Lung-delivered IL-10 Mitigates Lung Inflammation Induced by Repeated Endotoxin Exposures

Background: Therapeutic options capable of resolving inflammatory lung disease resulting from high-consequence occupational and environmental hazards are lacking. Objective: This study seeks to determine the therapeutic potential of direct lung-delivered interleukin (IL)-10 following repeated high concentration lipopolysaccharide exposures. Methods: C57BL/6 mice were intratracheally instilled with LPS (10µg) and then IL-10 (1µg) or vehicle (saline) control 5-hours later. This LPS exposure and treatment paradigm was then repeated daily for the next 2 days. Serum and lung tissues were collected and assessed for proinflammatory and profibrotic mediators. Lung cell infiltrates were enumerated by flow cytometry. Lung sections were stained for myeloperoxidase (MPO), CCR2, vimentin and post-translational protein citrullination (CIT) and malondialdehyde-acetaldehyde (MAA) modifications. Lung function testing and longitudinal in vivo micro-CT imaging were performed. Whole lungs were profiled using bulk RNA sequencing. Results: Repeated IL-10 therapy significantly (p<0.05) mitigated several LPS-induced adverse effects. IL-10 treatment reduced LPS-induced weight loss and serum pentraxin-2 and IL-6 levels. LPS-induced proinflammatory/profibrotic mediators (i.e., TNF-a, IL-6, CXCL1, CCL2, MMP-8, MMP-9, TIMP-1, fibronectin) were decreased with IL-10 treatment. IL-10 reduced LPS-induced influx of lung neutrophils, CD8+ T cells, NK cells, recruited monocyte-macrophages, and monocytes, as well as tissue expression of CCR2+ monocytes/macrophages, MPO+ neutrophils, vimentin, CIT, and MAA. IL-10 treatment reversed LPS-induced airway hyperresponsiveness. Micro-CT imaging confirmed reduction in LPS-induced lung density by IL-10 treatment. Lung-delivered IL-10 therapy administered after daily repeated endotoxin exposures strikingly reduces lung inflammatory and profibrotic processes and airway dysfunction to hasten lung recovery and resolution. Short-term, lung-localized IL-10 therapy following high-consequence environmental exposure events may represent a novel therapy to prevent chronic lung disease development. Overall design: C57BL/6 male mice (between 6 and 8 weeks of age) were purchased from The Jackson Laboratory (Bar Harbor, ME), randomized upon arrival, and allowed to acclimate for 1 week prior to initiation of experiments. 6 mice were intratracheally instilled with LPS (10µg). Half of these mice were then intratracheally instilled with IL-10 (1µg) and the other half was intratracheally instilled with vehicle (saline) control 5-hours later. This exposure paradigm was then repeated for the next 2 days so that each mouse received 3 total doses of LPS in the morning and 3 total doses of either IL-10 or the vehicle control 5 hours post-LPS. Animals were euthanized 24 hours following the final LPS exposure. Whole lungs were harvested and profiled using bulk RNA sequencing.