Flagellin nebulization enhances respiratory immune responses in the porcine model

Respiratory delivery of the Toll-like receptor 5 agonist FLAMOD, a recombinant flagellin, offers a promising approach for treating bacterial pneumonia. FLAMOD stimulates the airway epithelium, mobilizing and activating immune cells and effectors to combat infections. While previous evidences were obtained in mouse models, this study represents the first comprehensive assessment of FLAMOD delivered by nebulization in pigs. Our results demonstrate that a single nebulization of FLAMOD did not cause any adverse effects on clinical parameters. Histological analysis supported that FLAMOD treatment led to immune cell infiltration in the lung tissue, indicative of an active immune response. Flow cytometry confirmed granulocyte recruitment in conducting airways. RNA sequencing established immune activation across the respiratory tract, from the nose, trachea, bronchi to the lungs, highlighting innate immunity, bacterial defense, cytokine and chemokine signaling, and granulocyte chemotaxis as key biological pathways. These findings demonstrated FLAMOD’s capacity to induce a robust and common immune response throughout the porcine respiratory system as well as specific compartmentalized immune signature. This study establishes FLAMOD as a potent activator of innate immunity, providing a proof-of-concept for inhalation-based therapeutic strategies to combat bacterial pneumonia in clinical setting. Twelve male six-week-old pigs (Sus scrofa domestica, line LRxLW TOPIGS 20, Lelystad) were nebulized with diluent buffer for 24h, flagellin for 4h and flagellin for 24h (n=4 per group). Nose, trachea, bronchus, and lung tissue as well as blood were sampled and processed for RNA isolation and sequencing.