Ligand-Dependent Associations of the Bovine TLR2 R152Q Variant with Neutrophil Oxidative Burst, Extracellular DNA Release, and Innate Immune Gene Expression

The bovine Toll-like receptor 2 (TLR2) R152Q substitution lies in an extracellular leucine-rich repeat domain and may alter pathogen-associated molecular pattern recognition, but its association with neutrophil function remains unknown. We genotyped 267 Polish Holstein–Friesian cows and evaluated isolated polymorphonuclear neutrophils (PMNs) after stimulation with the TLR2 agonist peptidoglycan (PGN) and lipopolysaccharide (LPS)–a well-documented PMN activator. The oxidative burst was assessed by the nitroblue tetrazolium reduction assay, extracellular double-stranded DNA release as an indirect index of neutrophil extracellular trap (NET)-associated DNA release by PicoGreen fluorescence, and the abundance of eight innate immune transcripts by RT-qPCR. Genotype frequencies were 0.59 for GG (coding for arginine), 0.39 for GA (heterozygotes), and 0.02 for AA (coding for glutamine), showing a significant deviation from Hardy–Weinberg equilibrium. In silico structural modeling predicted an allosteric destabilizing effect of the R152Q substitution. Functional responses of PMNs were both ligand- and concentration-dependent. The heterozygous GA PMNs exhibited the highest oxidative activity and extracellular dsDNA release following PGN stimulation. Conversely, AA PMNs demonstrated altered effector responses, including lower dsDNA release under LPS stimulation, accompanied by elevated basal transcript levels of recognition genes (TLR2, TLR4, NCF1, PADI4) but reduced IRAK4 expression upon stimulation. These findings suggest that the TLR2 R152Q polymorphism is associated with ligand- and concentration-dependent variation in bovine PMN functional responses. Overall, the enhanced responses in GA PMNs point toward a potential heterozygote-associated functional advantage, whereas the predicted structural destabilization and altered effector responses in the rare AA genotype (n = 3) suggest possible selective constraints against mutant homozygotes, though these latter findings remain preliminary and require confirmation in larger cohorts.