Allergen Immunotherapy Enhances Airway Epithelial Antiviral Immunity in Patients with Allergic Asthma (VITAL Study): A Double-Blind Randomized Controlled Trial.

Rationale: Allergic asthma is linked to impaired bronchial epithelial secretion of IFNs, which may be causally linked to the increased risk of viral exacerbations. We have previously shown that allergen immunotherapy (AIT) effectively reduces asthma exacerbations and prevents respiratory infections requiring antibiotics; however, whether AIT alters antiviral immunity is still unknown. Objectives: To investigate the effect of house dust mite sublingual AIT (HDM-SLIT) on bronchial epithelial antiviral and inflammatory responses in patients with allergic asthma. Methods: In this double-blind, randomized controlled trial (VITAL [The Effect of Allergen Immunotherapy on Anti-viral Immunity in Patients with Allergic Asthma]), adult patients with HDM allergic asthma received HDM-SLIT 12-SQ or placebo for 24 weeks. Bronchoscopy was performed at baseline and at Week 24, which included sampling for human bronchial epithelial cells. Human bronchial epithelial cells were cultured at baseline and at Week 24 and stimulated with the viral mimic polyinosinic:polycytidylic acid (poly(I:C)). mRNA expression was quantified using qRT-PCR, and protein concentrations were measured using multiplex ELISA. Measurements and Main Results: Thirty-nine patients were randomized to HDM-SLIT (n = 20) or placebo (n = 19). HDM-SLIT resulted in increased polyinosinic:polycytidylic acid-induced expression of IFN-ß at both the gene (P = 0.009) and protein (P = 0.02) levels. IFN-λ gene expression was also increased (P = 0.03), whereas IL-33 tended to be decreased (P = 0.09). On the other hand, proinflammatory cytokines IL-6 (P = 0.009) and TNF-α (tumor necrosis factor-α) (P = 0.08) increased compared with baseline in the HDM-SLIT group. There were no significant changes in TSLP (thymic stromal lymphopoietin), IL-4, IL-13, and IL-10. Conclusions: HDM-SLIT improves bronchial epithelial antiviral resistance to viral infection. These results potentially explain the efficacy of HDM-SLIT in reducing exacerbations in allergic asthma. Clinical trial registered with www.clinicaltrials.gov (NCT04100902).

House dust mite sensitization and exposure affects bronchial epithelial anti-microbial response to viral stimuli in patients with asthma.

INTRODUCTION: Allergen exposure worsens viral-triggered asthma exacerbations and could predispose the host to secondary bacterial infections. We have previously demonstrated that exposure to house dust mite (HDM) reduced TLR-3-induced IFN-ß in human bronchial epithelial cells (HBECs) from healthy donors. We hypothesize that HDM sensitization in different ways may be involved in both viral and bacterial resistance of HBECs in asthma. In this study, the role of HDM sensitization and effects of HDM exposure on viral stimulus-challenged HBECs from asthmatic donors have been explored with regard to expression and release of molecules involved in anti-viral and anti-bacterial responses, respectively. METHODS: HBECs from HDM-sensitized (HDM+) and unsensitized (HDM-) patients with asthma were used. HBECs were exposed to HDM or heat inactivated (hi)-HDM (20 µg/ml) for 24 h prior to stimulation with the viral infection mimic, Poly(I:C), for 3 or 24 h. Samples were analyzed with ELISA and RT-qPCR for ß-defensin-2, IFN-ß, TSLP, and neutrophil-recruiting mediators: IL-8 and TNF-⍺. NFκB signaling proteins p105, p65, and IκB-⍺ were analyzed by Western blot. RESULTS: Poly(I:C)-induced IFN-ß expression was reduced in HBECs from HDM + compared to HDM- patients (p = 0.05). In vitro exposure of HBECs to HDM furthermore reduced anti-microbial responses to Poly(I:C) including ß-defensin-2, IL-8, and TNF-⍺, along with reduced NFκB activity. This was observed in HBECs from asthma patients sensitized to HDM, as well as in non-sensitized patients. By contrast, Poly (I:C)-induced release of TSLP, a driver of T2 inflammation, was not reduced with exposure to HDM. CONCLUSION: Using HBECs challenged with viral infection mimic, Poly(I:C), we demonstrated that allergic sensitization to HDM was associated with impaired anti-viral immunity and that HDM exposure reduced anti-viral and anti-bacterial defense molecules, but not TSLP, across non-allergic as well as allergic asthma. These data suggest a role of HDM in the pathogenesis of asthma exacerbations evoked by viral infections including sequential viral-bacterial and viral-viral infections.

Direct effects of mast cell proteases, tryptase and chymase, on bronchial epithelial integrity proteins and anti-viral responses.

BACKGROUND: Mast cells (MCs) are known to contribute to both acute and chronic inflammation. Bronchial epithelial cells are the first line of defence against pathogens and a deficient anti-viral response has been suggested to play a role in the pathogenesis of asthma exacerbations. However, effects of MC mediators on bronchial epithelial immune response have been less studied. The aim of this study is to investigate the direct effects of stimulation with MC proteases, tryptase and chymase, on inflammatory and anti-viral responses in human bronchial epithelial cells (HBECs). METHOD: Cultured BEAS-2b cells and primary HBECs from 3 asthmatic patients were stimulated with tryptase or chymase (0.1 to 0.5 µg/ml) for 1, 3, 6 and 24 h. To study the effects of MC mediators on the anti-viral response, cells were stimulated with 10 µg/ml of viral mimic Poly (I:C) for 3 and 24 h following pre-treatment with 0.5 µg/ml tryptase or chymase for 3 h. Samples were analysed for changes in pro-inflammatory and anti-viral mediators and receptors using RT-qPCR, western blot and Luminex. RESULTS: Tryptase and chymase induced release of the alarmin ATP and pro-inflammatory mediators IL-8, IL-6, IL-22 and MCP-1 from HBECs. Moreover, tryptase and chymase decreased the expression of E-cadherin and zonula occludens-1 expression from HBECs. Pre-treatment of HBECs with tryptase and chymase further increased Poly (I:C) induced IL-8 release at 3 h. Furthermore, tryptase significantly reduced type-I and III interferons (IFNs) and pattern recognition receptor (PRR) expression in HBECs. Tryptase impaired Poly (I:C) induced IFN and PRR expression which was restored by treatment of a serine protease inhibitor. Similar effects of tryptase on inflammation and anti-viral responses were also confirmed in primary HBECs from asthmatic patients. CONCLUSION: MC localization within the epithelium and the release of their proteases may play a critical role in asthma pathology by provoking pro-inflammatory and alarmin responses and downregulating IFNs. Furthermore, MC proteases induce downregulation of epithelial junction proteins which may lead to barrier dysfunction. In summary, our data suggests that mast cells may contribute towards impaired anti-viral epithelial responses during asthma exacerbations mediated by the protease activity of tryptase.

IL-1ß mediates lung neutrophilia and IL-33 expression in a mouse model of viral-induced asthma exacerbation.

BACKGROUND: Viral-induced asthma exacerbations, which exhibit both Th1-type neutrophilia and Th2-type inflammation, associate with secretion of Interleukin (IL)-1ß. IL-1ß induces neutrophilic inflammation. It may also increase Th2-type cytokine expression. We hypothesised that IL-1ß is causally involved in both Th1 and Th2 features of asthma exacerbations. This hypothesis is tested in our mouse model of viral stimulus-induced asthma exacerbation. METHOD: Wild-type (WT) and IL-1ß deficient (IL-1ß-/-) mice received house dust mite (HDM) or saline intranasally during three weeks followed by intranasal dsRNA (PolyI:C molecule known for its rhinovirus infection mimic) for three consecutive days to provoke exacerbation. Bronchoalveolar lavage fluid was analysed for inflammatory cells and total protein. Lung tissues were stained for neutrophilic inflammation and IL-33. Tissue homogenates were analysed for mRNA expression of Muc5ac, CXCL1/KC, TNF-α, CCL5, IL-25, TSLP, IL-33, IL-1ß, CCL11 and CCL2 using RT-qPCR. RESULTS: Expression of IL-1ß, neutrophil chemoattractants, CXCL1 and CCL5, the Th2-upstream cytokine IL-33, and Muc5ac were induced at exacerbation in WT mice and were significantly inhibited in IL-1ß-/- mice at exacerbation. Effects of HDM alone were not reduced in IL-1ß-deficient mice. CONCLUSION: Without being involved in the baseline HDM-induced allergic asthma, IL-1ß signalling was required to induce neutrophil chemotactic factors, IL-33, and Muc5ac expression at viral stimulus-induced exacerbation. We suggest that IL-1ß has a role both in neutrophilic and Th2 inflammation at viral-induced asthma exacerbations.

Mast Cell Tryptase Promotes Airway Remodeling by Inducing Anti-Apoptotic and Cell Growth Properties in Human Alveolar and Bronchial Epithelial Cells.

Bronchial and alveolar remodeling and impaired epithelial function are characteristics of chronic respiratory diseases. In these patients, an increased number of mast cells (MCs) positive for serine proteases, tryptase and chymase, infiltrate the epithelium and alveolar parenchyma. However, little is known regarding the implication of intraepithelial MCs on the local environment, such as epithelial cell function and properties. In this study, we investigated whether MC tryptase is involved in bronchial and alveolar remodeling and the mechanisms of regulation during inflammation. Using novel holographic live cell imaging, we found that MC tryptase enhanced human bronchial and alveolar epithelial cell growth and shortened the cell division intervals. The elevated cell growth induced by tryptase remained in a pro-inflammatory state. Tryptase also increased the expression of the anti-apoptotic protein BIRC3, as well as growth factor release in epithelial cells. Thus, our data imply that the intraepithelial and alveolar MC release of tryptase may play a critical role in disturbing bronchial epithelial and alveolar homeostasis by altering cell growth-death regulation.

C57Bl/6N mice have an attenuated lung inflammatory response to dsRNA compared to C57Bl/6J and BALB/c mice.

BACKGROUND: Lower respiratory infections caused by ssRNA viruses are a major health burden globally. Translational mouse models are a valuable tool for medical research, including research on respiratory viral infections. In in vivo mouse models, synthetic dsRNA can be used as a surrogate for ssRNA virus replication. However, studies investigating how genetic background of mice impacts the murine lung inflammatory response to dsRNA is lacking. Hence, we have compared lung immunological responses of BALB/c, C57Bl/6N and C57Bl/6J mice to synthetic dsRNA. METHODS: dsRNA was administered intranasally to BALB/c, C57Bl/6N and C57Bl/6J mice once/day for three consecutive days. Lactate dehydrogenase (LDH) activity, inflammatory cells, and total protein concentration were analyzed in bronchoalveolar lavage fluid (BALF). Pattern recognition receptors levels (TLR3, MDA5 and RIG-I) were measured in lung homogenates using RT-qPCR and western blot. Gene expression of IFN-ß, TNF-α, IL-1ß and CXCL1 was assessed in lung homogenates by RT-qPCR. ELISA was used to analyze protein concentrations of CXCL1 and IL-1ß in BALF and lung homogenates. RESULTS: BALB/c and C57Bl/6J mice showed infiltration of neutrophils to the lung, and an increase in total protein concentration and LDH activity in response to dsRNA administration. Only modest increases in these parameters were observed for C57Bl/6N mice. Similarly, dsRNA administration evoked an upregulation of MDA5 and RIG-I gene and protein expression in BALB/c and C57Bl/6J, but not C57Bl/6N, mice. Further, dsRNA provoked an increase in gene expression of TNF-α in BALB/c and C57Bl/6J mice, IL-1ß only in C57Bl/6N mice and CXCL1 exclusively in BALB/c mice. BALF levels of CXCL1 and IL-1ß were increased in BALB/c and C57Bl/6J mice in response to dsRNA, whereas the response of C57Bl/6N was blunt. Overall, inter-strain comparisons of the lung reactivity to dsRNA revealed that BALB/c, followed by C57Bl/6J, had the most pronounced respiratory inflammatory responses, while the responses of C57Bl/6N mice were attenuated. CONCLUSIONS: We report clear differences of the lung innate inflammatory response to dsRNA between BALB/c, C57Bl/6J and C57Bl/6N mice. Of particular note, the highlighted differences in the inflammatory response of C57Bl/6J and C57Bl/6N substrains underscore the value of strain selection in mouse models of respiratory viral infections.

Restoring myocardial infarction-induced long-term memory impairment by targeting the cystic fibrosis transmembrane regulator.

BACKGROUND: Cognitive impairment is a serious comorbidity in heart failure patients, but effective therapies are lacking. We investigated the mechanisms that alter hippocampal neurons following myocardial infarction (MI). METHODS: MI was induced in male C57Bl/6 mice by left anterior descending coronary artery ligation. We utilised standard procedures to measure cystic fibrosis transmembrane regulator (CFTR) protein levels, inflammatory mediator expression, neuronal structure, and hippocampal memory. Using in vitro and in vivo approaches, we assessed the role of neuroinflammation in hippocampal neuron degradation and the therapeutic potential of CFTR correction as an intervention. FINDINGS: Hippocampal dendrite length and spine density are reduced after MI, effects that associate with decreased neuronal CFTR expression and concomitant microglia activation and inflammatory cytokine expression. Conditioned medium from lipopolysaccharide-stimulated microglia (LCM) reduces neuronal cell CFTR protein expression and the mRNA expression of the synaptic regulator post-synaptic density protein 95 (PSD-95) in vitro. Blocking CFTR activity also down-regulates PSD-95 in neurons, indicating a relationship between CFTR expression and neuronal health. Pharmacologically correcting CFTR expression in vitro rescues the LCM-mediated down-regulation of PSD-95. In vivo, pharmacologically increasing hippocampal neuron CFTR expression improves MI-associated alterations in neuronal arborisation, spine density, and memory function, with a wide therapeutic time window. INTERPRETATION: Our results indicate that CFTR therapeutics improve inflammation-induced alterations in hippocampal neuronal structure and attenuate memory dysfunction following MI. FUNDING: Knut and Alice Wallenberg Foundation [F 2015/2112]; Swedish Research Council [VR; 2017-01243]; the German Research Foundation [DFG; ME 4667/2-1]; Hjärnfonden [FO2021-0112]; The Crafoord Foundation; Åke Wibergs Stiftelse [M19-0380], NMMP 2021 [V2021-2102]; the Albert Påhlsson Research Foundation; STINT [MG19-8469], Lund University; Canadian Institutes of Health Research [PJT-153269] and a Heart and Stroke Foundation of Ontario Mid-Career Investigator Award.

Mast cell tryptase enhances wound healing by promoting migration in human bronchial epithelial cells.

Epithelial damage and increase of intraepithelial mast cells (MC) are characteristics of asthma. The role of MC mediator tryptase and the protease-activated receptor-2 (PAR2) on epithelial wound healing is not fully investigated. Stimulation of bronchial epithelial cells (BECs) with tryptase promoted gap closure, migration and cellular speed compared to controls. Stimulated BECs had higher expression of migration marker CD151 compared to controls. Proliferation marker KI67 was upregulated in tryptase-stimulated BECs compared to controls. Treatment with PAR2 antagonist I-191 reduced gap closure, migration and cell speed compared to BECs stimulated with tryptase. We found that tryptase enhances epithelial wound healing by increased migration and proliferation, which is in part regulated via PAR2. Our data suggest that tryptase might be beneficial in tissue repair under baseline conditions. However, in a pathological context such as asthma with increased numbers of activated MCs, it might lead to epithelial remodeling and loss of function.

Imiquimod Boosts Interferon Response, and Decreases ACE2 and Pro-Inflammatory Response of Human Bronchial Epithelium in Asthma.

Background: Both anti-viral and anti-inflammatory bronchial effects are warranted to treat viral infections in asthma. We sought to investigate if imiquimod, a TLR7 agonist, exhibits such dual actions in ex vivo cultured human bronchial epithelial cells (HBECs), targets for SARS-CoV-2 infectivity. Objective: To investigate bronchial epithelial effects of imiquimod of potential importance for anti-viral treatment in asthmatic patients. Methods: Effects of imiquimod alone were examined in HBECs from healthy (N=4) and asthmatic (N=18) donors. Mimicking SARS-CoV-2 infection, HBECs were stimulated with poly(I:C), a dsRNA analogue, or SARS-CoV-2 spike-protein 1 (SP1; receptor binding) with and without imiquimod treatment. Expression of SARS-CoV-2 receptor (ACE2), pro-inflammatory and anti-viral cytokines were analyzed by RT-qPCR, multiplex ELISA, western blot, and Nanostring and proteomic analyses. Results: Imiquimod reduced ACE2 expression at baseline and after poly(I:C) stimulation. Imiquimod also reduced poly(I:C)-induced pro-inflammatory cytokines including IL-1ß, IL-6, IL-8, and IL-33. Furthermore, imiquimod increased IFN-ß expression, an effect potentiated in presence of poly(I:C) or SP1. Multiplex mRNA analysis verified enrichment in type-I IFN signaling concomitant with suppression of cytokine signaling pathways induced by imiquimod in presence of poly(I:C). Exploratory proteomic analyses revealed potentially protective effects of imiquimod on infections. Conclusion: Imiquimod triggers viral resistance mechanisms in HBECs by decreasing ACE2 and increasing IFN-ß expression. Additionally, imiquimod improves viral infection tolerance by reducing viral stimulus-induced epithelial cytokines involved in severe COVID-19 infection. Our imiquimod data highlight feasibility of producing pluripotent drugs potentially suited for anti-viral treatment in asthmatic subjects.

TLR3/TAK1 signalling regulates rhinovirus-induced interleukin-33 in bronchial smooth muscle cells.

BACKGROUND: Asthma exacerbations are commonly associated with rhinovirus (RV) infection. Interleukin-33 (IL-33) plays an important role during exacerbation by enhancing Type 2 inflammation. Recently we showed that RV infects bronchial smooth muscle cells (BSMCs) triggering production of interferons and IL-33. Here we compared levels of RV-induced IL-33 in BSMCs from healthy and asthmatic subjects, and explored the involvement of pattern-recognition receptors (PRRs) and downstream signalling pathways in IL-33 expression. METHOD: BSMCs from healthy and severe and non-severe asthmatic patients were infected with RV1B or stimulated with the PRR agonists poly(I:C) (Toll-like receptor 3 (TLR3)), imiquimod (TLR7) and poly(I:C)/LyoVec (retinoic acid-inducible gene 1 (RIG-I)/melanoma differentiation-associated protein 5 (MDA5)). Knockdown of TLR3, RIG-I and MDA5 was performed, and inhibitors targeting TBK1, nuclear factor-κB (NF-κB) and transforming growth factor (TGF)-ß-activated kinase 1 (TAK1) were used. Gene and protein expression were assessed. RESULTS: RV triggered IL-33 gene and protein expression in BSMCs. BSMCs from patients with non-severe asthma showed higher baseline and RV-induced IL-33 gene expression compared to cells from patients with severe asthma and healthy controls. Furthermore, RV-induced IL-33 expression in BSMCs from healthy and asthmatic individuals was attenuated by knockdown of TLR3. Inhibition of TAK1, but not NF-κB or TBK1, limited RV-induced IL-33. The cytokine secretion profile showed higher production of IL-33 in BSMCs from patients with non-severe asthma compared to healthy controls upon RV infection. In addition, BSMCs from patients with non-severe asthma had higher levels of RV-induced IL-8, TNF-α, IL-1ß, IL-17A, IL-5 and IL-13. CONCLUSION: RV infection caused higher levels of IL-33 and increased pro-inflammatory and Type 2 cytokine release in BSMCs from patients with non-severe asthma. RV-induced IL-33 expression was mainly regulated by TLR3 and downstream via TAK1. These signalling molecules represent potential therapeutic targets for treating asthma exacerbations.

Oxidative Stress Attenuates TLR3 Responsiveness and Impairs Anti-viral Mechanisms in Bronchial Epithelial Cells From COPD and Asthma Patients.

COPD and asthma exacerbations are commonly triggered by rhinovirus infection. Potentially promoting exacerbations, impaired anti-viral signaling and attenuated viral clearance have been observed in diseased bronchial epithelium. Oxidative stress is a feature of inflammation in asthma and COPD and is prominent during exacerbations. It is not known whether oxidative stress affects the anti-viral signaling capacity. Bronchial epithelial cells from asthmatic and COPD donors were infected with rhinovirus or treated with the oxidative stressor H2O2 followed by exposure to the synthetic viral replication intermediate poly(I:C). Poly(I:C) was used to ascertain a constant infection-like burden. Gene and protein levels of antioxidants as well as anti-viral responses were measured 3 and 24 h post poly(I:C) exposure. Rhinovirus infection and poly(I:C) stimulation induced protein levels of the antioxidants SOD1 and SOD2. In asthmatic bronchial epithelial cells pre-treatment with H2O2 dose-dependently decreased the antioxidant response to poly(I:C), suggesting exaggerated oxidative stress. Further, poly(I:C)-induced IFNß gene expression was reduced after pre-treatment with H2O2. This epithelial effect was associated with a reduced expression of the pattern recognition receptors RIG-I, MDA5 and TLR3 both on gene and protein level. Pre-treatment with H2O2 did not alter antioxidant responses in COPD bronchial epithelial cells and, more modestly than in asthma, reduced poly(I:C)-induced IFNß gene expression. Knockdown of TLR3 but not RIG-I/MDA5 abrogated impairment of poly(I:C)-induced IFNß gene expression by H2O2. We developed a method by which we could demonstrate that oxidative stress impairs anti-viral signaling in bronchial epithelial cells from asthmatic and COPD patients, most pronounced in asthma. The impairment apparently reflects reduced responsiveness of TLR3. These present findings shed light on molecular mechanisms potentially causing reduced interferon responses to rhinovirus infection at exacerbations in asthma and COPD. Together, our findings suggest a possible self-perpetuating vicious cycle underlying recurrent exacerbations, leading to an impaired anti-viral response, which in turn leads to viral-induced exacerbations, causing more airway inflammation.

Electrochemical reduction of quinones: interfacing experiment and theory for defining effective radii of redox moieties.

Using cyclic voltammetry, we examined the dependence of the reduction potentials of six quinones on the concentration of the supporting electrolyte. An increase in the electrolyte concentration, resulting in an increase in the solution polarity, caused positive shifts of the reduction potentials. We ascribed the observed changes in the potentials to the dependence of the solvation energy of the quinones and their anions on the media polarity. Analysis of the reduction potentials, using the Born solvation energy equation, yielded unfeasibly small values for the effective radii of the quinone species, that is, the experimentally obtained effective radii were up to 4-fold smaller than the radii of the solvation cavities that we calculated for the quinones. The nonspherical shapes of the quinones, along with the uneven charge density distribution in their anions, encompassed the underlying reasons for the discrepancies between the obtained experimental and theoretical values for the radii of these redox species. The generalized Born approach, which does not treat the solvated species as single spheres, provided means for addressing this discrepancy and yielded effective radii that were relatively close to the measured values.

Benefits of ICT adoption and use in regional general medical practices: a pilot study.

This paper presents a pilot study of benefits derived from information and communications technology(ICT) adoption and use in medical practices in regional Australia. The study involved 122 regional medical practitioners. The results show that like the more general small business sector, the perception of certain benefits is associated with the size of the practice (in terms of employee levels) and/or the gender of the respondent practitioner. The data also showed that the level of skill of certain software used within the practice was significantly associated with the level of perceived benefit derived from ICT adoption and use.