Ex Vivo and In Vitro Analysis Identify a Detrimental Impact of Neutrophil Extracellular Traps on Eye Structures in Equine Recurrent Uveitis

Abstract

Equine recurrent uveitis (ERU) is a common ocular disease of horses and described as a model for human autoimmune uveitis. This immune-mediated, inflammatory condition progressively destroys the eye, ultimately leading to blindness. Genetic and autoimmune factors, next to infections with Leptospira, are discussed as key factors in the pathogenesis. Furthermore, a release of neutrophil extracellular traps (NETs) by activated neutrophils is involved. NETs are composed of decondensed chromatin and proteins that can immobilize invading pathogens. However, if NETs accumulate, they can contribute to detrimental autoimmune processes. Thus, we aimed to investigate the impact of NETs in ERU patients. Therefore, we quantified several NET-markers (cell-free DNA, nucleosomes, citrullinated histone H3, histone-myeloperoxidase complexes, interleukin-17, equine cathelicidin 1 and DNase I activity) and NET-autoantibodies in sera and vitreous body fluids (VBF) of ERU-diseased horses and correlated the data with the disease status (signalment, ERU scores and Leptospira infection status). NET markers were detected to varying degrees in VBF of diseased horses, and partially correlated to disease severity and the presence of Leptospira spp. Cell-free DNA and nucleosomes as NET markers correlate with ERU severity in total and VBF scores, despite the presence of active DNases. Additionally, a significant correlation between fundus affection in the eye and NET autoantibodies was detectable. Therefore, we further investigated the influence of VBF samples from equine patients and isolated NETs on the blood-retina barrier in a cell culture model. VBF of diseased horses significantly induced cytotoxicity in retinal pigment epithelial cells. Moreover, partially digested NETs also resulted in cytotoxic effects. In the presence of lipopolysaccharide (LPS), the main component of the leptospiral surface, both undigested and completely digested NETs were cytotoxic. Correlations between the ERU-scores and Leptospira were also calculated. Detection of leptospiral DNA, and antibody titers of the serovar Grippotyphosa correlated with disease severity. In addition, a correlation between Leptospira and several NET markers was observed in VBF. Altogether, our findings suggest a positive correlation between NET markers with disease severity and involvement of Leptospira in the VBF of ERU-diseased horses, as well as a cytotoxic effect of NETs in eyes.

Impact of bronchoalveolar lavage from influenza A virus diseased pigs on neutrophil functions and growth of co-infecting pathogenic bacteria

“…Myeloperoxidase (MPO) is one of the main antimicrobial components that can be found attached to NETs. In the form of histone–MPO complexes, it serves as a very specific NET marker as these complexes are not formed under necrotic circumstances ( 26 , 32 ). Significantly higher amounts of histone–MPO complexes were detected in IAV-positive BALF compared with controls ( Figure 2C ).…”

“…The assays were performed according to the manufacturer’s recommendations. A sandwich ELISA for the detection of histone–MPO complexes was performed as described before ( 26 ).…”

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

“…16 Non-infectious and immune-mediated diseases can also be associated with NET release, 14 and in horses this has included equine asthma 32,72 and recurrent uveitis. 21,22 The presence of NETs in septic equine synovial fluid (SF) and peritoneal fluid (PF) samples is unknown, and therefore it is unclear if those fluids could be of use in the diagnosis of infection or septic inflammation and for predicting prognosis. Diagnosis of septic inflammation can be challenging using routine laboratory methods given overlapping results for non-septic and septic processes.…”

Neutrophil Extracellular Traps (NETs) in Ocular Diseases: An Update

“…There are investigations about vitreous body fluids (VBF), sera, and ocular histological sections from horses with or without ERU. Interestingly, several NETs markers in sera of ERU, such as free DNA, histone-complexes, and MPO, show a higher level in the samples from ERU horses compared to healthy horses, implying NETs are involved in the pathogenesis of ERU [ 93 , 94 ]. As the potential NETs-inducing factors, equine cathelicidins are up-regulated in the VBF of ERU-diseased horses.…”

“…These pro-inflammatory cytokines mediate the formation of NETs and the production of adenosine monophosphate (AMP), resulting in the occurrence of ERU. Leptospira, which induces NETs in a concentration-dependent manner in the VBF of ERU-diseased horses, is another contributor to the pathogenesis of ERU [ 93 , 96 ]. However, little is known about whether NETs contribute to the pathogenesis of ERU.…”