Genomic characterization and therapeutic utilization of IL-13-responsive sequences in asthma.

Epithelial responses to the cytokine interleukin-13 (IL-13) cause airway obstruction in asthma. Here we utilized multiple genomic techniques to identify IL-13-responsive regulatory elements in bronchial epithelial cells and used these data to develop a CRISPR interference (CRISPRi)-based therapeutic approach to downregulate airway obstruction-inducing genes in a cell type- and IL-13-specific manner. Using single-cell RNA sequencing (scRNA-seq) and acetylated lysine 27 on histone 3 (H3K27ac) chromatin immunoprecipitation sequencing (ChIP-seq) in primary human bronchial epithelial cells, we identified IL-13-responsive genes and regulatory elements. These sequences were functionally validated and optimized via massively parallel reporter assays (MPRAs) for IL-13-inducible activity. The top secretory cell-selective sequence from the MPRA, a novel, distal enhancer of the sterile alpha motif pointed domain containing E-26 transformation-specific transcription factor (SPDEF) gene, was utilized to drive CRISPRi and knock down SPDEF or mucin 5AC (MUC5AC), both involved in pathologic mucus production in asthma. Our work provides a catalog of cell type-specific genes and regulatory elements involved in IL-13 bronchial epithelial response and showcases their use for therapeutic purposes.

Early biomolecular changes in brain microvascular endothelial cells under Epstein-Barr virus influence: a Raman microspectroscopic investigation.

The brain microvascular endothelial cells (ECs) play an important role in protecting the brain from hazardous pathogens. However, some viral pathogens can smartly modulate the endothelial pathways to gain entry inside the brain. Further, these viruses can cause endothelial dysfunction which could develop serious neurological ailments. Epstein-Barr virus (EBV), an oncogenic virus, has also been linked to various neurological disorders. The virus primarily infects epithelial and B cells, however, it also has a tendency to infect ECs and cause endothelial activation. However, the impact of EBV influence on ECs is still underexplored. Studying the early events of virus-mediated cellular modulation could help in understanding the virus' infection strategy or aftermath. Raman microspectroscopy has been widely utilized in biomedical sciences to decipher cellular changes. To understand the EBV-influenced EC modulation by studying intracellular biomolecular changes at early time points, we utilized the Raman microspectroscopy tool. We treated the ECs with EBV and acquired the Raman spectra at different time points (2, 4, 6, 12, 24 and 36 h) and different sites (nucleus and periphery) to check changes in Raman intensities associated with specific biomolecules. In the EBV-treated cells, the status of various biomolecules in terms of Raman intensities was observed to be altered compared with uninfected cells. Specifically, the cholesterol, polysaccharide, nucleotides, nucleic acid and proline moieties were altered at different time points. We also investigated the possible correlation between these molecules using molecular network analysis and observed various associated factors. These factors could be influenced by EBV to alter the associated biomolecular levels. Our study paves the pathway to study EBV infection in human brain microvascular ECs and highlights specific biomolecular alterations, which can be focused for further mechanistic investigations.

Primary and Secondary Genotoxicity of Nanoparticles: Establishing a Co-Culture Protocol for Assessing Micronucleus Using Flow Cytometry.

Genotoxicity is an important endpoint to assess for understanding the risks associated with nanoparticles (NPs). Most genotoxicity studies performed on NPs have focused on primary genotoxicity analyzed by comet- or micronuclei (MN) assay using microscopic scoring. Here, we established a protocol for a more efficient version of MN assessment using flow cytometry and, importantly, both primary and secondary (inflammation-driven) genotoxicity was assessed. Human bronchial epithelial cells (HBEC-3kt) were exposed to nickel oxide (NiO) NPs directly or indirectly. The indirect exposure was done to assess secondary genotoxicity, and in this case immune cells (THP-1 derived macrophages) were exposed on inserts and the HBEC were cultured in the lower compartment. The results in monocultures showed that no increased MN formation was observed in the HBEC cells but instead a clear MN induction was noted in THP-1 cells indicating higher sensitivity. No MN formation was either observed when the HBEC were indirectly exposed, but an increase in DNA strand breaks was detected using the comet assay. Taken together, the present study emphasizes the feasibility of assessing primary and secondary genotoxicity and, furthermore, shows a clear MN induction in THP-1 monoculture following NiO NPs exposure.

The Induction of Alpha-1 Antitrypsin by Vitamin D in Human T Cells Is TGF-ß Dependent: A Proposed Anti-inflammatory Role in Airway Disease.

Background: Vitamin D upregulates anti-inflammatory and antimicrobial pathways that promote respiratory health. Vitamin D synthesis is initiated following skin exposure to sunlight, however nutritional supplementation can be required to address deficiency, for example during the winter months or due to cultural constraints. We recently reported that 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) treatment induced alpha-1 antitrypsin (AAT) expression in CD4+, but not CD8+ T cells, with evidence supporting an immunoregulatory role. Research Question: To understand the relationship between vitamin D, lung AAT levels and T lymphocytes further we investigated whether TGF-ß is required as a co-factor for 1,25(OH)2D3-induced upregulation of AAT by vitamin D in CD8+ T cells in vitro and correlated circulating vitamin D levels with lung AAT levels in vivo. Results: 1,25(OH)2D3 in combination with TGF-ß1 increased AAT expression by CD8+ T cells, as well as VDR and RXRα gene expression, which may partly explain the requirement for TGF-ß. CD4+ T cells may also require autocrine stimulation with TGF-ß as a co-factor since 1,25(OH)2D3 was associated with increased TGF-ß bioactivity and neutralisation of TGF-ß partially abrogated 1,25(OH)2D3-induced SERPINA1 gene expression. Neither CD4+ nor CD8+ T cells responded to the circulating vitamin D precursor, 25-hydroxyvitamin D3 for induction of SERPINA1, suggesting that local generation of 1,25(OH)2D3 is required. Transcriptional gene profiling studies previously demonstrated that human bronchial epithelial cells rapidly increased TGF-ß2 gene expression in response to 1,25(OH)2D3. Here, human epithelial cells responded to precursor 25(OH)D3 to increase bioactive TGF-ß synthesis. CD8+ T cells responded comparably to TGF-ß1 and TGF-ß2 to increase 1,25(OH)2D3-induced AAT. However, CD8+ T cells from adults with AAT-deficiency, homozygous for the Z allele of SERPINA1, were unable to mount this response. AAT levels in the airways of children with asthma and controls correlated with circulating 25(OH)D3. Conclusions: Vitamin D increases AAT expression in human T cells and this response is impaired in T cells from individuals homozygous for the Z allele of SERPINA1 in a clinic population. Furthermore, a correlation between circulating vitamin D and airway AAT is reported. We propose that vitamin D-induced AAT contributes to local immunomodulation and airway health effects previously attributed to vitamin D.

Synthetic hydrosilicate nanotubes induce low pro-inflammatory and cytotoxic responses compared to natural chrysotile in lung cell cultures.

Asbestos (Mg-hydrosilicate; chrysotile) is known to cause pleural diseases, pulmonary fibrosis and lung cancers, via mechanisms strongly depending on diameter-length ratio and possibly metal content. A critical question is whether synthetic hydrosilicate nanotubes (NTs) of short length possess little toxic potential compared to chrysotile. Five Mg- and two NiNTs of different lengths were assessed for cytotoxicity and pro-inflammatory responses in THP-1 macrophages and human bronchial epithelial lung cells (HBEC3-KT), in comparison with chrysotile. NT lengths/diameters were characterized by TEM, surface areas by BET- and BJH analysis, and chemical composition by XRD. The different Mg- and NiNTs induced little cytotoxicity in both cell models, in contrast to chrysotile that induced marked cytotoxicity. The two longest synthetic MgNTs, with median lengths of 3 and 5 µm, induced increased levels of pro-inflammatory cytokines in THP-1 macrophages, but much less than chrysotile (median length 15 µm) and silica nanoparticles (Si10). The shortest NTs did not induce any increase in cytokines. In HBEC3-KT cells, all synthetic NTs induced no or only small changes in cytokine responses, in contrast to chrysotile and Si10. The synthetic NTs induced lower TGF-ß responses than chrysotile in both cell models. In conclusion, the pro-inflammatory responses were associated with the length of synthetic hydrosilicate NTs in THP-1 macrophages, but not in HBEC3-KT cells. Notably, the shortest NTs showed no or little pro-inflammatory activity or cytotoxicity in both cell models. Such a safety by design approach is important for development of new materials being candidates for various new products.

Reduced expression of miR-146a in human bronchial epithelial cells alters neutrophil migration.

BACKGROUND: The role of miRNAs in the pathogenesis and determining the phenotypes of asthma is not fully elucidated. miR-146a has been previously shown to suppress inflammatory responses in different cells. In this study, we investigated the functions of miR-146a in human bronchial epithelial cells (HBECs) in association with neutrophilic, eosinophilic, and paucigranulocytic phenotypes of asthma. METHODS: Bronchial brushing specimens and brochial mucosal biopsy samples were collected from adult patients with asthma and from age- and gender-matched non-asthmatic individuals. The expression of miR-146a in bronchial brushing specimens, bronchial biopsy tissue sections or cultured primary bronchial epithelial cells was analyzed by RT-qPCR or by in situ hybridization. The expression of direct and indirect miR-146a target genes was determined by RT-qPCR or ELISA. The migration of neutrophils was studied by neutrophil chemotaxis assay and flow cytometry. For statistical analysis, unpaired two-way Student's t test, one-way ANOVA or linear regression analysis were used. RESULTS: Reduced expression of miR-146a was found in bronchial brushing specimens from asthma patients as compared to non-asthmatics and irrespective of the phenotype of asthma. In the same samples, the neutrophil attracting chemokines IL-8 and CXCL1 showed increased expression in patients with neutrophilic asthma and increased IL-33 expression was found in patients with eosinophilic asthma. Linear regression analysis revealed a significant negative association between the expression of miR-146a in bronchial brushings and neutrophil cell counts in bronchoalveolar lavage fluid of patients with asthma. In bronchial biopsy specimens, the level of miR-146a was highest in the epithelium as determined with in situ hybridization. In primary conventional HBEC culture, the expression of miR-146a was induced in response to the stimulation with IL-17A, TNF-α, and IL-4. The mRNA expression and secretion of IL-8 and CXCL1 was inhibited in both stimulated and unstimulated HBECs transfected with miR-146a mimics. Supernatants from HBECs transfected with miR-146a had reduced capability of supporting neutrophil migration in neutrophil chemotaxis assay. CONCLUSION: Our results suggest that decreased level of miR-146a in HBECs from patients with asthma may contribute to the development of neutrophilic phenotype of asthma.

Regional Differences in Airway Epithelial Cells Reveal Tradeoff between Defense against Oxidative Stress and Defense against Rhinovirus.

Rhinovirus is a leading cause of acute respiratory infections and asthma attacks, but infections are also frequently cleared from the nasal mucosa without causing symptoms. We sought to better understand host defense against rhinovirus by investigating antiviral defense in primary human nasal and bronchial airway epithelial cells cultured ex vivo. Surprisingly, upon rhinovirus infection or RIG-I stimulation, nasal-derived epithelial cells exhibited much more robust antiviral responses than bronchial-derived cells. Conversely, RIG-I stimulation triggered more robust activation of the NRF2-dependent oxidative stress response in bronchial cells compared to nasal cells. NRF2 activation dampened epithelial antiviral responses, whereas NRF2 knockdown enhanced antiviral responses and was protective during rhinovirus infection. These findings demonstrate a tradeoff in epithelial defense against distinct types of airway damage, namely, viral versus oxidative, and reveal differential calibration of defense responses in cells derived from different airway microenvironments.

Characterization of the proteome and lipidome profiles of human lung cells after low dose and chronic exposure to multiwalled carbon nanotubes.

The effects of long-term chronic exposure of human lung cells to multi-walled carbon nanotubes (MWCNT) and their impact upon cellular proteins and lipids were investigated. Since the lung is the major target organ, an in vitro normal bronchial epithelial cell line model was used. Additionally, to better mimic exposure to manufactured nanomaterials at occupational settings, cells were continuously exposed to two non-toxic and low doses of a MWCNT for 13-weeks. MWCNT-treatment increased ROS levels in cells without increasing oxidative DNA damage and resulted in differential expression of multiple anti- and pro-apoptotic proteins. The proteomic analysis of the MWCNT-exposed cells showed that among more than 5000 identified proteins; more than 200 were differentially expressed in the treated cells. Functional analyses revealed association of these differentially regulated proteins to cellular processes such as cell death and survival, cellular assembly, and organization. Similarly, shotgun lipidomic profiling revealed accumulation of multiple lipid classes. Our results indicate that long-term MWCNT-exposure of human normal lung cells at occupationally relevant low-doses may alter both the proteome and the lipidome profiles of the target epithelial cells in the lung.

Clusterin and neuropilin-2 as potential biomarkers of tumor progression in benzo[a]pyrene-transformed 16HBE cells xenografted nude mouse model.

Benzo[a]pyrene (BaP) is a ubiquitous environment contaminant and its exposure could increase incidence of human lung cancer. In order to confirm and compare potential biomarkers of BaP-induce carcinogenesis and tumor progression, time-dependent changes of clusterin (CLU) and neuropilin-2 (NRP2) levels were evaluated in sera of BaP-transformed 16HBE cell line T-16HBE-C1 cells xenografted nude mice. Performance of CLU and NRP2 on tissue classification and tumor progression forecast was also calculated. Levels of CLU and NRP2 were significant elevated in both culture supernatant of T-16HBE-C1 cells and sera of T-16HBE-C1 cells xenografted nude mice compared with control. CLU and NRP2 were both found positively stained in tumor tissue. CLU and NRP2 alone could well predicate tumor progression in nude mice and CLU appeared to be more sensitive than NRP2. When both of them combined, performance of predication would improve. In conclusion, CLU and NRP2 could serve as potential biomarkers of tumor progression in nude mice xenografted with T-16HBE-C1 cells.

Effects on human bronchial epithelial cells following low-dose chronic exposure to nanomaterials: A 6-month transformation study.

The most plausible exposure route to manufactured nanomaterials (MNM) remains pulmonary inhalation. Yet, few studies have attempted to assess carcinogenic properties in vitro following long-term exposure of human pulmonary cells to low and occupationally relevant doses. The most advanced in vitro tests for carcinogenicity, the cell transformation assay (CTA), rely mostly on rodent cells and short-term exposure. We hypothesized that long-term exposure of human bronchial epithelial cells with a normal phenotype could be a valuable assay for testing carcinogenicity of nanomaterials. Therefore, this study (performed within the framework of the FP7-NANoREG project) assessed carcinogenic potential of chronic exposure (up to 6months) to low doses of multi-walled carbon nanotubes (MWCNT, NM-400 and NM-401) and TiO2 materials (NM62002 and KC7000). In order to harmonize and standardize the experiments, standard operating protocols of MNM dispersion (NANOGENOTOX) were used by three different NANoREG project partners. All nanomaterials showed low cytotoxicity in short-term tests for the tested doses (0.96 and 1.92µg/cm2). During long-term exposure, however, NM-401 clearly affected cell proliferation. In contrast, no cell transformation was observed for NM-401 by any of the partners. NM-400 and NM62002 formed some colonies after 3months. We conclude that agglomerated NM-401 in low doses affect cell proliferation but do not cause cell transformation in the CTA assay used.

Human primary airway epithelial cells isolated from active smokers have epigenetically impaired antiviral responses.

BACKGROUND: Cigarette smoking (CS) is the main risk factor for the development of chronic obstructive pulmonary disease (COPD) and most COPD exacerbations are caused by respiratory infections including influenza. Influenza infections are more severe in smokers. The mechanism of the increased risk and severity of infections in smokers is likely multifactorial, but certainly includes changes in immunologic host defenses. METHODS: We investigated retinoic acid-inducible protein I (RIG-I) and interferon (IFN) induction by influenza A virus (IAV) in human bronchial epithelial cells (HBEC) isolated from smokers or nonsmokers. Subcultured HBEC cells were infected with A/Puerto Rico/8/1934 (PR8) IAV at an MOI of 1. After 24 h of infection, cells and supernatants were collected for qRT-PCR, immunoblot or ELISA to determine RIG-I, Toll-like receptor3 (TLR3) and IFN expression levels. RESULTS: IAV exposure induced a vigorous IFN-ß, IFN-λ 1 and IFN-λ 2/3 antiviral response in HBEC from nonsmokers and significant induction of RIG-I and TLR3. In cells from smokers, viral RIG-I and TLR3 mRNA induction was reduced 87 and 79 % compared to the response from nonsmokers. CS exposure history was associated with inhibition of viral induction of the IFN-ß, IFN-λ1 and IFN-λ 2/3 mRNA response by 85, 96 and 95 %, respectively, from that seen in HBEC from nonsmokers. The demethylating agent 5-Aza-2-deoxycytidine reversed the immunosuppressive effects of CS exposure in HBEC since viral induction of all three IFNs was restored. IFN-ß induction of RIG-I and TLR3 was also suppressed in the cells from smokers. CONCLUSION: Our results suggest that active smoking reduces expression of antiviral cytokines in primary HBEC cells. This effect likely occurs via downregulation of RIG-I and TLR3 due to smoke-induced epigenetic modifications. Reduction in lung epithelial cell RIG-I and TLR3 responses may be a major mechanism contributing to the increased risk and severity of viral respiratory infections in smokers and to viral-mediated acute exacerbations of COPD.

Label-free quantitative proteomic analysis of benzo(a)pyrene-transformed 16HBE cells serum-free culture supernatant and xenografted nude mice sera.

To screen potential biomarkers of benzo(a)pyrene (BaP)-induced lung cancer, the proteomic profiles of BaP-transformed 16HBE cell line T-16HBE-C1 cells serum-free culture supernatant and xenografted nude mice sera were compared with those of 16HBE group by utilizing label-free quantitative proteomic strategy. By employing nano-LC-MS/MS technology followed by MaxQuant and Perseus processing, 489 differentially expressed proteins were identified between T-16HBE-C1 and 16HBE cells serum-free culture supernatant, and 49 significantly up-regulated proteins were identified in T-16HBE-C1 xenografted nude mice sera. Three proteins neuropilin-2 (NRP2), clusterin (CLU) and A-kinase anchor protein 12 (AKAP12) were up-regulated in the serum-free culture supernatant of T-16HBE-C1 cells. These 3 human proteins were present in the sera of nude mice xenografted with T-16HBE-C1 cells, but were undetectable in mice xenografted with 16HBE cells. The proteomic results of NRP2 and AKAP12 were confirmed by Western blotting and enzyme-linked immunosorbent assays, respectively. Moreover, the serum NRP2 levels were significantly elevated at the 4th day after tumor cell implantation and showed good positive correlation with tumor growth characterized by tumor volume. In conclusion, serum NRP2, CLU and AKAP12 could be potential biomarkers of BaP-induced lung cancer. The proteomic results will gain deeper insights into the mechanisms of BaP-induced carcinogenesis.

Effect of N-arachidonoyl-l-serine on human cerebromicrovascular endothelium.

N-arachidonoyl-l-serine (ARA-S) is an endogenous lipid, chemically related to the endocannabinoid, N-arachidonoyl ethanolamine (i.e., anandamide) and with similar physiologic and pathophysiologic functions. Reports indicate that ARA-S possesses vasoactive and neuroprotective properties resembling those of cannabinoids. However, in contrast to cannabinoids, ARA-S binds weakly to its known classical receptors, CB1 and CB2, and is therefore considered to be a 'cannabinoid-like' substance. The originally described ARA-S induced-endothelial-dependent vasorelaxation was not abrogated by CB1, CB2 receptor antagonists or TRPV1 competitive inhibitor. The present report demonstrates that ARA-S enhances the fluorescence staining of both cannabinoid receptors (CB1 and CB2) in human brain endothelial cells (HBEC). This reaction is specific since it was reduced by respective selective receptor antagonist (SR141716A and SR141728A). ARA-S alone or in the presence of ET-1 was shown to alter the cytoskeleton (actin). Both ARA-S stimulated phosphorylation of various kinases (MAPK, Akt, JNK and c-JUN) and alteration of cytoskeleton are mediated via CB1, CB2 and TRPV1 receptors. The findings also showed the involvement of Rho/Rock and PI3/Akt/NO pathways in the ARA-S-induced phosphorylation of kinases and actin reorganization in HBEC. All of the above mentioned ARA-S-induced effects were reduced by the treatment with LY294002 (inhibitor of PI3/Akt kinase), except MAPK kinase. In addition, MAPK, JNK, c-JUN phosphorylation were inhibited by H1152 (inhibitor of Rho/ROCK kinase), except Akt kinase. Furthermore, PI3/Akt pathway was inhibited by pretreatment with l-NAME (inhibitor of NOS). The findings suggest that ARA-S is a modulator of Rho kinase and may play a critical role in the regulation of its activity and subsequent effects on the cytoskeleton and its role in supporting essential cell functions like vasodilation, proliferation and movement.

PARK2-mediated mitophagy is involved in regulation of HBEC senescence in COPD pathogenesis.

Cigarette smoke (CS)-induced mitochondrial damage with increased reactive oxygen species (ROS) production has been implicated in COPD pathogenesis by accelerating senescence. Mitophagy may play a pivotal role for removal of CS-induced damaged mitochondria, and the PINK1 (PTEN-induced putative kinase 1)-PARK2 pathway has been proposed as a crucial mechanism for mitophagic degradation. Therefore, we sought to investigate to determine if PINK1-PARK2-mediated mitophagy is involved in the regulation of CS extract (CSE)-induced cell senescence and in COPD pathogenesis. Mitochondrial damage, ROS production, and cell senescence were evaluated in primary human bronchial epithelial cells (HBEC). Mitophagy was assessed in BEAS-2B cells stably expressing EGFP-LC3B, using confocal microscopy to measure colocalization between TOMM20-stained mitochondria and EGFP-LC3B dots as a representation of autophagosome formation. To elucidate the involvement of PINK1 and PARK2 in mitophagy, knockdown and overexpression experiments were performed. PINK1 and PARK2 protein levels in lungs from patients were evaluated by means of lung homogenate and immunohistochemistry. We demonstrated that CSE-induced mitochondrial damage was accompanied by increased ROS production and HBEC senescence. CSE-induced mitophagy was inhibited by PINK1 and PARK2 knockdown, resulting in enhanced mitochondrial ROS production and cellular senescence in HBEC. Evaluation of protein levels demonstrated decreased PARK2 in COPD lungs compared with non-COPD lungs. These results suggest that PINK1-PARK2 pathway-mediated mitophagy plays a key regulatory role in CSE-induced mitochondrial ROS production and cellular senescence in HBEC. Reduced PARK2 expression levels in COPD lung suggest that insufficient mitophagy is a part of the pathogenic sequence of COPD.

Rhinovirus infection causes steroid resistance in airway epithelium through nuclear factor κB and c-Jun N-terminal kinase activation.

BACKGROUND: Although inhaled glucocorticoids are the mainstays of asthma treatment, they are poorly effective at treating and preventing virus-induced asthma exacerbations. The major viruses precipitating asthma exacerbations are rhinoviruses. OBJECTIVE: We sought to evaluate whether rhinovirus infection interferes with the mechanisms of action of glucocorticoids. METHODS: Cultured primary human bronchial or transformed (A549) respiratory epithelial cells were infected with rhinovirus 16 (RV-16) before dexamethasone exposure. Glucocorticoid receptor (GR) α nuclear translocation, glucocorticoid response element (GRE) binding, and transactivation/transrepression functional readouts were evaluated by using immunocytochemistry, Western blotting, DNA binding assays, real-time quantitative PCR, coimmunoprecipitation, and ELISA techniques. Specific inhibitors of c-Jun N-terminal kinase (JNK) and of IκB kinase (IKK) were used to investigate the involvement of intracellular signaling pathways. RESULTS: RV-16 infection impaired dexamethasone-dependent (1) inhibition of IL-1ß-induced CXCL8 release, (2) induction of mitogen-activated protein kinase phosphatase 1 gene expression, and (3) binding of GR to GREs in airway epithelial cells. This was associated with impaired GRα nuclear translocation, as assessed by means of both immunochemistry (54.0% ± 6.8% vs 24.7% ± 3.8% GR-positive nuclei after 10 nmol/L dexamethasone treatment in sham- or RV-16-infected cells, respectively; P < .01) and Western blotting. RV-16 infection induced nuclear factor κB activation and GRα phosphorylation, which were prevented by inhibitors of IKK2 and JNK, respectively. In rhinovirus-infected cells the combination of JNK and IKK2 inhibitors totally restored dexamethasone suppression of CXCL8 release, induction of mitogen-activated protein kinase phosphatase 1 gene expression, and GRα nuclear translocation. CONCLUSION: RV-16 infection of human airway epithelium induces glucocorticoid resistance. Inhibition of RV-16-induced JNK and nuclear factor κB activation fully reversed rhinovirus impairment of both GRα nuclear translocation and the transactivation/transrepression activities of glucocorticoids.

TNF-α-mediated bronchial barrier disruption and regulation by src-family kinase activation.

BACKGROUND: Because TNF-α is increased in severe asthma, we hypothesized that TNF-α contributes to barrier dysfunction and cell activation in bronchial epithelial cells. We further hypothesized that src-family kinase inhibition would improve barrier function in healthy cells in the presence of TNF-α and directly in cultures of severe asthmatic cells where the barrier is disrupted. OBJECTIVES: We assessed the effect of TNF-α, with or without src-family kinase inhibitor SU6656, on barrier properties and cytokine release in differentiated human bronchial epithelial cultures. Further, we tested the effect of SU6656 on differentiated primary cultures from severe asthma. METHODS: Barrier properties of differentiated human bronchial epithelial air-liquid interface cultures from healthy subjects and subjects with severe asthma were assessed with transepithelial electrical resistance and fluorescent dextran passage. Proteins were detected by immunostaining or Western blot analysis and cytokines by immunoassay. Mechanisms were investigated with src kinase and other inhibitors. RESULTS: TNF-α lowered transepithelial electrical resistance and increased fluorescent dextran permeability, caused loss of occludin and claudins from tight junctions with redistribution of p120 catenin and E-cadherin from adherens junctions, and also increased endogenous TNF-α, IL-6, IL-1ß, IL-8, thymic stromal lymphoprotein, and pro-matrix metalloprotease 9 release. SU6656 reduced TNF-α-mediated paracellular permeability changes, restored occludin, p120, and E-cadherin and lowered autocrine TNF-α release. Importantly, SU6656 improved the barrier properties of severe asthmatic air-liquid interface cultures. Redistribution of E-cadherin and p120 was observed in bronchial biopsies from severe asthmatic airways. CONCLUSIONS: Inhibiting TNF-α or src kinases may be a therapeutic option to normalize barrier integrity and cytokine release in airway diseases associated with barrier dysfunction.