DNA of neutrophil extracellular traps promote NF-κB-dependent autoimmunity via cGAS/TLR9 in chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) is characterised by persistent airway inflammation even after cigarette smoking cessation. Neutrophil extracellular traps (NETs) have been implicated in COPD severity and acute airway inflammation induced by short-term cigarette smoke (CS). However, whether and how NETs contribute to sustained airway inflammation in COPD remain unclear. This study aimed to elucidate the immunoregulatory mechanism of NETs in COPD, employing human neutrophils, airway epithelial cells (AECs), dendritic cells (DCs), and a long-term CS-induced COPD mouse model, alongside cyclic guanosine monophosphate-adenosine monophosphate synthase and toll-like receptor 9 knockout mice (cGAS--/-, TLR9-/-); Additionally, bronchoalveolar lavage fluid (BALF) of COPD patients was examined. Neutrophils from COPD patients released greater cigarette smoke extract (CSE)-induced NETs (CSE-NETs) due to mitochondrial respiratory chain dysfunction. These CSE-NETs, containing oxidatively-damaged DNA (NETs-DNA), promoted AECs proliferation, nuclear factor kappa B (NF-κB) activation, NF-κB-dependent cytokines and type-I interferons production, and DC maturation, which were ameliorated/reversed by silencing/inhibition of cGAS/TLR9. In the COPD mouse model, blocking NETs-DNA-sensing via cGAS-/- and TLR9-/- mice, inhibiting NETosis using mitoTEMPO, and degrading NETs-DNA with DNase-I, respectively, reduced NETs infiltrations, airway inflammation, NF-κB activation and NF-κB-dependent cytokines, but not type-I interferons due to IFN-α/ß receptor degradation. Elevated NETs components (myeloperoxidase and neutrophil elastase activity) in BALF of COPD smokers correlated with disease severity and NF-κB-dependent cytokine levels, but not type-I interferon levels. In conclusion, NETs-DNA promotes NF-κB-dependent autoimmunity via cGAS/TLR9 in long-term CS exposure-induced COPD. Therefore, targeting NETs-DNA and cGAS/TLR9 emerges as a potential strategy to alleviate persistent airway inflammation in COPD.

Inherent differences of small airway contraction and Ca2+ oscillations in airway smooth muscle cells between BALB/c and C57BL/6 mouse strains.

BALB/c and C57BL/6 mouse strains are widely used as animal model in studies of respiratory diseases, such as asthma. Asthma is characterized by airway hyperresponsiveness, which is eventually resulted from the excessive airway smooth muscle (ASM) contraction mediated by Ca2+ oscillations in ASM cells. It is reported that BALB/c mice have inherently higher airway responsiveness, but show no different contractive response of tracheal ring as compared to C57BL/6 mice. However, whether the different airway responsiveness is due to the different extents of small airway contraction, and what's underlying mechanism remains unknown. Here, we assess agonist-induced small airway contraction and Ca2+ oscillations in ASM cells between BALB/c and C57BL/6 mice by using precision-cut lung slices (PCLS). We found that BALB/c mice showed an intrinsically stronger extent of small airway narrowing and faster Ca2+ oscillations in ASM cells in response to agonists. These differences were associated with a higher magnitude of Ca2+ influx via store-operated Ca2+ entry (SOCE), as a result of increased expression of SOCE components (STIM1, Orai1) in the ASM cells of small airway of BALB/c mice. An established mathematical model and experimental results suggested that the increased SOC current could result in increased agonist-induced Ca2+ oscillations. Therefore, the inherently higher SOC underlies the increased Ca2+ oscillation frequency in ASM cells and stronger small airway contraction in BALB/c mice, thus higher airway responsiveness in BALB/c than C57BL/6 mouse strain.

EIF5A2 specifically regulates the transcription of aging-related genes in human neuroblastoma cells.

BACKGROUND: Post-transcriptional regulation plays a critical role in controlling biological processes such as aging. Previous studies have shown that eukaryotic initiation factor 5A (EIF5A) might play a crucial role in aging. It is unknown whether EIF5A2, a second isoform of EIF5A, could impact aging through post-transcriptional regulation. METHODS: In the present study, EIF5A2 overexpression (EIF5A2-OE) was induced in SH-SY5Y cells. RNA-seq, bioinformatics analysis and RT-qPCR validation experiments were then performed to explore the molecular mechanism of EIF5A2-mediated transcriptional regulation. Cell viability, proportion of senescent cells and the cell cycle were respectively determined by Cell Counting Kit-8, SA-ß­galactosidase and flow cytometry to evaluate the cell senescence. RESULTS: A total of 190 downregulated and 126 upregulated genes related to EIF5A2-OE were identified. Genes closely related to cellular aging processes such as unfolded protein response (UPR), cell adhesion and calcium signaling pathway were under global transcriptional regulation. Moreover, EIF5A2-OE promoted the viability of SH-SY5Y cells and reduced cell senescence in vitro. Among 30 genes with the most significant expression differences in EIF5A2-OE cells, we identified eight genes, including ASNS, ATF3, ATF4, CEBPB, DDIT3, HERPUD1, HSPA5 and XBP1, enriched in the UPR. Through EIF5A2-tanscription factors (TFs)-targets regulation network in EIF5A2-OE cells, we found three TFs, BHLHE40, RHOXF1 and TBX20, that targeted at these eight UPR-related genes. Verification test via the published database of human glial cell tissue showed only BHLHE40 and RHOXF1 were significantly associated with EIF5A2. CONCLUSIONS: Our findings suggest that EIF5A2 may alleviate cell senescence in vitro and mediate UPR-related genes via specific TFs. Thus, EIF5A2 could function as a regulator of aging via the regulation of transcription, which greatly expands the current understanding of the mechanisms of EIF5A2-mediated gene regulation.

Inhibition of neutrophil elastase prevents cigarette smoke exposure-induced formation of neutrophil extracellular traps and improves lung function in a mouse model of chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) is an important public health challenge worldwide, and is usually caused by significant exposure to noxious agents, particularly cigarette smoke. Recent studies have revealed that excessive production of neutrophil extracellular traps (NETs) in the airways is associated with disease severity in COPD patients. NETs are extracellular neutrophil-derived structures composed of chromatin fibers decorated with histones and granule proteases including neutrophil elastase (NE). However, the effective prevention of NET formation in COPD remains elusive. Here, we demonstrated that treatment with GW311616A, a potent and selective inhibitor of NE, prevented cigarette smoke extract (CSE)-induced NET formation in human neutrophils by blocking NE nuclear translocation and subsequent chromatin decondensation. Inhibition of NE also abrogated CSE-induced ROS production and migration impairment of neutrophils. Administration of GW311616A in vivo substantially reduced pulmonary generation of NETs while attenuating the key pathological changes in COPD, including airway leukocyte infiltration, mucus-secreting goblet cell hyperplasia, and emphysema-like alveolar destruction in a mouse model of COPD induced by chronic cigarette smoke exposure. Mice treated with GW311616A also showed significant attenuation of neutrophil numbers and percentages and the levels of neutrophil chemotactic factors (LTB4, KC, and CXCL5) and proinflammatory cytokines (IL-1ß, and TNF-α) in bronchoalveolar lavage fluid compared to mice treated with cigarette smoke exposure only. Furthermore, GW311616A treatment considerably improved lung function in the COPD mouse model, including preventing the decline of FEV100/FVC and delta PEF as well as inhibiting the increase in FRC, TLC, and FRC/TLC. Overall, our study suggests that NE plays a critical role in cigarette smoke-induced NET formation by neutrophils and that inhibition of NE is a promising strategy to suppress NET-mediated pathophysiological changes in COPD.

Lumican is elevated in the lung in human and experimental acute respiratory distress syndrome and promotes early fibrotic responses to lung injury.

BACKGROUND: Fibroproliferative repair starts early in the inflammatory phase of acute respiratory distress syndrome (ARDS) and indicates a poor prognosis. Lumican, a small leucine-rich proteoglycan, is implicated in homeostasis and fibrogenesis, but its role in ARDS is unclear. METHODS: Bronchoalveolar lavage fluid (BALF) samples were obtained from ARDS patients (n = 55) enrolled within 24 h of diagnosis and mechanically ventilated (n = 20) and spontaneously breathing (n = 29) control subjects. Lipopolysaccharide (LPS)-induced acute lung injury (ALI) mouse models were intratracheally administered an adeno-associated virus (AAV) vector expressing lumican shRNA. Primary human lung fibroblasts (HLF) and small airway epithelial cells (SAECs) were cultured with tumour necrosis factor (TNF)-α or lumican. Luminex/ELISA, histochemistry/immunohistochemistry, immunofluorescence microscopy, quantitative real-time PCR, and western blotting were performed. RESULTS: Lumican levels were significantly higher in the BALF of ARDS patients than in that of ventilated or spontaneously breathing controls (both p < 0.0001); they were correlated with the PaO2/FiO2 ratio and levels of proinflammatory cytokines (interleukin-6, interleukin-8, and TNF-α) and profibrotic factors (fibronectin, alpha-1 type I collagen [COL1A1], and alpha-1 type III collagen [COL3A1]). Lumican expression was enhanced in the alveolar walls and airway epithelium in the ALI mouse model. Murine lumican levels were also linked to proinflammatory and profibrotic cytokine levels in the BALF. In vitro, TNF-α induced the synthesis and secretion of lumican in HLF. In turn, lumican increased the expression of alpha-smooth muscle actin (α-SMA), COL1A1, and COL3A1 in HLF, upregulated α-SMA and COL3A1, downregulated E-cadherin, and caused spindle-shaped morphological changes in SAECs. Moreover, increased ERK phosphorylation and Slug were noted in both HLF and SAECs treated with lumican. In vivo, AAV-mediated knockdown of lumican inhibited the pulmonary production of fibronectin and COL3A1 and alleviated lung fibrotic lesions in LPS-challenged mice. CONCLUSIONS: Pulmonary lumican levels were increased early in human and experimental ARDS and linked to disease severity and inflammatory fibrotic processes. Lumican triggers the transdifferentiation of lung fibroblasts into myofibroblasts and epithelial-mesenchymal transition in SAECs, possibly via the ERK/Slug pathway. Knockdown of pulmonary lumican attenuated extracellular matrix deposition in ALI mice. Overall, lumican promotes fibrotic responses in the early phase of ARDS, suggesting its potential as a therapeutic target.

PKM2 regulates cigarette smoke-induced airway inflammation and epithelial-to-mesenchymal transition via modulating PINK1/Parkin-mediated mitophagy.

Cigarette smoke (CS) mediates inflammation and epithelial-mesenchymal transition (EMT) in bronchial epithelial cells, contributing to airway remodeling in chronic obstructive pulmonary disease (COPD). Cross-talk between metabolic pathways and cell signaling has emerged as an important focus of research in the field of inflammation. Here, we established in vitro and in vivo models of CS-induced COPD to elucidate the role of pyruvate kinase M2 (PKM2), a glycolytic enzyme, in CS-induced airway remodeling. Exposure to CS significantly increased PKM2 expression in lung tissues of C57BL/6 mice and BEAS-2B cells, which positively related to the levels of airway inflammation and EMT. Administering PKM2 inhibitor shikonin attenuated CS-induced airway inflammation and EMT process. Moreover, knockdown of PKM2 by small-interfering RNA (siRNA) decreased the release of TNF-α and IL-8, ROS and reversed the CS extract (CSE)-induced changes of N-cadherin and E-cadherin in BEAS-2B cells. In CSE-treated cells, we also observed enhancement of PINK1/Parkin-mediated mitophagy, which were decreased by PKM2 siRNA. Furthermore, pretreatment with mitophagy inducer CCCP before CSE stimulation led to increased expressions of both nuclear and cytosolic PKM2, accompanied by reduction of TGF-ß-induced factor homeobox 2 (TGIF2), a repressor of TGF-ß1/smad pathway and EMT, while PKM2 knockdown restored the expression of TGIF2. Our results imply that CS induces PKM2 upregulation in airway epithelial cells, acting in synergism with PINK/Parkin-mediated mitophagy, which may initiate and exaggerate airway inflammation and EMT process. Further studies will be required to elucidate more molecular details and other pathways by which PKM2-mitophagy signaling regulates the effector function of airway epithelial.

APEX1 regulates alternative splicing of key tumorigenesis genes in non-small-cell lung cancer.

BACKGROUND: Aberrant alternative splicing (AS) contributes to tumor progression. Previous studies have shown that apurinic-apyrimidinic endonuclease-1 (APEX1) is involved in tumor progression. It is unknown whether APEX1 functions in tumor progression by regulation of AS. It is also unknown whether APEX1 can regulate non-small-cell lung cancer (NSCLC) proliferation and apoptosis. We analyzed APEX1 expression levels in 517 lung NSCLC samples from the TCGA (Cancer Genome Atlas) database. The impact of APEX1 over expression on A549 cell proliferation and apoptosis was detected by the methyl thiazolyl tetrazolium assay and by flow cytometry. The transcriptome of A549 cells with and without APEX1 over expression was determined by Illumina sequencing, followed by analysis of AS. RT-qPCR validated expression of APEX1-related genes in A549 cells. We have successfully applied RNA-seq technology to demonstrate APEX1 regulation of AS. RESULTS: APEX1 expression was shown to be upregulated in NSCLC samples and to reduce cell proliferation and induce apoptosis of A549 cells. In addition, APEX1 regulated AS of key tumorigenesis genes involved in cancer proliferation and apoptosis within MAPK and Wnt signaling pathways. Each of these pathways are involved in lung cancer progression. Furthermore, validated AS events regulated by APEX1 were in key tumorigenesis genes; AXIN1 (axis inhibition protein 1), GCNT2 (N-acetyl glucosaminyl transferase 2), and SMAD3 (SMAD Family Member 3). These genes encode signaling pathway transcription regulatory factors. CONCLUSIONS: We found that increased expression of APEX1 was an independent prognostic factor related to NSCLC progression. Therefore, APEX1 regulation of AS may serve as a molecular marker or therapeutic target for NSCLC treatment.

Circadian clock dysfunction of epithelial cells in pulmonary diseases.

Highly-differentiated pulmonary epithelial cells are essential for maintaining lung homeostasis by exerting various physiological functions, which are regulated by circadian clock consisted of an autoregulatory feedback loop of clock genes, including Brain-Muscle Aryl-hydrocarbon Receptor Nuclear Translocator-Like 1 (BMAL1) and Nuclear Heme Receptor Reverse Erythroblastosis Virus α (REV-ERB-α). The circadian clock dysfunction of epithelial cells has been increasingly associated with the pulmonary diseases: BMAL1 and REV-ERB-α regulates inflammatory response of club cells induced by lipopolysaccharide and cigarette smoke (CS) respectively; the clock disfunction in alveolar epithelial type2 cells (AEC-II) has been implicated in CS-induced airway inflammation and early-life hyperoxia-related susceptibility to influenza infection; the ciliary beat frequency of ciliated cells also shows circadian rhythms. Here, we review the current knowledge on the circadian regulation of different epithelial-cell subtypes, attempting to provide insights into how clock dysfunction contributes to pulmonary diseases, and explore possible pharmacological therapies and future directions for fundamental studies.

Autotaxin levels in serum and bronchoalveolar lavage fluid are associated with inflammatory and fibrotic biomarkers and the clinical outcome in patients with acute respiratory distress syndrome.

BACKGROUND: Autotaxin (ATX) is a secreted glycoprotein that is widely present in extracellular biological fluids and has been implicated in many inflammatory and fibrotic diseases. However, the clinical impact of the release of ATX in patients with acute respiratory distress syndrome (ARDS) remains unclear. METHODS: Serum and bronchoalveolar lavage fluid (BALF) levels of ATX, interleukin (IL)-6, IL-8, tumor necrosis factor (TNF)-α, matrix metalloproteinase (MMP)-7, fibronectin, oncostatin M (OSM), and SPARC (secreted protein acidic and rich in cysteine) were collected from 52 patients with ARDS within 24 h of diagnosis. All cytokines were measured by Magnetic Luminex Assay. BALF albumin (BA) and serum albumin (SA) were measured by enzyme-linked immunosorbent assay. RESULTS: Serum ATX, MMP-7, and BALF IL-8 levels were significantly higher in patients who did not survive than in those who survived up to 28 days after diagnosis of ARDS (P < 0.05). BALF and serum ATX levels were correlated with IL-6, IL-8, and MMP-7 levels in BALF and serum, respectively. In addition, BALF ATX was positively correlated with BALF TNF-α, fibronectin, OSM, and SPARC as well as the BA/SA ratio, while serum ATX was correlated with severity of illness based on the SOFA score and PaO2/FIO2 ratio. Furthermore, serum ATX was better able to predict 28-day ARDS-related mortality (area under the curve 0.744, P < 0.01) than the SOFA score, APACHE II score, or PaO2/FIO2 ratio. Serum ATX independently predicted mortality in a univariate Cox regression model (P < 0.0001). CONCLUSION: The serum ATX level is a potential prognostic biomarker in patients with ARDS. BALF ATX is associated with pulmonary biomarkers of inflammation and fibrosis, suggesting a role of ATX in the pathogenesis of ARDS.

Prognostic Value of Angiopoietin-like 4 in Patients with Acute Respiratory Distress Syndrome.

BACKGROUND: Angiopoietin-like 4 (ANGPTL4) is a secreted glycoprotein that plays an important role in endothelial injury and the inflammatory response. Experimental models have implicated ANGPTL4 in acute respiratory distress syndrome (ARDS), but its impact on the progression of ARDS is unclear. METHODS: Paired bronchoalveolar lavage fluid (BALF) and serum samples were obtained from patients with ARDS (n = 56) within 24 h of diagnosis and from control subjects (n = 32). ANGPTL4, angiopoietin-2, interleukin (IL)-6, and TNF-α levels were measured by magnetic Luminex assay. BALF albumin (BA) and serum albumin (SA) were evaluated by enzyme-linked immunosorbent assay. RESULTS: BALF and serum ANGPTL4 concentrations were higher in patients with ARDS than in controls and were even higher in non-survivors than in survivors. The serum ANGPTL4 level was higher in indirect (extrapulmonary) ARDS than in direct (pulmonary) ARDS. Furthermore, BALF and serum ANGPTL4 levels correlated well with angiopoietin-2, IL-6, and TNF-α levels in BALF and serum. BALF ANGPTL4 was positively correlated with the BA/SA ratio (an indicator of pulmonary vascular permeability), and serum ANGPTL4 was associated with the severity of multiple organ dysfunction syndrome based on SOFA and APACHE II scores. Moreover, serum ANGPTL4 was better able to predict 28-day ARDS-related mortality (AUC 0.746, P < 0.01) than the APACHE II score or PaO2/FiO2 ratio. Serum ANGPTL4 was identified as an independent risk factor for mortality in a univariate Cox regression model (P < 0.001). CONCLUSION: ANGPTL4 levels were elevated in patients with ARDS and significantly correlated with disease severity and mortality. ANGPTL4 may be a novel prognostic biomarker in ARDS.

Specific killing of CCR9 high-expressing acute T lymphocytic leukemia cells by CCL25 fused with PE38 toxin.

We have previously demonstrated that CCR9 plays a pivotal role in drug resistance and invasion in human acute T-lymphocytic leukemia (T-ALL). In this study, we investigated whether the MOLT4 cells, which naturally express CCR9 at high levels, can be successfully killed by the specific ligand, CCL25 fused to Pseudomonas exotoxin 38 (PE38) toxin. Our results demonstrated that CCL25-PE38 was able to specifically kill MOLT4 cells via apoptosis induction, and suppress the growth of CCR9(+) tumors. This work shows that CCR9 high-expressing human T-ALL cells can be successfully killed by delivering PE38 toxin fused to the ligand CCL25.