Abstract
Neutrophil extracellular traps (NETs) are structures composed of DNA, histones, and antimicrobial proteins that are released extracellularly by neutrophils and other immune cells as a means for trapping and killing invading pathogens. Here, we describe NET formation in milk and in mammary alveoli of mastitic sheep, and provide a dataset of proteins found in association to these structures. Nucleic acid staining, immunomicroscopy and fluorescent in-situ hybridization of mastitic mammary tissue from sheep infected with Streptococcus uberis demonstrated the presence of extranuclear DNA colocalizing with antimicrobial proteins, histones, and bacteria. Then, proteomic analysis by LTQ-Orbitrap Velos mass spectrometry provided detailed information on protein abundance changes occurring in milk upon infection. As a result, 1095 unique proteins were identified, of which 287 being significantly more abundant in mastitic milk. Upon protein ontology classification, the most represented localization classes for upregulated proteins were the cytoplasmic granule, the nucleus, and the mitochondrion, while function classes were mostly related to immune defence and inflammation pathways. All known NET markers were massively increased, including histones, granule proteases, and antimicrobial proteins. Of note was the detection of protein arginine deiminases (PAD3 and PAD4). These enzymes are responsible for citrullination, the post-translational modification that is known to trigger NET formation by inducing chromatin decondensation and extracellular release of NETs. As a further observation, citrullinated residues were detected by tandem mass spectrometry in histones of samples from mastitic animals. In conclusion, this work provides novel microscopic and proteomic information on NETs formed in vivo in the mammary gland, and reports the most complete database of proteins increased in milk upon bacterial mastitis.Electronic supplementary materialThe online version of this article (doi:10.1186/s13567-015-0196-x) contains supplementary material, which is available to authorized users.
Highlights
Mastitis is an inflammation of the mammary gland typically consequent to bacterial infection
Confocal microscopy supports formation of Neutrophil extracellular traps (NETs) in vivo in mammary alveoli of acutely mastitic sheep Milk and tissues collected at necroscopy from the mammary gland of sheep that developed acute mastitis after experimental S. uberis infection were analyzed microscopically by SYTOX Orange staining
This structure was almost completely destroyed by treatment with nuclease (Figure 1B), and it was never observed in negative samples (Figure 1C)
Summary
Mastitis is an inflammation of the mammary gland typically consequent to bacterial infection. Other relevant causal agents of mastitis in sheep are mycoplasmas, but since these etiologic agents do cause other severe symptoms, including lameness, keratoconjunctivitis, and respiratory problems, some authors fail to consider them as Somatic cells are typically represented by epithelial cells, neutrophils, macrophages, and lymphocytes as the major cell types [5] Their physiological levels in sheep milk are still the subject of controversies; issues remain on the fluctuations of this parameter due to numerous factors other than mastitis, such as management practices, stage of lactation, parity, and presence of lentiviral infections, to name a few [6,7]. In milk of healthy sheep at the peak of lactation, epithelial cells and their fragments are the Pisanu et al Veterinary Research (2015) 46:59
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