Flagellin and R-848 co-stimulation induces early synergistic and delayed attenuated immune responses in the chicken peripheral blood

Understanding the interaction between distinct Toll-like receptors (TLRs) is crucial for optimizing combinatorial TLR ligands as vaccine adjuvants. In this study, we investigated the crosstalk between TLR-5 and TLR-7 in the chicken peripheral blood mononuclear cells (PBMCs) using their respective agonists, flagellin and R-848. The PBMCs were isolated from two-week-old specific pathogen-free chickens (n = 6) and stimulated with flagellin and/or resiquimod (R-848). Levels of gene activity governing key immune responses (IFN-β, IL-1β, IFN-γ, iNOS, IL-13) was measured at 3 h, 6 h, 12 h, 24 h, and 48 h post-stimulation using qRT-PCR, and nitric oxide (NO) levels were quantified at 24 and 48 h using the Griess assay. The combination of flagellin and resiquimod resulted in a synergistic up-regulation of IL-1β, IFN-β, IFN-γ, IL-13, and iNOS transcripts at 3h post-stimulation (P<0.05). This early peak was followed by attenuated expression profiles at later time points, with no corresponding increase in NO production, despite elevated iNOS transcripts. In conclusion, co-stimulation with flagellin and R-848 induced a potent, early-phase immune activation in the chicken PBMCs, highlighting its potential as an immunomodulatory strategy.