Investigating the IL-17 family and Th17-related cytokines in juvenile-onset systemic lupus erythematosus

Ong, Joanne SH
Investigating the IL-17 family and Th17-related cytokines in juvenile-onset systemic lupus erythematosus. Master of Philosophy thesis, University of Liverpool.

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Background: Juvenile-onset systemic lupus erythematosus (JSLE) is a multi-system autoimmune disease characterised by severe disease and poorer prognosis compared to adult-onset disease. Currently, its aetiopathogenesis is unknown but is postulated to be a result of a loss of peripheral tolerance. T helper 17 (Th17) cells produce pro-inflammatory cytokines Interleukin-17A (IL-17A) and IL-21 and are upregulated by IL-23. These cytokines have been shown to factor in the inflammatory manifestations of adult SLE. Six members of the IL-17 family have been identified so far, as well as IL-17 receptor subtypes. Although research on IL-17 family members is scarce, existing studies have implicated IL-17B, IL-17C and IL-17 receptor E (IL-17RE) in autoimmune pathology. Objective: To optimise the measurement of the IL-17 family (IL-17A, IL-17B, IL-17C and IL-17RE) and Th17-related cytokines (IL-21 and IL-23) in order to investigate their roles in the pathogenesis of JSLE. Methods: Peripheral blood components were isolated from JSLE, healthy paediatric control and juvenile idiopathic arthritis (JIA) patients. Spike-and-recovery validation experiments were conducted using JSLE and control serum and plasma in an IL-17A single plex and IL-17A, IL-21 and IL-23 ELISAs. mRNA expression of IL-17A was analysed by qPCR in CD4+ T cells from JSLE and control patients. Western blot analysis of IL-17A protein was optimised and measured in the PBMCs, CD4+ T cells and neutrophils of JSLE, control and JIA patients. JSLE and control PBMC mRNA expression of IL-17B, IL-17C and IL-17RE were quantified using qPCR, then mRNA expression of IL-17C and IL-17RE in CD3/CD28 stimulated JSLE and control PBMCs were analysed. Results: 102 patients (JSLE: n=31; healthy controls: n=66; JIA: n=4; lupus nephritis: n=1) were involved in this study, with multiple episode samples from JSLE and JIA patients. Poor recovery and matrix interference were detected in all IL-17A, IL-21 and IL-23 immunoassays. Protein IL-17A expression of neutrophils was significantly decreased in JSLE patients (p=0.03). IL-17A neutrophil expression was validated using purified neutrophils from healthy adult donors. IL-17B was not expressed in any samples. No significant differences were found between JSLE and control CD4+ cells or PBMCs at an mRNA and protein level for IL-17A, IL-17C or IL-17RE expression. IL-17RE expression was significantly decreased in stimulated JSLE PBMCs (p=0.007), with fold decreases in IL-17C and IL-17RE of JSLE relative to controls. Western blotting of IL-17C and IL-17RE protein expression was optimised in JSLE and control CD4+ T cells. Conclusions: Findings show IL-17A is not significantly increased in the peripheral blood of JSLE patients and that IL-17A is expressed by neutrophils. IL-17B is not expressed in human PBMCs while IL-17C and IL-17RE are, but are not significantly increased in JSLE PBMCs. Future immunoassays must be validated for recovery potential and existing data should be interpreted with caution. This study involved the validation and rigorous optimisation of novel methods investigating the IL-17 family and Th17-related cytokines, contributes significantly to limited research in the context of paediatric autoimmune disease, Th17 and the IL-17 family, and opens up new avenues for future directions in these fields.

Item Type: Thesis (Master of Philosophy)
Additional Information: Date: 2012-08 (completed)
Subjects: ?? RJ ??
Divisions: Faculty of Health and Life Sciences
Depositing User: Symplectic Admin
Date Deposited: 05 Sep 2013 10:51
Last Modified: 16 Dec 2022 04:38
DOI: 10.17638/00008733