Chen, Xujiao

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 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.

Clearance of protein-bound uremic toxins (PBUTs) by dialysis is a challenge in the treatment of uremic patients. Shen-Shuai-Ning (SSN), a traditional Chinese medicine formulation, has been used commonly in China to retard kidney disease progression and decrease uremic toxins in chronic kidney disease (CKD) patients, but the effects of SSN on serum PBUTs in dialysis patients were not investigated. We conducted a randomized controlled trial in patients on peritoneal dialysis (PD) at dialysis center of Changzheng Hospital to evaluate the effects of SSN on serum PBUTs. Participants with SSN intervention took 5 g SSN granule three times daily for 12 weeks, while the baseline medications and dialysis prescriptions remained during the study in all patients. The serum concentrations of indoxyl sulphate (IS) and p-cresol sulphate (PCS) were determined by HPLC/MS/MS and biochemical parameters were assessed during the study. Sixty PD patients were enrolled and randomly allocated into SSN group and control group. Total IS level was significantly lower in SSN group than in control group at week 4, 8, and 12 (27.28 ± 18.19, 29.73 ± 19.10, and 29.41 ± 17.61 mg/l compared with 39.25 ± 20.23, 44.86 ± 23.91, and 45.34 ± 33.52 mg/l, respectively). However, there were no statistical difference of total PCS, free forms of IS and PCS concentrations between SSN group and control group during 12 weeks follow-up. Administration of SSN granule orally decreased serum total IS level effectively in uremic patients on PD with good tolerance. Benefits of PD patients’ outcomes from IS reduction by SSN awaits further large size and long duration clinical trials to verify.