Effector responses of bovine blood neutrophils against Escherichia coli: Role of NOD1/NF-κB signalling pathway

Neutrophils use a broad array of pattern recognition receptors to sense and respond to invading pathogens and are important in the early control of acute bacterial infections. Nucleotide-binding oligomerizing domain-1 (NOD1) is a cytoplasmic receptor involved in recognizing bacterial peptidoglycan. Reduced neutrophil NOD1 expression has been reported in periparturient dairy cows. The aim of this study was to investigate the role of NOD1 signalling in the early responses of bovine neutrophils to bacterial infections. Blood neutrophils from healthy heifers were preincubated for 2h with ML130, a selective inhibitor of NOD1-dependent nuclear factor-κB (NF-κB) activation. Thereafter, cells were cultured with live Escherichia coli for additional 30 min or subjected to Boyden chamber cell migration assay with E. coli in the lower chamber. Results showed that ML130 inhibited E. coli-induced NF-κB nuclear translocation. There was an indication, although not significant, that ML130 down-regulated gene expression of proinflammatory cytokines interleukin (IL)-1β and tumour necrosis factor (TNF)-α, chemokines IL-8 and C-X-C motif ligand 2 (CXCL2), and adhesion molecule CD62L, in E. coli-challenged neutrophils. Flow cytometry-based Annexin V staining revealed a considerable increase in neutrophil survival upon E. coli infection, an effect that was attenuated in the presence of ML130. Additionally, inhibition of NOD1/NF-κB signalling resulted in reduced migration of neutrophils to E. coli, and impaired phagocytosis, intracellular bacterial killing and reactive oxygen species production by neutrophils. These results indicate that NOD1/NF-κB pathway plays a crucial role in modulating neutrophil responses that are important for early control of infections. Approaches aiming at restoring neutrophil NOD1 function could be beneficial for prevention or treatment of coliform mastitis.