Abstract
Mastitis caused by Escherichia coli and Staphylococcus aureus is a major pathology of dairy cows. To better understand the differential response of the mammary gland to these two pathogens, we stimulated bovine mammary epithelial cells (bMEC) with either E. coli crude lipopolysaccharide (LPS) or with S. aureus culture supernatant (SaS) to compare the transcriptomic profiles of the initial bMEC response. By using HEK 293 reporter cells for pattern recognition receptors, the LPS preparation was found to stimulate TLR2 and TLR4 but not TLR5, Nod1 or Nod2, whereas SaS stimulated TLR2. Biochemical analysis revealed that lipoteichoic acid, protein A and α-hemolysin were all present in SaS, and bMEC were found to be responsive to each of these molecules. Transcriptome profiling revealed a core innate immune response partly shared by LPS and SaS. However, LPS induced expression of a significant higher number of genes and the fold changes were of greater magnitude than those induced by SaS. Microarray data analysis suggests that the activation pathways and the early chemokine and cytokine production preceded the defense and stress responses. A major differential response was the activation of the type I IFN pathway by LPS but not by SaS. The higher upregulation of chemokines (Cxcl10, Ccl2, Ccl5 and Ccl20) that target mononuclear leucocytes by LPS than by SaS is likely to be related to the differential activation of the type I IFN pathway, and could induce a different profile of the initial recruitment of leucocytes. The MEC responses to the two stimuli were different, as LPS was associated with NF-κB and Fas signaling pathways, whereas SaS was associated with AP-1 and IL-17A signaling pathways. It is noteworthy that at the protein level secretion of TNF-α and IL-1β was not induced by either stimulus. These results suggest that the response of MEC to diffusible stimuli from E. coli and S. aureus contributes to the onset of the response with differential leucocyte recruitment and distinct inflammatory and innate immune reactions of the mammary gland to infection.
Highlights
Mastitis is ranked as the top disease for dairy cattle on the basis of incurred economic losses [1,2]
We investigated the presence of agonists of TLR2, TLR4, TLR5, Nod1, and Nod2 in the LPS preparation by incubating
Since the response of HEK293 -TLR2 cells indicated that S. aureus culture supernatant (SaS) contains at least one agonist of TLR2, we looked for the presence of lipoteichoic acid (LTA), which is supposed to be sensed by TLR2 in association with CD36 [35] and is able to activate bovine mammary epithelial cells (bMEC) [10,33]
Summary
Mastitis is ranked as the top disease for dairy cattle on the basis of incurred economic losses [1,2]. Escherichia coli and Staphylococcus aureus are two major pathogens causing mammary infections of dairy ruminants. Most cases of E. coli mastitis are clinical and of short duration, in general less than 10 days, because the inflammatory reaction is usually able to clear the infection [3]. In gland through the teat canal, these bacteria are able to replicate rapidly in milk. The prompt detection of pathogens by the innate immune system is of prime importance, and because mammary epithelial cells (MEC) are in the front line, they could play an important role as sentinels. MEC are equipped with several sensors of bacteria, and they are able to react by producing mediators of inflammation and local defense [7,8,9]. An important research issue is to define how MEC play their role of sentinel of the mammary gland
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