Background Anti-interleukin (IL)-4R alpha monoclonal antibodies (mAb) improve lung function and decrease the number of exacerbations in patients with COPD type (T)2 inflammation. However, the involvement of early innate immune responses underlying these treatment effects is not well known. We sought to understand the effect and mechanisms of IL-4R alpha mAb treatment on bronchial epithelial cells (BECs) from COPD patients under T2 inflammatory conditions with and without rhinoviral infection. Methods Primary BECs from healthy and COPD patients were grown at an air-liquid interface and stimulated with IL-4 or IL-13 cytokines in the presence of IL-4R alpha mAb. Cells were infected with human rhinovirus 1B and collected 24 h after infection. Antiviral mediators (i.e., interferons (IFNs) and pattern recognition receptors (PRRs)), as well as chemokine and alarmin expression, were measured by reverse transcriptase quantitative PCR and ELISA. Results Treatment with IL-4R alpha mAb (100 nM) inhibited the eotaxin-3 (CCL26) gene after IL-4/IL-13 induction (p<0.05) in COPD BECs. However, no significant changes in rhinovirus-induced IFN-beta, PRRs or thymic stromal lymphopoietin gene responses were observed with IL-4/IL-13 stimulation and IL-4R alpha mAb treatment. A significant increase in mucin 5AC gene expression was observed with both IL-4 and IL-13 stimulation, but it was not reduced with IL-4R alpha treatment in BECs. C Conclusions Inhibition of IL-4R alpha reduced CCL26 levels without affecting antiviral immune responses in BECs from COPD patients. Inhibition of IL-4R alpha reduced IL-4/IL-13 signalling without broadly suppressing the immune system, which might suggest that inhibition of the IL-4R alpha pathways may prevent COPD exacerbations through reduction of eosinophil chemotaxis.

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. 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-β, TNF-α, IL-1β and CXCL1 was assessed in lung homogenates by RT-qPCR. ELISA was used to analyze protein concentrations of CXCL1 and IL-1β in BALF and lung homogenates. 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-1β only in C57Bl/6N mice and CXCL1 exclusively in BALB/c mice. BALF levels of CXCL1 and IL-1β 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. 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.

Background: Azithromycin (AZM) is effective in reducing asthma exacerbations and augments in vitro epithelial anti-viral immunity. However, the clinical data in eosinophilic (eos) and non-eosinophilic (non-eos) asthma phenotypes are conflicting. Objectives: To compare the effect of in vitro AZM treatment on IFNβ response in rhinovirus (RV1B) infected bronchial epithelial cells (BECs) of eos and non-eos asthma patients. Methods: Twenty asthma patients, 10 eos (5 atopic) and 10 non-eos (5 atopic), underwent bronchoscopy to harvest BECs. BECs were pre-treated in vitro with and without AZM 24h before infection with RV1B. Gene expression levels of IFNβ was measured 24h after infection by real-time PCR and compared across asthma phenotypes using Mann-Whitney and Wilcoxon tests. Results: AZM increased RV1B-induced IFNβ expression in non-eos asthma (p=0.002, fig. 1a) and there was a trend in eos asthma (p=0.06). This response was similar in eos and non-eos groups (p=0.15). Interestingly, within eos asthma patients, non-atopic asthmatics had a better response to AZM induced IFNβ augmentation compared to atopic asthmatics (p=0.03). Whereas in non-eos asthma patients, no significant difference was found between atopic and non-atopic patients (p=0.88). Conclusions: In vitro AZM treatment augments BEC antiviral response to RV1B in both eosinophilic and non-eosinophilic asthma phenotypes.

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.

We have previously shown that the induction of anti-viral interferons (IFNs) upon TLR3 stimulation may be impaired in asthma patients. However, viral infections could activate several pattern recognition receptors (PRRs) simultaneously, including TLR3, but also TLR7 and the cytosolic MDA5 and RIGI receptors. Here, we aimed to study the effect of TLR3/TLR7 co-stimulation in the IFN response of the bronchial epithelium in asthma patients. Bronchial epithelial cells from asthma patients (n=9) were stimulated with a TLR3 agonist, poly(I:C), and/or imiquimod, a TLR7 agonist, for 24 hours. siRNA knockdown of MDA5 and RIG-I was also performed. Gene expression was analyzed by RT-qPCR and Nanostring analysis. Compared to unstimulated cells, imiquimod induced no IFN-β, but poly(I:C) a 17-fold induction. Interestingly, poly(I:C) and imiquimod co-stimulation induced a 43-fold induction of IFN-β, which was significantly higher when compared to the poly(I:C) (17-fold) induction. siRNA knockdown of MDA5/RIG-I in co-stimulated cells led to an abolishment of the IFN-β boost. Nanostring analysis of downstream IFN signaling genes in poly(I:C) vs. co-stimulated cells revealed differential expression of STAT2, JAK1, IRF1 and C1QBP. Downmodulation of the negative regulator of MDA5 signaling, C1QBP, was confirmed by RT-qPCR. Poly(I:C)-induced IFN signaling is boosted during simultaneous TLR7 stimulation, involving MDA5 and RIG-I receptors. Hence, the IFN response to viral infections in asthma patients with a deficient TLR7 function (previously described) could be compromised. However, further knockdown-studies of other PRRs are warranted in order to make firm conclusions.

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-1β, IL-6, IL-8, and IL-33. Furthermore, imiquimod increased IFN-β 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-β 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.

Introduction: Combining anti-viral and anti-inflammatory effects in a single drug may be beneficial in treating COVID-19. We hypothesized that the TLR7 agonist imiquimod (imq) may exert these actions in human bronchial epithelial cells (HBECs), which are targets in SARS-CoV-2 mediated lung injury. Methods: Using primary HBECs from asthmatic donors (N=18), we explored actions of imq related to airway viral resistance and tolerance. HBECs were treated with imq alone or in combination with the viral mimic poly (I:C) or the SARS-CoV-2 spike protein 1 (SP1). Anti-viral and pro-inflammatory mediators were analyzed by Luminex, RT-qPCR and mRNA gene pathway analysis. Results: imq treatment alone induced IFN-ß and CCL5 (p<0.05) mRNA and reduced transcription of IL-1ß (p<0.01) at 24h. In SP1 or poly (I:C) stimulated HBECs, treatment with imq augmented IFN-ß mRNA expression by a 2-fold, respectively (p<0.05). Imq in combination with poly (I:C) decreased protein release of IL-8, CCL5, IL-1ß and IL-6 (p<0.05). Furthermore, gene pathway analysis revealed that imq enriched poly(I:C)-induced IFN signaling, IL-20 family signaling (epithelial repair), antigen presentation and cytokine signaling. Enriched cytokine signaling genes included negative regulators such as IKBKG and SIGIRR. Conclusion: imq exerts distinct anti-viral resistance effects in HBECs by increasing anti-viral signaling and improves viral infection tolerance by diminishing epithelial cytokines potentially involved in severe COVID-19. Our findings highlight a possibility of developing dual action drugs suitable for anti-SARS-CoV-2 treatment.

BakgrundFor att tackla den radande SARS-COV-2-pandemin har det stallts hoga krav pa en snabb utveckling och utvardering av nya antivirala lakemedel. Bland nagra av de antivirala lakemedelskandidaterna finns imiquimod, en TLR7 agonist normalt administrerad for behandling av konsvartor och basalcellscarcinom. Eftersom det har pavisats att imiquimod kan oka den antivirala responsen, ar var hypotes att den ocksa skulle kunna anvandas som ett nyttpotentiellt lakemedel for att behandla COVID19.MetodHumana bronkepitelceller (BECs) fran 4 friska och 8 astmatiska patienter odlades fram efter borstprov i samband med bronkoskopi och behandlades med imiquimod ensamt, eller tillsammans med dsRNA samt SARS-COV-2 spike-protein (s-protein) for att harma en virusinfektion. SiRNA mot MDA5 och RIG-I anvandes ocksa. Cellerna analyserades efter 3 och 24 timmar for uttryck av angiotensin-converting-enzyme-2 (ACE2), interferon-beta (IFNb)samt andra cytokiner involverade i COVID19 med luminex, RT-qPCR och western blot.ResultatObehandlade astmatiska BECs uttrycker mindre ACE2 jamfort med BECs fran friska individer. I mekanistiska experiment med astmatiska BECs halverade behandling med imiquimod uttrycket av ACE2 (p<0.05). Imiquimod okade aven uttrycket av IFNb 2 ganger, samtidigt som IL-1b, involverade i cytokinstormar, minskade (p<0.05). Kombination av s-protein och imiquimod okade IFNb ytterligare 2 ganger (p<0.05). Dessa observationer gjordes aven i dsRNA-stimulerade celler som behandlats med imiquimod (p<0.05). SiRNA visade attimiquimods IFNb boostade effekt var reglerad via receptorerna MDA5 och RIG-I.SlutsatsBehandling av BECs med imiquimod visar pa tre effekter: minskat uttryck av SARS-COV-2 receptorn ACE2, okad antiviral kapacitet hos bronkepitelceller samt en minskning av cytokiner involverade i dodliga cytokinstormar.

BakgrundFor att tackla den radande SARS-COV-2-pandemin har det stallts hoga krav pa en snabb utveckling och utvardering av nya antivirala lakemedel. Bland nagra av de antivirala lakemedelskandidaterna finns imiquimod, en TLR7 agonist normalt administrerad for behandling av konsvartor och basalcellscarcinom. Eftersom det har pavisats att imiquimod kan oka den antivirala responsen, ar var hypotes att den ocksa skulle kunna anvandas som ett nyttpotentiellt lakemedel for att behandla COVID19.MetodHumana bronkepitelceller (BECs) fran 4 friska och 8 astmatiska patienter odlades fram efter borstprov i samband med bronkoskopi och behandlades med imiquimod ensamt, eller tillsammans med dsRNA samt SARS-COV-2 spike-protein (s-protein) for att harma en virusinfektion. SiRNA mot MDA5 och RIG-I anvandes ocksa. Cellerna analyserades efter 3 och 24 timmar for uttryck av angiotensin-converting-enzyme-2 (ACE2), interferon-beta (IFNb)samt andra cytokiner involverade i COVID19 med luminex, RT-qPCR och western blot.ResultatObehandlade astmatiska BECs uttrycker mindre ACE2 jamfort med BECs fran friska individer. I mekanistiska experiment med astmatiska BECs halverade behandling med imiquimod uttrycket av ACE2 (p<0.05). Imiquimod okade aven uttrycket av IFNb 2 ganger, samtidigt som IL-1b, involverade i cytokinstormar, minskade (p<0.05). Kombination av s-protein och imiquimod okade IFNb ytterligare 2 ganger (p<0.05). Dessa observationer gjordes aven i dsRNA-stimulerade celler som behandlats med imiquimod (p<0.05). SiRNA visade attimiquimods IFNb boostade effekt var reglerad via receptorerna MDA5 och RIG-I.SlutsatsBehandling av BECs med imiquimod visar pa tre effekter: minskat uttryck av SARS-COV-2 receptorn ACE2, okad antiviral kapacitet hos bronkepitelceller samt en minskning av cytokiner involverade i dodliga cytokinstormar.

Background: Increased mast cell (MC) densities in the airway epithelium is a hallmark of asthma. However, little is known regarding the role and effect of MC mediators on epithelium damaged by respiratory viruses. Aim: To investigate the density of MCs in biopsies in patients with different asthma phenotypes and the effect of MC mediator tryptase on wound-healing in bronchial epithelial cells. Methods: Intraepithelial MCs (IEMCs) were stained for tryptase (MCT) and tryptase/chymase (MCTC) in bronchial biopsies. In a BEAS-2B cell line, the effect of tryptase and the TLR3 agonist polyI:C on wound healing was studied using live cell imaging (Phase Holographic Imaging). Results: MCT, but not MCTC, were increased in the epithelium in ICS-naïve mild asthma and ICS-responsive asymptomatic moderate and severe asthma when compared to ICS-unresponsive symptomatic moderate and severe asthmatics (p=0.03). Within the asthma groups, patients with atopy had the highest numbers of IEMCs compared to non-atopic asthmatics and controls (p=0.002). Stimulation of BEAS-2B with polyI:C resulted in impaired wound gap closure compared to untreated cells (p=0.05). However, MC tryptase enhanced cell proliferation and wound healing responses when compared to cells stimulated with polyI:C (p<0.0001) and untreated control (p=0.0007). Conclusion: We showed that asymptomatic patients with different severities of asthma had a higher density of IEMCs regardless of treatment with ICS. Further, the MC mediator tryptase promoted epithelial proliferation and wound healing responses. Hence, IEMCs may have beneficial roles and MC localization may be an important determinant in asthma pathology.