| BackgroundInfectious keratitis is a common sight-threatening ocular disease all over the world, which has significant morbidity and always leads to severe vision dysfunction. The main pathogens contaminating cornea include bacteria, fungus, virus and so on. Researches out and in home show that the main pathogens in bacterial keratitis are Gram positive coccus (S. aureus most common) and then are the Gram negative bacilli (P.aeruginosa most common). As first line of innate immunity for corneal infection defense, neutrophils play crucial roles in eliminating pathogens, limiting inflammation, and in the development and prognosis of diseases at early term of corneal infection. Especially in the bacterial infection, neutrophils migrate to inflammatory sites, engulf pathogens, respiratory burst, produce reactive oxygen species, degranulate to release antimicrobial peptides and eliminate the pathogens, at the same time, inflammatory mediators are produced to chemotaxis more cells to infiltrate and amplify the inflammatory reactions, which facilitates the extinction of pathogens and plays a vital part in controlling early corneal infection. A novel PMN bactericidal mechanism named neutrophil extracellular traps (NETs) was discovered recently. In infective portion, PMN actively release DNA scaffold to entrap and limit the spread of pathogens and kill them by the histones and granular proteins adhering onto the DNA fibrous network. It has been found that NETs exist in amount of infectious diseases and take significant parts in the pathological development. How to modify the progress and prognosis of these diseases by modulating the formation of NETs has been the point of people’s relative researches.Glucocorticoids (GCs) have potent anti-inflammatory effect and are indispensable for the curing and alleviation of a lot of chronic inflammatory diseases, allergic or self-immune diseases. While GCs also have the potential to suppress the anti-infection immune responses, it is full of controversy whether or not to use GCs in clinical treatment of infectious diseases. How to judge the exact indication of GCs application becomes a giant challenge for many clinicians. Therefore to study deeply on the mechanisms of immune modulation of GCs is meaningful for rational selection and usage of medicine, and the design of effective treatment plan to achieve the goal of efficient elimination of pathogens with less injury to the tissues.As somewhat key elements of infecitve immunity, the modulation of NETs formation by GCs certainly influences the whole effect of GCs on treating infectious diseases. However the mechanism of NETs formation in vivo is still not full understood, not mention to its observation and function in ocular diseases. In this paper, we mainly focus on the modulation of dexamethasone (DXM) on the formation and bactericidal efficient of NETs along with its role in the progress and prognosis of infectious keratitis.MethodsThe part of in vitro experiment in this study was performed on the primary neutrophils separated from healthy volunteers and the HL-60cell line that has been differentiated. S. aureus and PMA was added to induce the formation of NETs. The morphology of NETs was observed by immunological fluorescent staining. On the impact of DXM to the formation of NETs, we mainly compared through photographs of sytox green staining, quantitative detection of free extracellular DNA by Picogreen dsDNA assay kit and the bacterial loads. The statistical analysis was performed by the software GraphPad Prism6.1.At the same time, we collected9patients with infectious keratitis, whose pathological corneal tisssue was scraped to slides and stained immunological fluorescence to observe the formation of NETs. In combination of his/her medical history (onset time of symptoms, and previous therapy), medical course, prognosis and the result of direct microscopic examination and pathogens culture from the laboratory, an preliminary analysis of the relationship between the formation of NETs and the progress of infectious keratitis was taken into an attempt.Results1. By immune fluorescent staining, we found that both S. aureus and PMA induced the formation of NETs in the PMN and the differentiated HL-60cell line as demonstrated by other experiments. This novel bactericidal mechanism was proved with the capacity of trapping and killing S. aureus. Furthermore, DXM pretreatment was found to inhibit the formation of NETs induced by S aureus as well as its bactericidal effect. It’s intriguing that DXM pretreatment seemed to have no effect on the NETs formation by PMA which somewhat disclosed the mechanism of NETs formation to us.2. We successfully detected the formation of NETs in the pathological corneal tissues of patients with infectious keratitis by immunological fluorescent staining. In combination of the medical history, we found that the formation of NETs did take a part in the progress and prognosis of infectious keratitis. Especially in the corneal fungal infected patients, more amount of NETs formation seemed to be accompanied with shorter treatment course. Thus we can preliminarily make the conclusion that the formation of NETs facilitates the end of the pathological progress of infectious keratitis.Conclusions1. DXM does inhibit the formation of NETs as well as its bactericidal effect.2. NETs may perform as an alternative investigative modality of infecous keratitis. We speculated that the NETs formation plays a part in the progress and prognosis of infectious keratitis. |
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