Epithelial IL-6 trans-signaling defines a new asthma phenotype with increased airway inflammation

Background: Although several studies link high levels of IL-6 and soluble IL-6 receptor (sIL-6R) to asthma severity and decreased lung function, the role of IL-6 trans-signaling (IL-6TS) in asthmatic patients is unclear. Objective: We sought to explore the association between epithelial IL-6TS pathway activation and molecular and clinical phenotypes in asthmatic patients. Methods: An IL-6TS gene signature obtained from air-liquid interface cultures of human bronchial epithelial cells stimulated with IL-6 and sIL-6R was used to stratify lung epithelial transcriptomic data (Unbiased Biomarkers in Prediction of Respiratory Disease Outcomes [U-BIOPRED] cohorts) by means of hierarchical clustering. IL-6TS-specific protein markers were used to stratify sputum biomarker data (Wessex cohort). Molecular phenotyping was based on transcriptional profiling of epithelial brushings, pathway analysis, and immunohistochemical analysis of bronchial biopsy specimens. Results: Activation of IL-6TS in air-liquid interface cultures reduced epithelial integrity and induced a specific gene signature enriched in genes associated with airway remodeling. The IL-6TS signature identified a subset of patients with IL-6TS-high asthma with increased epithelial expression of IL-6TS-inducible genes in the absence of systemic inflammation. The IL-6TS-high subset had an overrepresentation of frequent exacerbators, blood eosinophilia, and submucosal infiltration of T cells and macrophages. In bronchial brushings Toll-like receptor pathway genes were upregulated, whereas expression of cell junction genes was reduced. Sputum sIL-6R and IL-6 levels correlated with sputum markers of remodeling and innate immune activation, in particular YKL-40, matrix metalloproteinase 3, macrophage inflammatory protein 1 beta, IL-8, and IL-1 beta. Conclusions: Local lung epithelial IL-6TS activation in the absence of type 2 airway inflammation defines a novel subset of asthmatic patients and might drive airway inflammation and epithelial dysfunction in these patients.Peer reviewe

Epithelial IL-6 trans-signaling defines a new asthma phenotype with increased airway inflammation

Background: Although several studies link high levels of IL-6 and soluble IL-6 receptor (sIL-6R) with asthma severity and decreased lung function, the role of IL-6 trans-signaling (IL-6TS) in asthma is unclear. Objective: To explore the association between epithelial IL-6TS pathway activation and molecular and clinical phenotypes in asthma. Methods: An IL-6TS gene signature, obtained from air-liquid interface (ALI) cultures of human bronchial epithelial cells stimulated with IL-6 and sIL-6R, was used to stratify lung epithelium transcriptomic data (U-BIOPRED cohorts) by hierarchical clustering. IL-6TS-specific protein markers were used to stratify sputum biomarker data (Wessex cohort). Molecular phenotyping was based on transcriptional profiling of epithelial brushings, pathway analysis and immunohistochemical analysis of bronchial biopsies. Results: Activation of IL-6TS in ALI cultures reduced epithelial integrity and induced a specific gene signature enriched in genes associated with airway remodeling. The IL-6TS signature identified a subset of IL-6TS. High asthma patients with increased epithelial expression of IL-6TS inducible genes in absence of systemic inflammation. The IL-6TS High subset had an overrepresentation of frequent exacerbators, blood eosinophilia, and submucosal infiltration of T cells and macrophages. In bronchial brushings, TLR pathway genes were up-regulated while the expression of tight junction genes was reduced. Sputum sIL-6R and IL-6 levels correlated with sputum markers of remodeling and innate immune activation, in particular YKL-40, MMP3, MIP-1β, IL-8 and IL-1β. Conclusions: Local lung epithelial IL-6TS activation in absence of type 2 airway inflammation defines a novel subset of asthmatics and may drive airway inflammation and epithelial dysfunction in these patients

Emerging role of RIG-I-like receptors in antiviral innate immunity in airway structural cells

Respiratory viral infections, especially involving rhinovirus, are main triggers of exacerbations of asthma and COPD. These severe conditions are a great burden to both the individual and society and currently lack effective treatments. Hence, a clear need exists for research into the cellular and molecular mechanisms by which viral infections evoke exacerbations of airway disease in order to better design effective therapeutics in the future. The aim of this thesis work has been to explore the immune response of airway structural cells to rhinoviral infection. To this end, we have employed in vitro cultures of primary human bronchial epithelial cells (BECs) and bronchial smooth muscle cells (BSMCs) from healthy subjects and patients with COPD and asthma. In general, these cells were infected with rhinovirus (RV) or stimulated with agonists for TLR3 and RIG-I-like receptors, and expression and/or release of specific genes and proteins were analysed by RT-qPCR, Western Blot and ELISA. In summary, our results show that BECs from severe COPD patients overproduce an upstream Th2-driving cytokine, TSLP, in response to RV infection and the RV replication intermediate dsRNA in a TLR3-dependent manner. Exaggerated production of the proinflammatory mediators TNF-α and CXCL8 was also observed in viral-challenged COPD-BECs and involved activation of both endosomal TLR3 and cytosolic RIG-I-like receptors. We further provide novel data that BSMCs from healthy individuals express the RIG-I-like receptors, RIG-I and MDA5. RV infection and activation of these innate immune sensors as well as TLR3 also lead to production of the antiviral cytokines IFN-β and IFN-λ1, suggesting a role for BSMCs in antiviral host defence. Finally, we have explored expression and regulation of another Th2-promoting cytokine, IL-33, in BSMCs from both healthy and asthmatic subjects. Our results demonstrate that IL-33 is upregulated by soluble mediators released from cultured BECs, and that dsRNA stimulation enhances this response, especially in asthmatic BSMCs. We further show that BSMCs also produce IL-33 in response to RV infection and activation of TLR3 and RIG-I-like receptors, and that ATP, via purinergic receptor signalling, is potentially involved as a mediator of IL-33 induction in BSMCs upon challenge with both epithelial-derived mediators and viral stimuli. In conclusion, our findings have provided new insights into regulatory functions of viral-challenged airway epithelial and smooth muscle cells in relation to airway inflammation and antiviral innate immunity. Intrinsic differences in health and disease have been identified, encouraging future studies of involved mechanisms that may serve as targets for novel therapeutic interventions in viral-induced exacerbations of asthma and COPD

Rhinovirus and dsRNA Induce RIG-I-Like Receptors and Expression of Interferon β and λ1 in Human Bronchial Smooth Muscle Cells.

Rhinovirus (RV) infections cause exacerbations and development of severe asthma highlighting the importance of antiviral interferon (IFN) defence by airway cells. Little is known about bronchial smooth muscle cell (BSMC) production of IFNs and whether BSMCs have dsRNA-sensing receptors besides TLR3. dsRNA is a rhinoviral replication intermediate and necrotic cell effect mimic that mediates innate immune responses in bronchial epithelial cells. We have explored dsRNA-evoked IFN-β and IFN-λ1 production in human BSMCs and potential involvement of TLR3 and RIG-I-like receptors (RLRs). Primary BSMCs were stimulated with 0.1-10 µg/ml dsRNA, 0.1-1 µg/ml dsRNA in complex with the transfection agent LyoVec (dsRNA/LyoVec; selectively activating cytosolic RLRs) or infected with 0.05-0.5 MOI RV1B. Both dsRNA stimuli evoked early (3 h), concentration-dependent IFN-β and IFN-λ1 mRNA expression, which with dsRNA/LyoVec was much greater, and with dsRNA was much less, after 24 h. The effects were inhibited by dexamethasone. Further, dsRNA and dsRNA/LyoVec concentration-dependently upregulated RIG-I and MDA5 mRNA and protein. dsRNA and particularly dsRNA/LyoVec caused IFN-β and IFN-λ1 protein production (24 h). dsRNA- but not dsRNA/LyoVec-induced IFN expression was partly inhibited by chloroquine that suppresses endosomal TLR3 activation. RV1B dose-dependently increased BSMC expression of RIG-I, MDA5, IFN-β, and IFN-λ1 mRNA. We suggest that BSMCs express functional RLRs and that both RLRs and TLR3 are involved in viral stimulus-induced BSMC expression of IFN-β and IFN-λ1

The Airway Epithelium—A Central Player in Asthma Pathogenesis

Asthma is a chronic inflammatory airway disease characterized by variable airflow obstruction in response to a wide range of exogenous stimuli. The airway epithelium is the first line of defense and plays an important role in initiating host defense and controlling immune responses. Indeed, increasing evidence indicates a range of abnormalities in various aspects of epithelial barrier function in asthma. A central part of this impairment is a disruption of the airway epithelial layer, allowing inhaled substances to pass more easily into the submucosa where they may interact with immune cells. Furthermore, many of the identified susceptibility genes for asthma are expressed in the airway epithelium. This review focuses on the biology of the airway epithelium in health and its pathobiology in asthma. We will specifically discuss external triggers such as allergens, viruses and alarmins and the effect of type 2 inflammatory responses on airway epithelial function in asthma. We will also discuss epigenetic mechanisms responding to external stimuli on the level of transcriptional and posttranscriptional regulation of gene expression, as well the airway epithelium as a potential treatment target in asthma

RV1B induces BSMC mRNA expression of IFNs.

<p>BSMCs exposed to RV1B at increasing doses (MOI = multiplicity of infection) induced expression of IFN-β (A) and IFN-λ₁ (D) 24 h post-infection as determined by RT-qPCR. IFN-β and IFN-λ₁ expression was further enhanced after 48 h (B, E). Chloroquine (10 µg/ml) reduced RV1B-induced IFN-β expression after 24 and 48 h (C), whereas IFN- λ₁ expression was reduced after 48 h only (F). Data are presented as mean with SEM and n = 6 for 24 h RV1B infection, n = 3 for 48 h infection and n = 2–3 for chloroquine experiments (BSMCs from three individual donors). *p≤0.05 and **p≤0.01 compared to non-infected cells (control).</p

dsRNA and dsRNA/LyoVec upregulate expression of the RLRs RIG-I and MDA5 in BSMCs.

<p>BSMC were treated with dsRNA or the RLR ligand dsRNA/LyoVec (dsRNA/LV) for 24 h. mRNA (A, C) and protein (B, D) expression of RIG-I (A, B) and MDA5 (C, D) was increased concentration-dependently. n = 9 for mRNA analysis and n = 6 for protein analysis (BSMCs from three individual donors). mRNA data are presented as mean with SEM and representative western blot images of each receptor protein are shown. *p≤0.05, **p≤0.01 and ***p≤0.001 compared to non-stimulated cells (control). ^#p≤0.05, ^##p≤0.01 and ^###p≤0.001 compared to 10 µg/ml dsRNA.</p

Effect of dsRNA on BSMC expression and production of IFN-β and IFN-λ₁.

<p>BSMCs were stimulated with dsRNA and expression of IFN-β (A, B) and IFN-λ₁ (E, F) mRNA was analysed by RT-qPCR after 3 h (A, E) or 24 h (B, F). Generation of IFN-β (C) and IFN-λ₁ protein (G) was determined after 24 h in the cell supernatants by ELISA. Chloroquine (Cq) and dexamethasone (Dex) added 1 h prior to dsRNA stimulation inhibited the early (3 h) response, but only dexamethasone inhibited the late (24 h) mRNA response. IFN-β protein was reduced with chloroquine treatment (D). Data are presented as mean with SEM and n = 8–10 (BSMCs from three individual donors). *p≤0.05, **p≤0.01 and ***p≤0.001 compared to non-stimulated cells (control). ^##p≤0.01 and ^###p≤0.001 compared to 10 µg/ml dsRNA.</p

RLR activation by dsRNA/LyoVec evokes BSMC mRNA expression and protein production of IFN-β and IFN-λ₁.

<p>IFN-β and IFN-λ₁ mRNA expression was increased by dsRNA/LyoVec (dsRNA/LV) after 3 h (A, D) and 24 h (B, E) and so was secretion of IFN-β and IFN-λ₁ after 24 h (C, F). Data are presented as mean with SEM and n = 8–10 (BSMCs from three individual donors). *p≤0.05, **p≤0.01 and ***p≤0.001 compared to non-stimulated cells (control).</p