Methylation of selected CpG islands involved in the transcription of myeloperoxidase and superoxide dismutase 2 in neutrophils of periparturient and mid-lactation cows.

It is generally accepted that intracellular killing of microorganisms by production of reactive oxygen species (ROS) in the phagosome of the neutrophil is an important arm of innate defense. High-producing dairy cows are prone to periparturient metabolic and infectious diseases. Both myeloperoxidase (MPO) activity and ROS production decrease the day of parturition. Several studies have demonstrated changes in the expression of genes involved in, for example, metabolism and defense in the circulating neutrophil during peripartum. In this study, we wanted to further characterize the periparturient neutrophil in terms of its oxidative killing capacity by analyzing the oxidative burst at 3 levels. First, the ROS phenotype was evaluated using chemiluminescence. The cows (sampled within 24 h after parturition and at 135 d in milk) showed a significantly slower production of ROS at parturition. Both primiparous (n = 13) and multiparous (n = 12) cows were included in this study, but parity did not affect the kinetics of ROS production. Second, the expression of 11 genes involved in ROS production was measured in the same cows: cytochrome b-245 α and ß chain (CYBA, CYBB; coding for membrane-bound constituents of NADPH oxidase); neutrophil cytosolic factors 1, 2, and 4 (NCF1, NCF2, and NCF4); Rac family small GTPase 1 and 2 (RAC1 and RAC2; coding for regulatory proteins of NADPH oxidase); superoxide dismutase 2 (SOD2); catalase (CAT); myeloperoxidase (MPO; coding for enzymes involved in metabolizing downstream ROS); and spleen-associated tyrosine kinase (SYK; involved in signaling). During peripartum, a shift in expression in the oxidative killing pathway was observed, characterized by a downregulation of MPO and a simultaneous upregulation of the genes coding for NADPH oxidase. Third, as total DNA methylation is known to change during pregnancy, we investigated whether the observed differences were due to different methylation patterns. Promotor regions initiate transcription of particular genes; therefore, we analyzed the methylation status in annotated CpG islands of MPO and SOD2, 2 genes with a significant difference in expression between both lactation stages. The differences in methylation of these CpG islands were nonsignificant. High-throughput techniques may be necessary to obtain more detailed information on the total DNA methylation dynamics in bovine neutrophils and increase our understanding of how gene expression is controlled in neutrophils.

Reactive oxygen species generation by bovine blood neutrophils with different CXCR1 (IL8RA) genotype following Interleukin-8 incubation.

BACKGROUND: Associations between polymorphisms in the bovine CXCR1 gene, encoding the chemokine (C-X-C motif) receptor 1 (IL8RA), and neutrophil traits and mastitis have been described. In the present study, blood neutrophils were isolated from 20 early lactating heifers with different CXCR1 genotype at position 735 or 980. The cells were incubated with different concentrations of recombinant bovine IL-8 (rbIL-8) for 2 or 6 h and stimulated with phorbol 12-myristate 13-acetate (PMA) or opsonized zymosan particles (OZP). Potential association between CXCR1 genotype and production of reactive oxygen species (ROS) was studied. RESULTS: Although on single nucleotide polymorphisms (SNPs) may potentially affect CXCR1 function, SNPs c.735C > G and c.980A > G showed no association with ROS production with or without incubation of rbIL-8. Neutrophils incubated with rbIL-8 for 2 or 6 h showed higher PMA- and lower OZP-induced ROS production compared to control without rbIL-8. CONCLUSIONS: In the present study no association could be detected between superoxide production by isolated bovine neutrophils during early lactation and CXCR1 gene polymorphism. IL-8 showed to possess inhibitory effects on ROS generation in bovine neutrophils.

Compromised neutrophil function and bovine E. coli mastitis: is C5a the missing link?

During early lactation, dairy cow are prone to developing severe mastitis in responses to intramammary Escherichia coli infections. These severe inflammatory responses have been correlated with reduced neutrophil function during the periparturient period. However, the causative mechanism of neutrophil dysfunction has not been elucidated. Studies in murine sepsis models have shown that during sepsis neutrophils are functionally paralysed due to the presence of high concentrations of complement factor 5a (C5a). In this review, we hypothesize that C5a as a critical early mediator in the development of severe E. coli mastitis. Furthermore, preliminary data suggest that crosstalk between C5a and TLR4 signalling in neutrophils may provide a positive feedback mechanism that may be involved in the pathogenesis of a severe mastitis response. Finally, we focus on the therapeutic potential of disrupting the C5a signalling pathway as an important strategy for treatment of severe E. coli mastitis in dairy cattle.