Impact of the ADAM9 N267H missense variant on ligand-dependent NET-associated DNA release and reactive oxygen species production in bovine neutrophils

Although ADAM9 (A disintegrin and metalloproteinase 9) is a transmembrane metalloproteinase implicated in leukocyte activation, its functional genetic variation in bovine neutrophils remains poorly characterized. Given the role of ADAM9 in inflammatory responses, identifying polymorphisms that alter its structure is crucial for understanding individual differences in bovine innate immune function. A missense variant in bovine ADAM9 (rs210368040; A > C), encoding an N267H substitution, was confirmed via Sanger sequencing of cDNA amplicons from bovine polymorphonuclear leukocytes (PMNs), which also verified ADAM9 expression in these cells. In silico analysis localized residue 267 to a surface-exposed loop within the metalloproteinase domain (peptidase M12B), suggesting that the mutation introduces an amino acid with different properties that could destabilize the domain or lead to a loss of function. Genotyping of 450 Polish Holstein-Friesian cows revealed a genotype distribution of AA ( n = 234), AC ( n = 191), and CC ( n = 25). Ex vivo phenotyping of 27 cows (AA, n = 8; AC, n = 9; CC, n = 10) was conducted following Escherichia coli lipopolysaccharide (LPS, 12.5–50 µg/mL) or Staphylococcus aureus peptidoglycan (PGN, 10–60 µg/mL) stimulation, with within-family false discovery rate (FDR) correction applied ( q < 0.05). AA homozygotes exhibited a dose-dependent reduction in reactive oxygen species (ROS) production - assessed by nitroblue tetrazolium (NBT) reduction - but higher extracellular dsDNA release under LPS stimulation. Conversely, under PGN stimulation, the CC genotype demonstrated superior neutrophil extracellular trap (NET) formation. Reverse transcription quantitative PCR confirmed lower expression of TLR2 and IRF3 in CC neutrophils under selected LPS conditions, whereas differences under PGN stimulation were not maintained after FDR adjustment. Our findings suggest that the ADAM9 N267H polymorphism is associated with altered bovine PMN reactivity, influencing the pattern of ROS production, NET-associated DNA release and gene expression in a stimulus-dependent manner. These results support ADAM9 N267H as a candidate functional variant linked to ligand-dependent innate immune responses in cattle.