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2015 ; 46
(1
): 59
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Neutrophil extracellular traps in sheep mastitis
#MMPMID26088507
Pisanu S
; Cubeddu T
; Pagnozzi D
; Rocca S
; Cacciotto C
; Alberti A
; Marogna G
; Uzzau S
; Addis MF
Vet Res
2015[Jun]; 46
(1
): 59
PMID26088507
show ga
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.
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