A collagen IV matrikine inhibits neutrophil recruitment in a mouse model of asthma exacerbation

Background: Increased neutrophilic infiltration of the airways is a key feature of acute asthma exacerbation, which may lead to prolonged hospital admissions and an enhanced rate of decline of lung function. The increase in asthma severity in this subpobulation of patients is thought to be partially mediated by neutrophil granulocytes secreting active proteases that degrade the surrounding extracellular matrix (ECM) and contribute to remodeling. We have reported an ECM fragment of the non-collagenous domain of collagen IV α3 (tumstatin) is significantly decreased in the airways of asthmatics (vs. non-asthmatics). This study sought to elucidate the effects of an active fragment of tumstatin (CP17), in the regulation of neutrophilic airway inflammation. Methods: Neutrophils were prepared from non-asthmatics via density gradient centrifugation (controlled for viability, activity and eosinophil contamination), before pre-incubation for 1h with CP17, scrambled peptide or vehicle and then exposure to a gradient of IL-8 in 3D μSlides (IBIDI, Germany). Time-lapse analysis of migration trajectories was performed using a specialized algorithm for cell detection. The Forward Migration Index (FMI) was calculated as published previously (IBIDI, Germany). The FMI (arbitrary units) represents the distance neutrophils migrated towards the IL-8 in parallel to the linear gradient. Mice were sensitized to OVA i.p. on day 1, 14 and 21 and challenged with 1% OVA aerosol on days 26, 27 and 28. Acute exacerbation was induced by instilling polyI:C i.t. 12h after last challenge. Prior to polyI:C challenge on days 27 and 28, 1 nmol CP17 or scrambled peptide was applied i.t.. BAL, lung histology (mucus producing cells (number PAS+ cells) and mRNA expression (CXCL1, CCL20) were analyzed. Results: In vitro: CP17 significantly reduced the mean FMI towards IL-8 from 0.27 (vehicle) to 0.12 and 0.10 respectively (5 and 50μM CP17,