Airway hyperresponsiveness correlates with airway TSLP in asthma independent of eosinophilic inflammation.

BACKGROUND: Thymic stromal lymphopoietin (TSLP) is released from the airway epithelium in response to various environmental triggers, inducing a type-2 inflammatory response, and is associated with airway inflammation, airway hyperresponsiveness (AHR), and exacerbations. TSLP may also induce AHR via a direct effect on airway smooth muscle and mast cells, independently of type-2 inflammation, although association between airway TSLP and AHR across asthma phenotypes has been described sparsely. OBJECTIVES: This study sought to investigate the association between AHR and levels of TSLP in serum, sputum, and bronchoalveolar lavage in patients with asthma with and without type-2 inflammation. METHODS: A novel ultrasensitive assay was used to measure levels of TSLP in patients with asthma (serum, n = 182; sputum, n = 81; bronchoalveolar lavage, n = 85) and healthy controls (serum, n = 47). The distribution and association among airway and systemic TSLP, measures of AHR, type-2 inflammation, and severity of disease were assessed. RESULTS: TSLP in sputum was associated with AHR independently of levels of eosinophils and fractional exhaled nitric oxide (ρ = 0.49, P = .005). Serum TSLP was higher in both eosinophil-high and eosinophil-low asthma compared to healthy controls: geometric mean: 1600 fg/mL (95% CI: 1468-1744 fg/mL) and 1294 fg/mL (95% CI: 1167-1435 fg/mL) versus 846 fg/mL (95% CI: 661-1082 fg/mL), but did not correlate with the level of AHR. Increasing age, male sex, and eosinophils in blood were associated with higher levels of TSLP in serum, whereas lung function, inhaled corticosteroid dose, and symptom score were not. CONCLUSIONS: The association between TSLP in sputum and AHR to mannitol irrespective of markers of type-2 inflammation further supports a role of TSLP in AHR that is partially independent of eosinophilic inflammation.

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).

Airway hyperresponsiveness reflects corticosteroid-sensitive mast cell involvement across asthma phenotypes.

BACKGROUND: Airway hyperresponsiveness is a hallmark of asthma across asthma phenotypes. Airway hyperresponsiveness to mannitol specifically relates to mast cell infiltration of the airways, suggesting inhaled corticosteroids to be effective in reducing the response to mannitol, despite low levels of type 2 inflammation. OBJECTIVE: We sought to investigate the relationship between airway hyperresponsiveness and infiltrating mast cells, and the response to inhaled corticosteroid treatment. METHODS: In 50 corticosteroid-free patients with airway hyperresponsiveness to mannitol, mucosal cryobiopsies were obtained before and after 6 weeks of daily treatment with 1600 µg of budesonide. Patients were stratified according to baseline fractional exhaled nitric oxide (Feno) with a cutoff of 25 parts per billion. RESULTS: Airway hyperresponsiveness was comparable at baseline and improved equally with treatment in both patients with Feno-high and Feno-low asthma: doubling dose, 3.98 (95% CI, 2.49-6.38; P < .001) and 3.85 (95% CI, 2.51-5.91; P < .001), respectively. However, phenotypes and distribution of mast cells differed between the 2 groups. In patients with Feno-high asthma, airway hyperresponsiveness correlated with the density of chymase-high mast cells infiltrating the epithelial layer (ρ, -0.42; P = .04), and in those with Feno-low asthma, it correlated with the density in the airway smooth muscle (ρ, -0.51; P = .02). The improvement in airway hyperresponsiveness after inhaled corticosteroid treatment correlated with a reduction in mast cells, as well as in airway thymic stromal lymphopoietin and IL-33. CONCLUSIONS: Airway hyperresponsiveness to mannitol is related to mast cell infiltration across asthma phenotypes, correlating with epithelial mast cells in patients with Feno-high asthma and with airway smooth muscle mast cells in patients with Feno-low asthma. Treatment with inhaled corticosteroids was effective in reducing airway hyperresponsiveness in both groups.

Phenotype and severity of asthma determines bronchial epithelial immune responses to a viral mimic.

BACKGROUND: Asthma is characterised by an aggravated immune response to respiratory viral infections. This phenomenon is a clinically well-recognised driver of acute exacerbations, but how different phenotypes of asthma respond immunologically to viruses is unclear. OBJECTIVES: To describe the association between different phenotypes and severity of asthma and bronchial epithelial immune responses to viral stimulation. METHODS: In the Immunoreact study, healthy subjects (n=10) and 50 patients with asthma were included; 30 (60%) were atopic, and 34 (68%) were eosinophilic; 14 (28%) had severe asthma. All participants underwent bronchoscopy with collection of bronchial brushings. Bronchial epithelial cells (BECs) were expanded and stimulated with the viral replication mimic poly (I:C) (Toll-like receptor (TLR)3 agonist) in vitro. The expression of TLR3-induced pro-inflammatory and antiviral responses of BECs were analysed using reverse transcriptase quantitative PCR and multiplex ELISA and compared across asthma phenotypes and severity of disease. RESULTS: Patients with atopic asthma had increased induction of interleukin (IL)-4, interferon (IFN)-ß, IL-6, tumour necrosis factor-α, and IL-1ß after poly (I:C) stimulation compared to non-atopic patients, whereas in patients with eosinophilic asthma only IL-6 and IL-8 induction was higher than in non-eosinophilic asthma. Patients with severe asthma displayed a decreased antiviral IFN-ß, and increased expression of IL-8, most pronounced in atopic and eosinophilic asthmatics. Furthermore, induction of IL-33 in response to poly (I:C) was increased in severe atopic and in severe eosinophilic asthma, but thymic stromal lymphopoietin only in severe eosinophilic asthma. CONCLUSIONS: The bronchial epithelial immune response to a viral mimic stimulation differs between asthma phenotypes and severities, which may be important to consider when targeting novel asthma treatments.

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.

Human host defense peptide LL-37 facilitates double-stranded RNA pro-inflammatory signaling through up-regulation of TLR3 expression in vascular smooth muscle cells.

OBJECTIVE: The importance of human host defense peptide LL-37 in vascular innate immunity is not understood. Here, we assess the impact of LL-37 on double-stranded RNA (dsRNA) signaling in human vascular smooth muscle cells. MATERIALS AND METHODS: Cellular import of LL-37 and synthetic dsRNA (poly I:C) were investigated by immunocytochemistry and fluorescence imaging. Transcript and protein expression were determined by qPCR, ELISA and Western blot. Knockdown of TLR3 was performed by siRNA. RESULTS: LL-37 was rapidly internalized, suggesting that it has intracellular actions. Co-stimulation with poly I:C and LL-37 enhanced pro-inflammatory IL-6 and MCP-1 transcripts several fold compared to treatment with poly I:C or LL-37 alone. Poly I:C increased IL-6 and MCP-1 protein production, and this effect was potentiated by LL-37. LL-37-induced stimulation of poly I:C signaling was not associated with enhanced import of poly I:C. Treatment with poly I:C and LL-37 in combination increased expression of dsRNA receptor TLR3 compared to stimulation with poly I:C or LL-37 alone. In TLR3 knockdown cells, treatment with poly I:C and LL-37 in combination had no effect on IL-6 and MCP-1 expression, showing loss of function. CONCLUSIONS: LL-37 potentiates dsRNA-induced cytokine production through up-regulation of TLR3 expression representing a novel pro-inflammatory mechanism.

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.

Increased expression of upstream TH2-cytokines in a mouse model of viral-induced asthma exacerbation.

BACKGROUND: Exacerbations of asthma caused by respiratory viral infections are serious conditions in need of novel treatment. To this end animal models of asthma exacerbations are warranted. We have shown that dsRNA challenges or rhinoviral infection produce exacerbation effects in mice with ovalbumin (OVA)-induced allergic asthma. However, house dust mite (HDM) is a more human asthma-relevant allergen than OVA. We thus hypothesised that dsRNA challenges in mice with HDM-induced experimental asthma would produce important translational features of asthma exacerbations. METHOD: Mouse airways were challenged locally with HDM or saline three times a week for three weeks to establish experimental asthma. Then daily local dsRNA challenges were given for three consecutive days to induce exacerbation. Bronchoalveolar lavage fluid (BALF) was analysed for inflammatory cells, total protein, the necrosis marker LDH and the alarmin ATP. Lung homogenates were analysed for mRNA expression (RT-qPCR) of TNF-α, CCL2, CCL5, IL-1ß, IL-33, thymic stromal lymphopoietin (TSLP), and IL-25 as well as pattern recognition receptors (PRRs) RIG-I, MDA5 and TLR3. Lung tissue IL-33 was analysed with ELISA and PRRs were quantified by western blot. Immunohistochemistry indicated lung distribution of IL-33. RESULTS: HDM challenge alone caused sustained increase in BALF total protein, eosinophils, lymphocytes and neutrophils, and transient increase in lung tissue expression of TSLP, IL-33 and TNF-α. dsRNA-induced exacerbation markedly and dose-dependently exaggerated these effects. Further, BALF levels of LDH and ATP, and lung tissue expression of CCL2, CCL5, IL-1ß, IL-25 and PRRs were increased exclusively at the exacerbations. Lung protein levels of IL-33 were transiently increased by HDM and further increased at exacerbation. CONCLUSION: We demonstrate several novel aspects of HDM-induced experimental asthma and added exacerbation effects of dsRNA. General inflammatory parameters in BALF such as exuded proteins, mixed granulocytes, LDH and ATP were increased at the present exacerbations as they are in human asthma exacerbations. We suggest that this model of asthma exacerbation involving dsRNA challenges given to mice with established HDM-induced asthma has translational value and suggest that it may be particularly suited for in vivo studies involving pharmacological effects on exacerbation-induced expression of major upstream TH2-cytokines; IL-33, TSLP and IL-25, as well as PRRs.

Rhinoviral stimuli, epithelial factors and ATP signalling contribute to bronchial smooth muscle production of IL-33.

BACKGROUND: Bronchial smooth muscle cells (BSMCs) from severe asthmatics have been shown to overexpress the Th2-driving and asthma-associated cytokine IL-33. However, little is known regarding factors involved in BSMC production of IL-33. Rhinovirus (RV) infections cause asthma exacerbations, which exhibit features of Th2-type inflammation. Here, we investigated the effects of epithelial-derived media and viral stimuli on IL-33 expression in human BSMCs. METHODS: Primary human BSMCs from healthy (n = 3) and asthmatic (n = 3) subjects were stimulated with conditioned media from primary human bronchial epithelial cells (BECs), double-stranded (ds)RNA, dsRNA/LyoVec, or infected with RV. BSMCs were also pretreated with the purinergic receptor antagonist suramin. IL-33 expression was analysed by RT-qPCR and western blot and ATP levels were determined in cell supernatants. RESULTS: RV infection and activation of TLR3 by dsRNA increased IL-33 mRNA and protein in healthy and asthmatic BSMCs. These effects were inhibited by dexamethasone. BSMC expression of IL-33 was also increased by stimulation of RIG-I-like receptors using dsRNA/LyoVec. Conditioned media from BECs induced BSMC expression of IL-33, which was further enhanced by dsRNA. BEC-derived medium and viral-stimulated BSMC supernatants exhibited elevated ATP levels. Blocking of purinergic signalling with suramin inhibited BSMC expression of IL-33 induced by dsRNA and BEC-derived medium. CONCLUSIONS: RV infection of BSMCs and activation of TLR3 and RIG-I-like receptors cause expression and production of IL-33. Epithelial-released factor(s) increase BSMC expression of IL-33 and exhibit positive interaction with dsRNA. Increased BSMC IL-33 associates with ATP release and is antagonised by suramin. We suggest that epithelial-derived factors contribute to baseline BSMC IL-33 production, which is further augmented by RV infection of BSMCs and stimulation of their pathogen-recognising receptors.

ERS International Congress 2023: highlights from the Airway Diseases Assembly.

In this review, early career and senior members of Assembly 5 (Airway Diseases, Asthma, COPD and Chronic Cough) present key recent findings pertinent to airway diseases that were presented during the European Respiratory Society International Congress 2023 in Milan, Italy, with a particular focus on asthma, COPD, chronic cough and bronchiectasis. During the congress, an increased number of symposia, workshops and abstract presentations were organised. In total, 739 abstracts were submitted for Assembly 5 and the majority of these were presented by early career members. These data highlight the increased interest in this group of respiratory diseases.

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.

ERS International Congress 2022: highlights from the Airway Diseases Assembly.

The European Respiratory Society (ERS) celebrated the return of an in-person meeting in Barcelona, Spain, after 2 years of virtual congresses. The ERS Congress 2022 programme was replete with symposia, skills workshops and abstract presentations from all 14 assemblies, encompassing over 3000 abstracts presented in the form of thematic poster discussion and oral presentations. In this article, highlights from the ERS Congress 2022 (including from thematic poster sessions, oral presentations and symposia from keynote speakers), presented by Assembly 5 (Airway diseases, asthma, COPD and chronic cough), are reviewed by Early Career Members and experts in the field, with the aim of presenting key recent findings in the field.

LL-37 and Double-Stranded RNA Synergistically Upregulate Bronchial Epithelial TLR3 Involving Enhanced Import of Double-Stranded RNA and Downstream TLR3 Signaling.

The human host defense peptide LL-37 influences double-stranded RNA signaling, but this process is not well understood. Here, we investigate synergistic actions of LL-37 and synthetic double-stranded RNA (poly I:C) on toll-like receptor 3 (TLR3) expression and signaling, and examine underlying mechanisms. In bronchial epithelial BEAS-2B cells, LL-37 potentiated poly I:C-induced TLR3 mRNA and protein expression demonstrated by qPCR and Western blot, respectively. Interestingly, these effects were associated with increased uptake of rhodamine-tagged poly I:C visualized by immunocytochemistry. The LL-37/poly I:C-induced upregulation of TLR3 mRNA expression was prevented by the endosomal acidification inhibitor chloroquine, indicating involvement of downstream TLR3 signaling. The glucocorticoid dexamethasone reduced LL-37/poly I:C-induced TLR3 expression on both mRNA and protein levels, and this effect was associated with increased IκBα protein expression, suggesting that dexamethasone acts via attenuation of NF-κB activity. We conclude that LL-37 potentiates poly I:C-induced upregulation of TLR3 through a mechanism that may involve enhanced import of poly I:C and that LL-37/poly I:C-induced TLR3 expression is associated with downstream TLR3 signaling and sensitive to inhibition of NF-κB activity.

NFκB1 Dichotomously Regulates Pro-Inflammatory and Antiviral Responses in Asthma.

Asthma exacerbations are commonly triggered by rhinovirus infections. Viruses can activate the NFκB pathway resulting in airway inflammation and increased Th2 cytokine expression. NFκB signaling is also involved in early activation of IFNß, which is a central mediator of antiviral responses to rhinovirus infection. Using a mouse model, this study tests our hypothesis that NFκB signaling is involved in impaired IFNß production at viral-induced asthma exacerbations. C57BL/6 wild-type and NFκB1-/- mice were challenged with house dust mite for 3 weeks and were subsequently stimulated with the rhinoviral mimic poly(I:C). General lung inflammatory parameters and levels of the Th2 upstream cytokine IL-33 were measured after allergen challenge. At exacerbation, production of IFNß and antiviral proteins as well as gene expression of pattern recognition receptors and IRF3/IRF7 was assessed. In the asthma exacerbation mouse model, lack of NFκB1 resulted in lower levels of IL-33 after allergen challenge alone and was associated with reduced eosinophilia. At exacerbation, mice deficient in NFκB1 exhibited enhanced expression of IFNß and antiviral proteins. This was accompanied by increased IRF3/IRF7 expression and induction of pattern recognition receptor expression. In a human asthma dataset, a negative correlation between IRF3 and NFκB1 expression was observed. NFκB may impair antiviral responses at exacerbation, possibly by reducing expression of the transcription factors IRF3/IRF7. These findings suggest a therapeutic potential for targeting NFκB pathways at viral infection-induced exacerbations.

Short-term exposure to urban PM2.5 particles induces histopathological and inflammatory changes in the rat small intestine.

Air pollution and exposure to fine airborne particles with aerodynamic diameter <2.5 µm (PM2.5 ) negatively impacts human health. Airways constitute a primary route of exposure but PM2.5 -contaminated food, drinks as well as mucociliary and hepatobiliary clearance all constitute potential entry points into the intestine. This study evaluated intestinal histopathological and inflammatory changes as well as enteric neuronal numbers after short- or long-term exposure to urban PM2.5 . Using a nebulizer, male rats were exposed to a mist with a concentration of 5.3mg PM2.5 /m3 for 8 h (short term) or 1.8 mg PM2.5 /m3 for 3 h/day, 5 days/week for 8 weeks (long-term) with controls run in parallel. Samples were taken from three regions of the small intestine as well as the colon. Results showed that short-term exposure to PM2.5 induces mucosal lesions and reduces IL1ß levels in the small intestine but not colon. No significant changes were observed after long-term exposure, suggesting the presence of intestinal adaptation to environmental stressors in the PM2.5 . To our knowledge, this is the first study to systematically characterize regional effects along the intestine.