15LO1 dictates glutathione redox changes in asthmatic airway epithelium to worsen type 2 inflammation.

Altered redox biology challenges all cells, with compensatory responses often determining a cell's fate. When 15 lipoxygenase 1 (15LO1), a lipid-peroxidizing enzyme abundant in asthmatic human airway epithelial cells (HAECs), binds phosphatidylethanolamine-binding protein 1 (PEBP1), hydroperoxy-phospholipids, which drive ferroptotic cell death, are generated. Peroxidases, including glutathione peroxidase 4 (GPX4), metabolize hydroperoxy-phospholipids to hydroxy derivatives to prevent ferroptotic death, but consume reduced glutathione (GSH). The cystine transporter SLC7A11 critically restores/maintains intracellular GSH. We hypothesized that high 15LO1, PEBP1, and GPX4 activity drives abnormal asthmatic redox biology, evidenced by lower bronchoalveolar lavage (BAL) fluid and intraepithelial cell GSH:oxidized GSH (GSSG) ratios, to enhance type 2 (T2) inflammatory responses. GSH, GSSG (enzymatic assays), 15LO1, GPX4, SLC7A11, and T2 biomarkers (Western blot and RNA-Seq) were measured in asthmatic and healthy control (HC) cells and fluids, with siRNA knockdown as appropriate. GSSG was higher and GSH:GSSG lower in asthmatic compared with HC BAL fluid, while intracellular GSH was lower in asthma. In vitro, a T2 cytokine (IL-13) induced 15LO1 generation of hydroperoxy-phospholipids, which lowered intracellular GSH and increased extracellular GSSG. Lowering GSH further by inhibiting SLC7A11 enhanced T2 inflammatory protein expression and ferroptosis. Ex vivo, redox imbalances corresponded to 15LO1 and SLC7A11 expression, T2 biomarkers, and worsened clinical outcomes. Thus, 15LO1 pathway-induced redox biology perturbations worsen T2 inflammation and asthma control, supporting 15LO1 as a therapeutic target.

Up-regulation of ACE2, the SARS-CoV-2 receptor, in asthmatics on maintenance inhaled corticosteroids.

BACKGROUND: The first step in SARS-CoV-2 infection is binding of the virus to angiotensin converting enzyme 2 (ACE2) on the airway epithelium. Asthma affects over 300 million people world-wide, many of whom may encounter SARS-CoV-2. Epidemiologic data suggests that asthmatics who get infected may be at increased risk of more severe disease. Our objective was to assess whether maintenance inhaled corticosteroids (ICS), a major treatment for asthma, is associated with airway ACE2 expression in asthmatics. METHODS: Large airway epithelium (LAE) of asthmatics treated with maintenance ICS (ICS+), asthmatics not treated with ICS (ICS-), and healthy controls (controls) was analyzed for expression of ACE2 and other coronavirus infection-related genes using microarrays. RESULTS: As a group, there was no difference in LAE ACE2 expression in all asthmatics vs controls. In contrast, subgroup analysis demonstrated that LAE ACE2 expression was higher in asthmatics ICS+ compared to ICS‾ and ACE2 expression was higher in male ICS+ compared to female ICS+ and ICS‾ of either sex. ACE2 expression did not correlate with serum IgE, absolute eosinophil level, or change in FEV1 in response to bronchodilators in either ICS- or ICS+. CONCLUSION: Airway ACE2 expression is increased in asthmatics on long-term treatment with ICS, an observation that should be taken into consideration when assessing the use of inhaled corticosteroids during the pandemic.

PBDEs affect inflammatory and oncosuppressive mechanisms via the EZH2 methyltransferase in airway epithelial cells.

AIMS: We aimed to investigate the effect of PBDEs (47, 99, 209) on cellular events involved in epigenetic modification, inflammation, and epithelial mesenchymal transition (EMT). MATERIALS AND METHODS: We studied: 1) ERK1/2 phosphorylation; 2) Enhancer of Zester Homolog 2 (EZH2); 3) Histone H3 tri-methylated in lysine 27 (H3K27me3); 4) K-RAS; 5) silencing disabled homolog 2-interacting protein gene (DAB2IP), 6) let-7a; 7) Muc5AC/Muc5B, and 8) IL-8 in a 3D in vitro model of epithelium obtained with primary Normal Human Bronchial Epithelial cells (pNHBEs) or A549 cell line, chronically exposed to PBDEs (47, 99, 209). KEY FINDINGS: PBDEs (10 nM, 100 nM and 1 µM) increased ERK1/2 phosphorylation, and EZH2, H3K27me3, and K-RAS protein expression, while decreased DAB2IP and Let-7a transcripts in pNHBEs ALI culture. Furthermore PBDEs (47, 99) (100 nM) increased Muc5AC and Muc5B mRNA, and PBDE 47 (100 nM) IL-8 mRNA via EZH2 in pNHBEs. Finally, PBDEs (100 nM) affected EZH2, H3K27me3, K-RAS protein expression, and DAB2IP, Let-7a transcripts and cell invasion in A549 cells. Gsk343 (methyltransferase EZH2 inhibitor) (1 mM) and U0126 (inhibitor of MEK1/2) (10 µM) were used to show the specific effect of PBDEs. SIGNIFICANCE: PBDE inhalation might promote inflammation/cancer via EZH2 methyltransferase activity and H3K27me3, k-RAS and ERk1/2 involvement, generating adverse health outcomes of the human lung.

Comparative analysis of ACE2 protein expression in rodent, non-human primate, and human respiratory tract at baseline and after injury: A conundrum for COVID-19 pathogenesis.

Angiotensin converting enzyme 2 (ACE2) is the putative functional receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Current literature on the abundance and distribution of ACE2 protein in the human respiratory tract is controversial. We examined the effect of age and lung injury on ACE2 protein expression in rodent and non-human primate (NHP) models. We also examined ACE2 expression in human tissues with and without coronavirus disease 19 (COVID-19). ACE2 expression was detected at very low levels in preterm, but was absent in full-term and adult NHP lung homogenates. This pattern of ACE2 expression contrasted with that of transmembrane protease serine type 2 (TMPRSS2), which was significantly increased in full-term newborn and adult NHP lungs compared to preterm NHP lungs. ACE2 expression was not detected in NHP lungs with cigarette smoke-induced airway disease or bronchopulmonary dysplasia. Murine lungs lacked basal ACE2 immunoreactivity, but responded to hyperoxia, bacterial infection, and allergen exposure with new ACE2 expression in bronchial epithelial cells. In human specimens, robust ACE2 immunoreactivity was detected in ciliated epithelial cells in paranasal sinus specimens, while ACE2 expression was detected only in rare type 2 alveolar epithelial cells in control lungs. In autopsy specimens from patients with COVID-19 pneumonia, ACE2 was detected in rare ciliated epithelial and endothelial cells in the trachea, but not in the lung. There was robust expression of ACE2 expression in F344/N rat nasal mucosa and lung specimens, which authentically recapitulated the ACE2 expression pattern in human paranasal sinus specimens. Thus, ACE2 protein expression demonstrates a significant gradient between upper and lower respiratory tract in humans and is scarce in the lung. This pattern of ACE2 expression supports the notion of sinonasal epithelium being the main entry site for SARS-CoV-2 but raises further questions on the pathogenesis and cellular targets of SARS-CoV-2 in COVID-19 pneumonia.

Kallikrein 13 serves as a priming protease during infection by the human coronavirus HKU1.

Human coronavirus HKU1 (HCoV-HKU1) is associated with respiratory disease and is prevalent worldwide, but an in vitro model for viral replication is lacking. An interaction between the coronaviral spike (S) protein and its receptor is the primary determinant of tissue and host specificity; however, viral entry is a complex process requiring the concerted action of multiple cellular elements. Here, we found that the protease kallikrein 13 (KLK13) was required for the infection of human respiratory epithelial cells and was sufficient to mediate the entry of HCoV-HKU1 into nonpermissive RD cells. We also demonstrated the cleavage of the HCoV-HKU1 S protein by KLK13 in the S1/S2 region, suggesting that KLK13 is the priming enzyme for this virus. Together, these data suggest that protease distribution and specificity determine the tissue and cell specificity of the virus and may also regulate interspecies transmission.

Is amiloride a promising cardiovascular medication to persist in the COVID-19 crisis?

In the ongoing coronavirus diseases-2019 (COVID-19) crisis that caused immense suffering and deaths, the choice of therapy for the prevention and life-saving conditions must be based on sound scientific evidence. Uncertainty and apprehension are exacerbated in people using angiotensin-converting enzyme (ACE) inhibitors to control their comorbidities such as hypertension and diabetes. These drugs are reported to result in unfavorable outcome as they tend to increase the levels of ACE2 which mediates the entry of SARS-CoV-2. Amiloride, a prototypic inhibitor of epithelial sodium channels (ENaC) can be an ideal candidate for COVID-19 patients, given its ACE reducing and cytosolic pH increasing effects. Moreover, its potassium-sparing and anti-epileptic activities make it a promising alternative or a combinatorial agent.

The Protective Effect of HBO1 on Cigarette Smoke Extract-Induced Apoptosis in Airway Epithelial Cells.

Purpose: Epigenetic modification is one of most important mechanisms underlying the pathogenesis of chronic obstructive pulmonary disease (COPD). The purpose of this study was to determine whether histone acetyltransferase binding to ORC1 (HBO1) can protect against cigarette smoke (CS)-induced cell apoptosis and sustain normal histone acetylation in COPD. Methods: Human lung tissue samples were obtained from patients who underwent lung resection. The emphysema mouse model and HBO1 overexpressing mice were each established by intraperitoneal injection with cigarette smoke extract (CSE) or intratracheal lentiviral vectors instillation. TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assays were used to assess apoptotic ratio in mice. The apoptosis of human bronchial epithelial cells (HBECs) was assayed by flow cytometry. HBO1, B-cell lymphoma-2 (BCL-2), and H3K14ac protein expression were detected by Western blotting. HBO1 mRNA expression was measured by quantitative real-time polymerase chain reaction. Results: Protein expression of HBO1 was decreased significantly in lung tissue from COPD patients and CSE-treated emphysema mouse models. Overexpression of HBO1 attenuated CSE-induced emphysematous changes, as well as apoptosis in the lungs of COPD mice. In vitro, the HBO1 protein degraded in a time- and dose-dependent course with CSE treatment. With flow cytometry, we proved that HBO1 could reverse the apoptosis of HBECs induced by CSE. Furthermore, HBO1 overexpression promoted the expression of anti-apoptotic BCL-2 protein and enhanced H3K14 acetylation in airway epithelial cells. Conclusion: These findings demonstrate that the key histone modulator HBO1 plays a protective role in COPD pathogenesis that may shed light on potential therapeutic targets to inhibit the progress of COPD.

Cigarette smoke induces overexpression of active human cathepsin S in lungs from current smokers with or without COPD.

Cigarette smoking has marked effects on lung tissue, including induction of oxidative stress, inflammatory cell recruitment, and a protease/antiprotease imbalance. These effects contribute to tissue remodeling and destruction resulting in loss of lung function in chronic obstructive pulmonary disease (COPD) patients. Cathepsin S (CatS) is a cysteine protease that is involved in the remodeling/degradation of connective tissue and basement membrane. Aberrant expression or activity of CatS has been implicated in a variety of diseases, including arthritis, cancer, cardiovascular, and lung diseases. However, little is known about the effect of cigarette smoking on both CatS expression and activity, as well as its role in smoking-related lung diseases. Here, we evaluated the expression and activity of human CatS in lung tissues from never-smokers and smokers with or without COPD. Despite the presence of an oxidizing environment, CatS expression and activity were significantly higher in current smokers (both non-COPD and COPD) compared with never-smokers, and correlated positively with smoking history. Moreover, we found that the exposure of primary human bronchial epithelial cells to cigarette smoke extract triggered the activation of P2X7 receptors, which in turns drives CatS upregulation. The present data suggest that excessive CatS expression and activity contribute, beside other proteases, to the deleterious effects of cigarette smoke on pulmonary homeostasis.

Mesenchymal proteases and tissue fluidity remodel the extracellular matrix during airway epithelial branching in the embryonic avian lung.

Reciprocal epithelial-mesenchymal signaling is essential for morphogenesis, including branching of the lung. In the mouse, mesenchymal cells differentiate into airway smooth muscle that wraps around epithelial branches, but this contractile tissue is absent from the early avian lung. Here, we have found that branching morphogenesis in the embryonic chicken lung requires extracellular matrix (ECM) remodeling driven by reciprocal interactions between the epithelium and mesenchyme. Before branching, the basement membrane wraps the airway epithelium as a spatially uniform sheath. After branch initiation, however, the basement membrane thins at branch tips; this remodeling requires mesenchymal expression of matrix metalloproteinase 2, which is necessary for branch extension but for not branch initiation. As branches extend, tenascin C (TNC) accumulates in the mesenchyme several cell diameters away from the epithelium. Despite its pattern of accumulation, TNC is expressed exclusively by epithelial cells. Branch extension coincides with deformation of adjacent mesenchymal cells, which correlates with an increase in mesenchymal fluidity at branch tips that may transport TNC away from the epithelium. These data reveal novel epithelial-mesenchymal interactions that direct ECM remodeling during airway branching morphogenesis.

Lung epithelial protein disulfide isomerase A3 (PDIA3) plays an important role in influenza infection, inflammation, and airway mechanics.

Protein disulfide isomerases (PDI) are a family of redox chaperones that catalyze formation or isomerization of disulfide bonds in proteins. Previous studies have shown that one member, PDIA3, interacts with influenza A virus (IAV) hemagglutinin (HA), and this interaction is required for efficient oxidative folding of HA in vitro. However, it is unknown whether these host-viral protein interactions occur during active infection and whether such interactions represent a putative target for the treatment of influenza infection. Here we show that PDIA3 is specifically upregulated in IAV-infected mouse or human lung epithelial cells and PDIA3 directly interacts with IAV-HA. Treatment with a PDI inhibitor, LOC14 inhibited PDIA3 activity in lung epithelial cells, decreased intramolecular disulfide bonds and subsequent oligomerization (maturation) of HA in both H1N1 (A/PR8/34) and H3N2 (X31, A/Aichi/68) infected lung epithelial cells. These reduced disulfide bond formation significantly decreased viral burden, and also pro-inflammatory responses from lung epithelial cells. Lung epithelial-specific deletion of PDIA3 in mice resulted in a significant decrease in viral burden and lung inflammatory-immune markers upon IAV infection, as well as significantly improved airway mechanics. Taken together, these results indicate that PDIA3 is required for effective influenza pathogenesis in vivo, and pharmacological inhibition of PDIs represents a promising new anti-influenza therapeutic strategy during pandemic and severe influenza seasons.

SPX-101 is stable in and retains function after exposure to cystic fibrosis sputum.

BACKGROUND: In healthy lungs, epithelial sodium channel (ENaC) is regulated by short, palate, lung, and nasal clone 1 (SPLUNC1). In cystic fibrosis (CF), ENaC is hyperactivated in part due to a loss of SPLUNC1 function. We have developed SPX-101 to replace the lost function of SPLUNC1 in the CF lung. METHODS: Expression of SPLUNC1 was determined in sputum from healthy and CF donors. Stability of SPLUNC1, S18 (the ENaC regulatory domain of SPLUNC1), and SPX-101 was determined in sputum from CF donors and towards neutrophil elastase. Activity of SPX-101 after exposure to CF sputum was determined in airway epithelial cells from CF donors and in the ßENaC transgenic mouse model. RESULTS: SPLUNC1 protein expression is significantly reduced in CF as compared to healthy sputum. SPLUNC1 is rapidly degraded in CF sputum as well as by a number of individual proteases known to be found in the sputum. SPX-101, but not S18, is stable in CF sputum. Finally, SPX-101 retains its ability to internalize ENaC, regulate airway surface liquid height, and increase survival of ßENaC mice after exposure to CF sputum. CONCLUSIONS: Our results demonstrate that SPX-101, but not SPLUNC1 or S18, is stable in CF sputum. These results support the therapeutic development of SPX-101 for the treatment of cystic fibrosis.

Transporters MRP1 and MRP2 Regulate Opposing Inflammatory Signals To Control Transepithelial Neutrophil Migration during Streptococcus pneumoniae Lung Infection.

Streptococcus pneumoniae remains a source of morbidity and mortality in both developed and underdeveloped nations of the world. Disease can manifest as pneumonia, bacteremia, and meningitis, depending on the localization of infection. Interestingly, there is a correlation in experimental murine infections between the development of bacteremia and influx of neutrophils into the pulmonary lumen. Reduction of this neutrophil influx has been shown to improve survivability during infection. In this study, we use in vitro biotinylation and neutrophil transmigration and in vivo murine infection to identify a system in which two epithelium-localized ATP-binding cassette transporters, MRP1 and MRP2, have inverse activities dictating neutrophil transmigration into the lumen of infected mouse lungs. MRP1 effluxes an anti-inflammatory molecule that maintains homeostasis in uninfected contexts, thus reducing neutrophil infiltration. During inflammatory events, however, MRP1 decreases and MRP2 both increases and effluxes the proinflammatory eicosanoid hepoxilin A3. If we then decrease MRP2 activity during experimental murine infection with S. pneumoniae, we reduce both neutrophil infiltration and bacteremia, showing that MRP2 coordinates this activity in the lung. We conclude that MRP1 assists in depression of polymorphonuclear cell (PMN) migration by effluxing a molecule that inhibits the proinflammatory effects of MRP2 activity.IMPORTANCEStreptococcus pneumoniae is a Gram-positive bacterium that normally inhabits the human nasopharynx asymptomatically. However, it is also a major cause of pneumonia, bacteremia, and meningitis. The transition from pneumonia to bacteremia is critical, as patients that develop septicemia have ~20% mortality rates. Previous studies have shown that while neutrophils, a major bacterium-induced leukocyte, aid in S. pneumoniae elimination, they also contribute to pathology and may mediate the lung-to-blood passage of the bacteria. Herein, we show that epithelium-derived MRP1 and MRP2 efflux immunomodulatory agents that assist in controlling passage of neutrophils during infection and that limiting neutrophil infiltration produced less bacteremia and better survival during murine infection. The importance of our work is twofold: ours is the first to identify an MRP1/MRP2 axis of neutrophil control in the lung. The second is to provide possible therapeutic targets to reduce excess inflammation, thus reducing the chances of developing bacteremia during pneumococcal pneumonia.

Overexpression of the base excision repair NTHL1 glycosylase causes genomic instability and early cellular hallmarks of cancer.

Base excision repair (BER), which is initiated by DNA N-glycosylase proteins, is the frontline for repairing potentially mutagenic DNA base damage. The NTHL1 glycosylase, which excises DNA base damage caused by reactive oxygen species, is thought to be a tumor suppressor. However, in addition to NTHL1 loss-of-function mutations, our analysis of cancer genomic datasets reveals that NTHL1 frequently undergoes amplification or upregulation in some cancers. Whether NTHL1 overexpression could contribute to cancer phenotypes has not yet been explored. To address the functional consequences of NTHL1 overexpression, we employed transient overexpression. Both NTHL1 and a catalytically-dead NTHL1 (CATmut) induce DNA damage and genomic instability in non-transformed human bronchial epithelial cells (HBEC) when overexpressed. Strikingly, overexpression of either NTHL1 or CATmut causes replication stress signaling and a decrease in homologous recombination (HR). HBEC cells that overexpress NTHL1 or CATmut acquire the ability to grow in soft agar and exhibit loss of contact inhibition, suggesting that a mechanism independent of NTHL1 catalytic activity contributes to acquisition of cancer-related cellular phenotypes. We provide evidence that NTHL1 interacts with the multifunctional DNA repair protein XPG suggesting that interference with HR is a possible mechanism that contributes to acquisition of early cellular hallmarks of cancer.

Nominal carbonic anhydrase activity minimizes airway-surface liquid pH changes during breathing.

The airway-surface liquid pH (pHASL ) is slightly acidic relative to the plasma and becomes more acidic in airway diseases, leading to impaired host defense. CO2 in the large airways decreases during inspiration (0.04% CO2 ) and increases during expiration (5% CO2 ). Thus, we hypothesized that pHASL would fluctuate during the respiratory cycle. We measured pHASL on cultures of airway epithelia while changing apical CO2 concentrations. Changing apical CO2 produced only very slow pHASL changes, occurring in minutes, inconsistent with respiratory phases that occur in a few seconds. We hypothesized that pH changes were slow because airway-surface liquid has little carbonic anhydrase activity. To test this hypothesis, we applied the carbonic anhydrase inhibitor acetazolamide and found minimal effects on CO2 -induced pHASL changes. In contrast, adding carbonic anhydrase significantly increased the rate of change in pHASL . Using pH-dependent rates obtained from these experiments, we modeled the pHASL during respiration to further understand how pH changes with physiologic and pathophysiologic respiratory cycles. Modeled pHASL oscillations were small and affected by the respiration rate, but not the inspiratory:expiratory ratio. Modeled equilibrium pHASL was affected by the inspiratory:expiratory ratio, but not the respiration rate. The airway epithelium is the only tissue that is exposed to large and rapid CO2 fluctuations. We speculate that the airways may have evolved minimal carbonic anhydrase activity to mitigate large changes in the pHASL during breathing that could potentially affect pH-sensitive components of ASL.

Immunohistochemical study on the secretory host defense system with lysozyme and secretory phospholipase A2 throughout rat respiratory tract.

The host defense system with lysozyme and secretory phospholipase A2 (sPLA2) was immunohistochemically investigated in rat respiratory tract under healthy conditions. In the nasal epithelium, a large number of non-ciliated and non-microvillous cells (NC) and a small number of goblet cells (GC) were immunopositive for lysozyme and sPLA2. A few acinar cells and almost all epithelial cells of intercalated ducts were immunopositive for both bactericidal substances in the nasal glands. In the laryngeal and tracheal epithelia, few NC and GC were immunopositive for both bactericidal substances. In the laryngeal and tracheal glands, a few acinar cells and most ductal epithelial cells were immunopositive for both bactericidal substances. In extra-pulmonary bronchus, small numbers of NC and GC were immunopositive for lysozyme and sPLA2, whereas few NC and no GC were immunopositive in the intra-pulmonary bronchus. No secretory source of either bactericidal substance was located in the bronchioles. In the alveolus, many glandular epithelial cells and alveolar macrophages were immunopositive for lysozyme but immunonegative for sPLA2. Moreover, lysozyme and sPLA2 were detected in the mucus layer and in the periciliary layer from the nose to the extra-pulmonary bronchus. These findings suggest that secretory sources of lysozyme and sPLA2 are distributed in almost all the respiratory tract. Their secretory products are probably transported to the pharynx and contribute to form the first line of defense against inhaled bacteria throughout the respiratory tract.

A Novel Dynamic Model Describing the Spread of the MERS-CoV and the Expression of Dipeptidyl Peptidase 4.

The Middle East respiratory syndrome (MERS) coronavirus, a newly identified pathogen, causes severe pneumonia in humans. MERS is caused by a coronavirus known as MERS-CoV, which attacks the respiratory system. The recently defined receptor for MERS-CoV, dipeptidyl peptidase 4 (DPP4), is generally expressed in endothelial and epithelial cells and has been shown to be present on cultured human nonciliated bronchiolar epithelium cells. In this paper, a class of novel four-dimensional dynamic model describing the infection of MERS-CoV is given, and then global stability of the equilibria of the model is discussed. Our results show that the spread of MERS-CoV can also be controlled by decreasing the expression rate of DPP4.

The Helicobacter pylori type IV secretion system promotes IL-8 synthesis in a model of pediatric airway epithelium via p38 MAP kinase.

Epidemiologic studies have reported an inverse relationship between childhood Helicobacter pylori infection and development of allergic asthma. Because lung epithelium plays an important role in allergic asthma pathogenesis, we hypothesized that H. pylori may directly influence airway epithelial cell innate immune function, particularly in early childhood. To test our hypothesis, we established an in vitro H. pylori infection model using primary tracheobronchial epithelial cell cultures derived from infant, juvenile and adult rhesus monkeys. Airway epithelial cell cultures were infected with wild-type or cag pathogenicity island mutant H. pylori strains, followed by evaluation of IL-8 and IL-6 protein synthesis. We found that H. pylori primarily increased IL-8 synthesis in a MOI and age-dependent fashion, with a greater than 4-fold induction in infant versus adult cultures. H. pylori-induced IL-8 synthesis in infant and juvenile cultures was significantly reduced by cag pathogenicity island mutants, indicating a requirement for the type IV secretion system. Although peptidoglycan recognition of nucleotide binding oligomerization domain-containing protein 1 (NOD1) and NF-kappaB have been implicated as key cytokine signaling molecules for H. pylori infection in gastric epithelium, NOD1 (ML130) or NF-kappaB (JSH-23) inhibitors minimally affected IL-8 synthesis in airway epithelial cell cultures following H. pylori infection. In contrast, inhibition of the p38 MAP kinase pathway (SB203580) resulted in almost complete suppression of H. pylori-induced IL-8 synthesis. Collectively, these results indicate that H. pylori can preferentially elicit IL-8 synthesis in a model of pediatric airway epithelium using the type IV secretion system via p38 MAP kinase.

Epithelial Cells Induce a Cyclo-Oxygenase-1-Dependent Endogenous Reduction in Airway Smooth Muscle Contractile Phenotype.

Airway smooth muscle cells (ASMCs) are phenotypically regulated to exist in either a proliferative or a contractile state. However, the influence of other airway structural cell types on ASMC phenotype is largely unknown. Although epithelial cells are known to drive ASM proliferation, their effects on the contractile phenotype are uncertain. In the current study, we tested the hypothesis that epithelial cells reduce the contractile phenotype of ASMCs. To do so, we measured force production by traction microscopy, gene and protein expression, as well as calcium release by Fura-2 ratiometric imaging. ASMCs incubated with epithelial-derived medium produced less force after histamine stimulation. We observed reduced expression of myocardin, α-smooth muscle actin, and calponin within ASMCs after coculture with epithelial cells. Peak calcium release in response to histamine was diminished, and depended on the synthesis of cyclo-oxygenase-1 products by ASM and on prostaglandin E receptors 2 and 4. Together, these in vitro results demonstrate that epithelial cells have the capacity to coordinately reduce ASM contraction by functional antagonism and by reduction of the expression of certain contractile proteins.

The helicase, DDX3X, interacts with poly(A)-binding protein 1 (PABP1) and caprin-1 at the leading edge of migrating fibroblasts and is required for efficient cell spreading.

DDX3X, a helicase, can interact directly with mRNA and translation initiation factors, regulating the selective translation of mRNAs that contain a structured 5' untranslated region. This activity modulates the expression of mRNAs controlling cell cycle progression and mRNAs regulating actin dynamics, contributing to cell adhesion and motility. Previously, we have shown that ribosomes and translation initiation factors localise to the leading edge of migrating fibroblasts in loci enriched with actively translating ribosomes, thereby promoting steady-state levels of ArpC2 and Rac1 proteins at the leading edge of cells during spreading. As DDX3X can regulate Rac1 levels, cell motility and metastasis, we have examined DDX3X protein interactions and localisation using many complementary approaches. We now show that DDX3X can physically interact and co-localise with poly(A)-binding protein 1 and caprin-1 at the leading edge of spreading cells. Furthermore, as depletion of DDX3X leads to decreased cell motility, this provides a functional link between DDX3X, caprin-1 and initiation factors at the leading edge of migrating cells to promote cell migration and spreading.

Curcumin Inhibits Heat-Induced Apoptosis by Suppressing NADPH Oxidase 2 and Activating the Akt/mTOR Signaling Pathway in Bronchial Epithelial Cells.

BACKGROUND: Heat causes bronchial epithelial cell apoptosis, which is a known factor contributing to airway damage during inhalation injury. Accumulating evidence has shown the effect of curcumin on inhibiting apoptosis. In this study, we investigated whether curcumin suppresses heat-induced apoptosis in bronchial epithelial cells and the underlying mechanism. METHODS: Bronchial epithelial cell line 16HBE140 cells were incubated at either 42 °C, 47 °C, 52 °C, or 57 °C for 5 min in a cell incubator and then returned back to normal culture conditions (37 °C). An in vivo thermal inhalation injury rat model was established with a heat gun blowing hot air into the airway of rats. 16HBE140 cells and lung tissue were obtained for further study with or without curcumin treatment. Cell viability was determined by measuring the absorbance of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). 2',7'-dichlorofluorescein diacetate fluorescence was used as a measure of reactive oxygen species (ROS) production. Levels of Bcl2, Bax, α-ATP, cleaved Poly (ADP-ribose) polymerase (PARP), cleaved caspase-3, gp91phox, p47phox, p67phox, p22phox, p40phox, and Rac were determined by Western blotting. TUNEL staining was used to determine apoptosis. RESULTS: Heat treatment triggered the apoptosis of 16HBE140 cells as shown by the increase in apoptosis molecular markers, including Bcl-2, Bax, cleaved PARP, and cleaved caspase-3. Administration of curcumin significantly inhibited apoptosis of 16HBE140 cells and suppressed the membrane translocation of NADPH oxidase 2 cytosolic components, as well as ROS production. Downregulation of Akt and mTOR phosphorylation induced by heat was also reversed by curcumin. Furthermore, we demonstrated that NADPH oxidase 2 is upstream of Akt/mTOR in heat-induced apoptosis. The protective role of curcumin on bronchial epithelia apoptosis was also confirmed in vivo by a rat inhalation injury model. CONCLUSION: This study demonstrates that one of the critical mechanisms underlying curcumin inhibiting heat-induced apoptosis is through suppressing NADPH Oxidase 2 and activating the Akt/mTOR signaling pathway in bronchial epithelial cells.