Understanding the immune defenses of the mammary gland is instrumental in devising and developing measures to control mastitis, the major illness of dairy ruminants. Innate immunity is an extremely broad field for investigation, and despite decades of research, our present knowledge of the innate defenses of the udder is incomplete. Yet, information is being gained on the recognition of pathogens by the mammary gland, and on several locally inducible defenses. The contribution of mammary epithelial cells to local defenses and to the mobilization of leucocytes is under growing scrutiny. Interactions of mastitis-causing bacteria such as Escherichia coli or Staphylococcus aureus and the mammary gland represents a suitable model for studies on innate immunity at an epithelium frontier. Powerful new research tools are radically modifying the prospects for the understanding of the interplay between the mammary gland innate defenses and mastitis-causing bacteria: genetic dissection of the immune response, microarray gene technology, transcriptomic methodologies and gene silencing by RNA interference will make possible the discovery of several of the key defense mechanisms which govern the susceptibility/resistance to mastitis at the molecular and genetic levels. It should then be possible to enhance the resistance of dairy ruminants to mastitis through immunomodulation and genetic improvement.

Staphylococcus aureusandEscherichia coliare among the most prevalent species of gram-positive and gram-negative bacteria, respectively, that induce clinical mastitis. The innate immune system comprises the immediate host defense mechanisms to protect against infection and contributes to the initial detection of and proinflammatory response to infectious pathogens. The objective of the present study was to characterize the different innate immune responses to experimental intramammary infection withE. coliandS. aureusduring clinical mastitis. The cytokine response and changes in the levels of soluble CD14 (sCD14) and lipopolysaccharide-binding protein (LBP), two proteins that contribute to host recognition of bacterial cell wall products, were studied. Intramammary infection with eitherE. coliorS. aureuselicited systemic changes, including decreased milk output, a febrile response, and induction of the acute-phase synthesis of LBP. Infection with either bacterium resulted in increased levels of interleukin 1β (IL-1β), gamma interferon, IL-12, sCD14, and LBP in milk. High levels of the complement cleavage product C5a and the anti-inflammatory cytokine IL-10 were detected at several time points followingE. coliinfection, whereasS. aureusinfection elicited a slight but detectable increase in these mediators at a single time point. Increases in IL-8 and tumor necrosis factor alpha were observed only in quarters infected withE. coli. Together, these data demonstrate the variability of the host innate immune response toE. coliandS. aureusand suggest that the limited cytokine response toS. aureusmay contribute to the well-known ability of the bacterium to establish chronic intramammary infection.

Mastitis caused byEscherichia coliandStaphylococcus aureusis 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 eitherE. colicrude lipopolysaccharide (LPS) or withS. aureusculture 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,Ccl5andCcl20) 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 fromE. coliandS. aureuscontributes to the onset of the response with differential leucocyte recruitment and distinct inflammatory and innate immune reactions of the mammary gland to infection.

The prompt recruitment of neutrophils to the site of infection is essential for the defense of the bovine mammary gland against invading pathogens and is determinant for the outcome of the infection.Escherichia coliis known to induce clinical mastitis, characterized by an intense neutrophil recruitment leading to the eradication of the bacteria, whereasStaphylococcus aureusinduces subclinical mastitis accompanied by a moderate neutrophil recruitment and the establishment of chronic mastitis. To elicit the neutrophil recruitment into the udder, inflammatory mediators must be produced after recognition of the invading pathogen. To our knowledge, those mediators have never been studied duringS. aureusmastitis, although understanding of the neutrophil recruitment mechanisms could allow a better understanding of the differences in the pathogeneses elicited byE. coliandS. aureus. Therefore, we studied, at several time points, the accumulation of neutrophils and the presence of the chemoattractant complement fragment C5a and of the cytokines interleukin-1β (IL-1β), tumor necrosis factor alpha, and IL-8 in milk after inoculation ofE. coliorS. aureusin lactating bovine udders. The low levels of C5a and the absence of cytokines in milk fromS. aureus-infected cows, compared to the high levels found in milk fromE. coli-infected animals, mirror the differences in the severities of the two inflammatory reactions. The cytokine deficit in milk afterS. aureusinoculation in the lactating bovine mammary gland could contribute to the establishment of chronic mastitis. This result could help in the design of preventive or curative strategies against chronic mastitis.