Abstract
Mastitis has a high incidence in dairy cows. Experimental infection with Escherichia coli increased the number of leukocytes in milk and the gene expression of the chemokine receptor CXCR4 in mammary gland tissues. A link between CXCR4 expression and lipopolysaccharide sensing was demonstrated in other species using in vitro models. The receptor that binds the chemokine stomal cell-derived factor 1 might be associated with the inflammatory response in bovine mammary glands. However, studies in cows are rare, and data on the localization of CXCR4 in bovine mammary glands and its distribution in bovine leukocytes are lacking. Fatty acids (FA) affect the inflammatory response. In human peripheral blood monocytes, exposure to conjugated linoleic acids (CLA) decreases the expression of CXCR4, leading to a decreased inflammatory response in these cells. In this study, we analyzed the expression of CXCR4 in the mammary glands of dairy cows by immunohistochemistry (n = 5) and laser capture microdissection followed by qualitative PCR (n = 3). We characterized the surface expression of CXCR4 on bovine leukocytes, including monocyte subpopulations, first by flow cytometry (n = 5) and then confirmed these results by Western blotting (n = 3). Rumen fistulated dairy cows (n = 4; 126 ± 4 d in milk) were fitted with abomasal infusion tubes, arranged in a 4 × 4 Latin square design, and supplemented for 6 wk twice daily with rising doses of FA followed by a 3-wk washout period. Then, CXCR4 expression on leukocytes was analyzed. The cows received a corn-based diet and were supplemented with coconut oil delivering medium-chain FA (38 g/d), linseed-safflower oil mix delivering n-3 FA (EFA, 39 g of linseed oil and 2 g of safflower oil per day), Lutalin (cis-9,trans-11 and trans-10,cis-12 CLA, 5 g/d; BASF), and EFA + CLA. In the bovine mammary gland, the epithelial cells of the lactiferous duct, but not alveolar epithelial cells, showed clear CXCR4 protein and mRNA signals. Among the leukocyte subsets, monocytes displayed the highest percentage of CXCR4-positive cells (87%), whereas circulating neutrophils showed almost no CXCR4 surface expression (3%) but stored the receptor intracellularly. The percentage of CXCR4-positive leukocytes was not affected by the different FA supplements, but FA supplementation reduced the receptor abundance per cell (40% on average). In conclusion, CXCR4 was clearly detected in the lactiferous duct cells of the mammary gland but not in the alveolar epithelial cells. Compared with other leukocytes, bovine monocytes showed the highest signal intensity of CXCR4 on their surface, whereas granulocytes stored CXCR4 intracellularly. Supplementation with all the FA reduced the surface expression of CXCR4 per leukocyte and could therefore potentially affect the inflammatory status associated with the surface expression of CXCR4. The importance of our observations should be verified in cows with mastitis in the future.
Highlights
Mastitis is an economically relevant disease with a high incidence in dairy cows
The CXCR4 protein was detected in the epithelial cells of the lactiferous duct epithelial cells (LD), and on a small scale, we observed a signal within the Alveolar epithelial cells (AV)
We demonstrated that supplementation with UFA and coconut oil rich in lauric acid led to a reduction in CXCR4 expression on individual leukocytes
Summary
Mastitis is an economically relevant disease with a high incidence in dairy cows. Mastitis is the main reason for administering antimicrobial therapy to this species (Jamali et al, 2018). In addition to inflammatory stimuli, hormones such as progesterone can affect the expression of CXCR4, as demonstrated in mouse mammary glands In this species, the expression of CXCR4 increases during pregnancy, and CXR4 is involved in the functional differentiation of alveoli in the adult mammary gland (Shiah et al, 2015). The expression of CXCR4 increases during pregnancy, and CXR4 is involved in the functional differentiation of alveoli in the adult mammary gland (Shiah et al, 2015) Bovine chemokine receptors such as CXR4 were first characterized by Widdison et al (2010), but studies on the function of CXCR4 in cows are rare (Mitterhuemer et al, 2010; Weiner et al, 2012; Sayasith and Sirois, 2014), and concrete data on the expression of the receptor on bovine leukocytes are missing
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