Background: Rhinovirus (RV)-induced chronic obstructive pulmonary disease (COPD) exacerbations exhibit TH₂-like inflammation. We hypothesized that RV-infected bronchial epithelial cells (BEC) overproduce TH₂-switching hub cytokine, thymic stromal lymphopoietin (TSLP) in COPD. Methods: Primary BEC from healthy (HBEC) and from COPD donors (COPD-BEC) were grown in 12-well plates, infected with RV16 (0.5–5 MOI) or stimulated with agonists for either toll-like receptor (TLR) 3 (dsRNA, 0.1–10 µg/ml) or RIG-I-like helicases (dsRNA-LyoVec, 0.1–10 µg/ml). Cytokine mRNA and protein were determined (RTqPCR; ELISA). Results: dsRNA dose-dependently evoked cytokine gene overproduction of TSLP, CXCL8 and TNF-α in COPD-BEC compared to HBEC. This was confirmed using RV16 infection. IFN-β induction did not differ between COPD-BEC and HBEC. Endosomal TLR3 inhibition by chloroquine dose-dependently inhibited dsRNA-induced TSLP generation and reduced generation of CXCL8, TNF-α, and IFN-β. Stimulation of cytosolic viral sensors (RIG-I-like helicases) with dsRNA-LyoVec increased production of CXCL8, TNF-α, and IFN-β, but not TSLP. Conclusions: Endosomal TLR3-stimulation, by dsRNA or RV16, induces overproduction of TSLP in COPD-BEC. dsRNA- and RV-induced overproduction of TNF-α and CXCL8 involves endosomal TLR3 and cytosolic RIG-I-like helicases and so does the generation of IFN-β in COPD-BEC. RV16 and dsRNA-induced epithelial TSLP may contribute to pathogenic effects at exacerbations and development of COPD.

BackgroundRhinovirus infection or dsRNA stimulation increased thymic stromal lymphopoietin (TSLP), an upstream pro-allergic cytokine, in asthmatic bronchial epithelial cells. We hypothesized that dsRNA challenges superimposed on established experimental allergic asthma constitute a useful exacerbation model. We further hypothesized that TSLP is induced at dsRNA- and rhinoviral infection-induced exacerbations.MethodsAllergic mice were challenged with OVA followed by three daily intranasal challenges with dsRNA or saline. Bronchoalveolar lavage fluid (BALF) was analysed for total protein, lactate dehydrogenase (LDH), CXCL1/KC, CCL2/MCP-1 and differential cell counts. Lung tissue histology, neutrophils and TSLP, TNF-α, IFN-β and IFN-λ mRNA were examined. Alternatively, allergen-challenged mice received intranasal rhinovirus-(RV)-1B followed by lung TSLP immunostaining.ResultsIn mice with allergic airway inflammation, dsRNA challenges caused a significant exacerbation increasing lung tissue inflammation score and tissue neutrophilia. Bronchoalveolar lavage fluid neutrophils, total protein, LDH, CXCL1/KC and CCL2/MCP-1 were also increased (P < 0.01), and so were lung tissue expressions of TNF-α, IFN-λ and TSLP (P < 0.01), but IFN-β was not increased. TSLP, IFN-λ and LDH were not increased by allergen or dsRNA challenges alone, but increased exclusively at exacerbations. RV1B infection-induced exacerbation also increased lung tissue TSLP (P < 0.05).ConclusionsdsRNA-induced exacerbation in mice with experimental asthma involved general inflammation, cytokines and interferons, in agreement with previous observations in exacerbating human asthma. Additionally, both dsRNA and RV1B infection increased lung TSLP exclusively at exacerbations. Our data suggest that dsRNA challenges superimposed on allergic inflammation are suited for pharmacological studies of asthma exacerbations including the regulation of lung tissue TSLP, TNF-α, IFN-β and IFN-λ.

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BackgroundExacerbations 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.MethodMouse 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-1β, 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.ResultsHDM 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-1β, 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.ConclusionWe 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-016-0808-x) contains supplementary material, which is available to authorized users.

BACKGROUND AND PURPOSEStatin treatment may ameliorate viral infection-induced exacerbations of chronic obstructive pulmonary disease (COPD), which exhibit Th2-type bronchial inflammation. Thymic stromal lymphopoietin (TSLP), a hub cytokine switching on Th2 inflammation, is overproduced in viral and dsRNA-stimulated bronchial epithelial cells from COPD donors. Hence, TSLP may be causally involved in exacerbations. This study tests the hypothesis that simvastatin inhibits dsRNA-induced TSLP. EXPERIMENTAL APPROACHEpithelial cells, obtained by bronchoscopy from COPD (n = 7) and smoker control (n = 8) donors, were grown and stimulated with a viral infection and danger signal surrogate, dsRNA (10 mg·mL ) before dsRNA. Cytokine expression and production, and transcription factor (IRF3 and NF-kB) activation were determined. KEY RESULTSdsRNA induced TSLP, TNF-a, CXCL8 and IFN-b. TSLP was overproduced in dsRNA-exposed COPD cells compared with control. Simvastatin, but not dexamethasone, concentration-dependently inhibited dsRNA-induced TSLP. Unexpectedly, simvastatin acted independently of mevalonate and did not affect dsRNA-induced NF-kB activation nor did it reduce production of TNF-a and CXCL8. Instead, simvastatin inhibited dsRNA-induced IRF3 phosphorylation and generation of IFN-b. CONCLUSIONS AND IMPLICATIONSIndependent of mevalonate and NF-kB, previously acknowledged anti-inflammatory mechanisms of pleiotropic statins, simvastatin selectively inhibited dsRNA-induced IRF3 activation and production of TSLP and IFN-b in COPD epithelium. These data provide novel insight into epithelial generation of TSLP and suggest paths to be exploited in drug discovery aimed at inhibiting TSLP-induced pulmonary immunopathology. AbbreviationsCOPD, chronic obstructive pulmonary disease; DAMP, danger-associated molecular patterns; dsRNA, double stranded RNA; GOLD, global initiative for obstructive lung disease; HMG-CoA, 3-hydroxy-3-methylglutaryl-coenzyme A; IKK, IkB kinase; IRF3, IFN regulatory factor 3; RV, rhinoviral; TANK, TRAF family member-associated NF-kB activator; TBK-1, TANK-binding kinase-1; Th, T helper cells; TLR3, Toll receptor 3; TRPV1, transient receptor potential cation channel subfamily V member 1; TSLP, thymic stromal lymphopoietin; UBC, Ubiquitin C