C57Bl/6N mice have an attenuated lung inflammatory response to dsRNA compared to C57Bl/6J and BALB/c mice.

BACKGROUND: Lower respiratory infections caused by ssRNA viruses are a major health burden globally. Translational mouse models are a valuable tool for medical research, including research on respiratory viral infections. In in vivo mouse models, synthetic dsRNA can be used as a surrogate for ssRNA virus replication. However, studies investigating how genetic background of mice impacts the murine lung inflammatory response to dsRNA is lacking. Hence, we have compared lung immunological responses of BALB/c, C57Bl/6N and C57Bl/6J mice to synthetic dsRNA. METHODS: dsRNA was administered intranasally to BALB/c, C57Bl/6N and C57Bl/6J mice once/day for three consecutive days. Lactate dehydrogenase (LDH) activity, inflammatory cells, and total protein concentration were analyzed in bronchoalveolar lavage fluid (BALF). Pattern recognition receptors levels (TLR3, MDA5 and RIG-I) were measured in lung homogenates using RT-qPCR and western blot. Gene expression of IFN-ß, TNF-α, IL-1ß and CXCL1 was assessed in lung homogenates by RT-qPCR. ELISA was used to analyze protein concentrations of CXCL1 and IL-1ß in BALF and lung homogenates. RESULTS: BALB/c and C57Bl/6J mice showed infiltration of neutrophils to the lung, and an increase in total protein concentration and LDH activity in response to dsRNA administration. Only modest increases in these parameters were observed for C57Bl/6N mice. Similarly, dsRNA administration evoked an upregulation of MDA5 and RIG-I gene and protein expression in BALB/c and C57Bl/6J, but not C57Bl/6N, mice. Further, dsRNA provoked an increase in gene expression of TNF-α in BALB/c and C57Bl/6J mice, IL-1ß only in C57Bl/6N mice and CXCL1 exclusively in BALB/c mice. BALF levels of CXCL1 and IL-1ß were increased in BALB/c and C57Bl/6J mice in response to dsRNA, whereas the response of C57Bl/6N was blunt. Overall, inter-strain comparisons of the lung reactivity to dsRNA revealed that BALB/c, followed by C57Bl/6J, had the most pronounced respiratory inflammatory responses, while the responses of C57Bl/6N mice were attenuated. CONCLUSIONS: We report clear differences of the lung innate inflammatory response to dsRNA between BALB/c, C57Bl/6J and C57Bl/6N mice. Of particular note, the highlighted differences in the inflammatory response of C57Bl/6J and C57Bl/6N substrains underscore the value of strain selection in mouse models of respiratory viral infections.

Allergen Immunotherapy Enhances Airway Epithelial Antiviral Immunity in Patients with Allergic Asthma (VITAL Study): A Double-Blind Randomized Controlled Trial.

Rationale: Allergic asthma is linked to impaired bronchial epithelial secretion of IFNs, which may be causally linked to the increased risk of viral exacerbations. We have previously shown that allergen immunotherapy (AIT) effectively reduces asthma exacerbations and prevents respiratory infections requiring antibiotics; however, whether AIT alters antiviral immunity is still unknown. Objectives: To investigate the effect of house dust mite sublingual AIT (HDM-SLIT) on bronchial epithelial antiviral and inflammatory responses in patients with allergic asthma. Methods: In this double-blind, randomized controlled trial (VITAL [The Effect of Allergen Immunotherapy on Anti-viral Immunity in Patients with Allergic Asthma]), adult patients with HDM allergic asthma received HDM-SLIT 12-SQ or placebo for 24 weeks. Bronchoscopy was performed at baseline and at Week 24, which included sampling for human bronchial epithelial cells. Human bronchial epithelial cells were cultured at baseline and at Week 24 and stimulated with the viral mimic polyinosinic:polycytidylic acid (poly(I:C)). mRNA expression was quantified using qRT-PCR, and protein concentrations were measured using multiplex ELISA. Measurements and Main Results: Thirty-nine patients were randomized to HDM-SLIT (n = 20) or placebo (n = 19). HDM-SLIT resulted in increased polyinosinic:polycytidylic acid-induced expression of IFN-ß at both the gene (P = 0.009) and protein (P = 0.02) levels. IFN-λ gene expression was also increased (P = 0.03), whereas IL-33 tended to be decreased (P = 0.09). On the other hand, proinflammatory cytokines IL-6 (P = 0.009) and TNF-α (tumor necrosis factor-α) (P = 0.08) increased compared with baseline in the HDM-SLIT group. There were no significant changes in TSLP (thymic stromal lymphopoietin), IL-4, IL-13, and IL-10. Conclusions: HDM-SLIT improves bronchial epithelial antiviral resistance to viral infection. These results potentially explain the efficacy of HDM-SLIT in reducing exacerbations in allergic asthma. Clinical trial registered with www.clinicaltrials.gov (NCT04100902).

LL-37 and Double-Stranded RNA Synergistically Upregulate Bronchial Epithelial TLR3 Involving Enhanced Import of Double-Stranded RNA and Downstream TLR3 Signaling.

The human host defense peptide LL-37 influences double-stranded RNA signaling, but this process is not well understood. Here, we investigate synergistic actions of LL-37 and synthetic double-stranded RNA (poly I:C) on toll-like receptor 3 (TLR3) expression and signaling, and examine underlying mechanisms. In bronchial epithelial BEAS-2B cells, LL-37 potentiated poly I:C-induced TLR3 mRNA and protein expression demonstrated by qPCR and Western blot, respectively. Interestingly, these effects were associated with increased uptake of rhodamine-tagged poly I:C visualized by immunocytochemistry. The LL-37/poly I:C-induced upregulation of TLR3 mRNA expression was prevented by the endosomal acidification inhibitor chloroquine, indicating involvement of downstream TLR3 signaling. The glucocorticoid dexamethasone reduced LL-37/poly I:C-induced TLR3 expression on both mRNA and protein levels, and this effect was associated with increased IκBα protein expression, suggesting that dexamethasone acts via attenuation of NF-κB activity. We conclude that LL-37 potentiates poly I:C-induced upregulation of TLR3 through a mechanism that may involve enhanced import of poly I:C and that LL-37/poly I:C-induced TLR3 expression is associated with downstream TLR3 signaling and sensitive to inhibition of NF-κB activity.

House dust mite sensitization and exposure affects bronchial epithelial anti-microbial response to viral stimuli in patients with asthma.

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-ß 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 ß-defensin-2, IFN-ß, 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-ß 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 ß-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.

Phenotype and severity of asthma determines bronchial epithelial immune responses to a viral mimic.

BACKGROUND: Asthma is characterised by an aggravated immune response to respiratory viral infections. This phenomenon is a clinically well-recognised driver of acute exacerbations, but how different phenotypes of asthma respond immunologically to viruses is unclear. OBJECTIVES: To describe the association between different phenotypes and severity of asthma and bronchial epithelial immune responses to viral stimulation. METHODS: In the Immunoreact study, healthy subjects (n=10) and 50 patients with asthma were included; 30 (60%) were atopic, and 34 (68%) were eosinophilic; 14 (28%) had severe asthma. All participants underwent bronchoscopy with collection of bronchial brushings. Bronchial epithelial cells (BECs) were expanded and stimulated with the viral replication mimic poly (I:C) (Toll-like receptor (TLR)3 agonist) in vitro. The expression of TLR3-induced pro-inflammatory and antiviral responses of BECs were analysed using reverse transcriptase quantitative PCR and multiplex ELISA and compared across asthma phenotypes and severity of disease. RESULTS: Patients with atopic asthma had increased induction of interleukin (IL)-4, interferon (IFN)-ß, IL-6, tumour necrosis factor-α, and IL-1ß after poly (I:C) stimulation compared to non-atopic patients, whereas in patients with eosinophilic asthma only IL-6 and IL-8 induction was higher than in non-eosinophilic asthma. Patients with severe asthma displayed a decreased antiviral IFN-ß, and increased expression of IL-8, most pronounced in atopic and eosinophilic asthmatics. Furthermore, induction of IL-33 in response to poly (I:C) was increased in severe atopic and in severe eosinophilic asthma, but thymic stromal lymphopoietin only in severe eosinophilic asthma. CONCLUSIONS: The bronchial epithelial immune response to a viral mimic stimulation differs between asthma phenotypes and severities, which may be important to consider when targeting novel asthma treatments.

Imiquimod Boosts Interferon Response, and Decreases ACE2 and Pro-Inflammatory Response of Human Bronchial Epithelium in Asthma.

Background: Both anti-viral and anti-inflammatory bronchial effects are warranted to treat viral infections in asthma. We sought to investigate if imiquimod, a TLR7 agonist, exhibits such dual actions in ex vivo cultured human bronchial epithelial cells (HBECs), targets for SARS-CoV-2 infectivity. Objective: To investigate bronchial epithelial effects of imiquimod of potential importance for anti-viral treatment in asthmatic patients. Methods: Effects of imiquimod alone were examined in HBECs from healthy (N=4) and asthmatic (N=18) donors. Mimicking SARS-CoV-2 infection, HBECs were stimulated with poly(I:C), a dsRNA analogue, or SARS-CoV-2 spike-protein 1 (SP1; receptor binding) with and without imiquimod treatment. Expression of SARS-CoV-2 receptor (ACE2), pro-inflammatory and anti-viral cytokines were analyzed by RT-qPCR, multiplex ELISA, western blot, and Nanostring and proteomic analyses. Results: Imiquimod reduced ACE2 expression at baseline and after poly(I:C) stimulation. Imiquimod also reduced poly(I:C)-induced pro-inflammatory cytokines including IL-1ß, IL-6, IL-8, and IL-33. Furthermore, imiquimod increased IFN-ß expression, an effect potentiated in presence of poly(I:C) or SP1. Multiplex mRNA analysis verified enrichment in type-I IFN signaling concomitant with suppression of cytokine signaling pathways induced by imiquimod in presence of poly(I:C). Exploratory proteomic analyses revealed potentially protective effects of imiquimod on infections. Conclusion: Imiquimod triggers viral resistance mechanisms in HBECs by decreasing ACE2 and increasing IFN-ß expression. Additionally, imiquimod improves viral infection tolerance by reducing viral stimulus-induced epithelial cytokines involved in severe COVID-19 infection. Our imiquimod data highlight feasibility of producing pluripotent drugs potentially suited for anti-viral treatment in asthmatic subjects.

Direct effects of mast cell proteases, tryptase and chymase, on bronchial epithelial integrity proteins and anti-viral responses.

BACKGROUND: Mast cells (MCs) are known to contribute to both acute and chronic inflammation. Bronchial epithelial cells are the first line of defence against pathogens and a deficient anti-viral response has been suggested to play a role in the pathogenesis of asthma exacerbations. However, effects of MC mediators on bronchial epithelial immune response have been less studied. The aim of this study is to investigate the direct effects of stimulation with MC proteases, tryptase and chymase, on inflammatory and anti-viral responses in human bronchial epithelial cells (HBECs). METHOD: Cultured BEAS-2b cells and primary HBECs from 3 asthmatic patients were stimulated with tryptase or chymase (0.1 to 0.5 µg/ml) for 1, 3, 6 and 24 h. To study the effects of MC mediators on the anti-viral response, cells were stimulated with 10 µg/ml of viral mimic Poly (I:C) for 3 and 24 h following pre-treatment with 0.5 µg/ml tryptase or chymase for 3 h. Samples were analysed for changes in pro-inflammatory and anti-viral mediators and receptors using RT-qPCR, western blot and Luminex. RESULTS: Tryptase and chymase induced release of the alarmin ATP and pro-inflammatory mediators IL-8, IL-6, IL-22 and MCP-1 from HBECs. Moreover, tryptase and chymase decreased the expression of E-cadherin and zonula occludens-1 expression from HBECs. Pre-treatment of HBECs with tryptase and chymase further increased Poly (I:C) induced IL-8 release at 3 h. Furthermore, tryptase significantly reduced type-I and III interferons (IFNs) and pattern recognition receptor (PRR) expression in HBECs. Tryptase impaired Poly (I:C) induced IFN and PRR expression which was restored by treatment of a serine protease inhibitor. Similar effects of tryptase on inflammation and anti-viral responses were also confirmed in primary HBECs from asthmatic patients. CONCLUSION: MC localization within the epithelium and the release of their proteases may play a critical role in asthma pathology by provoking pro-inflammatory and alarmin responses and downregulating IFNs. Furthermore, MC proteases induce downregulation of epithelial junction proteins which may lead to barrier dysfunction. In summary, our data suggests that mast cells may contribute towards impaired anti-viral epithelial responses during asthma exacerbations mediated by the protease activity of tryptase.

SARS-CoV-2 spike protein binds to bacterial lipopolysaccharide and boosts proinflammatory activity.

There is a link between high lipopolysaccharide (LPS) levels in the blood and the metabolic syndrome, and metabolic syndrome predisposes patients to severe COVID-19. Here, we define an interaction between SARS-CoV-2 spike (S) protein and LPS, leading to aggravated inflammation in vitro and in vivo. Native gel electrophoresis demonstrated that SARS-CoV-2 S protein binds to LPS. Microscale thermophoresis yielded a KD of ∼47 nM for the interaction. Computational modeling and all-atom molecular dynamics simulations further substantiated the experimental results, identifying a main LPS-binding site in SARS-CoV-2 S protein. S protein, when combined with low levels of LPS, boosted nuclear factor-kappa B (NF-κB) activation in monocytic THP-1 cells and cytokine responses in human blood and peripheral blood mononuclear cells, respectively. The in vitro inflammatory response was further validated by employing NF-κB reporter mice and in vivo bioimaging. Dynamic light scattering, transmission electron microscopy, and LPS-FITC analyses demonstrated that S protein modulated the aggregation state of LPS, providing a molecular explanation for the observed boosting effect. Taken together, our results provide an interesting molecular link between excessive inflammation during infection with SARS-CoV-2 and comorbidities involving increased levels of bacterial endotoxins.

Human host defense peptide LL-37 facilitates double-stranded RNA pro-inflammatory signaling through up-regulation of TLR3 expression in vascular smooth muscle cells.

OBJECTIVE: The importance of human host defense peptide LL-37 in vascular innate immunity is not understood. Here, we assess the impact of LL-37 on double-stranded RNA (dsRNA) signaling in human vascular smooth muscle cells. MATERIALS AND METHODS: Cellular import of LL-37 and synthetic dsRNA (poly I:C) were investigated by immunocytochemistry and fluorescence imaging. Transcript and protein expression were determined by qPCR, ELISA and Western blot. Knockdown of TLR3 was performed by siRNA. RESULTS: LL-37 was rapidly internalized, suggesting that it has intracellular actions. Co-stimulation with poly I:C and LL-37 enhanced pro-inflammatory IL-6 and MCP-1 transcripts several fold compared to treatment with poly I:C or LL-37 alone. Poly I:C increased IL-6 and MCP-1 protein production, and this effect was potentiated by LL-37. LL-37-induced stimulation of poly I:C signaling was not associated with enhanced import of poly I:C. Treatment with poly I:C and LL-37 in combination increased expression of dsRNA receptor TLR3 compared to stimulation with poly I:C or LL-37 alone. In TLR3 knockdown cells, treatment with poly I:C and LL-37 in combination had no effect on IL-6 and MCP-1 expression, showing loss of function. CONCLUSIONS: LL-37 potentiates dsRNA-induced cytokine production through up-regulation of TLR3 expression representing a novel pro-inflammatory mechanism.

Interferon-ß deficiency at asthma exacerbation promotes MLKL mediated necroptosis.

Defective production of antiviral interferon (IFN)-ß is thought to contribute to rhinovirus-induced asthma exacerbations. These exacerbations are associated with elevated lung levels of lactate dehydrogenase (LDH), indicating occurrence of cell necrosis. We thus hypothesized that reduced lung IFN-ß could contribute to necrotic cell death in a model of asthma exacerbations. Wild-type and IFN-ß-/- mice were given saline or house dust mite (HDM) intranasally for 3 weeks to induce inflammation. Double-stranded RNA (dsRNA) was then given for additional 3 days to induce exacerbation. HDM induced an eosinophilic inflammation, which was not associated with increased expression of cleaved caspase-3, cleaved PARP or elevated bronchoalveolar lavage fluid (BALF) LDH levels in wild-type. However, exacerbation evoked by HDM + dsRNA challenges increased BALF levels of LDH, apoptotic markers and the necroptotic markers receptor-interacting protein (RIP)-3 and phosphorylation of mixed linage kinase domain-like protein (pMLKL), compared to HDM + saline. Absence of IFN-ß at exacerbation further increased BALF LDH and protein expression of pMLKL compared to wild-type. We demonstrate that cell death markers are increased at viral stimulus-induced exacerbation in mouse lungs, and that absence of IFN-ß augments markers of necroptotic cell death at exacerbation. Our data thus suggest a novel role of deficient IFN-ß production at viral-induced exacerbation.

IL-1ß mediates lung neutrophilia and IL-33 expression in a mouse model of viral-induced asthma exacerbation.

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