title: Neutrophil function in juvenile systemic Lupus Erythematosus (JSLE)
Creators: name: Chiewchengchol, Direkrit
Publisher: name: University of Liverpool Repository
Year: 2014

description: Objectives This project aimed to investigate the abnormal neutrophil functions in terms of cell survival, phagocytosis, ROS production and chemotaxis in JSLE patients. Furthermore, factors that could affect normal neutrophil functions (such as patient sera, recombinant human cytokines and commonly-used medications) were investigated. Methods Neutrophils and sera were isolated from whole blood of JSLE patients, healthy juvenile controls and healthy adult controls. Neutrophils from healthy adult controls were incubated with 10% serum from either JSLE patients or juvenile controls. Different recombinant cytokines or hydroxychloroquine were added in 10% human AB serum to neutrophils from healthy adult controls. Neutrophil apoptosis was measured using flow cytometry using annexin V-FITC and propidium iodide. The expression of proteins (Mcl-1, caspases 3,7,8 and 9) was measured by Western blotting. Phagocytosis and ROS production from neutrophils incubated with S. aureus opsonised with JSLE serum were quantified. Levels of cytokines in JSLE serum were measured using a Luminex assay and effects of GM-CSF and TNF-α on neutrophil apoptosis induced by JSLE serum were measured. JSLE neutrophil apoptosis was measured and Mcl-1 mRNA expression from JSLE neutrophils was quantified using real-time PCR. Phagocytosis, ROS production and neutrophil chemotaxis by JSLE neutrophils were investigated. Lastly, the effects of hydroxychloroquine on neutrophil functions were explored. Results The results showed that neutrophils incubated with inactive and active JSLE sera had significantly increased apoptosis at 6 h compared to control sera. Cleaved (active) forms of caspases 3,7,8 were identified in neutrophils incubated with inactive and active JSLE sera (that showed high rates of apoptosis) compared to control sera. Decreased bacterial opsonisation leading to defective phagocytosis and ROS production was observed in neutrophils incubated with S. aureus opsonised with JSLE serum. Serum analysis showed IL-8 levels in active JSLE patients were significantly increased. GM-CSF was the most potent cytokine in delaying apoptosis and significantly saved neutrophil apoptosis induced by JSLE serum. Low concentrations of TNF-α significantly protected neutrophils against apoptosis by down-regulating several genes and proteins involved in death receptor signaling pathway (e.g. TNFR, FADD, TRADD, caspases 8 and 10). No significant differences were detected in apoptosis, phagocytosis, ROS production and chemotaxis of neutrophils isolated from JSLE patients compared to healthy juvenile controls, and healthy adult neutrophils treated with hydroxychloroquine compared to untreated cells. Conclusions This study demonstrated that JSLE serum played an important role in regulating the functions of JSLE neutrophils. Factor(s) in JSLE serum induced neutrophil apoptosis and caused decreased bacterial opsonisation, leading to defective neutrophil phagocytosis and ROS production. Increased IL-8 levels could be used as an indicator of disease activity. GM-CSF was the most protective cytokine and overcame the pro-apoptotic effects of JSLE serum; thus, GM-CSF could potentially be used as an alternative treatment in JSLE patients. Effects of TNF-α are probably tissue-specific and the clinical application of TNF blockers in JSLE patients needs to be carefully considered. The functions of JSLE neutrophils were unimpaired and hydroxychloroquine showed no effects on neutrophil functions.