Mastitis Increases Mammary mRNA Abundance of β-Defensin 5, Toll-Like-Receptor 2 (TLR2), and TLR4 but Not TLR9 in Cattle

Abstract

Coordination of the primary defense mechanisms against pathogens relies on the appropriate expression of pathogen recognition receptors (PRRs) triggering the early release of effector molecules of the innate immune system. To analyze the impact of this system on the counteraction of infections of the mammary gland (mastitis), we characterized the bovine gene encoding the key PRR Toll-like receptor 9 (TLR9) and mapped its precise position on chromosome BTA22. The sequence information was used to establish real-time PCR quantification assays to measure the mRNA abundances of TLR9, TLR2, and TLR4 together with those of ␤-defensin 5 (BNBD5), an early bactericidal effector molecule of the innate system, in healthy and infected mammary glands. Mastitis strongly increased (4-to 13-fold) the mRNA abundances of all of these genes except TLR9. Slight subclinical infections already caused a substantial increase in the copy numbers, though they did so the least for TLR9. Induction was not systemic, since mRNA abundance was low in uninfected control quarters of the udder but high in the severely infected quarters of the same animal. The number of TLR2 copies correlated well with those of TLR4, indicating coordinated regulation of these two PRRs during infection of the udder. Their coordinated regulation explains our unexpected observation that pure Staphylococcus aureus infections caused a strong increase also in TLR4 mRNA abundance. In situ hybridizations revealed that BNBD5 is expressed predominantly in the mammary epithelial cells (MEC) of the infected gland. Our data therefore suggest a significant contribution of the innate immune system to counteract mastitis and attribute a prominent effector function to the MEC.

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

“…Also, it is possible that the mammary epithelia cells or gland may have its own specific manner of responding to the presence of pathogenic products which may be different from other cell types and organs. In a recent study, Goldammer et al [17] experimentally induced Staphylococcus aureus mastitis in bovine mammary gland and observed a coordinated up-regulation of the mRNAs of both TLR2 and TLR4. Staphylococcus aureus is a species of Gram-positive bacteria, which is expected to up-regulate only TLR2.…”

“…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.…”

“…Similarly, Boudjellab et al [7] observed that MAC-T cells produce IL (interleukin)-8 in response to Escherichia coli derived LPS. Furthermore, Goldammer et al [17] noted a coordinated up-regulation of TLR4 and TLR2 mRNAs during intramammary infection induced by Staphylococcus aureus, a Gram-positive bacterium. However, little is known about the roles of TLR4 or TLR2 signaling adaptors in the translocation of NF-κB and IRF3 transcription factors which are crucial initiators of the expression of the necessary pro-inflammatory cytokines required by the mammary epithelial cells of cattle to fight invading pathogens.…”

Staphylococcus aureuslipoteichoic acid triggers inflammation in the lactating bovine mammary gland

“…The recognition of LTA by leucocytes requires TLR2 and is improved by interaction with CD14 [45]. Bovine mammary tissue and bovine MEC express transcripts of the gene coding TLR2 [16,51], and the protein is expressed on bovine MEC [24]. Bovine MEC express transcripts for CD14 [51], and the protein is found on the milk fat globule membrane [42], which derives from the apical face of MEC.…”

“…Each one received at time zero RPMI in one quarter, 10 g LTA in another one, and 100 g LTA in a third quarter. The infused quarters were aseptically sampled just before infusion after the morning milking, then 4,8,12,16,24,32,48,72 and 96 h post-infusion. At each sampling time up to 24 h post-infusion, the rectal temperature was taken and a clinical examination was performed (appearance of gland and milk).…”