Visualizing neutrophil extracellular traps in septic equine synovial and peritoneal fluid samples using immunofluorescence microscopy

Abstract

Septic synovitis and peritonitis are routinely diagnosed in horses based on clinical examination findings and laboratory assessment of synoviocentesis and abdominocentesis samples, respectively. Diagnosis is difficult in some cases because of an overlap in laboratory results for septic and non-septic inflammation. Neutrophil extracellular trap (NET) formation is part of the innate immune response against pathogens. Identifying and quantifying NETs, which have not been explored in clinical samples from horses with septic synovitis and peritonitis, to our knowledge, may be helpful in detecting infectious processes. Our main objective was to determine whether NETs could be visualized in septic equine synovial and peritoneal fluid cytology samples using immunofluorescence with antibodies against citrullinated histone H3 (Cit-H3) and myeloperoxidase (MPO). We analyzed 9 synovial and 4 peritoneal fluid samples. NET percentages were quantified using a simple counting technique, which is suitable for high-quality, well-preserved, and stained cytospin smears. NETs were evident in all septic samples and were absent in a non-septic sample; NETs were better visualized with Cit-H3 than with MPO immunolabeling. Overall, we believe that there is the potential for NETs and associated markers to be used to investigate and understand septic inflammation in horses.

Keywords: NETs; histones; horses; immunofluorescence; infection; peritoneal fluid; synovial fluid.

Figure 1.

Immunofluorescent images of synovial fluid cytospins prepared from horse 2 with a septic distal interphalangeal joint. The blue DAPI filter (A, D) was used to detect DNA, and the green FITC filter to visualize citrullinated histone 3 (Cit-H3, monoclonal antibody; B) and myeloperoxidase (MPO; E). Co-localization of extracellular DNA fibers with Cit-H3 or MPO, evident in the merged images (C, F, respectively) were consistent with neutrophil extracellular traps. 400× (A–C); 1,000× (D–F).

Figure 2.

Neutrophil extracellular traps (NETs) were visualized in cytospins of septic synovial and peritoneal fluid samples from horses using immunofluorescence. A polyclonal (A, B) and monoclonal (C–F) antibody was directed against citrullinated histone 3 (Cit-H3, green), and DAPI was used to stain DNA (blue). The images have all been merged. Cit-H3 immunofluorescence staining was similar for both antibodies and varied from being localized to swollen nuclei, consistent with early NET formation (D), to forming slender extracellular fibers (A, B, E, F) or NET aggregates (C). Images were of synovial fluid from horses 6 (A, B, F), 7 (D), and 5 (E), and peritoneal fluid from horse 9 (C). 400× (A–C); 1,000× (D–F).

Figure 3.

Comparison of neutrophil (NEUT) counts (× 10⁹/L) with neutrophil extracellular trap (NET) counts as percentages in synovial fluid (SF) samples from 7 horses. Neutrophil counts were calculated from the total nucleated cell count of the SF samples multiplied by the differential cell count for neutrophils. The numbers on the x-axis are the horse IDs. All samples met septic inflammation criteria, apart from horse 1 (tarsocrural joint), which had moderate suppurative inflammation.