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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.

SUBMITTER: Birckhead EM

PROVIDER: S-EPMC10621558 | biostudies-literature | 2023 Nov

REPOSITORIES: biostudies-literature

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

Birckhead Emily M EM Das Shubhagata S Tidd Naomie N Raidal Sharanne L SL Raidal Shane R SR

Journal of veterinary diagnostic investigation : official publication of the American Association of Veterinary Laboratory Diagnosticians, Inc 20230903 6

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 sample ...[more]

PMID: 37661696

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Project description:BackgroundOsteoarthritis remains one of the greatest causes of morbidity and mortality in the equine population. The inability to detect pre-clinical changes in osteoarthritis has been a significant impediment to the development of effective therapies against this disease. Synovial fluid represents a potential source of disease-specific small non-coding RNAs (sncRNAs) that could aid in the understanding of the pathogenesis of osteoarthritis. We hypothesised that early stages of osteoarthritis would alter the expression of sncRNAs, facilitating the understanding of the underlying pathogenesis and potentially provide early biomarkers.MethodsSmall RNA sequencing was performed using synovial fluid from the metacarpophalangeal joints of both control and early osteoarthritic horses. A group of differentially expressed sncRNAs was selected for further validation through qRT-PCR using an independent cohort of synovial fluid samples from control and early osteoarthritic horses. Bioinformatic analysis was performed in order to identify putative targets of the differentially expressed microRNAs and to explore potential associations with specific biological processes.ResultsResults revealed 22 differentially expressed sncRNAs including 13 microRNAs; miR-10a, miR-223, let7a, miR-99a, miR-23b, miR-378, miR-143 (and six novel microRNAs), four small nuclear RNAs; U2, U5, U11, U12, three small nucleolar RNAs; U13, snoR38, snord96, and one small cajal body-specific RNA; scarna3. Five sncRNAs were validated; miR-223 was significantly reduced in early osteoarthritis and miR-23b, let-7a-2, snord96A and snord13 were significantly upregulated. Significant cellular actions deduced by the differentially expressed microRNAs included apoptosis (P < 0.0003), necrosis (P < 0.0009), autophagy (P < 0.0007) and inflammation (P < 0.00001). A conservatively filtered list of 57 messenger RNA targets was obtained; the top biological processes associated were regulation of cell population proliferation (P < 0.000001), cellular response to chemical stimulus (P < 0.000001) and cell surface receptor signalling pathway (P < 0.000001).ConclusionsSynovial fluid sncRNAs may be used as molecular biomarkers for early disease in equine osteoarthritic joints. The biological processes they regulate may play an important role in understanding early osteoarthritis pathogenesis. Characterising these dynamic molecular changes could provide novel insights on the process and mechanism of early osteoarthritis development and is critical for the development of new therapeutic approaches.

Project description:Articular conditions are common in horses and can result in loss of function, chronic pain and/or inability to work. Common conditions include osteoarthritis, osteochondrosis and synovial sepsis, which can be life-threatening, but despite the high clinical prevalence of these conditions, rapid and specific diagnosis, monitoring and prognostication remains a challenge for practicing veterinarians. Synovial fluid from a range of arthropathies was enriched for low abundance proteins using combinatorial peptide ligand ProteoMiner™ beads and analysed via liquid chromatography-tandem mass spectrometry. Changes in protein abundances were analysed using label-free quantification. Principle component analysis of differentially expressed proteins identified groupings associated with joint pathology. Findings were validated using ELISA. Lactotransferrin (LTF) abundance was increased in sepsis compared to all other groups and insulin-like growth factor-binding protein 6 (IGFBP6) abundance decreased in sepsis compared to other disease groups. Pathway analysis identified upregulation of the complement system in synovial joint sepsis and the downregulation of eukaryotic translation initiation factors and mTOR signalling pathways in both OA and OC compared to the healthy group. Overall, we have identified a catalogue of proteins which we propose to be involved in osteoarthritis, osteochondrosis and synovial sepsis pathogenesis. SIGNIFICANCE: Osteoarthritis, osteochondrosis and synovial sepsis, which can be life-threatening, are common articular conditions in which rapid and specific diagnosis, monitoring and prognostication remains a challenge for practicing veterinarians. This study has identified that the equine synovial fluid proteome exhibits distinctive profile changes between osteoarthritis, osteochondrosis, synovial sepsis and healthy joints. Elevated synovial abundance of lactotransferrin and decreased insulin-like growth factor-binding protein 6 were both found to distinguish synovial sepsis from all other study groups. Thus, these protein markers may have a future role in clinical practice to enable an earlier and reliable diagnosis of synovial sepsis.

Dataset Information

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

Publications

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

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