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Since microRNA (miR)-223-3p modulates inflammatory responses and chronic obstructive pulmonary disease (COPD) is associated with amplified pulmonary inflammation, we hypothesized that miR-223-3p plays a role in COPD pathogenesis. Expression of miR-223-3p was measured in lung tissue of two independent cohorts with patients with GOLD stage II–IV COPD, never smokers, and smokers without COPD. The functional role of miR-223-3p was studied in deficient mice and on overexpression in airway epithelial cells from COPD and controls. We observed higher miR-223-3p levels in patients with COPD stage II–IV compared with (non)-smoking controls, and levels were associated with higher neutrophil numbers in bronchial biopsies of patients with COPD. MiR-223-3p expression was also increased in lungs and bronchoalveolar lavage of cigarette smoke (CS)-exposed mice. CS-induced neutrophil and monocyte lung infiltration was stronger in miR-223-deficient mice on acute (5 days) exposure, but attenuated on subchronic (4 wk) exposure. Additionally, miR-223 deficiency attenuated acute and subchronic CS-induced lung infiltration of dendritic cells and T lymphocytes. Finally, in vitro overexpression of miR-223-3p in non-COPD airway epithelial cells suppressed C-X-C motif chemokine ligand 8 (CXCL8) and granulocyte monocyte-colony stimulation factor (GM-CSF) secretion and gene expression of the proinflammatory transcription factor TRAF6. Importantly, this suppressive effect of miR-223-3p was compromised in COPD-derived cultures. In conclusion, we demonstrate that miR-223-3p is increased in lungs of patients with COPD and CS-exposed mice and is associated with neutrophilic inflammation. In vivo data indicate that miR-223 acts as negative regulator of acute CS-induced neutrophilic and monocytic inflammation. In vitro data suggest that miR-223-3p does so by suppressing proinflammatory airway epithelial responses, which is less effective in COPD epithelium.

Tissue-resident natural killer cells have been identified in numerous organs, but little is known about their functional contribution to respiratory immunity, in particular during chronic lung diseases such as COPD.To investigate the phenotype and antiviral responses of trNK cells in murine cigarette smoke-induced experimental COPD and in human lung parenchyma from COPD donors.Mice were exposed to cigarette smoke for 10 weeks to induce COPD-like lung disease. Lung tissue resident NK cell phenotypes and function were analysed by flow cytometry in both murine and human disease with and without challenge with influenza A virus.In the mouse lung CD49a+CD49b+EOMES+ and CD49a+CD49b-EOMESlo NK cell populations had a distinct phenotype compared with CD49a- circulating NK cells. CD49a+ NK cells were more extensively altered earlier in disease onset than circulating NK cells and increased proportions of CD49a+ NK cells correlated with worsening disease in both murine and human COPD. Furthermore, the presence of lung disease delayed both circulating and tissue-resident NK cell functional responses to influenza infection. CD49a+ NK cells markedly increased their NKG2D, CD103 and CD69 expression in experimental COPD following influenza infection, and human CD49a+ NK cells were hyperactive to ex vivo influenza infection in COPD donors.Collectively, these results demonstrate that tissue-resident NK cell function is altered in cigarette smoke-induced disease and suggests that smoke exposure may aberrantly prime tissue-resident NK cell responsiveness to viral infection. This may contribute to excess inflammation during viral exacerbations of COPD.

BackgroundReceptor-interacting protein kinase 1 (RIPK1) is a key mediator of regulated cell death (including apoptosis and necroptosis) and inflammation, both drivers of COPD pathogenesis. We aimed to define the contribution of RIPK1 kinase-dependent cell death and inflammation in the pathogenesis of COPD.MethodsWe assessedRIPK1expression in single-cell RNA sequencing (RNA-seq) data from human and mouse lungs, and validated RIPK1 levels in lung tissue of COPD patientsviaimmunohistochemistry. Next, we assessed the consequences of genetic and pharmacological inhibition of RIPK1 kinase activity in experimental COPD, usingRipk1S25D/S25Dkinase-deficient mice and the RIPK1 kinase inhibitor GSK′547.ResultsRIPK1expression increased in alveolar type 1 (AT1), AT2, ciliated and neuroendocrine cells in human COPD. RIPK1 protein levels were significantly increased in airway epithelium of COPD patients compared with never-smokers and smokers without airflow limitation. In mice, exposure to cigarette smoke (CS) increasedRipk1expression similarly in AT2 cells, and further in alveolar macrophages and T-cells. Genetic and/or pharmacological inhibition of RIPK1 kinase activity significantly attenuated airway inflammation upon acute and subacute CS exposure, as well as airway remodelling, emphysema, and apoptotic and necroptotic cell death upon chronic CS exposure. Similarly, pharmacological RIPK1 kinase inhibition significantly attenuated elastase-induced emphysema and lung function decline. Finally, RNA-seq on lung tissue of CS-exposed mice revealed downregulation of cell death and inflammatory pathways upon pharmacological RIPK1 kinase inhibition.ConclusionsRIPK1 kinase inhibition is protective in experimental models of COPD and may represent a novel promising therapeutic approach.

RNA interference (RNAi) enables highly specific silencing of potential target genes for treatment of pulmonary pathologies. The intracellular RNAi pathway can be activated by cytosolic delivery of small interfering RNA (siRNA), inducing sequence-specific gene knockdown on the post-transcriptional level. Although siRNA drugs hold many advantages over currently applied therapies, their clinical translation is hampered by inefficient delivery across cellular membranes. We previously developed hybrid nanoparticles consisting of an siRNA-loaded nanosized hydrogel core (nanogel) coated with Curosurf®, a clinically used pulmonary surfactant (PS). The latter enhances both particle stability as well as intracellular siRNA delivery, which was shown to be governed by the PS-associated surfactant protein B (SP-B). Despite having a proven in vitro and in vivo siRNA delivery potential when prepared ex novo, clinical translation of this liquid nanoparticle suspension requires the identification of a long-term preservation strategy that maintains nanoparticle stability and potency. In addition, to achieve optimal pulmonary deposition of the nanocomposite, its compatibility with state-of-the-art pulmonary administration techniques should be evaluated. Here, we demonstrate that PS-coated nanogels can be lyophilized, reconstituted and subsequently nebulized via a vibrating mesh nebulizer. The particles retain their physicochemical integrity and their ability to deliver siRNA in a human lung epithelial cell line. The latter result suggests that the functional integrity of SP-B in the PS coat towards siRNA delivery might be preserved as well. Of note, successful lyophilization was achieved without the need for stabilizing lyo- or cryoprotectants. Our results demonstrate that PS-coated siRNA-loaded nanogels can be lyophilized, which offers the prospect of long-term storage. In addition, the formulation was demonstrated to be suitable for local administration with a state-of-the-art nebulizer for human use upon reconstitution. Hence, the data presented in this study represent an important step towards clinical application of such nanocomposites for treatment of pulmonary disease.

BackgroundChanges in microRNA (miRNA) expression can contribute to the pathogenesis of many diseases, including asthma. We aimed to identify miRNAs that are differentially expressed between asthma patients and healthy controls, and explore their association with clinical and inflammatory parameters of asthma.MethodsDifferentially expressed miRNAs were determined by small RNA sequencing on bronchial biopsies of 79 asthma patients and 82 healthy controls using linear regression models. Differentially expressed miRNAs were associated with clinical and inflammatory asthma features. Potential miRNA–mRNA interactions were analysed using mRNA data available from the same bronchial biopsies, and enrichment of pathways was identified with Enrichr and g:Profiler.ResultsIn total, 78 differentially expressed miRNAs were identified in bronchial biopsies of asthma patients compared with controls, of which 60 remained differentially expressed after controlling for smoking and inhaled corticosteroid treatment. We identified several asthma-associated miRNAs, including miR-125b-5p and miR-223-3p, based on a significant association with multiple clinical and inflammatory asthma features and their negative correlation with genes associated with the presence of asthma. The most enriched biological pathway(s) affected by miR-125b-5p and miR-223-3p were inflammatory response and cilium assembly/organisation. Of interest, we identified that lower expression of miR-26a-5p was linked to more severe eosinophilic inflammation as measured in blood, sputum as well as bronchial biopsies.ConclusionCollectively, we identified miR-125b-5p, miR-223-3p and miR-26a-5p as potential regulators that could contribute to the pathogenesis of asthma.

Background Exposure to diisocyanates is an important cause of occupational asthma (OA) in the industrialized world. Since OA occurs after long-term exposure to diisocyanates, we developed a chronic mouse model of chemical-induced asthma where toluene diisocyanate (TDI) was administered at two different exposure sites. Objectives Evaluating the effect of long-term respiratory isocyanate exposure - with or without prior dermal exposure- on sensitization, inflammatory responses and airway hyperreactivity (AHR). Methods On days 1 and 8, BALB/c mice were dermally treated (20 μl/ear) with 0.5% 2,4-toluene diisocyanate TDI or the vehicle acetone olive oil (AOO) (3:2). Starting from day 15, mice received intranasal instillations with 0.1% TDI of vehicle five times in a week, for five successive weeks. One day after the last instillation airway hyperreactivity (AHR) to methacholine was assessed, followed by an evaluation of pulmonary inflammation and structural lung changes. Immune-related parameters were assessed in the lungs (BAL and tissue), blood, cervical- and auricular lymph nodes. Results Mice repeatedly intranasally exposed to TDI showed systemic sensitization and a mixed Th1/Th2 type immune response, without the presence of AHR. However, when mice are first dermally sensitized with TDI, followed by repeated intranasal TDI challenges, this results in a pronounced Th2 response and AHR. Conclusion Dermal exposure to TDI determines airway hyperreactivity after repeated airway exposure to TDI.

Objectives Innate lymphoid cells (ILCs) secrete cytokines, such as IFN‐γ, IL‐13 and IL‐17, which are linked to chronic obstructive pulmonary disease (COPD). Here, we investigated the role of pulmonary ILCs in COPD pathogenesis. Methods Lung ILC subsets in COPD and control subjects were quantified using flow cytometry and associated with clinical parameters. Tissue localisation of ILC and T‐cell subsets was determined by immunohistochemistry. Mice were exposed to air or cigarette smoke (CS) for 1, 4 or 24 weeks to investigate whether pulmonary ILC numbers and activation are altered and whether they contribute to CS‐induced innate inflammatory responses. Results Quantification of lung ILC subsets demonstrated that ILC1 frequency in the total ILC population was elevated in COPD and was associated with smoking and severity of respiratory symptoms (COPD Assessment Test [CAT] score). All three ILC subsets localised near lymphoid aggregates in COPD. In the COPD mouse model, CS exposure in C57BL/6J mice increased ILC numbers at all time points, with relative increases in ILC1 in bronchoalveolar lavage (BAL) fluid. Importantly, CS exposure induced increases in neutrophils, monocytes and dendritic cells that remained elevated in Rag2/Il2rg‐deficient mice that lack adaptive immune cells and ILCs. However, CS‐induced CXCL1, IL‐6, TNF‐α and IFN‐γ levels were reduced by ILC deficiency. Conclusion The ILC1 subset is increased in COPD patients and correlates with smoking and severity of respiratory symptoms. ILCs also increase upon CS exposure in C57BL/6J mice. In the absence of adaptive immunity, ILCs contribute to CS‐induced pro‐inflammatory mediator release, but are redundant in CS‐induced innate inflammation., In this article, we investigated the role of pulmonary innate lymphoid cells (ILCs) in the pathogenesis of chronic obstructive pulmonary disease (COPD). We found that the ILC1 subset is increased in COPD patients and correlates with smoking and severity of respiratory symptoms. In C57BL/6 mice, ILCs increase upon cigarette smoke (CS) exposure. In the absence of adaptive immunity, ILCs contribute to CS‐induced pro‐inflammatory mediator release, but they are redundant in CS‐induced innate inflammation.

In past 10 years, microRNAs (miRNAs) have gained scientific attention due to their importance in the pathophysiology of allergic diseases and their potential as biomarkers in liquid biopsies. They act as master post‐transcriptional regulators that control most cellular processes. As one miRNA can target several mRNAs, often within the same pathway, dysregulated expression of miRNAs may alter particular cellular responses and contribute, or lead, to the development of various diseases. In this review, we give an overview of the current research on miRNAs in allergic diseases, including atopic dermatitis, allergic rhinitis, and asthma. Specifically, we discuss how individual miRNAs function in the regulation of immune responses in epithelial cells and specialized immune cells in response to different environmental factors and respiratory viruses. In addition, we review insights obtained from experiments with murine models of allergic airway and skin inflammation and offer an overview of studies focusing on miRNA discovery using profiling techniques and bioinformatic modeling of the network effect of multiple miRNAs. In conclusion, we highlight the importance of research into miRNA function in allergy and asthma to improve our knowledge of the molecular mechanisms involved in the pathogenesis of this heterogeneous group of diseases.

PURPOSE: Exposure to low concentrations of toluene diisocyanate (TDI) leads to immune-mediated chemical-induced asthma. The role of the adaptive immune system has already been thoroughly investigated; nevertheless, the involvement of innate immune cells in the pathophysiology of chemical-induced asthma is still unresolved. The aim of the study is to investigate the role of innate lymphoid cells (ILCs) and dendritic cells (DCs) in a mouse model for chemical-induced asthma. METHODS: On days 1 and 8, BALB/c mice were dermally treated (20 μL/ear) with 0.5% TDI or the vehicle acetone olive oil (AOO; 2:3). On days 15, 17, 19, 22 and 24, the mice received an oropharyngeal challenge with 0.01% TDI or AOO (1:4). One day after the last challenge, airway hyperreactivity (AHR) to methacholine was assessed, followed by an evaluation of pulmonary inflammation and immune-related parameters, including the cytokine pattern in bronchoalveolar lavage fluid, lymphocyte subpopulations of the lymph nodes and their ex vivo cytokine production profile, blood immunoglobulins and DC and ILC subpopulations in the lungs. RESULTS: Both DC and ILC2 were recruited to the lungs after multiple airway exposures to TDI, regardless of the prior dermal sensitization. However, prior dermal sensitization with TDI alone results in AHR and predominant eosinophilic airway inflammation, accompanied by a typical type 2 helper T (Th2) cytokine profile. CONCLUSIONS: TDI-induced asthma is mediated by a predominant type 2 immune response, with the involvement of adaptive Th2 cells. However, from our study we suggest that the innate ILC2 cells are important additional players in the development of TDI-induced asthma. ispartof: ALLERGY ASTHMA & IMMUNOLOGY RESEARCH vol:13 issue:2 pages:295-311 ispartof: location:Korea (South) status: published

Alterations in miRNA expression may play a role in the pathogenesis of asthma. Our aim was to identify miRNAs that are differentially expressed in bronchial biopsies of asthmatics compared to healthy controls and to explore their association with clinical asthma parameters. Small RNA sequencing data from bronchial biopsies of 79 asthmatics and 82 healthy controls was used to determine differential miRNA expression. The analysis was repeated in 30 non-smoking, ICS negative asthmatics and 42 non-smoking healthy controls. The differentially expressed miRNAs were associated with clinical parameters, including FEV1% predicted, neutrophil and eosinophil counts in blood, sputum and bronchial biopsies. In total 78 miRNAs were differentially expressed in bronchial biopsies from asthmatics compared to healthy controls. Of those 78 miRNAs, 62 were also differentially expressed in non-smoking, ICS negative asthmatics compared to non-smoking healthy controls. The most significant miRNAs were miR-16-5p, miR-451a, miR-125a/b and the let-7 family. Of the 62 asthma-associated miRNAs, 25 miRNAs correlated with one or more clinical asthma parameters. Lower levels of let-7a and let-7c were significantly associated with higher levels of blood and tissue eosinophils within asthma, while lower levels of miR-125a/b were associated with lower FEV1% predicted and higher blood eosinophils and neutrophils within asthma. In conclusion, various miRNAs including miR-125a/b and the let-7 family are differentially expressed in bronchial biopsies from asthmatics compared to healthy controls and are associate with multiple clinical asthma features. Based on the literature and our data, we suggest that those miRNAs might serve as potential asthma biomarker.

We previously showed that miR-320d is increased in bronchial biopsies from COPD patients upon corticosteroid treatment for 6 or 30 months and is negatively correlated with pro-inflammatory gene expression (Faiz et al. Eur Respir J 2019; 53:4). We hypothesize that miR-320d has an anti-inflammatory role in airway epithelium. Our aim was to investigate the role of miR-320d in the pathogenesis of COPD and to investigate the effect of miR-320d overexpression on cigarette smoke extract (CSE)-induced pro-inflammatory responses in airway epithelial cells (AECs). MiR-320d expression was assessed in lung tissue from two cohorts of mild to severe COPD patients (n=38-40) and smoking matched controls (n=33-52) and in AECs from 6 severe COPD and 6 non-COPD subjects exposed to 20% CSE using qPCR. AECs were transfected with 1nM miR-320d mimic, treated with 20% CSE for 24h and the secretion of CXCL8 was measured in cell supernatants using ELISA. MiR-320d expression was significantly lower in lung tissue from COPD patients compared to controls. Upon 6 hours CSE exposure, a trend towards increased levels of miR-320d was observed in non-COPD-derived AECs, while a significant increase in miR-320d expression was measured in COPD-derived AECs. 24 hours exposure to CSE induced miR-320d expression in non-COPD-derived AECs, but no longer in COPD-derived AECs. Furthermore, overexpression of miR-320d reduced CXCL8 levels in non-COPD-derived AECs, but not in COPD-derived AECs. In conclusion, miR-320d expression is lower in lung tissue of COPD patients compared to controls. The induction and the anti-inflammatory effect of miR-320d on epithelial cytokine release differed in AECs derived from non-COPD compared to COPD, suggesting that this could contribute to the dysregulated inflammatory response in COPD.

Airway epithelial barrier dysfunction is frequently observed in asthma and may have important implications. The physical barrier function of the airway epithelium is tightly interwoven with its immunomodulatory actions, while abnormal epithelial repair responses may contribute to remodelling of the airway wall. We propose that abnormalities in the airway epithelial barrier play a crucial role in the sensitization to allergens and pathogenesis of asthma. Many of the identified susceptibility genes for asthma are expressed in the airway epithelium, supporting the notion that events at the airway epithelial surface are critical for the development of the disease. However, the exact mechanisms by which the expression of epithelial susceptibility genes translates into a functionally altered response to environmental risk factors of asthma are still unknown. Interactions between genetic factors and epigenetic regulatory mechanisms may be crucial for asthma susceptibility. Understanding these mechanisms may lead to identification of novel targets for asthma intervention by targeting the airway epithelium. Moreover, exciting new insights have come from recent studies using single‐cell RNA sequencing (scRNA‐Seq) to study the airway epithelium in asthma. This review focuses on the role of airway epithelial barrier function in the susceptibility to develop asthma and novel insights in the modulation of epithelial cell dysfunction in asthma.

Chronic obstructive pulmonary disease (COPD) is a highly prevalent respiratory disease characterized by airflow limitation and chronic inflammation. MiR-155 is described as an ancient regulator of the immune system. Our objective was to establish a role for miR-155 in cigarette smoke (CS)-induced inflammation and COPD. We demonstrate increased miR-155 expression by RT-qPCR in lung tissue of smokers without airflow limitation and patients with COPD compared to never smokers and in lung tissue and alveolar macrophages of CS-exposed mice compared to air-exposed mice. In addition, we exposed wild type and miR-155 deficient mice to CS and show an attenuated inflammatory profile in the latter. Alveolar macrophages were sorted by FACS from the different experimental groups and their gene expression profile was analyzed by RNA sequencing. This analysis revealed increased expression of miR-155 targets and an attenuation of the CS-induced increase in inflammation-related genes in miR-155 deficient mice. Moreover, intranasal instillation of a specific miR-155 inhibitor attenuated the CS-induced pulmonary inflammation in mice. Finally, elastase-induced emphysema and lung functional changes were significantly attenuated in miR-155 deficient mice. In conclusion, we highlight a role for miR-155 in CS-induced inflammation and the pathogenesis of COPD, implicating miR-155 as a new therapeutic target in COPD.

Background Clinical and experimental studies have identified a crucial role for IL-33 and its receptor ST2 in allergic asthma. Inhalation of traffic-related pollutants, such as diesel exhaust particles (DEP), facilitates the development of asthma and can cause exacerbations of asthma. However, it is unknown whether IL-33/ST2 signalling contributes to the enhancing effects of air pollutants on allergic airway responses. Objective We aim to investigate the functional role of IL-33/ST2 signalling in DEP-enhanced allergic airway responses, using an established murine model. Methods C57BL/6J mice were exposed to saline, DEP alone, house dust mite (HDM) alone or combined DEP+HDM. To inhibit IL-33 signalling, recombinant soluble ST2 (r-sST2) was given prophylactically (ie, during the whole experimental protocol) or therapeutically (ie, at the end of the experimental protocol). Airway hyperresponsiveness and the airway inflammatory responses were assessed in bronchoalveolar lavage fluid (BALF) and lung. Results Combined exposure to DEP+HDM increased IL-33 and ST2 expression in lung, elevated inflammatory responses and bronchial hyperresponsiveness compared to saline, sole DEP or sole HDM exposure. Prophylactic interference with the IL-33/ST2 signalling pathway impaired the DEP-enhanced allergic airway inflammation in the BALF, whereas effects on lung inflammation and airway hyperresponsiveness were minimal. Treatment with r-sST2 at the end of the experimental protocol did not modulate the DEP-enhanced allergic airway responses. Conclusion Our data suggest that the IL-33/ST2 pathway contributes to the onset of DEP-enhanced allergic airway inflammation.

Aberrant expression of microRNAs (miRNAs) can have a detrimental role in disease pathogenesis.To identify dysregulated miRNAs in lung tissue of patients with chronic obstructive pulmonary disease (COPD).We performed miRNA and mRNA profiling using high throughput stem-loop reverse-transcriptase quantitative polymerase chain reaction and mRNA microarray, respectively, on lung tissue of 30 patients (screening cohort) encompassing 8 never-smokers, 10 smokers without airflow limitation, and 12 smokers with COPD. Differential expression of miRNA-218-5p (miR-218-5p) was validated by reverse-transcriptase quantitative polymerase chain reaction in an independent cohort of 71 patients, an in vivo murine model of COPD, and primary human bronchial epithelial cells. Localization of miR-218-5p was assessed by in situ hybridization. In vitro and in vivo perturbation of miR-218-5p combined with RNA sequencing and gene set enrichment analysis was used to elucidate its functional role in COPD pathogenesis.Several miRNAs were differentially expressed among the different patient groups. Interestingly, miR-218-5p was significantly down-regulated in smokers without airflow limitation and in patients with COPD compared with never-smokers. Decreased pulmonary expression of miR-218-5p was validated in an independent validation cohort, in cigarette smoke-exposed mice, and in human bronchial epithelial cells. Importantly, expression of miR-218-5p strongly correlated with airway obstruction. Furthermore, cellular localization of miR-218-5p in human and murine lung revealed highest expression of miR-218-5p in the bronchial airway epithelium. Perturbation experiments with a miR-218-5p mimic or inhibitor demonstrated a protective role of miR-218-5p in cigarette smoke-induced inflammation and COPD.We highlight a role for miR-218-5p in the pathogenesis of COPD.

Air pollution has adverse effects on the onset and morbidity of respiratory diseases, including asthma. In this review, we discuss recent insights into the effects of air pollution on the incidence and exacerbation of asthma. We focus on epidemiological studies that describe the association between air pollution exposure and development, mortality, persistence and exacerbations of asthma among different age groups. Moreover, we also provide an update on translational studies describing the mechanisms behind this association.Mechanisms linking air pollutants such as particulate matter, nitrogen dioxide (NO2) and ozone to the development and exacerbation of asthma include the induction of both eosinophilic and neutrophilic inflammation driven by stimulation of airway epithelium and increase of pro-inflammatory cytokine production, oxidative stress and DNA methylation changes. Although exposure during foetal development is often reported as a crucial timeframe, exposure to air pollution is detrimental in people of all ages, thus influencing asthma onset as well as increase in asthma prevalence, mortality, persistence and exacerbation.In conclusion, this review highlights the importance of reducing air pollution levels to avert the progressive increase in asthma incidence and morbidity.

Background: Whereas occupational asthma, induced by workplace exposures to allergens or respiratory sensitizers, such as toluene 2,4-diisocyanate (TDI), causes a significant burden to patients and society, its pathogenesis still needs to be fully elucidated. Although the adaptive responses have been characterized previously, there is little information about the role of innate lymphoid cells (ILC) in TDI-induced asthma. A critical regulator of ILC function is microRNA-155 (miR-155), a miR associated with asthma pathogenesis. In a mouse model we investigated changes in ILC subsets upon isocyanate exposure and assessed whether miR-155 contributes to TDI-induced airway inflammation and hyperresponsiveness (AHR). Methods: C57BL/6 and miR-155 knockout (KO) mice received 2%TDI or vehicle (acetone/olive oil) on each ear (day 1, 8), followed by oropharyngeal instillations of 0.01%TDI or vehicle (day 15, 22, 29). On day 31, inflammation was evaluated in bronchoalveolar lavage fluid (BALF) by flow cytometry. AHR was measured with the forced oscillation technique. Lung sections were stained with Congo Red or periodic acid-Schiff. Results: TDI-exposed mice had higher numbers of BALF cells, eosinophils, neutrophils, CD11b+ DCs, CD8+ and CD4+ T cells and ILC. All T helper and ILC populations (IFN-?+ ILC1, IL-13+ ILC2, IL-17+ ILC3) were elevated, with a predominant type 2 response. TDI-exposed mice had higher numbers of lung eosinophils and goblet cells and tended to have AHR. The inflammation and AHR was attenuated in the TDI-exposed miR-155 KO mice. Conclusion: In a model of TDI-induced asthma, the inflammation is attenuated in miR-155 KO mice, suggesting a pro-inflammatory role of this miR in the pathogenesis of occupational asthma.

Since novel treatments to target eosinophilic inflammation in Type 2 asthma are emerging, we aimed to evaluate and meta-analyze the efficacy of monoclonal antibodies to reduce exacerbation rate. PubMed and Web of Science were searched for phase II and phase III randomized clinical trials with monoclonal antibodies targeting key mediators of type 2-associated asthma. Thirty trials were selected involving biologics that target the IL-5 pathway, IL-13, the common IL-4 and IL-13 receptor, IL-9, IL-2 and TSLP. As no head-to-head trials were retrieved from literature, we performed an arm-based network meta-analysis to compare effects on exacerbation rate between the different treatments. Mepolizumab, reslizumab and benralizumab significantly reduced the risk of exacerbations compared to placebo (by 47–52%, 50–60%, and 28–51% respectively). Reslizumab and benralizumab also improved lung function. Dupilumab and tezepelumab improved lung function in frequent exacerbators. Lebrikizumab had no significant effect on the number of exacerbations, symptom control or health-related quality of life. Tralokinumab improved lung function compared to placebo. Network meta-analysis of all treatment and placebo arms, showed no superiority of one biologic over the others. Large reductions in exacerbation rates were observed compared to placebo, though only benralizumab was sufficiently powered (n = 2051) to demonstrate significantly decreased exacerbation rates in the subgroup analysis of IL-5 acting agents compared to placebo. Monoclonal antibodies such as mepolizumab, reslizumab and benralizumab have proven their benefit to reduce exacerbation rates in severe persistent eosinophilic asthma in the published trials. However, no statistically significant superiority was observed of one biologic over the other in the network meta-analysis. More studies with direct head to head comparisons and better defined endotypes are required. Electronic supplementary material The online version of this article (10.1186/s12931-019-1138-3) contains supplementary material, which is available to authorized users.

BackgroundOccupational asthma, induced by workplace exposures to low molecular weight agents such as toluene 2,4-diisocyanate (TDI), causes a significant burden to patients and society. Little is known about innate lymphoid cells (ILCs) in TDI-induced asthma. A critical regulator of ILC function is microRNA-155, a microRNA associated with asthma.ObjectiveTo determine whether TDI exposure modifies the number of ILCs in the lung and whether microRNA-155 contributes to TDI-induced airway inflammation and hyperresponsiveness.MethodsC57BL/6 wild-type and microRNA-155 knockout mice were sensitised and challenged with TDI or vehicle. Intracellular cytokine expression in ILCs and T-cells was evaluated in bronchoalveolar lavage (BAL) fluid using flow cytometry. Peribronchial eosinophilia and goblet cells were evaluated on lung tissue, and airway hyperresponsiveness was measured using the forced oscillation technique. Putative type 2 ILCs (ILC2) were identified in bronchial biopsies of subjects with TDI-induced occupational asthma using immunohistochemistry. Human bronchial epithelial cells were exposed to TDI or vehicle.ResultsTDI-exposed mice had higher numbers of airway goblet cells, BAL eosinophils, CD4+T-cells and ILCs, with a predominant type 2 response, and tended to have airway hyperresponsiveness. In TDI-exposed microRNA-155 knockout mice, inflammation and airway hyperresponsiveness were attenuated. TDI exposure induced IL-33 expression in human bronchial epithelial cells and in murine lungs, which was microRNA-155 dependent in mice. GATA3+CD3−cells, presumably ILC2, were present in bronchial biopsies.ConclusionTDI exposure is associated with increased numbers of ILCs. The proinflammatory microRNA-155 is crucial in a murine model of TDI asthma, suggesting its involvement in the pathogenesis of occupational asthma due to low molecular weight agents.

Background: Whereas occupational asthma, induced by workplace exposures to allergens or respiratory sensitizers, such as toluene 2,4-diisocyanate (TDI), causes a significant burden to patients and society, its pathogenesis still needs to be fully elucidated. Although the adaptive responses have been characterized previously, there is little information about the role of innate lymphoid cells (ILC) in TDI-induced asthma. A critical regulator of ILC function is microRNA-155 (miR-155), a miR associated with asthma pathogenesis. In a mouse model we investigated changes in ILC subsets upon isocyanate exposure and assessed whether miR-155 contributes to TDI-induced airway inflammation and hyperresponsiveness (AHR). Methods: C57BL/6 and miR-155 knockout (KO) mice received 2%TDI or vehicle (acetone/olive oil) on each ear (day 1, 8), followed by oropharyngeal instillations of 0.01%TDI or vehicle (day 15, 22, 29). On day 31, inflammation was evaluated in bronchoalveolar lavage fluid (BALF) by flow cytometry. AHR was measured with the forced oscillation technique. Lung sections were stained with Congo Red or periodic acid-Schiff. Results: TDI-exposed mice had higher numbers of BALF cells, eosinophils, neutrophils, CD11b+ DCs, CD8+ and CD4+ T cells and ILC. All T helper and ILC populations (IFN-?+ ILC1, IL-13+ ILC2, IL-17+ ILC3) were elevated, with a predominant type 2 response. TDI-exposed mice had higher numbers of lung eosinophils and goblet cells and tended to have AHR. The inflammation and AHR was attenuated in the TDI-exposed miR-155 KO mice. Conclusion: In a model of TDI-induced asthma, the inflammation is attenuated in miR-155 KO mice, suggesting a pro-inflammatory role of this miR in the pathogenesis of occupational asthma.

Suppression of airway inflammation with inhaled corticosteroids has been the key therapeutic approach for asthma for many years. Identification of inflammatory phenotypes in asthma has moreover led to important breakthroughs, e.g. with specific targeting of the IL-5 pathway as add-on treatment in difficult-to-treat eosinophilic asthma. However, the impact of interfering with the neutrophilic component in asthma is less documented and understood. This review provides an overview of established and recent insights with regard to the role of neutrophils in asthma, focusing on research in humans. We will describe the main drivers of neutrophilic responses in asthma, the heterogeneity in neutrophils and how they could contribute to asthma pathogenesis. Moreover we will describe findings from clinical trials, in which neutrophilic inflammation was targeted. It is clear that neutrophils are important actors in asthma development and play a role in exacerbations. However, more research is required to fully understand how modulation of neutrophil activity could lead to a significant benefit in asthma patients with airway neutrophilia.

Extracellular RNAs present in biofluids have emerged as potential biomarkers for disease. Where most studies focus on plasma or serum, other biofluids may contain more informative RNA molecules, depending on the type of disease. Here, we present an unprecedented atlas of messenger, circular and small RNA transcriptomes of a comprehensive collection of 20 different human biofluids. By means of synthetic spike-in controls, we compared RNA content across biofluids, revealing a more than 10 000-fold difference in RNA concentration. The circular RNA fraction is increased in nearly all biofluids compared to tissues. Each biofluid transcriptome is enriched for RNA molecules derived from specific tissues and cell types. In addition, a subset of biofluids, including stool, sweat, saliva and sputum, contains high levels of bacterial RNAs. Our atlas enables a more informed selection of the most relevant biofluid to monitor particular diseases. To verify the biomarker potential in these biofluids, four validation cohorts representing a broad spectrum of diseases were profiled, revealing numerous differential RNAs between case and control subjects. Taken together, our results reveal novel insights in the RNA content of human biofluids and may serve as a valuable resource for future biomarker studies. All spike-normalized data is publicly available in the R2 web portal and serve as a basis to further explore the RNA content in biofluids.

Inflammatory bowel disease (IBD) is characterized by severe gastrointestinal inflammation and results from a complex interplay between genetic and environmental factors. IBD includes two prominent subtypes: Crohn's disease (CD) and ulcerative colitis (UC). One of the main risk factors for the development of CD is cigarette smoking, while UC is rather a disease of ex-smokers. To date, many of the mechanisms underlying the immune imbalance in IBD and the involvement of cigarette smoke (CS) are incompletely understood. Transient receptor potential (TRP) proteins are non-selective cation channels that, upon activation, lead to plasma membrane depolarization and, in general, to Ca2+ influx. TRP channels of the ankyrin and vanilloid family, expressed by sensory neurons in the central and enteric nervous systems, have been extensively studied in the context of intestinal inflammation. Moreover, recent advances made on the role of non-neuronal expressed TRP channels shed light on the involvement of epithelial cells in inflammatory processes. This review focuses on how CS may impact TRP channel function in intestinal inflammation. Firstly, we discuss the current knowledge on neuronal TRP channels, known to be linked to IBD, in health, immune homeostasis and intestinal inflammation. Subsequently, we address how TRP channels are activated by CS and its components in other organ systems and also hypothesize on the potential implications for CS-mediated TRP channel activation in gut inflammation.

Inhalation of traffic-related particulate matter (e.g., diesel exhaust particles [DEPs]) is associated with acute inflammatory responses in the lung, and it promotes the development and aggravation of allergic airway diseases. We previously demonstrated that exposure to DEP was associated with increased recruitment and maturation of monocytes and conventional dendritic cells (DCs), resulting in TH2 polarization. Monocytes and immature DCs express the G-protein coupled receptor chemR23, which binds the chemoattractant chemerin. Using chemR23 knockout (KO) and corresponding wild-type (WT) mice, we determined the role of chemR23 signaling in response to acute exposure to DEPs and in response to DEP-enhanced house dust mite (HDM)-induced allergic airway inflammation. Exposure to DEP alone, as well as combined exposure to DEP plus HDM, elevated the levels of chemerin in the bronchoalveolar lavage fluid of WT mice. In response to acute exposure to DEPs, monocytes and monocyte-derived DCs accumulated in the lungs of WT mice, but this response was significantly attenuated in chemR23 KO mice. Concomitant exposure to DEP plus HDM resulted in allergic airway inflammation with increased eosinophilia, goblet cell metaplasia, and TH2 cytokine production in WT mice, which was further enhanced in chemR23 KO mice. In conclusion, we demonstrated an opposing role for chemR23 signaling depending on the context of DEP-induced inflammation. The chemR23 axis showed proinflammatory properties in a model of DEP-induced acute lung inflammation, in contrast to anti-inflammatory effects in a model of DEP-enhanced allergic airway inflammation.

Crohn's disease is a pathological condition of the gastro-intestinal tract, causing severe transmural inflammation in the ileum and/or colon. Cigarette smoking is one of the best known environmental risk factors for the development of Crohn's disease. Nevertheless, very little is known about the effect of prolonged cigarette smoke exposure on inflammatory modulators in the gut. We examined the effect of cigarette smoke on cytokine profiles in the healthy and inflamed gut of human subjects and in the trinitrobenzene sulphonic acid mouse model, which mimics distal Crohn-like colitis. In addition, the effect of cigarette smoke on epithelial expression of transient receptor potential channels and their concurrent increase with cigarette smoke-augmented cytokine production was investigated. Active smoking was associated with increased IL-8 transcription in ileum of controls (p < 0,001; n = 18-20/ group). In the ileum, TRPV1 mRNA levels were decreased in never smoking Crohn's disease patients compared to healthy subjects (p

Liquid biopsies offer a minimally invasive alternative to tissue biopsies for both diagnosis and monitoring of treatment response. Extracellular RNAs present in biofluids have emerged as potential biomarkers in health and disease, including cancer. While current studies typically focus on plasma or serum, other biofluids may contain more informative RNA molecules for particular disease types. Here, we present an unprecedented atlas of messenger, circular, and small RNA transcriptomes of a comprehensive collection of 20 different human biofluids (amniotic fluid, aqueous humor, ascites, bile, bronchial lavage fluid, breast milk, cerebrospinal fluid, colostrum, gastric fluid, pancreatic cyst fluid, plasma, saliva, seminal fluid, serum, sputum, stool, synovial fluid, sweat, tear fluid, and urine). By means of 173 synthetic RNA spike-in controls, we compared RNA content across biofluids, revealing a more than 10,000-fold difference in RNA concentration. Of interest, the circular RNA fraction is significantly increased in nearly all biofluids compared to various solid tissues. Each biofluid transcriptome is enriched for RNA molecules derived from specific tissues and cell types. In addition, a subset of biofluids, including stool, sweat, saliva, and sputum, contain high levels of bacterial RNAs. These findings enable a more informed selection of the most relevant biofluid to monitor individual cancer types. To verify the biomarker potential in these biofluids, four validation cohorts representing a broad spectrum of diseases, including urine from bladder cancer patients and CSF from glioblastoma patients, were profiled, revealing differential RNAs between case and control subjects. Taken together, our results reveal novel insights in the RNA content of human biofluids and may serve as a valuable resource for future biomarker studies. This abstract is also being presented as Poster B01. Citation Format: Eva Hulstaert, Annelien Morlion, Francisco Avila Cobos, Kimberly Verniers, Justine Nuytens, Eveline Vanden Eynde, Nurten Yigit, Jasper Anckaert, Anja Geerts, Pieter Hindryckx, Peggy Jacques, Guy Brusselle, Ken Bracke, Tania Maes, Thomas Malfait, Thierry Derveaux, Virginie Ninclaus, Caroline Van Cauwenbergh, Kristien Roelens, Ellen Roets, Dimitri Hemelsoet, Kelly Tilleman, Lieve Brochez, Scott Kuersten, Lukas Simon, Sebastian Karg, Christa Nöhammer, Alexandra Kautzky-Willers, Michael Leutner, Ondrej Slaby, Gary Schroth, Jo Vandesompele, Pieter Mestdagh. Charting extracellular transcriptomes in The Human Biofluid RNA Atlas [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr PR15.

Background: Clinical and experimental studies have demonstrated an important role for IL-33 and its receptor ST2 in allergic asthma. Inhalation of traffic-related pollutants, such as diesel exhaust particles (DEP), facilitates the development of asthma and can cause exacerbations of asthma. However, it is unknown whether IL-33/ST2 signaling contributes to the enhancing effects of air pollutants on allergic airway responses. Aims: To investigate the functional role of IL-33/ST2 signaling in DEP-enhanced allergic airway responses, using an established murine model of combined DEP and allergen exposure. Methods: C57BL/6J mice were exposed to saline, DEP alone, house dust mite (HDM) alone, or combined DEP+HDM. To inhibit IL-33 signaling, recombinant soluble ST2 (r-sST2) was given prophylactically (i.e. during the whole experimental protocol) or therapeutically (i.e. at the end of the experimental protocol). Airway hyperresponsiveness and the airway inflammatory responses were assessed in bronchoalveolar lavage fluid (BALF) and lung. Results: Combined exposure to DEP+HDM increased IL-33 and ST2 expression in lung, elevated inflammatory responses and bronchial hyperresponsiveness compared to saline, sole DEP or sole HDM exposure. Prophylactic interference with the IL-33/ST2 signaling pathway impaired the DEP-enhanced allergic airway inflammation in the BALF, whereas effects on lung inflammation and airway hyperresponsiveness were minimal. Treatment with r-sST2 at the end of the experimental protocol did not modulate the DEP-enhanced allergic airway responses. Conclusion: Our data suggest that the IL-33/ST2 pathway contributes to the onset of DEP-enhanced allergic airway inflammation.

We recently found higher miR-223-3p levels in sputum of asthma patients compared to controls. We hypothesized that miR-223-3p regulates pro-inflammatory responses to well-known risk factors for asthma, i.e. allergic sensitization and viral infection and could thus contribute to the regulation of airway inflammation in asthma. The aim was to validate miR-223-3p expression in bronchial biopsies from an independent asthma cohort and to study whether miR-223-3p increases pro-inflammatory activity of human bronchial epithelial cells (16HBE). MiR-223-3p expression was investigated in bronchial biopsies of asthma patients (n=46) and healthy controls (n=82) using RNA-sequencing. 16HBE cells were exposed to house dust mite (HDM) or viral mimic poly-(I:C) and miR-223-3p expression levels were measured by qPCR. 16HBE cells were transfected with miR-223-3p mimic, incubated with HDM or poly-(I:C) and secretion of pro-inflammatory cytokine CCL20 was measured using ELISA. Significantly higher levels of miR-223-3p were validated in bronchial biopsies from asthma patients compared to controls. In vitro, miR-223-3p was hardly detectable in 16HBE cells. Overexpression of miR-223-3p in 16HBE cells resulted in significantly higher basal and poly-(I:C)-induced CCL20 secretion, with a similar trend for HDM-induced CCL20 levels. In conclusion, we confirmed increased expression of miR-223-3p in airways of asthma patients compared to healthy controls. Overexpression of miR-223-3p in human bronchial epithelial cells resulted in higher CCL20 levels. This suggests that increased levels of miR-223-3p in asthma, e.g. when secreted in exosomes may contribute to the release of the pro-inflammatory CCL20 by airway epithelial cells.