Chicken surfactant protein A1 activates macrophages phagocytosis and attenuates LPS-induced inflammatory response through the TLR4-mediated NF-кB pathway

Chicken surfactant protein A1 (cSP-A1) is a soluble C-type lectin found primarily in chicken lungs. Its function and other potential bioactivities are unclear. This study aimed to express, purify, and identify recombinant cSP-A1 (RcSP-A1), investigate its effects on chicken macrophage HD11 cells, and evaluate its ability to regulate the LPS-induced inflammatory response. The results showed that RcSP-A1 was produced in HEK 293F cells and could be purified using a Ni²⁺ affinity column. The RcSP-A1 purified concentration was 7.5 µg/mL. Functional examinations showed that RcSP-A1 could aggregate all tested bacterial strains and led to a macrophage phagocytosis rate significantly higher than in the control (p < 0.01). Subsequently, HD11 cells, preincubated with various RcSP-A1 concentrations (12.5, 25, and 50 μg/mL) and 5 mM CaCl₂ for 2 h, were stimulated by LPS (1 μg/mL) for 24 h. The results showed that RcSP-A1 significantly attenuated the stimulating effects of LPS on the transcription and protein expression levels of proinflammatory cytokines (IL-1β, IL-6, and TNF-α) and inhibited nitric oxide production. Mechanism studies demonstrated that RcSP-A1 exerted an anti-inflammatory effect on LPS-stimulated cells by down-regulating the expression of TLR4, MyD88, and p65, up-regulating the expression of IкB-α, and inhibiting the activation of the NF-кB signaling pathway. These findings suggested that RcSP-A1 promoted bacterial aggregation and phagocytosis and inhibited the LPS-induced inflammatory response in HD11 cells through the TLR4/NF-κB signaling pathway, displaying an important role in innate immune defense.