Immunodetection of NETs in Paraffin-Embedded Tissue

Abstract

The pathogenic potential of neutrophil extracellular traps (NETs) was recently described, and their detection in tissue could serve as a prognostic marker. NETs are delicate and filigree structures; hence good tissue preservation is essential for their detection. Indeed, analysis of paraffin-embedded tissue has proven superior to the study of cryo sections. Though, under favorable conditions, the presence of NETs can be detected in tissue sections stained with histological dyes, definitive identification of NETs needs the colocalization of immunofluorescent signals for both nuclear and granular (or cytoplasmic) NET components. We tested diverse antigen retrieval methods and various combinations of commercially available antibodies and present here staining protocols to detect NETs in human and murine tissue sections.

Induction of neutrophil extracellular traps during tissue injury: Involvement of STING and Toll‐like receptor 9 pathways

“…3 Mitochondrial DNA is the major structural component of NETs, 4,5 and it induced the postinjury inflammation and activated neutrophils. [7][8][9] The NETosis neutrophilproduced products, including extracellular DNA, histones and granular enzymes, act as damage-associated molecular pattern molecules (DAMPs), inducing a subsequent immune response and also contributing to coagulopathy, tissue injury, cancer and barrier dysfunction. The identification of NETs in tissues could serve as a prognostic marker.…”

First observation of neutrophil extracellular traps in human leptospirosis

“…[3] In the past, the presence of NETs in tissues, particularly in paraffin embedded samples, was hardly identified, and staining for just one component, e.g., DNA, was considered not sufficient. [5] Our observation of colocalization of granular proteins in the nucleus and the citrullination of histones provide unique features for neutrophils undergoing NETosis that can be exploited for identification of these cells in tissue sections as it has been recently described. [5] In this regard, we have recently described that Leptospira interrogans may induce NETs in human neutrophils and that pathogenic Leptospira has the ability to escape from NETs.…”

“…[5] Our observation of colocalization of granular proteins in the nucleus and the citrullination of histones provide unique features for neutrophils undergoing NETosis that can be exploited for identification of these cells in tissue sections as it has been recently described. [5] In this regard, we have recently described that Leptospira interrogans may induce NETs in human neutrophils and that pathogenic Leptospira has the ability to escape from NETs. [8] However, the presence of NETs in human leptospirosis was not described previously.…”

“…Search for leptospiral antigen in lung tissues using antibodies against Leptospira interrogans was intended using an immunohistochemical procedure that have been described before. [7] 2.2 NETs detection As regarding NETs detection, a few modifications for a better visualization in paraffin embedded tissues using a immunofluorescence (IF) technique were recently published [5] and applied in this case. Rabbit polyclonal against human neutrophil elastase (NE) (Calbiochem-Merk Millipore -Darmstad, Germany), or citrulinated histone 3 (H3cit) (Santa Cruz, Ca, USA), followed by anti species antibody labeled with different fluorochromes were used.…”

“…[4] These DNA structures, named neutrophil extracellular traps (NETs), are composed of chromatin, associated with several proteins with antimicrobial properties such as citrullinated histones, elastase and myeloperoxidase among others, and serve as a physical barrier that prevents the further spreading of pathogens. [5] Although it was originally proposed that NETs are formed exclusively in tissues at sites of bacterial or fungal infection, NETs have also been found within blood vessels where they ensnare circulating bacteria during experimental sepsis. [6] The presence of NETs in human leptospirosis is unknown.…”