Innate immunity of the bovine mammary gland

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

Identification and functional characterization of a novel IL-8 receptor in cattle

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Identification and characterization of a new interleukin-8 receptor in bovine species

International audienceMastitis is an inflammation of the mammary gland, most of the time caused by invading pathogens. Phagocytosis by neutrophils is a crucial defense of the mammary gland and the prompt recruitment of these phagocytes from blood to milk compartments is essential for the outcome of the infection. ELR+ CXC chemokines, ligands of the two interleukin-8 receptors (IL-8R), CXCR1 and CXCR2, are likely to be involved in the initiation of the inflammatory response and also in the migration of neutrophils. Recently, the polymorphism of bovine CXCR2 has been associated with resistance to mastitis. However, as the bovine IL-8R are not functionally defined, their contribution to the recruitment of neutrophils remains undetermined. In this study, the RNA ligase-mediated (RLM)-RACE method was used to clone a novel bovine interleukin-8 receptor (nIL-8R) of the bovine species. We showed that both bovine IL-8R (nIL-8R and the published CXCR2) are functional since bovine IL-8 induced migration of HEK-293 cells expressing either IL-8R. In addition, comparisons of full-length sequences suggested that the published CXCR2 sequence was improperly annotated and that the sequences of the nIL-8R and the published CXCR2 are homologous to human CXCR2 and CXCR1, respectively. This was confirmed by binding assays with labeled IL-8 and GRO-beta and calcium (Ca) flux responses of transfected cells. Moreover, the C-terminal of both bovine IL-8R showed 100% identity, whereas they differ in most other species, suggesting that the two bovine IL-8R initiate similar signal transduction. These results constitute a basis to improve our understanding of the molecular mechanisms implicated in the recruitment of bovine neutrophils

Differential cytokine and chemokine expression from mammary epithelial cells stimulated by Staphylococcus aureus or Escherichia coli strains isolated from mastitis

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Determination and characterization of bovine interleukin-17 cDNA

International audienceInterleukin-17 (IL-17) is a proinflammatory cytokine produced by activated memory T cells, and it appears to play an upstream role in T cell-triggered inflammation by stimulating stromal cells to secrete other cytokines. We hypothesize that IL-17 plays a role in the recruitment of neutrophils in the bovine mammary gland during infection or immune-mediated inflammation. The rapid amplification of cDNA ends (RACE) method was used to obtain a cDNA of bovine IL-17 (BoIL-17) containing a 462-bp open reading frame (ORF) encoding a protein of 153 amino acids (aa) with a molecular mass of 17.2 kDa, a 23-residue NH2-terminal signal peptide, a single potential N-linked glycosylation site, and 6 cysteine residues. BoIL-17 protein shared 73.5% identity with the human protein and 67% with the mouse and rat proteins. Sf9 insect cells were transfected with BoIL-17 cDNA, and supernatant was tested for biologic activity on a primary culture of bovine mammary epithelial cells (MECs). mRNA synthesis of IL-6, IL-8, and growth-related oncogene alpha (Gro alpha) was induced, suggesting a functional role for IL-17 in mammary immunity

Characterization of the PS Cells, a mammary epithelial cell line for the study of mammary gland immunity

International audienceDecryption of diseases such as mastitis or breast cancer relies on our knowledge of the physiology of mammary epithelial cells. These cells produce milk, but they are also supposed to be sentinels for the detection of infections and the triggering of inflammation within the udder. Despite their pivotal role, none of the existing cell lines is sufficient to provide a steady and unfailing deciphering of the general physiology of mammary cells and their implication in immune responses against diseases. Here, we describe the PS cell line, especially its cellular purity and specificity, and its ability to respond to mastitis pathogens. Contrary to bovine primary cell cultures, the PS cell line is exclusively composed of luminal cytokeratin18-positive mammary epithelial cells. It expresses α and β-caseins, and it is able to secrete cytokines, chemokines (CCL20, CXCL8) and anti-microbial peptides (SAA3, TAP) in response to bacterial agonists in the absence of serum, unlike other mammary epithelial cell lines. This immune capacity is allowed by expression of functional receptors implicated in the recognition of components synthetized by mastitis pathogens, in particular lipopeptides (recognized by toll-like receptors (TLR) 1, 2 and 6). Other Microbe-Associated Molecular Patterns (MAMPs) such as peptidoglycan subunits (NOD 1 or NOD2), flagellin (TLR5), and LPS (TLR4-CD14-MD2 complex) induce weaker responses. This may result from a low expression of TLR5 and CD14, in keeping with some in vivo data but at variance with some data obtained with primary cells. The PS cell line will be useful to specify the part played by mammary epithelial cells in the innate defense against mastitis, and to investigate their interplay with the other cell populations constituting the udder during mastitis

Proteomics of the response of the mammary gland to a bacterial infection

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