Lara Ravanetti

An early innate response underlies severe influenza-induced exacerbations of asthma in a novel steroid-insensitive and anti-IL-5-responsive mouse model

Acute worsening of asthma symptoms (exacerbation) is predominantly triggered by respiratory viruses, with influenza causing the most severe exacerbations. The lack of an adequate animal model hampers mechanistic insight and the development of new therapeutics.

IL-33 drives influenza-induced asthma exacerbations by halting innate and adaptive anti-viral immunity

Background Influenza virus triggers severe asthma exacerbations for which no adequate treatment is available. It is known that IL‐33 levels correlate with exacerbation severity, but its role in the immunopathogenesis of exacerbations has remained elusive. Objective We hypothesized that IL‐33 is necessary to drive asthma exacerbations. We intervened with the IL‐33 cascade and sought to dissect its role, also in synergy with thymic stromal lymphopoietin (TSLP), in airway inflammation, antiviral activity, and lung function. We aimed to unveil the major source of IL‐33 in the airways and IL‐33–dependent mechanisms that underlie severe asthma exacerbations. Methods Patients with mild asthma were experimentally infected with rhinovirus. Mice were chronically exposed to house dust mite extract and then infected with influenza to resemble key features of exacerbations in human subjects. Interventions included the anti–IL‐33 receptor ST2, anti–TSLP, or both. Results We identified bronchial ciliated cells and type II alveolar cells as a major local source of IL‐33 during virus‐driven exacerbation in human subjects and mice, respectively. By blocking ST2, we demonstrated that IL‐33 and not TSLP was necessary to drive exacerbations. IL‐33 enhanced airway hyperresponsiveness and airway inflammation by suppressing innate and adaptive antiviral responses and by instructing epithelial cells and dendritic cells of house dust mite–sensitized mice to dampen IFN‐&bgr; expression and prevent the TH1‐promoting dendritic cell phenotype. IL‐33 also boosted luminal NETosis and halted cytolytic antiviral activities but did not affect the TH2 response. Conclusion Interventions targeting the IL‐33/ST2 axis could prove an effective acute short‐term therapy for virus‐induced asthma exacerbations. Graphical abstract Figure. No Caption available.

IL33 and TSLP involvements in influenza-induced exacerbation in a murine model of chronic asthma

Introduction: Viral airway infections cause acute worsening of asthma symptoms and additional accumulation of inflammatory cell subsets in the airways. Here we aimed to dissect the innate immune response that might underlie virus-induced exacerbations in an adequate murine model of exacerbating asthma. Methods: Mice were sensitized i.n. to HDM extract for 5 days/week for 5 consecutive weeks. On day 29 mice were infected with low dose of influenza A/X31; 3 day before and after the virus infection the animals have been treated with anti TSLP and IL33 receptors (T1/ST2 and TSLPR, respectively) mAbs. The airways cellular influx was assessed, cytokines were measured in BALF, lung function and histology were determined in different time points after infection. Results: Infected sensitized mice infected showed an exacerbation of allergic inflammation as reflected by a prolonged AHR and a boosted inflammatory influx into the airways, in comparison to infected non-sensitized mice. As an early event upon influenza infection, sensitized mice showed the induction of IL-33 and TSLP paralleled by the increased influx into the airways of ILC2, basophils, NK and NKT cells. Blocking the effect of IL-33 and TSLP using anti T1/ST2 and TSLPR mAbs the NKT cells, basophils, ILC2 recruitments into the airways were attenuated; the eosinophilic and neutrophilic influxes were reduced in BALF. Conclusions: Influenza infection of sensitized mice boosted the induction of crucial effectors of the innate immune response, leading the worsening of a broad inflammatory response into the airways. Limiting IL33 and TSLP can dampen airway inflammation and virus-induced changes in HDM-sensitized mice.

Su1252 Gastric Juice Composition and Acid Suppression in Pediatric Gastroesophageal Reflux Disease

INTRODUCTION: Gastric acid suppression is justified to prevent severe gastro esophageal reflux (GER) disease related complications. However, it does not reduce the total amount and proximal extent of GER in the esophagus and non-acid components are able to induce (extra-esophageal) GER symptoms as well. No data on the composition of gastric juice (GJ) in children using gastric acid suppression exists. We therefore aimed to assess whether the composition of gastric juice in children using proton pump inhibitors (PPIs) differs, compared to that of their controls. METHODS: Infants and children (0-18 years) on proton pump inhibitors (PPIs) for at least six weeks and a control group not using anti reflux medication, were included. GJ was obtained through an existing nasogastric or a percutaneous endoscopic gastrostomy tube/ Mic-key gastrostomy. In the collected GJ (5 ml), pH, pepsin activity, bile salts and endotoxin (LPS) levels were determined. Pepsin was measured using a fluorometric assay using 4-Methyl-Coumaryl-7-Amide (MCA) substrate with/without pepstatin. Concentrations of deconjugated and taurine/glycine-conjugated bile salts were assessed by reversephase HPLC. Levels of LPS were determined using the spectrophotometric Limulus Amebocyte Lysate assay. RESULTS: GJ was analyzed from 16 children with (median: 3.8 yrs, range: 17.6 years) and 16 children (4.0 yrs, range: 16.0) without PPI therapy. Median duration of PPI treatment was 24 weeks (range: 514 weeks). Gastric pH was 5.0 (range: 5.0) and 1.0 (range: 4.5) in the PPI and control group respectively (p <0.001). Pepsin, unconjugated bile salts, and endotoxin were not significantly different in the two groups. Total taurine conjugated bile salts, and specifically taurocholate, was significantly higher in the PPI group (p=0.01 and p=0.005). pH and concentration of deconjugated bile and conjugated bile salts were significantly associated (p=0.006 and p=0.02). Endotoxin and bile salts were not significantly associated. CONCLUSION: Taurine conjugated bile acids are significantly higher in children chronically using PPIs compared to controls. Moreover acidity of gastric pH correlated negatively with deconjugated and conjugated bile salts. These findings imply that GJ under chronic proton pump inhibition contains nonacid components potentially harmful to esophageal mucosa and bronchial tissue.

Influenza exacerbations in an acute and chronic house dust mite ‘asthma’ model

Methods In the acute asthma model, mice were sensitised intranasally with HDM or PBS on three consecutive days and challenged two weeks later. Two days after the challenge the mice were infected intranasally with 10 TCID50 influenza (A/PR/8/34) or PBS and sacrificed 4, 8 or 14 days after infection. In the chronic asthma model the mice receive influenza in the 5th week of sensitisation (intranasally, 5 days a week for 5 weeks). In both models we assess(ed) cellular influx by analysing bronchoalveolar lavage fluid (BALF), and cytokines and viral load in lung lysate. PenH is determined as a measure of airway hyperresponsiveness (AHR).