three concentrations of ACh (1, 2.6, and 10 μM). Both maximum [Ca2+]cyt and maximum force responses were ACh concentration dependent, but force increased disproportionately, thereby increasing static Ca2+ sensitivity. The dynamic properties of E-C coupling were assessed in several ways. The temporal delay between the onset of ACh-induced [Ca2+]cyt and onset force responses was not affected by ACh concentration. The rates of rise of the ACh-induced [Ca2+]cyt and force responses increased with increasing ACh concentration. The integral of the phase-loop plot of [Ca2+]cyt and force from onset to steady state also increased with increasing ACh concentration, whereas the rate of relaxation remained unchanged. Although these results suggest an ACh concentration-dependent increase in the rate of cross-bridge recruitment and in the rate of rise of [Ca2+]cyt, the extent of regulatory myosin light-chain (rMLC20) phosphorylation was not dependent on ACh concentration. We conclude that the dynamic properties of [Ca2+]cyt and force responses in ASM are dependent on ACh concentration but reflect more than changes in the extent of rMLC20 phosphorylation.

The effects of airway inflammation on airway smooth muscle (ASM) are mediated by pro-inflammatory cytokines such as tumor necrosis factor alpha (TNFα). In this review article, we will provide a unifying hypothesis for a homeostatic response to airway inflammation that mitigates oxidative stress and thereby provides resilience to ASM. Previous studies have shown that acute exposure to TNFα increases ASM force generation in response to muscarinic stimulation (hyper-reactivity) resulting in increased ATP consumption and increased tension cost. To meet this increased energetic demand, mitochondrial O2 consumption and oxidative phosphorylation increases but at the cost of increased reactive oxygen species (ROS) production (oxidative stress). TNFα-induced oxidative stress results in the accumulation of unfolded proteins in the endoplasmic reticulum (ER) and mitochondria of ASM. In the ER, TNFα selectively phosphorylates inositol-requiring enzyme 1 alpha (pIRE1α) triggering downstream splicing of the transcription factor X-box binding protein 1 (XBP1s); thus, activating the pIRE1α/XBP1s ER stress pathway. Protein unfolding in mitochondria also triggers an unfolded protein response (mtUPR). In our conceptual framework, we hypothesize that activation of these pathways is homeostatically directed towards mitochondrial remodeling via an increase in peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC1α) expression, which in turn triggers: (1) mitochondrial fragmentation (increased dynamin-related protein-1 (Drp1) and reduced mitofusin-2 (Mfn2) expression) and mitophagy (activation of the Phosphatase and tensin homolog (PTEN)-induced putative kinase 1 (PINK1)/Parkin mitophagy pathway) to improve mitochondrial quality; (2) reduced Mfn2 also results in a disruption of mitochondrial tethering to the ER and reduced mitochondrial Ca2+ influx; and (3) mitochondrial biogenesis and increased mitochondrial volume density. The homeostatic remodeling of mitochondria results in more efficient O2 consumption and oxidative phosphorylation and reduced ROS formation by individual mitochondrion, while still meeting the increased ATP demand. Thus, the energetic load of hyper-reactivity is shared across the mitochondrial pool within ASM cells.

Airway inflammation plays a key role in asthma. In various diseases, inflammation leads to an increase in reactive oxygen species (ROS) generation and is associated with accumulation of unfolded proteins leading to endoplasmic reticulum (ER) stress. In response to ER stress, a homeostatic signaling cascade is triggered to restore normal function by halting protein translation and activating protein degradation and expression of molecular chaperones involved in protein folding. Three ER stress protein markers located at the ER membrane are involved in this signaling cascade: protein kinase RNA‐like endoplasmic reticulum kinase (PERK), activating transcription factor 6 (ATF6) and inositol‐requiring enzyme 1 (IRE1α). In particular, phosphorylation of IRE1α catalyzes the alternative splicing of XBP1 mRNA leading to expression of the active XBP1 transcription factor. In this study, we hypothesize that pro‐inflammatory cytokine TNFα induces ROS production triggering ER stress in human airway smooth muscle (hASM). hASM cells were isolated from lung specimen incidental to patient surgery and exposed to TNFα (20 ng/ml) for 1, 3, 6, 12 or 24 h. To measure ROS production, hASM cells were loaded with the general oxidative stress indicator CM‐H2DCFDA or with the mitochondrial superoxide indicator MitoSOX™ Red. The CM‐H2DCFDA and MitoSOX™ Red signals were visualized using a Nikon A1R confocal imaging system. Images were analyzed using ImageJ. Exposure of hASM to tunicamycin (2 μg/ml) was used as a positive control for ER stress. Expression of IRE1α and PERK were determined by Western blot. Phosphorylation of IRE1α was also evaluated by immunoprecipitation and Western blot (Wes Simple Western™ system). The expression of the spliced isoform of the transcription factor XBP1 was examined by quantitative real‐time PCR (RT‐PCR). We found that ROS production increased in hASM within 1 h of TNFα exposure, followed by an increase in IRE1α phosphorylation within 1–3 h. Finally after 3–6 h, there was an increase in alternative splicing of XBP1 mRNA. Together these results show that the pro‐inflammatory cytokine TNFα increases ROS generation in hASM cells and induces an ER stress response that includes an increase in IRE1α phosphorylation and splicing of XBP1. This ER stress response may affect many downstream targets, among which we believe Mfn2 is a major target which affects mitochondrial Ca 2+ buffering, with specific effects on hASM force and/or increased cell proliferation associated with asthma. Support or Funding Information NIH grant HL126451 This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

In human airway smooth muscle (hASM), mitochondrial volume density is greater in asthmatic patients compared with normal controls. There is also an increase in mitochondrial fragmentation in hASM of moderate asthmatics associated with an increase in dynamin-related protein 1 (Drp1) and a decrease in mitofusin 2 (Mfn2) expression, mitochondrial fission, and fusion proteins, respectively. Proinflammatory cytokines such TNFα contribute to hASM hyperreactivity and cell proliferation associated with asthma. However, the involvement of proinflammatory cytokines in mitochondrial remodeling is not clearly established. In nonasthmatic hASM cells, mitochondria were labeled using MitoTracker Red and imaged in three dimensions using a confocal microscope. After 24-h TNFα exposure, mitochondria in hASM cells were more fragmented, evidenced by decreased form factor and aspect ratio and increased sphericity. Associated with increased mitochondrial fragmentation, Drp1 expression increased while Mfn2 expression was reduced. TNFα also increased mitochondrial biogenesis in hASM cells reflected by increased peroxisome proliferator-activated receptor-γ coactivator 1α expression and increased mitochondrial DNA copy number. Associated with mitochondrial biogenesis, TNFα exposure also increased mitochondrial volume density and porin expression, resulting in an increase in maximum O 2 consumption rate. However, when normalized for mitochondrial volume density, O 2 consumption rate per mitochondrion was reduced by TNFα exposure. Associated with mitochondrial fragmentation and biogenesis, TNFα also increased hASM cell proliferation, an effect mimicked by siRNA knockdown of Mfn2 expression and mitigated by Mfn2 overexpression. The results of this study support our hypothesis that in hASM cells exposed to TNFα mitochondria are more fragmented, with an increase in mitochondrial biogenesis and mitochondrial volume density resulting in reduced O 2 consumption rate per mitochondrion.

Abstract Interleukin‐6 (IL‐6) is a pleiotropic cytokine that has been shown to be produced acutely by skeletal muscle in response to exercise, yet chronically elevated with obesity and aging. The mechanisms by which IL‐6 influences skeletal muscle mitochondria acutely and chronically are unclear. To better understand the influence of extramyocellular IL‐6 on skeletal muscle mitochondrial physiology, we treated differentiated myotubes with exogenous IL‐6 to evaluate the dose‐ and duration‐dependent effects of IL‐6 on salient aspects of mitochondrial biology and the role of canonical IL‐6 signaling in muscle cells. Acute exposure of myotubes to IL‐6 increased the mitochondrial reactive oxygen species (mtROS) production and oxygen consumption rates ( J O 2 ) in a manner that was dependent on activation of the JAK/STAT pathway. Furthermore, STAT3 activation by IL‐6 was partly attenuated by MitoQ, a mitochondrial‐targeted antioxidant, suggesting that mtROS potentiates STAT3 signaling in skeletal muscle in response to IL‐6 exposure. In concert with effects on mitochondrial physiology, acute IL‐6 exposure induced several mitochondrial adaptations, consistent with the stress‐induced mitochondrial hyperfusion. Exposure of myotubes to chronically elevated IL‐6 further increased mtROS with eventual loss of respiratory capacity. These data provide new evidence supporting the interplay between cytokine signaling and mitochondrial physiology in skeletal muscle.

Airway inflammation is a key aspect of diseases such as asthma. Proinflammatory cytokines such as TNFα mediate the inflammatory response. In various diseases, inflammation leads to endoplasmic reticulum (ER) stress, the accumulation of unfolded proteins, which triggers homeostatic responses to restore normal cellular function. We hypothesized that TNFα triggers ER stress through an increase in reactive oxygen species generation in human airway smooth muscle (hASM) with a downstream effect on mitofusin 2 (Mfn2). In hASM cells isolated from lung specimens incidental to patient surgery, dose- and time-dependent effects of TNFα exposure were assessed. Exposure of hASM to tunicamycin was used as a positive control. Tempol (500 μM) was used as superoxide scavenger. Activation of three ER stress pathways were evaluated by Western blotting: 1) autophosphorylation of inositol-requiring enzyme1 (IRE1α) leading to splicing of X-box binding protein 1 (XBP1); 2) autophosphorylation of protein kinase RNA-like endoplasmic reticulum kinase (PERK) leading to phosphorylation of eukaryotic initiation factor 2α; and 3) translocation and cleavage of activating transcription factor 6 (ATF6). We found that exposure of hASM cells to tunicamycin activated all three ER stress pathways. In contrast, TNFα selectively activated the IRE1α/XBP1 pathway in a dose- and time-dependent fashion. Our results indicate that TNFα does not activate the PERK and ATF6 pathways. Exposure of hASM cells to TNFα also decreased Mfn2 protein expression. Concurrent exposure to TNFα and tempol reversed the effect of TNFα on IRE1α phosphorylation and Mfn2 protein expression. Selective activation of the IRE1α/XBP1 pathway in hASM cells after exposure to TNFα may reflect a unique homeostatic role of this pathway in the inflammatory response of hASM cells.