Characterization of cytokine expression in milk somatic cells during intramammary infections withEscherichia coliorStaphylococcus aureusby real-time PCR

Abstract

-The expression of inflammatory cytokines, including interleukin (IL)-6, IL-8, IL-12, granulocyte macrophage-colony stimulating factor (GM-CSF), tumor necrosis factor (TNF)-α, and interferon (IFN)-γ, by milk somatic cells was characterized by real-time polymerase chain reaction (PCR) in dairy cows experimentally challenged with either E. coli (n = 8) or S. aureus (n = 8). The mRNA abundance of a target gene was calibrated with that of a reference gene (β-actin) and expressed as fold of induction over the control quarter at each time point. At no single time point did all eight quarters challenged with the same type of bacteria demonstrated increased expression of a target gene and there was large variation among animals at each given time. As a consequence, most tested comparisons were not statistically significant except the peak time points of IL-8 expression (75-and 29-fold in glands challenged with E. coli and S. aureus, respectively). However, the average fold induction of all targeted cytokines was increased in response to both bacterial challenges with the exception of IFN-γ. The expression of IFN-γ was only increased in milk somatic cells isolated from E. coli, but not S. aureus, challenged mammary glands. Moreover, upregulated expression of cytokine genes had higher magnitudes and/or faster responses in glands challenged with E. coli in comparison with those challenged with S. aureus. We propose that the compromised upregulation of inflammatory cytokines in S. aureus infected glands may, at least partially, contribute to the chronic course of infection caused by this pathogen. Further research on identifying factors responsible for the differentially expressed cytokine profiles may be fundamental to developing strategies that mitigate the outcome of bovine mastitis. mastitis / cytokine / Escherichia coli / Staphylococcus aureus / SCC

Morphometric analysis of proinflammatory cytokines in mammary glands of sows suggests an association between clinical mastitis and local production of IL-1beta, IL-6 and TNF-alpha

“…Possibly, in these sows there may be abundant and regulated release of anti-inflammatory mediators [40,53], limiting the production of the inflammatory cytokines. However, in accordance with our results, previous studies in cows have shown a significant increase in the proinflammatory cytokines at the level of both mRNA [33,37] and protein [6,11,26,32,45,48,54] in either mammary tissues or milk collected from the infected glands following intramammary inoculation with E. coli. The results here indicate that the development of clinical symptoms of coliform mastitis in the sow is associated with a locally increased proinflammatory cytokine production in response to intramammary E. coli infection.…”

Bacterial lipopolysaccharide induces increased expression of toll-like receptor (TLR) 4 and downstream TLR signaling molecules in bovine mammary epithelial cells

“…IRF3 activation is responsible for the expression of IFN-β and IFN inducible genes (e.g IP10, glucocorticoid-attenuated response gene 16). The up-regulation of these two transcription factors (NF-κB and IRF3) in the TLR4 signaling pathway in MAC-T cells supports the findings of several studies characterizing cytokine production by the mammary gland following bacterial infections [4,30] and thus confirming the ability of the mammary gland to mount a robust innate immune response to these infections.…”

“…Available lines of evidence indicate that bovine mammary epithelial cells respond to bacterial lipopolysaccharide and other microbial products by producing proinflammatory cytokines required to combat invading pathogens [4,7,17,30,32,38]. Other epithelial cell types (e.g.…”

“…In addition, mRNA sequences and chromosomal location of most genes of TLR4 signaling molecules in cattle were not available until recently [10]. The role of mammary epithelial signaling of the immune system in response to LPS has been proven by several investigations in cattle [4,7,30,32,38]. Persson Waller et al [32] observed a marked increase in milk and lymph interleukin (IL)-8 concentrations and milk somatic cell counts in mammary epithelial cells challenged with LPS.…”

Bacterial lipopolysaccharide stimulates bovine neutrophil production of TNF-α, IL-1β, IL-12 and IFN-γ

“…However, during mastitis PMN far outnumber macrophages by several orders of magnitude, and can therefore be major sources of cytokines like TNF-α. It was recently demonstrated by real-time PCR that mRNA expression of TNF-α, IL-6, IFN-γ, IL-8 and IL-12 was up-regulated in milk PMN following experimental intramammary injection of either E. coli, Staphylococcus aureus or S. aureus α-toxin [3,24,37,38]. Combined with the results from the present study it would appear that increased mRNA expression in milk PMN correlates with cytokine secretion.…”