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Investigating the role of Toll-like receptors in juvenile onset systemic lupus erythematosus

Thorbinson, Colin (2010) Investigating the role of Toll-like receptors in juvenile onset systemic lupus erythematosus. Masters thesis, University of Liverpool.

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Abstract

Background: Juvenile-onset Systemic Lupus Erythematosus (JSLE) is a chronic debilitating multi-system autoimmune condition characterised by auto-antibody production directed against nuclear antigens. It is associated with a more severe onset and more aggressive clinical course than in adults, with poor prognostic factors presenting earlier in childhood. It has been proposed that dysregulated neutrophil apoptosis may be a source of autoantigen in JSLE. Toll-like receptors (TLRs) are essential in the function of the innate immune system recognising pathogenic material. Upon stimulation they initiate a non-specific immune response leading to the production of an antigen-specific immune defence and autoantibody production. TLRs have been implicated in the development of autoimmunity. TLRs 3, 7, 8 and 9 are capable of recognising nucleic autoantigens typical of SLE and their expression has been shown to positively correlate with anti-dsDNA titres and disease activity in adult-onset SLE. To date there have been no studies examining the role of TLRs in JSLE. Aim: To assess whether apoptotic neutrophils in JSLE are providing a source of nuclear autoantigen which are being detected through TLRs 3, 7, 8 and 9 resulting in an inflammatory response through activation of an adaptive autoimmune response. Methods: Peripheral Blood Mononuclear Cells (PBMCs) and B cells were isolated from JSLE patients, Juvenile Idiopathic Arthritis (JIA) (inflammatory controls) and non-inflammatory controls. TLR 3, 7-9 mRNA and protein expression was measured using quantitative PCR (qPCR) and flow cytometry respectively. PBMCs were incubated with TLR agonists, activation was measured by IFN-a protein and mRNA expression, by ELISA and qPCR respectively. Neutrophils were isolated from healthy controls and incubated with JSLE serum to induce apoptosis. Apoptotic neutrophils were incubated with PBMCs at varying concentrations for 6 hours. After which IFN-a mRNA expression was measured by qPCR. PBMCs were treated with a MyD88 inhibitor to block TLRs 7-9 or left untreated. MyD88 treated and untreated PBMCs were incubated with TLR9 agonist or apoptotic neutrophils for 6 hours. TLR activation was measured using IFN-a mRNA expression by qPCR. Results: JSLE patients have a significantly increased PBMC TLR 3, 8 and 9 protein and mRNA expression compared to controls (p<0.05). B cells also showed increased TLR 7 and 9 mRNA expression compared to controls (p<0.05). Stimulation of TLR 3, 7, 8 and 9 in PBMCs leads to a significantly increased IFN-a protein and mRNA expression (p<0.05). Incubation of PBMCs with varying concentrations of apoptotic neutrophils demonstrated a dose-response relationship as measured by IFN-a mRNA expression and TLR expression positively correlated with increasing apoptosis (p<0.05). MyD88 inhibition of PBMCs was found to effectively inhibit IFN-a mRNA expression in PBMCs incubated with TLR9 agonist and apoptotic neutrophils. Conclusions: This study has demonstrated increased TLR expression in JSLE PBMCs and B cells. We have shown TLR stimulation to result in IFN-a production and using this criteria have shown apoptotic neutrophils to be potent stimulators of TLRs and therefore a likely source of autoantigen in JSLE. The role of TLRs in this inflammatory response was demonstrated by a dose-response relationship to apoptotic neutrophil concentration and TLR expression being shown to positively correlate with apoptosis. MyD88 inhibition was shown to be an effective strategy in halting this inflammatory response to apoptotic neutrophils.

Item Type:Thesis (Masters)
Subjects:R Medicine > RC Internal medicine
R Medicine > RC Internal medicine > RC0254 Neoplasms. Tumors. Oncology (including Cancer)
Departments, Research Centres and Related Units:Academic Faculties, Institutes and Research Centres > Faculty of Medicine > School of Reproductive & Developmental Medicine
Refereed:Yes
Status:Unpublished
ID Code:1452
Deposited On:11 Jan 2011 10:58
Last Modified:19 May 2011 12:08

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