A novel approach to identifying and quantifying neutrophil extracellular trap formation in septic dogs using immunofluorescence microscopy

Abstract

BackgroundCanine neutrophils release neutrophil extracellular traps (NETs) in response to lipopolysaccharide but NETs from clinical septic dogs had not been identified. The primary aim is to describe the methodology of identifying and quantifying neutrophil extracellular traps (NETs) in cytology samples of septic foci in dogs with sepsis using immunofluorescence microscopy. Cytology samples including endotracheal tracheal wash (ETW), bronchoalveolar lavage (BAL), abdominal and pleural effusion collected from 5 dogs (3 septic, 2 non-septic) were fixed, permeabilized and stained for myeloperoxidase (MPO), citrullinated histone H3 (citH3) and cell-free DNA (cfDNA). Fluorescence microscopy was used to identify and quantify NETs in 10 random views at 40× magnification. NETs were identified based on co-localization of MPO, citH3 and cfDNA. NETs were quantified as a ratio (number of NETs: number of neutrophils). Neutrophils were identified based on cytoplasmic MPO, cellular diameter and nuclear morphology.ResultsNETs were identified and quantified in all cytology samples collected from septic dogs. A small number of NETs was documented in one dog with sterile chronic bronchitis. No NETs were found in sterile abdominal effusion collected from one dog with congestive heart failure.ConclusionsImmunofluorescence microscopy could be a useful tool for the study of NETs in dogs with clinical sepsis.

Biomarker Guided Diagnosis of Septic Peritonitis in Dogs

“…It could also suggest that adaptive immune responses and collaborative interactions between leukocytes reacting to the presence of bacteria leads to a distinct, or a greater inflammatory response than tissue damage alone. The high concentrations of cfDNA and nucleosomes in the peritoneal effusions of SP dogs is also very suggestive that NET release was occurring in the peritoneal cavities of these dogs, as has been previously reported (89). The increased blood PCT concentrations relative to those in effusion identified in the present study is consistent with previous findings of high PCT concentrations in dogs with sepsis (20, 21).…”

DAMPs and NETs in Sepsis

“…NETs have been detected in bronchoalveolar lavage samples from septic humans or canines with ARDS, indicating that, even after transmigration, neutrophils are capable of undergoing NETosis (174, 175). A recent study utilizing samples from different models of ALI in mice and from patients with ALI revealed increased levels of NETs and histones H3 and H4 in the bronchoalveolar lavage fluids (BALF) (176).…”

A Comparative Review of Neutrophil Extracellular Traps in Sepsis

“…For example, Streptococcus pneumoniae , a Gram-positive bacteria commonly found in the human respiratory tract, can express the virulence factor, endA, which degrades DNA, allowing for bacteremia and sepsis to occur ( 71 ). We recently demonstrated that NETs within the septic foci of clinical septic dogs can bind directly to bacteria suggesting that entrapment of bacteria by NETs may occur in dogs (Figures 1B,C ) ( 72 ).…”

“…Migration of neutrophils from the vasculature into the interstitium and bronchoalveolar space is a key feature of ARDS in sepsis. NETs in bronchoalveolar lavage fluid collected from septic people and dogs with ARDS have recently been documented indicating that transmigrated neutrophils undergo NETosis in naturally occurring ARDS ( 56 , 57 , 72 , 92 ). Serine proteases released via NETosis can have a direct pathophysiological role in the progression of ARDS.…”