Viral-induced asthma exacerbations, which exhibit both Th1-type neutrophilia and Th2-type inflammation, associate with secretion of Interleukin (IL)-1β. IL-1β induces neutrophilic inflammation. It may also increase Th2-type cytokine expression. We hypothesised that IL-1β 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. Wild-type (WT) and IL-1β deficient (IL-1β−/−) 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-1β, CCL11 and CCL2 using RT-qPCR. Expression of IL-1β, 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-1β−/− mice at exacerbation. Effects of HDM alone were not reduced in IL-1β-deficient mice. Without being involved in the baseline HDM-induced allergic asthma, IL-1β signalling was required to induce neutrophil chemotactic factors, IL-33, and Muc5ac expression at viral stimulus-induced exacerbation. We suggest that IL-1β has a role both in neutrophilic and Th2 inflammation at viral-induced asthma exacerbations.

Background: Degraded extracellular matrix can stimulate the innate immune system via the Toll-Like Receptor-4 (TLR4). In the pancreas, syndecan-anchored heparan sulphate (HS) on the ductal epithelium can be cleaved off its protein cores by the proteases (trypsin and elastase) and potentially activate TLR4 signalling. Methods: To investigate this signalling event, a low sulphated HS (500 μg/ml) was infused into the biliary-pancreatic duct of C57BL/6J wild-type mice. Phosphate buffered saline (PBS) and lipopolysaccharide (LPS) were used as negative and positive controls, respectively. Mice were sacrificed after 1, 3, 6, 9, and 48 hours and tissues were analysed for neutrophil and cytokine contents. In order to study the TLR4 signalling pathway of HS in the pancreas, genetically engineered mice lacking TLR4, Myeloid Differentiation primary response gene (88) (MyD88) or Interferon Regulatory Factor 3 (IRF3) were subjected to pancreatic infusion of HS. Results: Neutrophil sequestration and corresponding myeloperoxidase (MPO) activity in the pancreas were increased 9 hours following HS challenge. In wild-type mice, the monocyte chemoattractant protein-1(MCP-1) increased at 3 hours after infusion, while RANTES increased after 9 hours. TLR4, MyD88, and IRF3 knockout mice showed an abrogated neutrophil recruitment and myeloperoxidase activity in the HS group, while the LPS response was only abolished in TLR4 and MyD88 knockouts. Conclusions: The results of this study show that HS is capable of initiating a TLR4-dependent innate immune response in the pancreas which is distinctly different from that induced by LPS. This inflammatory response was mediated predominantly through IRF3- dependent pathway. Release of HS into the pancreatic duct may be one important mediator in the pancreatic ductal defence.

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.

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. 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. 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. 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.

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‐β 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 β‐defensin‐2, IFN‐β, 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‐β 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 β‐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.