TLR5 activation in respiratory epithelial cells orchestrate mucosal Th17 response through both indirect and direct pathways.

Background: Flagellin, a potent mucosal adjuvant administered via the intranasal route, has been widely recognized for its capacity to enhance immune responses against diverse pathogens. However, the effects and the underlying mechanisms by which flagellin modulates CD4⁺ T cell differentiation remain incompletely understood.

Methods: Recombinant flagellin proteins, including full-length flagellin (SF) and a TLR5-binding deficient variant (SFΔ90-97), were produced and purified. An OT-II derived CD4⁺ T cell adoptive transfer model, a classical intranasal immunization model and dendritic cell (DC)-CD4⁺ T co-culturing system were used. The proliferation and differentiation of CD4⁺ T cells were analyzed using flow cytometry analysis. RNA sequencing and neutralizing antibody blocking experiments were performed to determine the essential cytokines involved in flagellin modulated Th17 differentiation.

Results: Flagellin preferentially promotes Th17 cells differentiation. Respiratory epithelial cells (RECs), acting as sentinel cells, are the first to encounter exogenous stimuli during intranasal immunization. Flagellin stimulates the secretion of various soluble cytokines by binding to TLR5 on the surface of RECs, with GM-CSF facilitating the functional activation of airway DCs. GM-CSF-conditioned DCs exhibit upregulated IL-6 expression which in turn drives the polarization of naïve CD4⁺ T cells toward the Th17 phenotype. Furthermore, TLR5-regulated REC-derived IL-6 synergizes with TLR5-modulated DCs to amplify Th17 polarization signals, thereby enhancing the Th17 induction.

Conclusion: Flagellin preferentially induced a Th17-enhanced immune response and RECs were highlighted its essential roles during this process through both indirect and direct pathways. For indirect pathway, RECs modulate DC function through GM-CSF. Moreover, RECs directly contribute to Th17 differentiation by secreting IL-6.