Delmotte, Philippe

Airway inflammation occurs as a defense mechanism in response to pathogens, allergens, toxins, or pollutants. The physiological responses of airway inflammation are mediated by proinflammatory cytokines such as tumor necrosis factor alpha (TNFα), that causes airway remodeling, characterized by airway smooth muscle (ASM) hyperreactivity and proliferation. The molecular mechanism underlying TNFα-induced ASM cell proliferation is not fully understood. Previous studies from our lab demonstrated that TNFα exposure induces accumulation of unfolded proteins in the endoplasmic reticulum (ER) triggering an ER stress response that involves autophosphorylation of inositol requiring enzyme 1α at Serine 724 (pIRE1α S724 ). pIRE1α S724 promotes alternative splicing of X-box binding protein 1 (XBP1s) mRNA. XBP1s acts as a ubiquitous transcription factor in ASM cells, targets a number of genes associated with different cellular responses. In the present study, we hypothesized that TNFα promotes XBP1s mediated transcriptional activation of the Cyclin B1, a protein essential for the control of the cell cycle, thus promoting ASM cell proliferation. Human ASM (hASM) cells were dissociated from bronchiolar tissue samples collected during lung surgery from female and male patients (49 to 61 years of age) with no history of chronic respiratory diseases or current smoking. Isolated hASM cells from the same patient were serum-deprived for 48 h and separated into two groups: 1) untreated controls, and 2) treated with TNFα (20 ng/ ml) for 6 h. To confirm the effect of TNFα on hASM cell proliferation, loss of function experiments were employed by: 1) transfecting hASM cells with a non-spliceable XBP1 mutant, and 2) treating the cells with 4μ8C, a pharmacological inhibitor for IRE1α mediated XBP1 splicing. hASM cell proliferation was measured using CyQuant cell proliferation assay. The binding site sequence of XBP1s to the Cyclin B1 promoter was identified by bioinformatic analysis and confirmed by chromatin immunoprecipitation (ChIP) assay. mRNA and protein expressions of Cyclin B1 were determined by qRT-PCR and Western blot respectively. Nuclear localization of Cyclin B1 was measured by nuclear fractionation, followed by western blot of nuclear and cytosolic fraction. Results were analyzed by paired t-tests. TNFα treatment for 6 h induced pIRE1α S724 phosphorylation and XBP1s splicing. Bioinformatic analysis revealed the presence of an XBP1s binding site on the Cyclin B1 promoter. The ChIP assay confirmed that XBP1s binds to the promoter region of the Cyclin B1 genes, and the binding affinity was increased in TNFα-treated hASM cells. Consistent with these results, we found that TNFα treatment increased both mRNA and protein expressions of Cyclin B1 in hASM cells. Nuclear localization of Cyclin B1 increased significantly in TNFα treated cells consistent with an increase in hASM cell proliferation. Inhibition of XBP1 splicing reduced the expression of Cyclin B1 in hASM cells and hASM cell proliferation. In summary, TNFα induces an ER stress response involving pIRE1α S724 phosphorylation and XBP1s splicing. In turn, XBP1s transcriptionally targets expression of Cyclin B1 that mediates hASM cell proliferation. Supported by NIH grant HL157984 This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Airway inflammation is increasing worldwide. Proinflammatory cytokines mediate an adaptive mechanism to overcome inflammation-induced cellular stress. Previously, we reported that TNFα mediates hASM cellular responses, leading to increased force and ATP consumption associated with increased O2 consumption, and oxidative stress. This study indicates that TNFα induces ER stress, which induces mitochondrial fragmentation via pIRE1αS724/XBP1s mediated CDK1/5 upregulation and pDRP1S616 phosphorylation. Mitochondrial fragmentation may promote hASM mitochondrial biogenesis to maintain healthy mitochondrial pool.

Airway inflammation is a key aspect of diseases such as asthma. Several inflammatory cytokines (e.g., TNFα and IL‐13) enhance cytosolic Ca 2+ concentration ([Ca 2+ ] cyto ) triggering airway smooth muscle (ASM) hyperreactivity (enhanced contractile state). Airway inflammation also induces ASM proliferation and remodeling (enhanced ASM synthetic state). In various diseases inflammation induces sarcoplasmic/endoplasmic reticulum (SR/ER) stress, which decreases SR/ER‐mitochondria interactions, with downstream effects on Ca 2+ regulation. In vascular smooth muscle cells, SR/ER stress promotes smooth muscle contraction and proliferation by disrupting SR/ER‐mitochondrial coupling. We hypothesized that inflammation induces SR/ER stress in human ASM, 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 specimen incidental to patient surgery and exposed to TNFα or IL‐13 for 24h. Expression of SR/ER stress markers including PERK (protein kinase‐like endoplasmic reticulum kinase), Bip (Binding immunoglobulin protein also known as 78 kDa glucose‐regulated protein), PDI (protein disulfide isomerase), and mitochondrial fission/fusion proteins Drp1 and Mfn2 were evaluated by Western blot. The expression of the spliced isoform of the transcription factor XBP1 was investigated by quantitative real‐time PCR (RT‐PCR). For mitochondrial morphology (fission/fusion), ASM cells were loaded with Mitotracker Green and images analyzed using ImageJ and Matlab to quantify mitochondrial Form Factor and Aspect Ratio. The effect of inflammation induced SR/ER stress on human ASM proliferation was evaluated using CyQUANT assay. We found that after 24 h exposure to TNFα and IL‐13: 1) expression of SR/ER stress protein markers (PERK, BiP and XBP1) increased; 2) Drp1 expression increased and 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) ASM 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. Support or Funding Information NIH grant HL126451 (GCS).

Airway inflammation is a key aspect of asthma and is associated with airway smooth muscle (ASM) hyperreactivity (airway narrowing due to increased Ca 2+ and force responses to agonist stimulation) as well as airway remodeling (ASM cell proliferation and altering extracellular matrix composition). Proinflammatory cytokines, such as TNF‐α mediate this airway inflammatory response. Previously, we found that the TNF‐α induces an increase ASM force, which is associated with an increase in ATP hydrolysis, imposing a metabolic stress on ASM. Mitochondria play an important role in providing ATP to support contraction in ASM. Mitochondria are dynamically remodelling within ASM cells displaying changing filamentous or fragmented tubular networks. Changing expression of Mitofusin 2 (Mfn2), which is associated with mitochondria fusion, or Dynamin Related Protein 1 (Drp1), which is associated with fission mediate this mitochondrial remodelling. The stressors that induce mitochondrial remodelling in ASM are unknown, but mitochondrial fragmentation may be an initial step in mitochondrial biogenesis to meet increasing energy demand. We hypothesized that human ASM (hASM) cells exposed to TNF‐α will display reduced Mfn2 and increased Drp1 expression resulting in mitochondrial fragmentation and mitochondrial biogenesis. To test our hypothesis, serum‐deprived primary hASM cells were exposed to 20 ng/ml TNF‐α for 24 h after which they were loaded with 500 μM MitoTracker Green to visualize mitochondria and imaged using confocal microscopy. Images were analyzed using MATLAB Image J software to quantify mitochondrial morphometry: form factor and aspect ratio. An increase in form factor and aspect ratio indicates greater mitochondrial filamentous networks. Expression of Mfn2 and Drp1 were determined by Western blot analysis. In hASM exposed to TNF‐α, Mfn2 expression decreased by ~40%, while Drp1 expression increased by ~75% compared to controls. These changes in fusion/fission proteins were associated with mitochondrial fragmentation (reduced form factor and aspect ratio) and an increase in mitochondria number. Our results indicate that hASM cells exposed to TNF‐α display mitochondrial fragmentation due to changes in Mfn2 and Drp1 expression. Mitochondrial fragmentation may be involved in mitochondrial biogenesis in response to increased metabolic demands imposed by airway hyperreactivity. Support or Funding Information APS fellowship (EK) and NIH grant HL126451 (GCS)

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 activates the inositol-requiring enzyme 1α (IRE1α)/X-box binding protein 1 spliced (XBP1s) endoplasmic reticulum (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-phenylbutyric acid (4-PBA) may reduce ER stress activation. In the present study, we hypothesized that chemical chaperone 4-PBA mitigates TNFα-induced ER stress in hASM cells. hASM cells were isolated from bronchiolar tissue obtained from five patients with no history of smoking or respiratory diseases. The hASM cells’ phenotype was confirmed via the expression of alpha-smooth muscle actin and elongated morphology. hASM cells from the same patient sample were then separated into three 12 h treatment groups: (1) TNFα (20 ng/mL), (2) TNFα + 4-PBA (1 μM, 30 min pretreatment), and (3) untreated control. The expressions of total IRE1α and phosphorylated IRE1α (pIRE1αS724) were determined through Western blotting. The splicing of XBP1 mRNA was analyzed using RT-PCR. We found that TNFα induced an increase in pIRE1αS724 phosphorylation, which was mitigated by treatment with chemical chaperone 4-PBA. We also found that TNFα induced an increase in XBP1s mRNA, which was also mitigated by treatment with chemical chaperone 4-PBA. These results support our hypothesis and indicate that chemical chaperone 4-PBA treatment mitigates TNFα-induced ER stress in hASM cells.

Cellular mitochondrial function can be assessed using high-resolution respirometry that measures the O2 consumption rate (OCR) across a number of cells. However, a direct measurement of cellular mitochondrial function provides valuable 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, which participates in both the tricarboxylic acid (TCA) cycle and electron transport chain (ETC) as Complex II. In this study, we determine the maximum velocity of the SDH reaction (SDHmax) in individual human airway smooth muscle (hASM) cells. To measure SDHmax, hASM cells were exposed to a solution containing 80 mM succinate and 1.5 mM nitroblue tetrazolium (NBT, reaction indicator). As the reaction proceeded, the change in optical density (OD) due to the reduction of NBT to its diformazan (peak absorbance wavelength of 570 nm) was measured using a confocal microscope with the pathlength for light absorbance tightly controlled. SDHmax was determined during the linear period of the SDH reaction and expressed as mmol fumarate/liter of cell/min. We determine that this technique is rigorous and reproducible, and reliable for the measurement of mitochondrial function in individual cells.

Airway inflammation is the central driver behind the pathogenesis of respiratory diseases such as asthma, chronic obstructive pulmonary disease (COPD), chronic bronchitis, and the very recent pandemic outbreak of COVID-19. Previous studies reported that proinflammatory cytokine TNFa plays a significant role in airway inflammation by inducing hyper-reactivity of airway smooth muscle (ASM) and airway remodeling. TNFa also increases ASM force generation and ATP hydrolysis that requires energy. The excess energy demand is met by an increase in the level of PGC1a protein, mitochondrial biogenesis and subsequently increased mitochondrial volume density. Based on these previous findings, we hypothesized that TNFa promotes CREB phosphorylation at a specific serine 133 residue (pCREBS133) thereby transcriptionally activating PGC1a expression leading to mitochondrial biogenesis and increased mitochondrial volume density. In the study, human ASM cells were dissociated from bronchiolar tissue samples collected during bronchiolar surgery from female and male donors (34 to 71 years of age) with no history of respiratory diseases or current smoking. The dissociated ASM cells were serum-deprived for 48 h before experimentation. The ASM cells from each donor were separated into two groups: 1) time-matched untreated controls, and 2) TNFa treated (20 ng/ml for 6 h). Expression of CREB, pCREBS133, PCG1a, VDAC (an indicator of mitochondrial volume), and downstream signaling molecules (NRFs, TFAM), which regulate transcription and replication of the mitochondrial genome, were determined by quantitative real-time PCR (qPCR) and/or Western blot analysis. The binding of pCREBS133 to the PGC1a promoter was confirmed by bioinformatics analysis and chromatin immunoprecipitation (ChIP) assay. Mitochondria within ASM cells were labeled using MitoTracker green and imaged by 3D confocal microscopy for the assessment of mitochondrial volume density. Mitochondrial biogenesis was quantified by mitochondrial DNA (mtDNA) copy number relative to nuclear DNA using qPCR. Results were analyzed by one-way analysis of variance (ANOVA). Human ASM cells treated with TNFa showed increased pCREBS133 phosphorylation without any significant change in total CREB. The ChIP assay using a CREB-specific antibody revealed that pCREBS133 binds to the promoter of PGC1a and TNFa treatment further increased the binding affinity. Consistent with these findings, TNFa treatment increased PCG1a expression in ASM cells, and subsequently activated its downstream signaling pathway revealed by increased expression of its target genes, NRF1, NRF2 and TFAM. TNFa-induced activation of the PCG1a signaling pathway in ASM cells resulted in increased mitochondrial biogenesis as indicated by increased mitochondrial DNA copy number and increased mitochondrial volume density. Supported by NIH grant HL157984 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.

Mitophagy is a regulated multistep process of selective removal of dysfunctional/damaged mitochondria by autophagy and is critical for the regulation of mitochondrial quality control in many physiological processes. The best characterized pathway of mitophagy involves PTEN-induced kinase 1 (PINK1) and E3-ubiquitin ligase Parkin. Upon mitochondrial depolarization, PINK1 stabilizes on the outer mitochondrial membrane (OMM) and recruits Parkin. After recruitment, PINK1 phosphorylates Parkin at Ser65, thus activating Parkin which promotes the ubiquitination of OMM proteins resulting in the subsequent lysosomal degradation of the mitochondria. Previously, we reported that tumor necrosis factor α (TNFα), a proinflammatory cytokine produced during inflammatory airway diseases, increases force generation and ATP hydrolysis, and results in increased mitochondrial fragmentation in human airway smooth muscle (hASM) cells. We also showed that TNFα induces PGC1α signaling, thus promoting mitochondrial biogenesis and increased mitochondrial volume density. In the present study, we hypothesize that TNFα induces mitophagy to promote selective clearance of dysfunctional mitochondria, thus maintaining a healthy mitochondrial pool to meet the increased energy demand in hASM cells. Experiments were performed in hASM cells dissociated from bronchiolar tissue samples collected during bronchiolar surgery from female and male donors with no current history of smoking or respiratory diseases. The hASM cells were serum deprived for 48 h and the phenotype was confirmed prior to experimentation. hASM cells were separated into three 6-h treatment groups: 1) TNFα (20 ng/mL), 2) FCCP (5 μM; positive control), and 3) untreated controls. The mitochondria and lysosomes in hASM cells were labelled with MitoTracker green (200 nM) and LysoTracker red (100 nM), respectively and 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 3D images were used to determine the coefficient for colocalization of mitochondria with lysosomes, lysosomal volume density, mitochondrial volume density, and mitochondrial complexity index (MCI). Protein expression and phosphorylation status of PINK1, Parkin, phospho-Parkin, VDAC (voltage-dependent anion channel), and LAMP1 (Lysosomal-associated membrane protein 1) were quantified by western blot. We observed that TNFα and FCCP treatment increased lysosomal volume density and mitochondrial colocalization with lysosomes in hASM cells, indicating the formation of mitophagosomes, characteristic of mitophagy. Consistent with previous findings, TNFα treatment increased mitochondrial volume density and decreased MCI, indicative of increased mitochondrial fragmentation. In addition, TNFα treatment increased the expression and mitochondrial translocation of PINK1 and Parkin. The results suggest that TNFα activates PINK1/Parkin-mediated mitophagy in hASM cells. Supported by NIH grants HL157984 (GCS) and HL105355 (OARR) 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.

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.