Differential toll-like receptor signaling pathways shape IL-1β-dependent inflammatory responses in chicken macrophages

Macrophages are primary regulators of the innate immune system, integrating pattern recognition receptor (PRR) signaling and cytokine production to shape early inflammatory processes. However, the molecular mechanisms governing macrophage-driven responses in chickens remain poorly understood, despite their importance in controlling major infectious diseases. Among these pathways, interleukin (IL)-1β functions as a central mediator of inflammatory signaling, yet its integration with Toll-like receptor (TLR) pathways in chicken macrophages remains unclear. Here, lipoteichoic acid (LTA)-Toll-like receptor (TLR)2, poly(I:C)-TLR3, and lipopolysaccharide (LPS)-TLR4 pathways were investigated in the chicken macrophage-like cell line HD11 and an IL1β-deficient HD11 mutant, with PBMC-derived macrophages included for comparison. HD11 cells exhibited minimal responsiveness to LTA-TLR2 stimulation, whereas LPS-TLR4 stimulation elicited a limited response primarily characterized by strong induction of IL-1β and IL-8 but minimal or undetectable IL-6, tumor necrosis factor (TNF)-α, IL-18, and interferon (IFN)-β. In contrast, poly(I:C)-TLR3 stimulation induced IL-1β, IL-18, and IFN-β with limited induction of other cytokines. A genetically engineered CRISPR/Cas9 IL1B knockout (KO) HD11 model has been established to evaluate molecular interactions of the inflammatory response, which appears to be modulated by IL-1β in a TLR ligand-specific context. The present study highlights distinct pattern recognition profiles on chicken macrophages that may offer a molecular basis for IL-1β-targeted signaling networks and immunomodulation in chickens.