title: Neutrophil extracellular traps formation by bacteria causing endometritis in the mare.
abstract: Besides the classical functions, neutrophils (PMNs) are able to release DNA in response to infectious stimuli, forming neutrophil extracellular traps (NETs) and killing pathogens. The pathogenesis of endometritis in the mare is not completely understood. The aim was to evaluate the in vitro capacity of equine PMNs to secrete NETs by chemical activation, or stimulated with Streptococcus equi subspecies zooepidemicus (Szoo), Escherichia coli (Ecoli) or Staphylococcus capitis (Scap) strains obtained from mares with endometritis. Ex vivo endometrial mucus from mares with bacterial endometritis were evaluated for the presence of NETs. Equine blood PMNs were used either without or with stimulation by phorbol-myristate-acetate (PMA), a strong inducer of NETs, for 1-3h. To evaluate PMN ability to produce NETs when phagocytosis was impaired, the phagocytosis inhibitor cytochalasin (Cyt) was added after PMA. After the addition of bacteria, a subsequent 1-h incubation was carried out in seven groups. NETs were visualized by 4',6-diamidino-2-phenylindole (DAPI) and anti-histone. Ex vivo samples were immunostained for myeloperoxidase and neutrophil elastase. A 3-h incubation period of PMN + PMA increased NETs (p < 0.05). Bacteria + 25 nM PMA and bacteria + PMA + Cyt increased NETs (p<0.05). Szoo induced fewer NETs than Ecoli or Scap (p < 0.05). Ex vivo NETs were present in mares with endometritis. Scanning electron microscopy showed the spread of NETs formed by smooth fibers and globules that can be aggregated in thick bundles. Formation of NETs and the subsequent entanglement of bacteria suggest that equine NETs might be a complementary mechanism in fighting some of the bacteria causing endometritis in the mare.
authors.affiliation: CIISA, Faculty of Veterinary Medicine, University of Lisbon, 1300-477 Lisbon, Portugal; Coimbra College of Agriculture, Coimbra, Portugal.
authors.forename: M R
authors.initials: MR
authors.lastname: Rebordão
authors.affiliation: CIISA, Faculty of Veterinary Medicine, University of Lisbon, 1300-477 Lisbon, Portugal.
authors.forename: C
authors.initials: C
authors.lastname: Carneiro
authors.affiliation: CIISA, Faculty of Veterinary Medicine, University of Lisbon, 1300-477 Lisbon, Portugal.
authors.forename: G
authors.initials: G
authors.lastname: Alexandre-Pires
authors.affiliation: CIISA, Faculty of Veterinary Medicine, University of Lisbon, 1300-477 Lisbon, Portugal.
authors.forename: P
authors.initials: P
authors.lastname: Brito
authors.affiliation: CIISA, Faculty of Veterinary Medicine, University of Lisbon, 1300-477 Lisbon, Portugal.
authors.forename: C
authors.initials: C
authors.lastname: Pereira
authors.affiliation: Faculty of Sciences, Microscopy Center, University of Lisbon, Portugal.
authors.forename: T
authors.initials: T
authors.lastname: Nunes
authors.affiliation: CIISA, Faculty of Veterinary Medicine, University of Lisbon, 1300-477 Lisbon, Portugal.
authors.forename: A
authors.initials: A
authors.lastname: Galvão
authors.affiliation: Instituto de Investigação Científica Tropical, CVZ, CIISA, Lisbon, Portugal.
authors.forename: A
authors.initials: A
authors.lastname: Leitão
authors.affiliation: CIISA, Faculty of Veterinary Medicine, University of Lisbon, 1300-477 Lisbon, Portugal.
authors.forename: C
authors.initials: C
authors.lastname: Vilela
authors.affiliation: CIISA, Faculty of Veterinary Medicine, University of Lisbon, 1300-477 Lisbon, Portugal. Electronic address: gmlfdias@fmv.ulisboa.pt.
authors.forename: G
authors.initials: G
authors.lastname: Ferreira-Dias
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D003572:Cytochalasins / Q000494:pharmacology; D004716:Endometritis / Q000276:immunology* / Q000382:microbiology*; D004926:Escherichia coli / Q000276:immunology; D065206:Extracellular Traps / Q000276:immunology*; D005260:Female; D006736:Horses; D007249:Inflammation / Q000276:immunology; D009504:Neutrophils / Q000276:immunology*; D009195:Peroxidase / Q000378:metabolism; D010587:Phagocytosis / Q000276:immunology; D013210:Staphylococcus / Q000276:immunology; D018502:Streptococcus equi / Q000276:immunology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
chemical_list: D003572:Cytochalasins; D009195:Peroxidase; D013755:Tetradecanoylphorbol Acetate
keywords: Endometritis; Escherichia coli; Mare; Neutrophil extracellular traps (NETs); Staphylococcus capitis; Streptococcus equi subspecies zooepidemicus