Airway Epithelial Cells In Response Research Articles

Cystic Fibrosis Clinical Isolates of Aspergillus fumigatus Induce Similar Muco-inflammatory Responses in Primary Airway Epithelial Cells.

Aspergillus is increasingly associated with lung inflammation and mucus plugging in early cystic fibrosis (CF) disease during which conidia burden is low and strains appear to be highly diverse. It is unknown whether clinical Aspergillus strains vary in their capacity to induce epithelial inflammation and mucus production. We tested the hypothesis that individual colonising strains of Aspergillus fumigatus would induce different responses. Ten paediatric CF Aspergillus isolates were compared along with two systemically invasive clinical isolates and an ATCC reference strain. Isolates were first characterised by ITS gene sequencing and screened for antifungal susceptibility. Three clusters (A−C) of Aspergillus isolates were identified by ITS. Antifungal susceptibility was variable, particularly for itraconazole. Submerged CF and non-CF monolayers as well as differentiated primary airway epithelial cell cultures were incubated with conidia for 24 h to allow germination. None of the clinical isolates were found to significantly differ from one another in either IL-6 or IL-8 release or gene expression of secretory mucins. Clinical Aspergillus isolates appear to be largely homogenous in their mucostimulatory and immunostimulatory capacities and, therefore, only the antifungal resistance characteristics are likely to be clinically important.

Characterization of the SARS-CoV-2 Host Response in Primary Human Airway Epithelial Cells from Aged Individuals.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19), a global pandemic characterized by an exaggerated immune response and respiratory illness. Age (>60 years) is a significant risk factor for developing severe COVID-19. To better understand the host response of the aged airway epithelium to SARS-CoV-2 infection, we performed an in vitro study using primary human bronchial epithelial cells from donors >67 years of age differentiated on an air–liquid interface culture. We demonstrate that SARS-CoV-2 infection leads to early induction of a proinflammatory response and a delayed interferon response. In addition, we observed changes in the genes and pathways associated with cell death and senescence throughout infection. In summary, our study provides new and important insights into the temporal kinetics of the airway epithelial innate immune response to SARS-CoV-2 in older individuals.

Comprehensive Transcriptomic Analysis Identifies Novel Antiviral Factors Against Influenza A Virus Infection.

Influenza A virus (IAV) has a higher genetic variation, leading to the poor efficiency of traditional vaccine and antiviral strategies targeting viral proteins. Therefore, developing broad-spectrum antiviral treatments is particularly important. Host responses to IAV infection provide a promising approach to identify antiviral factors involved in virus infection as potential molecular drug targets. In this study, in order to better illustrate the molecular mechanism of host responses to IAV and develop broad-spectrum antiviral drugs, we systematically analyzed mRNA expression profiles of host genes in a variety of human cells, including transformed and primary epithelial cells infected with different subtypes of IAV by mining 35 microarray datasets from the GEO database. The transcriptomic results showed that IAV infection resulted in the difference in expression of amounts of host genes in all cell types, especially those genes participating in immune defense and antiviral response. In addition, following the criteria of P<0.05 and |logFC|≥1.5, we found that some difference expression genes were overlapped in different cell types under IAV infection via integrative gene network analysis. IFI6, IFIT2, ISG15, HERC5, RSAD2, GBP1, IFIT3, IFITM1, LAMP3, USP18, and CXCL10 might act as key antiviral factors in alveolar basal epithelial cells against IAV infection, while BATF2, CXCL10, IFI44L, IL6, and OAS2 played important roles in airway epithelial cells in response to different subtypes of IAV infection. Additionally, we also revealed that some overlaps (BATF2, IFI44L, IFI44, HERC5, CXCL10, OAS2, IFIT3, USP18, OAS1, IFIT2) were commonly upregulated in human primary epithelial cells infected with high or low pathogenicity IAV. Moreover, there were similar defense responses activated by IAV infection, including the interferon-regulated signaling pathway in different phagocyte types, although the differentially expressed genes in different phagocyte types showed a great difference. Taken together, our findings will help better understand the fundamental patterns of molecular responses induced by highly or lowly pathogenic IAV, and the overlapped genes upregulated by IAV in different cell types may act as early detection markers or broad-spectrum antiviral targets.

Krüppel-like factor 5 regulates wound repair and the innate immune response in human airway epithelial cells

A complex network of transcription factors regulates genes involved in establishing and maintaining key biological properties of the human airway epithelium. However, detailed knowledge of the contributing factors is incomplete. Here we characterize the role of Krüppel-like factor 5 (KLF5), in controlling essential pathways of epithelial cell identity and function in the human lung. RNA-seq following siRNA-mediated depletion of KLF5 in the Calu-3 lung epithelial cell line identified significant enrichment of genes encoding chemokines and cytokines involved in the proinflammatory response and also components of the junctional complexes mediating cell adhesion. To determine direct gene targets of KLF5, we defined the cistrome of KLF5 using ChIP-seq in both Calu-3 and 16HBE14o− lung epithelial cell lines. Occupancy site concordance analysis revealed that KLF5 colocalized with the active histone modification H3K27ac and also with binding sites for the transcription factor CCAAT enhancer-binding protein beta (C/EBPβ). Depletion of KLF5 increased both the expression and secretion of cytokines including IL-1β, a response that was enhanced following exposure to Pseudomonas aeruginosa lipopolysaccharide. Calu-3 cells exhibited faster rates of repair after KLF5 depletion compared with negative controls in wound scratch assays. Similarly, CRISPR-mediated KLF5-null 16HBE14o− cells also showed enhanced wound closure. These data reveal a pivotal role for KLF5 in coordinating epithelial functions relevant to human lung disease.

Atractylodin attenuates the expression of MUC5AC and extracellular matrix in lipopolysaccharide-induced airway inflammation by inhibiting the NF-κB pathway.

This study aimed to explore the effects of atractylodin (ATR) on lipopolysaccharide (LPS)-induced inflammatory response in human airway epithelial cells. The cytotoxicity was assessed by CCK-8 assay. The mRNA expression and concentration of interleukin (IL)-6, IL-8, and mucin 5AC (MUC5AC) were measured by qRT-PCR and ELISA, respectively. Western blotting was performed to determine protein expression. We found that LPS stimulation increased the mRNA expression and concentrations of IL-6, IL-8, and MUC5AC, as well as the expression of Col-I and FN in 16HBE cells, but this effect of LPS was attenuated by ATR treatment. Mechanistically, ATR suppressed LPS-induced activation of the NF-κB pathway in 16HBE cells. Moreover, ATR repressed ovalbumin-induced airway inflammation and NF-kB pathway in mice. In conclusion, ATR attenuated the expression of MUC5AC and ECM in LPS-induced airway inflammation by inhibiting the NF-κB pathway.

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.

Microbial interaction: Prevotella spp. reduce P. aeruginosa induced inflammation in cystic fibrosis bronchial epithelial cells

BackgroundIn Cystic Fibrosis (CF) airways, the dehydrated, thick mucus promotes the establishment of persistent polymicrobial infections and drives chronic airways inflammation. This also predisposes the airways to further infections, the vicious, self-perpetuating cycle causing lung damage and progressive lung function decline. The airways are a poly-microbial environment, containing both aerobic and anaerobic bacterial species. Pseudomonas aeruginosa (P. aeruginosa) infections contribute to the excessive inflammatory response in CF, but the role of anaerobic Prevotella spp., frequently found in CF airways, is not known. MaterialsWe assessed innate immune signalling in CF airway epithelial cells in response to clinical strains of P. histicola, P. nigresens and P. aeruginosa. CFBE41o- cells were infected with P. aeruginosa (MOI 100, 2h) followed by infection with P. histicola or P. nigrescens (MOI 100, 2h). Cells were incubated under anaerobic conditions for the duration of the experiments. ResultsOur study shows that P. histicola and P. nigresens can reduce the growth of P. aeruginosa and dampen the inflammatory response in airway epithelial cells. We specifically illustrate that the presence of the investigated Prevotella spp. reduces Toll-like-receptor (TLR)-4, MAPK, NF-κB(p65) signalling and cytokine release (Interleukin (IL)-6, IL-8) in mixed infections. ConclusionOur work, for the first time, strongly indicates a relationship between P. aeruginosa and anaerobic Prevotella spp.. The observed modified NF-κB and MAPK signalling indicates some mechanisms underlying this interaction that could offer a novel therapeutic approach to combat chronic P. aeruginosa infection in people with CF.

ORMDL3 regulates poly I:C induced inflammatory responses in airway epithelial cells

BackgroundOroscomucoid 3 (ORMDL3) has been linked to susceptibility of childhood asthma and respiratory viral infection. Polyinosinic-polycytidylic acid (poly I:C) is a synthetic analog of viral double-stranded RNA, a toll-like receptor 3 (TLR3) ligand and mimic of viral infection.MethodsTo investigate the functional role of ORMDL3 in the poly I:C-induced inflammatory response in airway epithelial cells, ORMDL3 knockdown and over-expression models were established in human A549 epithelial cells and primary normal human bronchial epithelial (NHBE) cells. The cells were stimulated with poly I:C or the Th17 cytokine IL-17A. IL-6 and IL-8 levels in supernatants, mRNA levels of genes in the TLR3 pathway and inflammatory response from cell pellets were measured. ORMDL3 knockdown models in A549 and BEAS-2B epithelial cells were then infected with live human rhinovirus (HRV16) followed by IL-6 and IL-8 measurement.ResultsORMDL3 knockdown and over-expression had little influence on the transcript levels of TLR3 in airway epithelial cells. Time course studies showed that ORMDL3-deficient A549 and NHBE cells had an attenuated IL-6 and IL-8 response to poly I:C stimulation. A549 and NHBE cells over-expressing ORMDL3 released relatively more IL-6 and IL-8 following poly I:C stimulation. IL-17A exhibited a similar inflammatory response in ORMDL3 knockdown and over-expressing cells, but co-stimulation of poly I:C and IL-17A did not significantly enhance the IL-6 and IL-8 response. Transcript abundance of IFNB following poly I:C stimulation was not significantly altered by ORMDL3 knockdown or over-expression. Dampening of the IL-6 response by ORMDL3 knockdown was confirmed in HRV16 infected BEAS-2B and A549 cells.ConclusionsORMDL3 regulates the viral inflammatory response in airway epithelial cells via mechanisms independent of the TLR3 pathway.

Endoplasmic reticulum stress/XBP1 promotes airway mucin secretion under the influence of neutrophil elastase.

Endoplasmic reticulum (ER) stress is an important reaction of airway epithelial cells in response to various stimuli, and may also be involved in the mucin secretion process. In the present study, the effect of ER stress on neutrophil elastase (NE)-induced mucin (MUC)5AC production in human airway epithelial cells was explored. 16HBE14o-airway epithelial cells were cultured and pre-treated with the reactive oxygen species (ROS) inhibitor, N-acetylcysteine (NAC), or the ER stress chemical inhibitor, 4-phenylbutyric acid (4-PBA), or the cells were transfected with inositol-requiring kinase 1α (IRE1α) small interfering RNA (siRNA) or X-box-binding protein 1 (XBP1) siRNA, respectively, and subsequently incubated with NE. The results obtained revealed that NE increased ROS production in the 16HBE14o-cells, with marked increases in the levels of ER stress-associated proteins, such as glucose-regulated protein 78 (GRP78), activating transcription factor 6 (ATF6), phosphorylated protein kinase R-like endoplasmic reticulum kinase (pPERK) and phosphorylated (p)IRE1α. The protein and mRNA levels of spliced XBP1 were also increased, and the level of MUC5AC protein was notably increased. The ROS scavenger NAC and ER stress inhibitor 4-PBA were found to reduce ER stress-associated protein expression and MUC5AC production and secretion. Further analyses revealed that MUC5AC secretion was also attenuated by IRE1α and XBP1 siRNAs, accompanied by a decreased mRNA expression of spliced XBP1. Taken together, these results demonstrate that NE induces ER stress by promoting ROS production in 16HBE14o-airway epithelial cells, leading to increases in MUC5AC protein production and secretion via the IRE1α and XBP1 signaling pathways.

The SWI/SNF-Related, Matrix Associated, Actin-Dependent Regulator of Chromatin A4 Core Complex Represses Respiratory Syncytial Virus-Induced Syncytia Formation and Subepithelial Myofibroblast Transition.

Epigenetics plays an important role in the priming the dynamic response of airway epithelial cells to infectious and environmental stressors. Here, we examine the epigenetic role of the SWI/SNF Related, Matrix Associated, Actin Dependent Regulator of Chromatin A4 (SMARCA4) in the epithelial response to RSV infection. Depletion of SMARCA4 destabilized the abundance of the SMARCE1/ARID1A SWI/SNF subunits, disrupting the innate response and triggering a hybrid epithelial/mesenchymal (E/M) state. Assaying SMARCA4 complex-regulated open chromatin domains by transposase cleavage -next generation sequencing (ATAC-Seq), we observed that the majority of cleavage sites in uninfected cells have reduced chromatin accessibility. Paradoxically, SMARCA4 complex-depleted cells showed enhanced RSV-inducible chromatin opening and gene expression in the EMT pathway genes, MMP9, SNAI1/2, VIM, and CDH2. Focusing on the key MMP9, we observed that SMARCA4 complex depletion reduced basal BRD4 and RNA Polymerase II binding, but enhanced BRD4/Pol II binding in response to RSV infection. In addition, we observed that MMP9 secretion in SMARCA4 complex deficient cells contributes to mesenchymal transition, cellular fusion (syncytia) and subepithelial myofibroblast transition. We conclude the SMARCA4 complex is a transcriptional repressor of epithelial plasticity, whose depletion triggers a hybrid E/M state that affects the dynamic response of the small airway epithelial cell in mucosal remodeling via paracrine MMP9 activity.

Specific Assay of Negative Strand Template to Quantify Intracellular Levels of Rhinovirus Double-Stranded RNA.

Human rhinovirus infections are a major trigger for acute exacerbations of lower airway diseases, including asthma and chronic obstructive pulmonary disease. Disease exacerbation is thought to be regulated via double-stranded RNA (dsRNA)-mediated signaling of proinflammatory and host defense responses in airway epithelial cells. Despite the central role of dsRNA in regulating host cell responses, no method for the quantitative assessment of dsRNA levels during HRV infections has been developed. Conventional RT-PCR for the negative strand template is not effective as self-priming results in apparent signals, even in the absence of primer during reverse transcription. To avoid these issues, we developed a selective assay for the negative strand template that uses a chimeric primer containing a 5′ non-viral sequence for reverse transcription and a primer using the non-viral sequence during subsequent PCR. We established that this assay avoided issues of self-priming and is strand specific, as it is unaffected even in the presence of a 1000-fold excess of positive strand. Assays in primary human airway epithelial cells showed that negative strand was detectable within 6 h of virus exposure and peaked at 18 h after virus exposure. The temporal pattern of negative strand induction mirrored that of genomic RNA but was always 1000-fold lower than positive strand, indicating that the negative strand levels regulate levels of dsRNA formation. This assay will permit relative quantification of dsRNA during studies of HRV regulation of epithelial cell function.

ACE2 expression is related to the interferon response in airway epithelial cells but is that functional for SARS-CoV-2 entry?

In vitro interferon (IFN)α treatment of primary human upper airway basal cells has been shown to drive ACE2 expression, the receptor of SARS-CoV-2. The protease furin is also involved in mediating SARS‐CoV‐2 and other viral infections, although its association with early IFN response has not been evaluated yet. In order to assess the in vivo relationship between ACE2 and furin expression and the IFN response in nasopharyngeal cells, we first examined ACE2 and furin levels and their correlation with the well-known marker of IFNs’ activation, ISG15, in children (n = 59) and adults (n = 48), during respiratory diseases not caused by SARS-CoV-2. A strong positive correlation was found between ACE2 expression, but not of furin, and ISG15 in all patients analyzed. In addition, type I and III IFN stimulation experiments were performed to examine the IFN-mediated activation of ACE2 isoforms (full-length and truncated) and furin in epithelial cell lines. Following all the IFNs treatments, only the truncated ACE2 levels, were upregulated significantly in the A549 and Calu3 cells, in particular by type I IFNs. If confirmed in vivo following IFNs’ activation, the induction of the truncated ACE2 isoform only would not enhance the risk of SARS‐CoV‐2 infection in the respiratory tract.

MicroRNA miR-146a-5p inhibits the inflammatory response and injury of airway epithelial cells via targeting TNF receptor-associated factor 6

ABSTRACT Bronchial asthma is a common respiratory disease, which is characterized by airway inflammation, remodeling and hyperresponsiveness. MicroRNAs (miRNAs), as reported, are implicated in the pathogenesis of many diseases, but how miRNAs-146a-5p (miR-146a-5p) works in asthma remains inconclusive. In this work, we proved that miR-146a-5p expression was inhibited in asthma patients’ plasma and platelet activating factor (PAF)-induced human small airway epithelial cells (HSAECs). MiR-146a-5p up-regulation ameliorated the inflammatory reaction and cell barrier damage of HSAECs induced by PAF, and inhibited the apoptosis; besides, miR-146a-5p down-regulation functioned oppositely. In addition, miR-146a-5p could target TNF receptor-associated factor 6 (TRAF6) and negatively regulate its expression. TRAF6 overexpression could counterract the impact of miR-146a-5p up-regulation on PAF-induced inflammation, cell barrier damage and apoptosis of HSAECs. Collectively, miR-146a-5p may protect airway epithelial cells and inhibit the pathogenesis of asthma via targeting TRAF6.

Bitter Taste Receptors in the Airway Cells Functions.

G protein-coupled receptors (GPCRs) play a central role in regulating the functions of a diverse range of cell types in the airway. Taste 2 receptor (T2R) family of GPCRs is responsible for the transduction of bitter taste; however, recent studies have demonstrated that different subtypes of T2Rs and key components of T2R signaling are expressed in several extra-oral tissues including airways with many physiological roles. In the lung, expression of T2Rs has been confirmed in multiple airway cell types including airway smooth muscle (ASM) cells, various epithelial cell subtypes, and on both resident and migratory immune cells. Most importantly, activation of T2Rs with a variety of putative agonists elicits unique signaling in ASM and specialized airway epithelial cells resulting in the inhibition of ASM contraction and proliferation, promotion of ciliary motility, and innate immune response in chemosensory airway epithelial cells. Here we discuss the expression of T2Rs and the mechanistic basis of their function in the structural cells of the airways with some useful insights on immune cells in the context of allergic asthma and other upper airway inflammatory disorders. Emphasis on T2R biology and pharmacology in airway cells has an ulterior goal of exploiting T2Rs for therapeutic benefit in obstructive airway diseases.

Comprehensive Transcriptomic Analysis Identifies Cellular Immune Response Against Influenza A Virus Infection

Influenza A virus (IAV) has the higher genetic variation and reassortment, the traditional vaccine and virus-directed antiviral strategies have hardly shown successful efficacy and even lead to serious resistance, therefor developing the broad-spectrum antiviral treatments is particularly important. Host response to IAV infection provides a promising alternative approach to find host factors that are involved in the replication of viruses as molecular drug target. In this study, in order to better illustrate how IAV infection triggers cellular response and understand virus-host interaction, we systematically studied the expression profiles of host gene in a variety of human cells (including epithelial cells, primary epithelial cells) infected with different subtypes of IAV in this research by collecting and excavating 40 microarray data from Gene Expression Omnibus (GEO). The transcriptome analysis results showed that each of influenza strains led to the certain amounts of genes differentially regulated in all cell types, especially those genes belonging to immune defense and antiviral response, but we found that the overlapping regulated genes accounted for only a few in all cell types with different subtypes of IAV infection following the the criteria of P<0.05 and |logFC|≥1.5, because of the difference of IAV strains and infected cell types. Integrative gene network analysis found that IFI6, IFIT2, ISG15, HERC5, RSAD2, GBP1, IFIT3, IFITM1, LAMP3, USP18 and CXCL10 may act as key signatures in alveolar basal epithelial cells for IAV infection, while BATF2, CXCL10, IFI44L, IL6 and OAS2 played a key role in airway epithelial cell response to different subtypes of IAV infection. In addition, we also revealed that some overlaps (BATF2, IFI44L, IFI44, HERC5, OASL, CXCL10, MX2, OAS2, XAF1, IFIT3, USP18, IFIH1, OAS1, IFIT2 and IFIT1) were commonly upregulated in human primary epithelial cells infected by different influenza strains of high or low pathogenicity. Additionally, in the phagocytes, the difference gene expression pattern in different subtypes of IAV infection was found, but phagocytes could elicit the similar defence response against virus infection. Our findings will help us understand the fundamental patterns of molecular responses induced by highly or lowly pathogenic IAV originating from different species and the overlapped genes upregulated by IAV in different cell types may be as key biomarkers to improve effective early detection and treatment. Funding Statement: This work was supported by Key Lab of Process Analysis and Control of Sichuan Universities (No.2018001), The Project-sponsored by Sichuan Province for ROCS (0903/00021728) and NSFC (81902073). Declaration of Interests: No potential conflict of interest was reported by the authors.

A long noncoding RNA antisense to ICAM-1 is involved in allergic asthma associated hyperreactive response of airway epithelial cells.

Epithelial cells of the conducting airways are a pivotal first line of defense against airborne pathogens and allergens that orchestrate inflammatory responses and mucociliary clearance. Nonetheless, the molecular mechanisms responsible for epithelial hyperreactivity associated with allergic asthma are not completely understood. Transcriptomic analysis of human airway epithelial cells (HAECs), differentiated in-vitro at air-liquid interface (ALI), showed 725 differentially expressed immediate-early transcripts, including putative long noncoding RNAs (lncRNAs). A novel lncRNA on the antisense strand of ICAM-1 or LASI was identified, which was induced in LPS-primed HAECs along with mucin MUC5AC and its transcriptional regulator SPDEF. LPS-primed expression of LASI, MUC5AC, and SPDEF transcripts were higher in ex-vivo cultured asthmatic HAECs that were further augmented by LPS treatment. Airway sections from asthmatics with increased mucus load showed higher LASI expression in MUC5AC+ goblet cells following multi-fluorescent in-situ hybridization and immunostaining. LPS- or IL-13-induced LASI transcripts were mostly enriched in the nuclear/perinuclear region and were associated with increased ICAM-1, IL-6, and CXCL-8 expression. Blocking LASI expression reduced the LPS or IL-13-induced epithelial inflammatory factors andMUC5AC expression, suggesting that the novel lncRNA LASI could play a key role in LPS-primed trained airway epithelial responses that are dysregulated in allergic asthma.

Baicalin suppresses type 2 immunity through breaking off the interplay between mast cell and airway epithelial cell

Ethnopharmacological relevanceThe traditional Japanese herbal medicine Shin'iseihaito was reported to ameliorate the airway type 2 inflammatory response in clinical and experimental studies. Airway type 2 inflammatory diseases, including bronchial asthma and eosinophilic chronic rhinosinusitis (ECRS), often coexist and interact with each other. However, it is still unclear how Shin'iseihaito exerts its pharmacological effects on cells involved in airway mucosa. Aim of the studyThis study aims to examine the direct effect of baicalin, a representative bioactive compound of Shin'iseihaito, on type 2 immune responses in human airway epithelial cells and mast cells. Material and methodsWe measured the plasma pharmacokinetics of flavonoids derived from Shin'iseihaito and investigated the effects of baicalin on type 2 immune responses in human airway epithelial cells and human mast cells. ResultsBaicalin, wogonin, and wogonoside were detected in the plasma. The maximum plasma concentration of baicalin was highest at 1610 ng/ml (3.6 μM). In the normal human bronchial epithelial cells treated with baicalin, with or without stimulation by IFN-γ, the IL-33 expression was significantly downregulated. However, baicalin treatment did not affect the levels of thymic stromal lymphopoietin and IL-25. We noted that IL-33-dependent expression of tryptase mRNA in mast cells was significantly inhibited by baicalin. Also, the expression of IL-5 in mast cells enhanced by stimulation with TSLP plus IL-1β was significantly downregulated by baicalin treatment. Moreover, the enhancement of IL-13 expression in mast cells by IL-33 simulation was also significantly inhibited by baicalin. ConclusionsOur results prove that by breaking off the vicious circle of mast cells and airway epithelial cells, baicalin may be an effective alternative therapeutic option for the treatment of type 2 inflammatory diseases, such as ECRS and comorbid asthma.

ABCF1 Regulates dsDNA-induced Immune Responses in Human Airway Epithelial Cells.

Background: The airway epithelium represents a critical component of the human lung that helps orchestrate defenses against respiratory tract viral infections, which are responsible for more than 2.5 million deaths/year globally. Innate immune activities of the airway epithelium rely on Toll-like receptors (TLRs), nucleotide binding and leucine-rich-repeat pyrin domain containing (NLRP) receptors, and cytosolic nucleic acid sensors. ATP Binding Cassette (ABC) transporters are ubiquitous across all three domains of life—Archaea, Bacteria, and Eukarya—and expressed in the human airway epithelium. ABCF1, a unique ABC family member that lacks a transmembrane domain, has been defined as a cytosolic nucleic acid sensor that regulates CXCL10, interferon-β expression, and downstream type I interferon responses. We tested the hypothesis that ABCF1 functions as a dsDNA nucleic acid sensor in human airway epithelial cells important in regulating antiviral responses.Methods: Expression and localization experiments were performed using in situ hybridization and immunohistochemistry in human lung tissue, while confirmatory transcript and protein expression was performed in human airway epithelial cells. Functional experiments were performed with siRNA methods in a human airway epithelial cell line. Complementary transcriptomic analyses were performed to explore the contributions of ABCF1 to gene expression patterns.Results: Using archived human lung and human airway epithelial cells, we confirm expression of ABCF1 gene and protein expression in these tissue samples, with a role for mediating CXCL10 production in response to dsDNA viral mimic challenge. Although, ABCF1 knockdown was associated with an attenuation of select genes involved in the antiviral responses, Gene Ontology analyses revealed a greater interaction of ABCF1 with TLR signaling suggesting a multifactorial role for ABCF1 in innate immunity in human airway epithelial cells.Conclusion: ABCF1 is a candidate cytosolic nucleic acid sensor and modulator of TLR signaling that is expressed at gene and protein levels in human airway epithelial cells. The precise level where ABCF1 protein functions to modulate immune responses to pathogens remains to be determined but is anticipated to involve IRF-3 and CXCL10 production.

Respiratory syncytial virus infection-induced mucus secretion by down-regulation of miR-34b/c-5p expression in airway epithelial cells.

Severe RSV infection is the main cause of hospitalization to children under the age of five. The regulation of miRNAs on the severity of RSV infection is unclear. The aim of the study was to identify the critical differential expression miRNAs (DE miRNAs) that can regulate the pathological response in RSV‐infected airway epithelial cells. In this study, miRNA and mRNA chips of RSV‐infected airway epithelia from Gene Expression Omnibus (GEO) were screened and analysed, separately. DE miRNAs‐targeted genes were performed for further pathway and process enrichment analysis. DE miRNA‐targeted gene functional network was constructed on the basis of miRNA‐mRNA interaction. The screened critical miRNA was also investigated by bioinformatics analysis. Then, RSV‐infected human bronchial epithelial cells (HBECs) were constructed to verify the expression of the DE miRNAs. Finally, specific synthetic DE miRNAs mimics were used to confirm the effect of DE miRNAs on the RSV‐infected HBECs. 45 DE miRNAs were identified from GEO62306 dataset. Our results showed that hsa‐mir‐34b‐5p and hsa‐mir‐34c‐5p decreased significantly in HBECs after RSV infection. Consistent with the biometric analysis, hsa‐mir‐34b/c‐5p is involved in the regulation of mucin expression gene MUC5AC. In RSV‐infected HBECs, the inducement of MUC5AC production by decreased hsa‐mir‐34b/c‐5p was partly mediated through activation of c‐Jun. These findings provide new insights into the mechanism of mucus obstruction after RSV infection and represent valuable targets for RSV infection and airway obstruction treatment.

Association between Two Single Nucleotide Polymorphisms of Thymic Stromal Lymphopoietin (TSLP) Gene and Asthma in Iranian Population.

Thymic stromal lymphopoietin (TSLP) is a cytokine similar to IL-7, which is released by airway epithelial cells in response to injury and inflammation. Current literature is contradictory about the association between different single nucleotide polymorphisms (SNPs) of the TSLP gene and asthma development in different countries. We aimed to evaluate the association between two common TSLP SNPs (rs2289276 and rs2289278) and the risk of asthma in the Iranian population. Genotyping of the TSLP gene was performed in 126 adult asthmatic patients and 300 controls; using the TaqMan genotyping assay. Moreover, total serum IgE level and eosinophil count were assessed. The results indicated that the TT genotype of rs2289276 was inversely associated with the risk of asthma (p=0.002). A similar inverse association was detected in subgroups of atopic (p=0.001) and non-atopic (p=0.005) asthma. Moreover, the TT genotype of this SNP was more prevalent in severe and late-onset categories of asthma. In subgroup analysis, a significant sex-specific association between rs2290276 and asthma was observed in women (p=0.004). The prevalence of rs2289276 was extremely low, which made it infeasible to perform any further analysis. Overall, our findings indicated that rs2290276 SNP of the TSLP gene has a protective phenotype against asthma development in the Iranian population.