Mouse Airway Epithelial Cells Research Articles

Cigarette Smoke Extract-Treated Mouse Airway Epithelial Cells-Derived Exosomal LncRNA MEG3 Promotes M1 Macrophage Polarization and Pyroptosis in Chronic Obstructive Pulmonary Disease by Upregulating TREM-1 via m6A Methylation.

Cigarette smoke extract (CSE)-treated mouse airway epithelial cells (MAECs)-derived exosomes accelerate the progression of chronic obstructive pulmonary disease (COPD) by upregulating triggering receptor expressed on myeloid cells 1 (TREM-1); however, the specific mechanism remains unclear. We aimed to explore the potential mechanisms of CSE-treated MAECs-derived exosomes on M1 macrophage polarization and pyroptosis in COPD. In vitro, exosomes were extracted from CSE-treated MAECs, followed by co-culture with macrophages. In vivo, mice exposed to cigarette smoke (CS) to induce COPD, followed by injection or/and intranasal instillation with oe-TREM-1 lentivirus. Lung function and pathological changes were evaluated. CD68+ cell number and the levels of iNOS, TNF-α, IL-1β (M1 macrophage marker), and pyroptosis-related proteins (NOD-like receptor family pyrin domain containing 3, apoptosis-associated speck-like protein containing a caspase-1 recruitment domain, caspase-1, cleaved-caspase-1, gasdermin D [GSDMD], and GSDMD-N) were examined. The expression of maternally expressed gene 3 (MEG3), spleen focus forming virus proviral integration oncogene (SPI1), methyltransferase 3 (METTL3), and TREM-1 was detected and the binding relationships among them were verified. MEG3 increased N6-methyladenosine methylation of TREM-1 by recruiting SPI1 to activate METTL3. Overexpression of TREM-1 or METTL3 negated the alleviative effects of MEG3 inhibition on M1 polarization and pyroptosis. In mice exposed to CS, EXO-CSE further aggravated lung injury, M1 polarization, and pyroptosis, which were reversed by MEG3 inhibition. TREM-1 overexpression negated the palliative effects of MEG3 inhibition on COPD mouse lung injury. Collectively, CSE-treated MAECs-derived exosomal long non-coding RNA MEG3 may expedite M1 macrophage polarization and pyroptosis in COPD via the SPI1/METTL3/TREM-1 axis.

Shuttle peptide delivers base editor RNPs to rhesus monkey airway epithelial cells in vivo

Gene editing strategies for cystic fibrosis are challenged by the complex barrier properties of airway epithelia. We previously reported that the amphiphilic S10 shuttle peptide non-covalently combined with CRISPR-associated (Cas) ribonucleoprotein (RNP) enabled editing of human and mouse airway epithelial cells. Here, we derive the S315 peptide as an improvement over S10 in delivering base editor RNP. Following intratracheal aerosol delivery of Cy5-labeled peptide in rhesus macaques, we confirm delivery throughout the respiratory tract. Subsequently, we target CCR5 with co-administration of ABE8e-Cas9 RNP and S315. We achieve editing efficiencies of up-to 5.3% in rhesus airway epithelia. Moreover, we document persistence of edited epithelia for up to 12 months in mice. Finally, delivery of ABE8e-Cas9 targeting the CFTR R553X mutation restores anion channel function in cultured human airway epithelia. These results demonstrate the therapeutic potential of base editor delivery with S315 to functionally correct the CFTR R553X mutation in respiratory epithelia.

Ambroxol-Enhanced Frequency and Amplitude of Beating Cilia Controlled by a Voltage-Gated Ca2+ Channel, Cav1.2, via pHi Increase and [Cl-]i Decrease in the Lung Airway Epithelial Cells of Mice.

Ambroxol (ABX), a frequently prescribed secretolytic agent which enhances the ciliary beat frequency (CBF) and ciliary bend angle (CBA, an index of amplitude) by 30%, activates a voltage-dependent Ca2+ channel (CaV1.2) and a small transient Ca2+ release in the ciliated lung airway epithelial cells (c-LAECs) of mice. The activation of CaV1.2 alone enhanced the CBF and CBA by 20%, mediated by a pHi increasei and a [Cl-]i decrease in the c-LAECs. The increase in pHi, which was induced by the activation of the Na+-HCO3- cotransporter (NBC), enhanced the CBF (by 30%) and CBA (by 15-20%), and a decrease in [Cl-]i, which was induced by the Cl- release via anoctamine 1 (ANO1), enhanced the CBA (by 10-15%). While a Ca2+-free solution or nifedipine (an inhibitor of CaV1.2) inhibited 70% of the CBF and CBA enhancement using ABX, CaV1.2 enhanced most of the CBF and CBA increases using ABX. The activation of the CaV1.2 existing in the cilia stimulates the NBC to increase pHi and ANO1 to decrease the [Cl-]i in the c-LAECs. In conclusion, the pHi increase and the [Cl-]i decrease enhanced the CBF and CBA in the ABX-stimulated c-LAECs.

A Novel Sandwich Method for Serial Block Face SEM Imaging of Airway Multiciliated Epithelium.

Volume electron microscopy technologies such as serial block face scanning electron microscopy (SBF-SEM) allow the characterization of tissue organization and cellular content in three dimensions at nanoscale resolution. Here, we describe the procedure to process and image an air-liquid interface culture of human or mouse airway epithelial cells for visualization of the multiciliated epithelium by SBF-SEM in vertical or horizontal cross section.

Hypermethylation of RNF125 promotes autophagy-induced oxidative stress in asthma by increasing HMGB1 stability

Asthma is a global chronic airway disease. The expression and role of RNF125, an E3 ubiquitin ligase, in asthma remain uncertain. In this study, we revealed that RNF125 was downregulated in the bronchial epithelium of mice and patients with asthma. Rnf125 hypermethylation was responsible for the low expression of RNF125 in primary airway epithelial cells of mice treated with OVA. Moreover, we demonstrated that RNF125 could attenuate autophagy, oxidative stress, and protect epithelial barrier invivo and invitro. Additionally, we identified HMGB1 as a substrate of RNF125, which interacted with the HMG B-box domain of HMGB1 and induced degradation via the ubiquitin proteasome system, reducing autophagy and oxidative stress. Overall, our findings elucidated that hypermethylation of Rnf125 reduced its expression, which promoted autophagy-induced oxidative stress in asthma by increasing HMGB1 stability. These findings offer a theoretical and experimental basis for the pathogenesis of asthma.

Dickkopf-1 inhibits the secretion of MUC5AC induced by Mycoplasma pneumoniae P1-C in mouse lung epithelial cells

Mycoplasma pneumoniae is the most common pathogen of respiratory tract infection in children and adults. Clinical observation shows that M. pneumoniae infection can cause massive mucus secretion in the respiratory tract, which makes the breathing of patients difficult. Studies have shown that M. pneumoniae infection can cause massive secretion of mucin 5AC (MUC5AC). Adhesin P1 plays an important role in the pathogenesis of M. pneumoniae infection by mediating the adhesion of pathogens to host cells, and the C-terminal residues of P1 (P1-C) are immunogenic. This study investigated the molecular mechanism of Wnt/β-catenin signaling pathway inhibitor Dickkopf-1 (DKK1) in the secretion of MUC5AC in mouse airway epithelial cells (MAECs) induced by P1-C. Scanning electron microscope and hematoxylin-eosin staining were used to observe the effect of P1-C on mucus secretion of MAECs. Protein chip was used to detect the secretion of cytokines and analyse the enrichment of related signaling pathways induced by P1-C in MAECs. Periodic acid schiff stain (PAS) staining, Tunel staining and Masson staining were used to detect the damage of the lungs of mouse exposed to P1-C. Immunohistochemistry was used to detect the secretion of MUC5AC expression, and Western blotting was used to reveal the molecular mechanism of DKK1-regulated secretion of MUC5AC induced by P1-C protein in MACES. The results showed that P1-C induced the massive secretion of mucus and inflammatory factors in MAECs. During P1-C infection, DKK1 down-regulated janus kinase 2 (JAK2), phosphorylation signaling and transcription activator 1 (p-STAT1) and phosphorylation signaling and activator of transcription 3 (p-STAT3) expression. Overexpression of DKK1 significantly up-regulated the expression of MUC5AC repressor transcription factor fork-head box protein A2 (FOXA2). At the same time, the expression of MUC5AC induced by P1-C was inhibited significantly. It is speculated that DKK1 can effectively reduce the secretion of MUC5AC in MAECs induced by P1-C by inhibiting the JAK/STAT1-STAT3 signaling pathway and up-regulating the expression of FOXA2.

Scorpio and centipede ameliorate asthma by inhibiting the crosstalk between ferroptosis and inflammation in airway epithelial cells.

We previously revealed that scorpio and centipede (SC) improve the inflammatory response in asthma, whereas it is unclear whether ferroptosis is involved in this process. The asthmatic mouse model was established and lung tissues were collected for histopathological examination. The levels of tumor necrosis factor-α (TNF-α), interleukin- (IL-)1β, Fe2+, malondialdehyde (MDA), glutathione peroxidase 4 (GPX4), ferritin heavy chain 1(FTH1), and reactive oxygen species (ROS) were assessed in asthmatic mice and mouse airway epithelial cells. Our results showed that ferroptosis was induced in asthmatic mice, as evidenced by the reduction of FTH1 and GPX4 expression and the increase of MDA and Fe2+ levels (all P<0.05). Ferrostatin-1 repressed inflammation and ferroptosis of asthmatic mice. Additionally, SC significantly inhibited the levels of TNF-α, IL-1β, MDA, and Fe2+, while enhancing FTH1 and GPX4 expression. However, SC plus erastin showed the reverse results. Moreover, ferroptosis remarkably increased in asthmatic airway epithelial cells, while SC suppressed ferroptosis of the cells (all P<0.05). SC ameliorated asthma by inhibiting the crosstalk between ferroptosis and inflammation in airway epithelial cells.

Reserve of Wnt/β-catenin Signaling Alleviates Mycoplasma pneumoniae P1-C-induced Inflammation in airway epithelial cells and lungs of mice

Mycoplasma pneumoniae (M. pneumoniae) is the most common pathogen of respiratory tract infections in both children and adults. M. pneumoniae P1 adhesin plays an important role in the pathogenesis of M. pneumoniae infection by mediating the attachment of pathogen to host cells. The inoculation of C-terminal residuals of P1 (P1-C) showed a protective role from M. pneumoniae infection. Accumulated evidence suggests that the Wnt/β-Catenin signaling is implicated in regulation of inflammatory responses to bacterial infections. However, mechanisms underlying the regulatory roles of Wnt signaling in host cells in response to M. pneumoniae infections are incompletely understood. In the present study, the impact and molecular mechanism of Wnt/β-catenin signaling in immune responses induced by M. pneumoniae P1-C were investigated. The results demonstrated that the P1-C could activate Wnt/β-catenin and Toll-like receptor (TLR) signaling in primary mouse airway epithelial cells cultured in an air-liquid interface (ALI) state. Interestingly, the inhibition of Wnt/β-catenin signaling by an adenovirus-mediated Wnt inhibitor Dickkopf-1 (Dkk1) gene transduction alleviated the P1-C induced inflammation fibrosis in mouse lung, accompanied by the reduced expression of epithelial mesenchymal transition (EMT) markers. Mechanistical analysis further demonstrated that the Dkk1 could suppress the expression of JAK2/STAT1-STAT3 and Caspase3, 8/Bax signaling in mouse lung tissues. In vitro study further revealed that XAV939, a small molecule of Wnt/β-catenin inhibitor, inhibited the P1-C-activated TLR4/MyD88 signaling and cytokine productions in primary mouse airway ALI epithelial cells. This study thus provides an insight into the function of Wnt/β-catenin signaling in regulation of the pathogenesis of M. pneumoniae infection, suggesting that targeting Wnt/β-catenin signaling by gene transduction of Dkk1, or pharmacological molecules of inhibitor may be a promised approach that worthy of further investigation in the treatment of M. pneumoniae pneumonia.

Ketone body augmentation decreases methacholine hyperresponsiveness in mouse models of allergic asthma.

Individuals with allergic asthma exhibit lung inflammation and remodeling accompanied by methacholine hyperresponsiveness manifesting in proximal airway narrowing and distal lung tissue collapsibility, and they can present with a range of mild-to-severe disease amenable or resistant to therapeutic intervention, respectively. There remains a need for alternatives or complements to existing treatments that could control the physiologic manifestations of allergic asthma. Our aim was to examine the hypothesis that because ketone bodies elicit anti-inflammatory activity and are effective in mitigating the methacholine hyperresponsiveness associated with obese asthma, increasing systemic concentrations of ketone bodies would diminish pathologic outcomes in asthma-relevant cell types and in mouse models of allergic asthma. We explored the effects of ketone bodies on allergic asthma-relevant cell types (macrophages, airway epithelial cells, CD4 T cells, and bronchial smooth muscle cells) in vitro as well as in vivo by using preclinical models representative of several endotypes of allergic asthma to determine whether promotion of ketosis through feeding a ketogenic diet or providing a ketone precursor or a ketone ester dietary supplement could affect immune and inflammatory parameters as well as methacholine hyperresponsiveness. In a dose-dependent manner, the ketone bodies acetoacetate and β-hydroxybutyrate (BHB) decreased proinflammatory cytokine secretion from mouse macrophages and airway epithelial cells, decreased house dust mite (HDM) extract-induced IL-8 secretion from human airway epithelial cells, and decreased cytokine production from polyclonally and HDM-activated T cells. Feeding a ketogenic diet, providing a ketone body precursor, or supplementing the diet with a ketone ester increased serum BHB concentrations and decreased methacholine hyperresponsiveness in several acute HDM sensitization and challenge models of allergic asthma. A ketogenic diet or ketone ester supplementation decreased methacholine hyperresponsiveness in an HDM rechallenge model of chronic allergic asthma. Ketone ester supplementation synergized with corticosteroid treatment to decrease methacholine hyperresponsiveness in an HDM-driven model of mixed-granulocytic severe asthma. HDM-induced morphologic changes in bronchial smooth muscle cells were inhibited in a dose-dependent manner by BHB, as was HDM protease activity. Increasing systemic BHB concentrations through dietary interventions could provide symptom relief for several endotypes of allergic asthmatic individuals through effects on multiple asthma-relevant cells.

Pre-existing atopy prevents mortality from respiratory viral infection via induction of neuregulin-1

Abstract Respiratory viral infections are associated with significant morbidity and mortality. RNA viruses, such as Respiratory Syncytial Virus (RSV) have been implicated in the development of asthma. We have a high-fidelity model of respiratory viral infection. Our mouse model utilizes a natural rodent pathogen, Sendai virus (SeV) similar to RSV, which faithfully replicates in mouse airway epithelial cells. We have shown that making these mice atopic (i.e, sensitized and challenged with house dust mite extracts) before viral infection prevents development of disease and more importantly prevents mice from dying of a normally lethal dose of SeV. Further, lungs and bronchoalveolar lavage fluid of atopic mice have significant increased levels of Neuregulin-1 (NRG1), potentially due to high expression of Nrg1 in dendritic cells as determined by RNAseq of CD11c+ cells. To determine a temporal relationship between NRG1 expression and respiratory viral infection, we utilized an in vitro system of well-differentiated human bronchial epithelial cells and mouse tracheal epithelial cells. Cells were treated on the basolateral side with NRG1 (10 to 100ng) for six-days before 4000 pfu of RSV-GFP or SeV-GFP inoculation, respectively. There was marked reduction in viral spreading (quantified by reduced GFP expression) with NRG1 treatment. Interleukin-6 (IL6) expression was reduced in NRG1 treated cells at 48h PI, as well. IL6 has been implicated with cellular stress and is involved in disease pathology. Therefore, reducing IL6 might affect the clinical presentation of serious infections. In conclusion our studies show that atopy induced NRG1 plays a novel role with a therapeutic potential in severe respiratory viral infections. Supported by Abigail Wexner Research Institute at Nationwide Children’s Hospital, Start-up fund to MHG

Targeting the P2Y13 Receptor Suppresses IL-33 and HMGB1 Release and Ameliorates Experimental Asthma.

Rationale: The alarmins IL-33 and HMGB1 (high mobility group box 1) contribute to type 2 inflammation and asthma pathogenesis. Objectives: To determine whether P2Y13-R (P2Y13 receptor), a purinergic GPCR (G protein-coupled receptor) and risk allele for asthma, regulates the release of IL-33 and HMGB1. Methods: Bronchial biopsy specimens were obtained from healthy subjects and subjects with asthma. Primary human airway epithelial cells (AECs), primary mouse AECs, or C57Bl/6 mice were inoculated with various aeroallergens or respiratory viruses, and the nuclear-to-cytoplasmic translocation and release of alarmins was measured by using immunohistochemistry and an ELISA. The role of P2Y13-R in AEC function and in the onset, progression, and exacerbation of experimental asthma was assessed by using pharmacological antagonists and mice with P2Y13-R gene deletion. Measurements and Main Results: Aeroallergen exposure induced the extracellular release of ADP and ATP, nucleotides that activate P2Y13-R. ATP, ADP, and aeroallergen (house dust mite, cockroach, or Alternaria antigen) or virus exposure induced the nuclear-to-cytoplasmic translocation and subsequent release of IL-33 and HMGB1, and this response was ablated by genetic deletion or pharmacological antagonism of P2Y13. In mice, prophylactic or therapeutic P2Y13-R blockade attenuated asthma onset and, critically, ablated the severity of a rhinovirus-associated exacerbation in a high-fidelity experimental model of chronic asthma. Moreover, P2Y13-R antagonism derepressed antiviral immunity, increasing IFN-λ production and decreasing viral copies in the lung. Conclusions: We identify P2Y13-R as a novel gatekeeper of the nuclear alarmins IL-33 and HMGB1 and demonstrate that the targeting of this GPCR via genetic deletion or treatment with a small-molecule antagonist protects against the onset and exacerbations of experimental asthma.

Lrig1 expression identifies airway basal cells with high proliferative capacity and restricts lung squamous cell carcinoma growth

BackgroundLung squamous cell carcinoma (LUSC) accounts for a significant proportion of cancer deaths worldwide, and is preceded by the appearance of progressively disorganised pre-invasive lesions in the airway epithelium. Yet the biological mechanisms underlying progression of pre-invasive lesions into invasive LUSC are not fully understood. LRIG1 (leucine-rich repeats and immunoglobulin-like domains 1) is downregulated in pre-invasive airway lesions and invasive LUSC tumours and this correlates with decreased lung cancer patient survival.Methods and resultsUsing an Lrig1 knock-in reporter mouse and human airway epithelial cells collected at bronchoscopy, we show that during homeostasis LRIG1 is heterogeneously expressed in the airway epithelium. In basal airway epithelial cells, the suspected cell of origin of LUSC, LRIG1 identifies a subpopulation of progenitor cells with higher in vitro proliferative and self-renewal potential in both the mouse and human. Using the N-nitroso-tris-chloroethylurea (NTCU)-induced murine model of LUSC, we find that Lrig1 loss-of-function leads to abnormally high cell proliferation during the earliest stages of pre-invasive disease and to the formation of significantly larger invasive tumours, suggesting accelerated disease progression.ConclusionTogether, our findings identify LRIG1 as a marker of basal airway progenitor cells with high proliferative potential and as a regulator of pre-invasive lung cancer progression. This work highlights the clinical relevance of LRIG1 and the potential of the NTCU-induced LUSC model for functional assessment of candidate tumour suppressors and oncogenes.

Obese adipose tissue modulates proinflammatory responses of mouse airway epithelial cells.

Although recognized as an important endocrine organ, little is known about the mechanisms through which adipose tissue can regulate inflammatory responses in distant tissues, such as lung that are affected by obesity. To explore potential mechanisms, male C57BL/6J mice were provided either high-fat diet, low-fat diet, or were provided a high-fat diet then switched to the low-fat diet to promote weight loss. Visceral adipocytes were then cultured in vitro to generate conditioned media (CM) that was used to treat both primary (mouse tracheal epithelial cells; MTECs) and immortalized (mouse-transformed club cells; MTCCs) airway epithelial cells. Adiponectin levels were greatly depressed in the CM from both obese and diet-switched adipocytes relative to mice continually fed the low-fat diet. MTECs from mice with obesity secreted higher baseline levels of inflammatory cytokines than MTECs from lean or diet-switched mice. MTECs treated with obese adipocyte CM increased their secretion of these cytokines compared with MTECs treated with lean CM. Diet-switched CM modestly decreased the production of cytokines compared with obese CM, and these effects were recapitulated when the CM was used to treat MTCCs. Adipose stromal vascular cells from mice with obesity expressed genes consistent with an M1 macrophage phenotype and decreased eosinophil abundance compared with lean stromal vascular fraction, a profile that persisted in the lean diet-switched mice despite substantial weight loss. Soluble factors secreted from obese adipocytes exert a proinflammatory effect on airway epithelial cells, and these alterations are attenuated by diet-induced weight loss, which could have implications for the airway dysfunction related to obese asthma and its mitigation by weight loss.

Cigarette smoke extract-treated airway epithelial cells-derived exosomes promote M1 macrophage polarization in chronic obstructive pulmonary disease

Chronic obstructive pulmonary disease (COPD) is a persistent respiratory disorder that is primarily caused by exposure to cigarette smoke (CS). Exosomes have emerged as crucial mediators of intercellular communication, but their role in CS-induced COPD is not fully understood. Here, we investigated whether exosomes derived from cigarette smoke extract (CSE)-treated mouse airway epithelial cells (MAECs) promote M1 macrophage polarization by upregulating triggering receptor expressed on myeloid cells-1 (TREM-1) expression during COPD pathogenesis. The exosomes isolated from PBS- or CSE-treated MAECs were named as ExoPBS or ExoCSE, respectively. Macrophages were transfected with si-TREM-1 to explore the role of TREM-1 in ExoCSE-induced M1 macrophage polarization. The lentivirus expressing shTREM-1 was injected into COPD model mice by intranasal instillation, which was carried out to explore the in vivo role of TREM-1 in ExoCSE-induced M1 macrophage polarization and CS-induced lung injury. We isolated ExoPBS and ExoCSE successfully, and found that ExoCSE promoted M1 macrophage polarization. Furthermore, we found that the promotion of ExoCSE to M1 macrophage polarization was partly reversed by TREM-1 knockdown. The results of animal experiments showed that ExoCSE administration aggravated CS-induced impairment in pulmonary function, lung injury and M1 macrophage polarization, which were partly rescued by TREM-1 silencing. Overall, ExoCSE promoted M1 macrophage polarization by upregulating TREM-1 expression, thereby aggravating the development of COPD.

Epithelial cell-derived CD83 restores immune tolerance in the airway mucosa by inducing regulatory T-cell differentiation.

The mechanism of generation of regulatory T cells (Treg) remains incompletely understood. Recent studies show that CD83 has immune regulatory functions. This study aims to investigate the role of epithelial cell-derived CD83 in the restoration of immune tolerance in the airway mucosa by inducing the Treg differentiation. In this study, CD83 and ovalbumin (OVA)-carrying exosomes were generated from airway epithelial cells. An airway allergy mouse model was developed to test the role of CD83/OVA-carrying exosomes in the suppression of airway allergy by inducing Treg generation. We observed that mouse airway epithelial cells expressed CD83 that could be up-regulated by CD40 ligand. The CD83 deficiency in epithelial cells retarded the Treg generation in the airway mucosa. CD83 up-regulated transforming growth factor-β-inducible early gene 1 expression in CD4+ T cells to promote Foxp3 expression. Exposure of primed CD4+ T cells to CD83/OVA-carrying exosomes promoted antigen-specific Treg generation. Administration of CD83/OVA-carrying exosomes inhibited experimental airway allergic response. In summary, airway epithelial cells express CD83 that is required in the Treg differentiation in the airway mucosa. Administration of CD83/OVA-carrying exosomes can inhibit airway allergy that has the translation potential in the treatment of airway allergic disorders.

Inducible expression of heat shock protein 20 protects airway epithelial cells against oxidative injury involving the Nrf2-NQO-1 pathway

BackgroundHeat shock protein (HSP) 20 is a molecular chaperone that exerts multiple protective functions in various kinds of tissues. However, the expression of HSP20 and its specific functions in airway epithelial cells (AECs) remain elusive.ResultsIn current study, we first confirmed the inducible expression of HSP20 in mouse AECs and in a human bronchial epithelial cell line BEAS-2B cells, under different oxidant stressors. Then by establishing a HSP20-abundant mouse model with repeated low-level-ozone exposures and stimulating this model with a single high-level ozone exposure, we found that the HSP20 abundance along with its enhanced phosphorylation potentially contributed to the alleviation of oxidative injuries, evidenced by the decreases in the bodyweight reduction, the BAL neutrophil accumulation, the AECs shedding, and the BAL concentrations of albumin and E-cadherin. The biological function of HSP20 and its molecular mechanisms were further investigated in BEAS-2B cells that were transfected with HSP20-, unphosphorylatable HSP20(Ala) or empty vector plasmids prior to the stimulation of H2O2, of which its oxidant capacity has been proved to be similar with those of ozone in an air–liquid culture system. We found that the H2O2-induced intracellular ROS level and the early cell apoptosis were attenuated in the HSP20- but not HSP20(Ala)- transfected cells. The intracellular expression of NQO-1 (mRNA and protein) and the intranuclear content of Nrf2 were significantly increased in the HSP20- transfected cells but not in the HSP20(Ala)- and empty vector-transfected cells after the stimulation of H2O2.ConclusionsThe inducible expression of HSP20 in AECs by oxidative stress exerts protective roles against oxidative damages, which may involve the activation of the Nrf2-NQO-1 pathway.

Transient immunosuppression blocks iBALT formation in the lung induced by helper-dependent adenoviral vector during gene delivery

Abstract Lung is an immune sensitive organ with presence of all immune cells at the frontline of immunity. Under chronic infection conditions, inducible Bronchus-associated lymphoid tissue (iBALT) can be formed to defend foreign infectious organisms. We wonder whether viral vectors for gene therapy could induce iBALT formation and whether this process can be controlled through immune modulation. To test these ideas, we repeatedly administered a helper-dependent adenoviral (HD-Ad) vector to mouse lungs and examined iBALT formation. HD-Ad vectors are based on adenovirus but with all viral coding sequences deleted and is one option for large gene delivery. In our previous studies, HD-Ad vectors have exhibited high in vivo efficiency in transduction of mouse, rabbit and pig airway epithelial cells, including stem/progenitor cells such as basal cells. HD-Ad vectors expressing therapeutic gene encoding cystic fibrosis transmenbrane conductance regulator (CFTR) rescue CFTR function in CF patient primary cells. In our mouse studies, we found that HD-Ad vectors induced iBALT formation and antibody production. We used cyclophosphamide or HD-Ad expressing IL-10 to modulate the host immune responses and to examine the effects on iBALT formation. Comparing with control group (HD-Ad vectors only), the mice treated with cyclophosphamide or IL-10 expressing HD-Ad vector showed a significant reduction of iBALT formation and infiltration of inflammatory cells, including CD4+ and B220+ as well as production of anti-Ad antibodies and neutralizing antibodies. Our results indicate that transient modulation significantly mitigated immunopathology and extended transgene expression in mouse airways following readministration of HD-Ad vectors.

EphA2 phosphorylates NLRP3 and inhibits inflammasomes in airway epithelial cells.

Inflammasomes are intracellular complexes that form in the cytosol of inflammatory cells. NLRP3 is one of the sensor proteins in the complex that can recognize a wide variety of stimuli ranging from microbial components to environmental particulates. Here, we report that in mouse airway epithelial cells (AECs), inflammasome activation is inhibited by EphA2, a member of the transmembrane tyrosine kinase receptor family, via tyrosine phosphorylation of NLRP3 in a model of reovirus infection. We find that EphA2 depletion markedly enhances interleukin-1β (IL-1β) and interleukin-18 (IL-18) production in response to the virus. EphA2-/- mice show stronger inflammatory infiltration and enhanced inflammasome activation upon viral infection, and aggravated asthma symptoms upon ovalbumin (ova) induction. Mechanistically, EphA2 binds to NLRP3 and induces its phosphorylation at Tyr132, thereby interfering with ASC speck formation and blocking the activation of the NLRP3-inflammasome. These data demonstrate that reovirus employs EphA2 to suppress inflammasome activation in AECs and that EphA2 deficiency causes a pathological exacerbation of asthma in an ova-induced asthma model.

Alternaria induces airway epithelial cytokine expression independent of protease‐activated receptor

A novel fungal allergen, Alternaria (Alt), has been previously shown to associate with the pathogenesis of allergic rhinitis and bronchial asthma, particularly in arid and semi-arid regions. Airway epithelial cells are among the first to encounter Alt, and epithelial cytokine production and subsequent airway inflammation are early events in the response to Alt exposure. However, the underlying mechanism is unclear. As protease-activated receptor 2 (PAR2) has been implicated in most of the Alt-induced biological events, we investigated the regulation of airway inflammation and epithelial cytokine expression by PAR2. Wild-type (WT) and Par2 knockout (Par2-KO) mice were used to evaluate the in vivo role of PAR2. Primary human and mouse airway epithelial cells were used to examine the mechanistic basis of epithelial cytokine regulation in vitro. Surprisingly, Par2 deficiency had no negative impact on the change of lung function, inflammation and cytokine production in the mouse model of Alt-induced asthma. Alt-induced cytokine production in murine airway epithelial cells from Par2-KO mice was not significantly different from the WT cells. Consistently, PAR2 knockdown in human cells also had no effect on cytokine expression. In contrast, the cytokine expressions induced by synthetic PAR2 agonist or other asthma-related allergens (e.g. cockroach extracts) were indeed mediated via a PAR2-dependent mechanism. Finally, we found that EGFR pathway was responsible for Alt-induced epithelial cytokine expression. The activation of EGFR, but not PAR2, was likely to drive the airway inflammation and epithelial cytokine production induced by Alt.

MicroRNA-145-5p promotes asthma pathogenesis by inhibiting kinesin family member 3A expression in mouse airway epithelial cells.

BackgroundMicroRNA (miR)-145-5p is a respiratory disease biomarker, and is upregulated in asthma pathogenesis. However, its underlying mechanisms were unclear, so were investigated in the present study.MethodsA mouse model of asthma was established by challenge with house dust mite (HDM) extract. An miR-145-5p antagomir was administered nasally and expression of kinesin family member 3A (KIF3A) and miR-145-5p was measured by immunohistochemistry, PCR, and western blot. Eosinophils in lavage fluid and levels of interleukin (IL)-4, IL-5, and IL-13 were quantified. Airway hyper-responsiveness was measured and KIF3A expression was tested following miR-145-5p overexpression or interference in the 16HBE14o- airway epithelial cell line. The effects of miR-145-5p and KIF3A co-transfection in 16HBE14o- cells were examined on cytokine release, epithelial barrier dysfunction, and epithelial repair in HDM-exposed cells.ResultsKIF3A downregulation and miR-145-5p upregulation were noted in airway epithelial cells of HDM-exposed asthmatic mice, while miR-145-5p antagonism significantly improved symptoms. MiR-145-5p promoted the HDM-induced release of chemokines and inflammatory factors and epithelial barrier dysfunction, and suppressed epithelial repair by directly targeting KIF3A.ConclusionmiR-145-5p influenced HDM-induced epithelial cytokine release and epithelial barrier dysfunction via regulating KIF3 expression. It also affected epithelial repair, exacerbating the HDM-induced T helper 2-type immune response in mice.