Caul-Futy, M. | Search bioRxiv

bioRxiv2025

Local tolerance and innate immune activation of primary human respiratory cells exposed to flagellin

Antibiotic-resistant respiratory infections can lead to treatment failure, highlighting the need for alternative strategies. FLAMOD, a recombinant flagellin, stimulates innate immunity via Toll-like receptor 5 when delivered intranasally. In mice, FLAMOD protects against bacterial pneumonia. The protection is associated with activation of airway epithelial cells. This study aimed to assess the tolerance of human primary respiratory epithelium to FLAMOD administered apically as liquid droplets or by nebulization, to measure the innate immune response, and the pharmacokinetics of FLAMOD. We used epithelia reconstituted from human nasal and bronchial (MucilAir), small airways (SmallAir) and alveolar (AlveolAir) primary epithelial cells, cultured at the air-liquid interface. We report that daily administration of escalating doses of FLAMOD for 5 days was well tolerated by epithelia as barrier integrity, cilia motion and cell viability were not affected. FLAMOD was rapidly degraded without leakage into the basal compartment. Each epithelial model exhibited responses involving pathways of innate defense and immune cell infiltration, which were dose-dependent, with an effective concentration of FLAMOD in the picomolar range. Similar tolerance profile and immune responses were obtained with airway epithelium from cystic fibrosis and chronic obstructive pulmonary disease patients. In conclusion, this study supports the stimulation of epithelial Toll-like receptor 5 signaling to fight against infections of vulnerable patients.

cell biology↗

bioRxiv2025

Flagellin-mediated TLR5 activation enhances innate immune responses in healthy and diseased human airway epithelium

Bacterial pneumonia poses a significant challenge to public health, often leading to antibiotic treatment failure. Enhancing innate immunity represents a promising adjunctive strategy to conventional antibiotic therapy. Bacterial flagellin, a Toll-like receptor 5 (TLR5) agonist, has been shown to stimulate innate immune defenses when delivered via the respiratory route, demonstrating efficacy in both preventing and treating bacterial pneumonia in murine models. This protective effect is primarily mediated through TLR5-driven activation of airway epithelial cells. This study aimed to characterize the immunomodulatory effects of flagellin on human primary respiratory epithelium. Using the MucilAir air-liquid interface model and RNA sequencing, we demonstrated that apical administration of flagellin induced robust immune responses in airway epithelium derived from healthy individuals, as well as patients with chronic obstructive pulmonary disease (COPD) and cystic fibrosis (CF). TLR5-mediated epithelial signaling triggered key immune-related pathways, including cytokine production, leukocyte chemotaxis, neutrophil recruitment, and antimicrobial defense, with strong commonalities across healthy and diseased airway epithelia. Furthermore, we demonstrated that flagellin effectively activated epithelial immune responses even in the presence of the bacteria Pseudomonas aeruginosa or Streptococcus pneumoniae. However, epithelial activation alone was insufficient to directly limit bacterial colonization or replication, highlighting the potential role of epithelial-immune cell interactions in achieving effective bacterial clearance. These findings support TLR5 activation as a promising therapeutic strategy to enhance host defense mechanisms and improve treatment outcomes for bacterial pneumonia in both healthy individuals and patients with COPD or CF.

immunology↗