Supplementary Material for: Progression of Acute Lung Injury in Intratracheal LPS Rat Model: Efficacy of Fluticasone, Dexamethasone, and Pirfenidone

Introduction: We investigated the potential of LPS (10-300 µg/rat) administered i.t. to induce reproducible features of acute lung injury (ALI) and compared the pharmacological efficacy of anti-inflammatory glucocorticoids and antifibrotic to reduce disease. Additionally, we studied the time dependent progression of ALI in this LPS rat model. Methods: We conducted 1. Dose effect studies of LPS administered i.t. at 10, 30, 100, 300 µg/rat on ALI at 4 hr timepoint; 2. Pharmacological interventions using i.t. fluticasone (100, 300 µg/rat), i.t. pirfenidone (4000 µg/rat), and peroral dexamethasone (1 mg/kg) at 4 hr timepoint; 3. Kinetic studies at 0, 2, 4, 6, 8, 10 and 24 hr post LPS challenge. Phenotype or pharmacological efficacy was assessed using predetermined ALI features such as pulmonary inflammation, edema, and inflammatory mediators. Results: All LPS doses induced a similar increase of inflammation, edema, and inflammatory mediators e.g., IL6, IL1β, TNFα and CINC-1. In pharmacological intervention studies, we showed fluticasone and dexamethasone ameliorated ALI by inhibiting inflammation (>60-80%), edema (>70-100%), and the increase of cytokines IL6, IL1β, TNFα (≥70-90%). We also noticed some inhibition of CINC-1 (25-35%) and TIMP1 (57%) increase with fluticasone and dexamethasone. Conversely, pirfenidone failed to inhibit inflammation, edema, and mediators of inflammation. Lastly, in ALI kinetic studies, we observed progressive pulmonary inflammation and TIMP1 levels, which peaked at 6 hr and remained elevated up to 24 hr. Progressive pulmonary edema started between 2-4 hr and was sustained at later timepoints. On average, levels of IL6 (peak at 6-8 hr), IL1β (peak at 2-10 hr), TNFα (peak at 2 hr), CINC-1 (peak at 2-6 hr), and TGFβ1 (peak at 8 hr) were elevated between 2-10 hr and declined towards 24 hr post LPS challenge. Discussion/Conclusion: Our data show that 10 µg/rat LPS achieved a robust, profound, and reproducible experimental ALI phenotype. Glucocorticoids ameliorated key ALI features at the 4 hr timepoint but the antifibrotic pirfenidone failed. Progressive inflammation and sustained pulmonary edema were present up to 24 hr, whereas levels of inflammatory mediators were dynamic during ALI progression. This study data might be helpful in designing appropriate experiments to test the potential of new therapeutics to cure ALI.