Exacerbations of asthma, typically caused by viral infections, are serious conditions in need of effective treatment. Animal models mimicking clinical features of asthma exacerbations including lung interferon responses are needed. Here we examined effects of dsRNA and the expression of two interferons using a model of allergic asthma additionally challenged with the rhinovirus infection intermediate dsRNA.
Experimental asthma in mice was established by immunisation (ip) with OVA-alum and 2 weeks later exposure to aerosolised OVA (30 min daily for 4 days). Mice with already established allergic asthma or control animals received intranasal challenge with dsRNA for 3 days. BALF was analysed for total- and differential cell counts as well as total protein. Lung tissue was obtained for lung histology and RT-qPCR analysis for IFN-β and IFN-λ.
Results: dsRNA-induced exacerbations in mice with established allergic asthma were recorded as increased BALF levels of total protein (p<0.05), increased lung Htx-inflammation score (p<0.001), and increased BALF- and lung tissue neutrophilia (p<0.001). Allergen challenge alone increased IFN-β (p<0.001) but not IFN-λ. dsRNA-induced exacerbation increased IFN-λ (p<0.05) but produced no further increase in IFN-β.
Conclusions: Interestingly, mRNA expression of IFN-β was increased after allergen challenge but was not further enhanced after dsRNA challenge. Lung tissue expression of IFN-λ was increased exclusively in animals with both allergic asthma and dsRNA-induced exacerbation. This model is suited for studies of inflammatory and antiviral defence aspects of asthma exacerbations, including pharmacology of lung expression of IFN-β and IFN-λ.
Rhinovirus (RV)-induced chronic obstructive pulmonary disease (COPD) exacerbations exhibit TH(2)-like inflammation. We hypothesized that RV-infected bronchial epithelial cells (BEC) overproduce TH(2)-switching hub cytokine, thymic stromal lymphopoietin (TSLP) in COPD.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).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.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.
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. 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-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. 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-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. 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.
Background Rhinovirus 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. Methods Allergic 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. Results In 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). Conclusions dsRNA-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.
Possibly reflecting anti-inflammatory properties, statin treatment may ameliorate viral-induced exacerbations. We have shown that simvastatin but not dexamethasone inhibits viral-induced, potentially pathogenic thymic stromal lymphopoietin production. It is important that treatments of exacerbations spare the host defence to viral infections. IFN-beta, IFN-lambda and IL-32 are major antiviral components of innate immunity. Objective: Explore effects of simvastatin and dexamethasone on TLR3-induced IFN-beta, IFN-lambda and IL-32 in asthmatic epithelium. Methods: Primary bronchial epithelial cells, obtained by fibre optic bronchoscopy from allergic asthma patients (n=6) were grown in 12-well plates and treated with simvastatin (0,2-5μg), dexamethasone (1μM) or vehicle control prior to TLR3 stimulation for 3 and 24 h and samples analysed by RTqPCR and ELISA. Results: TLR3 stimulation induced early (3h) expression of IFN-beta (1000-fold) that was dose-dependently inhibited by simvastatin but not by dexamethasone. IFN-lambda was induced (6000-fold) at 3h and further sustained at 24 h (2000-fold). Only the early IFN-lambda expression was reduced by the highest dose of simvastatin. Dexamethasone was without effect. IL-32 was induced (2-fold) at 3h and further increased at 24h. At both time-points the highest dose of simvastatin reduced IL-32 but dexamethasone was without effect. Conclusion: Dexamethasone treatment spared TLR3-induced expression of all three anti-viral cytokines in bronchial epithelial cells from asthma donors. Simvastatin completely inhibited TLR3-induced IFN-beta whereas the inducement of IFN-lambda and IL-32 were largely spared by simvastatin.
Respiratory viral infections cause exacerbations of asthma and COPD that cannot be effectively treated today. Thymic Stromal Lymphopoietin (TSLP) is an upstream epithelial cytokine linking the innate and adaptive immune system. Viral stimuli induce epithelial overexpression of TSLP in asthma and COPD. We hypothesise that TSLP switches on Th2-type inflammation in severe asthma/COPD. A deficient antiviral interferon-response has also been showed in asthmatic epithelium.
The aim of this thesis was to study effects of selected compounds on viral induced epithelial TSLP and antiviral proteins.
We show that capsazepine, a small airway relaxant, inhibits TSLP induced by the viral infection surrogate dsRNA in human bronchial epithelial cells (HBECs) from asthmatic and healthy donors. Surprisingly, simvastatin, a cholesterol-lowering compound with pleiotrophic effects, inhibits dsRNA-induced IRF3 phosphorylation and TSLP but not NFkB in HBECs from COPD-patients, healthy smokers and asthmatics. TSLP-inhibitory effects of capsazepine and simvastatin are superior to effects produced by the glucocorticoid dexamethasone. However, simvastatin, but not dexamethasone, inhibits antiviral interferon-beta (IFNβ) and IL-32. We developed a method by which repeated topical nasal dsRNA for the first time induces effects in vivo on human respiratory mucosa. We found that IFNβ, IFNλ and IL-32 were upregulated by dsRNA during but not outside birch pollen season. dsRNA challenges were below threshold for TSLP induction.
In conclusion, using HBEC we discovered that different classes of compounds were effective inhibitors of viral induced TSLP. With pharmacological tools we discovered that IRF3 phosphorylation is involved in TSLP production meaning also that difficulties arise regarding the aim of inhibiting TSLP without inhibiting interferons.
For future studies we devise novel human in vivo methods for study of pharmacology of airway antiviral protein production.
Background: Possibly reflecting anti-inflammatory properties, statin treatment may ameliorate COPD exacerbations. Viral infections apparently cause TH2 type COPD exacerbations. We have shown previously that thymic stromal lymphopoietin (TSLP), a cytokine linking innate and adaptive immunity and switching on TH2 type inflammation, is overproduced in viral stimulated epithelial cells from GOLD stage IV COPD donors. Objective: Explore effects of simvastatin on viral-induced TSLP in epithelial cells from patients with GOLD stage II COPD. Methods: Primary bronchial epithelial cells, obtained by fibre optic bronchoscopy from COPD (n=4) and healthy individual (n=3) donors, were grown in 12-well plates and stimulated with a synthetic viral surrogate, double-stranded RNA (dsRNA, 10μg/ml) to induce cytokine expression (3h, RT-qPCR) and production (24 h, ELISA). Simvastatin (0,2-5μg) with or without mevalonate (13μg/ml) was added 18 h prior to dsRNA. Alternatively, dexamethasone (1μM) was added 1 h prior to dsRNA. Results: dsRNA induced TSLP, TNF-alpha and IL-8 mRNA and protein expression (p 0.05). Conclusion: Independent of the mevalonic pathway, simvastatin selectively inhibited dsRNA-induced TSLP-production in COPD cells. These data support exploration of statin treatment in viral-induced COPD exacerbations. The pharmacology of simvastatin may unravel paths of selective inhibition of TSLP-production in COPD epithelium.
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