title: Neutrophil extracellular traps in sheep mastitis.
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.
authors.affiliation: Porto Conte Ricerche, Alghero, (SS), Italy. pisanu@portocontericerche.it.
authors.forename: Salvatore
authors.initials: S
authors.lastname: Pisanu
authors.affiliation: Dipartimento di Medicina Veterinaria, Università di Sassari, Sassari, Italy. tcubeddu@uniss.it.
authors.forename: Tiziana
authors.initials: T
authors.lastname: Cubeddu
authors.affiliation: Porto Conte Ricerche, Alghero, (SS), Italy. pagnozzi@portocontericerche.it.
authors.forename: Daniela
authors.initials: D
authors.lastname: Pagnozzi
authors.affiliation: Dipartimento di Medicina Veterinaria, Università di Sassari, Sassari, Italy. rocca@uniss.it.
authors.forename: Stefano
authors.initials: S
authors.lastname: Rocca
authors.affiliation: Dipartimento di Medicina Veterinaria, Università di Sassari, Sassari, Italy. ccacciotto@uniss.it.
authors.forename: Carla
authors.initials: C
authors.lastname: Cacciotto
authors.affiliation: Dipartimento di Medicina Veterinaria, Università di Sassari, Sassari, Italy. alberti@uniss.it.
authors.forename: Alberto
authors.initials: A
authors.lastname: Alberti
authors.affiliation: Istituto Zooprofilattico Sperimentale G, Pegreffi, Sassari, Italy. gavino.marogna@izs-sardegna.it.
authors.forename: Gavino
authors.initials: G
authors.lastname: Marogna
authors.affiliation: Porto Conte Ricerche, Alghero, (SS), Italy. uzzau@portocontericerche.it.
authors.forename: Sergio
authors.initials: S
authors.lastname: Uzzau
authors.affiliation: Porto Conte Ricerche, Alghero, (SS), Italy. addis@portocontericerche.it.
authors.forename: Maria Filippa
authors.initials: MF
authors.lastname: Addis
mesh_terms: D000818:Animals; D065206:Extracellular Traps / Q000378:metabolism* / Q000382:microbiology; D005260:Female; D006801:Humans; D008321:Mammary Glands, Animal / Q000382:microbiology / Q000473:pathology; D008413:Mastitis / Q000276:immunology / Q000382:microbiology / Q000662:veterinary*; D008892:Milk / Q000166:cytology / Q000382:microbiology; D009504:Neutrophils / Q000378:metabolism* / Q000382:microbiology; D012756:Sheep; D012757:Sheep Diseases / Q000276:immunology* / Q000382:microbiology; D013290:Streptococcal Infections / Q000276:immunology / Q000382:microbiology / Q000662:veterinary*; D013291:Streptococcus / Q000502:physiology*