Document Type

Theses, Ph.D

Rights

Available under a Creative Commons Attribution Non-Commercial Share Alike 4.0 International Licence

Disciplines

1.6 BIOLOGICAL SCIENCES

Publication Details

Successfully submitted for the award of Doctor of Philosophy (Ph.D) to the Technological University Dublin, January, 2006.

Abstract

Members of the TNF superfamily play important roles in the development and maintenance of an effective immune response. One such member of this superfamily, LIGHT, can act as a ligand for three receptors, HVEM, LT-R and DcR3. The engagement of LIGHT and HVEM, an important secondary signal for the full activation of T cells, results in a strong Th1 type response with increased production of cytokines such as INF--. The LIGHT-LT-R pathway plays a role in the recruitment of immune cells to sites of inflammation and can induce apoptosis in certain cells. DcR3, a soluble receptor, is speculated to function in modulating LIGHT’s activity by preventing it from binding with HVEM and LT-R. Two studies published in 2001 highlighted the in vivo effects of constitutive expression of LIGHT on T cells in transgenic mice. The phenotype observed in these mice indicated that there was a loss of self-tolerance leading to systemic autoimmunity as characterised by the presence of multiple autoantibodies and inflammation of numerous organs. The aim of this thesis is to investigate the expression of LIGHT and associated receptors in systemic lupus erythematosus (SLE) and coeliac disease (CD). Based on the disease manifestations seen in LIGHT transgenic mice we speculated that patients with these conditions would have altered mRNA expression patterns of LIGHT or possibly its receptors. To achieve our aim, quantitative Real-time PCR was to be employed to perform a gene expression study using a control group of healthy volunteers as well as cohorts of CD and SLE patients. A cohort of Wegener’s granulomatosis (WG) patients was also included in this study. Real-time PCR has emerged as a powerful technique for quantifying the expression of genes. However, before embarking on a logistically complex and expensive gene expression study it was felt that gaining hands-on experience in developing Real-time PCR assays for simpler studies, where the end results could be predetermined, would yield benefits in the long-term. To this end, we chose two models to study various aspects of Real-time PCR assay design. II Cystic fibrosis is the most common lethal recessive genetic disease in Caucasian populations and thus serves as a good model for developing a Real-time PCR assay for mutation detection. After identifying insufficiencies with how mutation detection is traditionally performed, we developed a novel approach on the LightCycler instrument by combining ARMS PCR with melting curve analysis. This approach allowed better design of hybridisation probes and facilitated more than one mutation to be detected per fluorescent colour channel on the LightCycler. Our optimised assays allow the detection of the five most common mutations, accounting for 90% of mutant alleles, in approximately 35 mins, which is significantly quicker than other traditional techniques. As a model for examining the capabilities of quantitative Real-time PCR, we examined the gene dosage of peripheral myelin protein 22 (PMP22). Alterations in gene copy number of PMP22 can result in two distinct neurological diseases Charcot-Marie-Tooth disease type 1A (CMT1A) and hereditary neuropathy with liability to pressure palsies (HNPP), respectively caused by a gain or loss of a copy of the PMP22 gene. In this study, we successfully developed a quantitative multiplex Real-time PCR that can diagnose CMT1A and HNPP patients with altered PMP22 gene copy numbers. Our assay provides a rapid alternative to traditional techniques used for gene dosage quantification and could be adapted for use in the diagnosis of many genetic diseases. Having gained knowledge in the use of Real-time PCR we proceeded to performing the gene expression study. During this study we have provided strong evidence that LIGHT and its associated receptors may play an important role in the pathogenesis of both SLE and CD and less so in WG. Our results demonstrated that LIGHT is elevated in both peripheral blood and the small intestine of patients with active CD. In SLE, there is a more profound dysregulation of LIGHT and associated receptors. Elevated levels of LIGHT and its three receptors, HVEM, LT-R and DcR3, were identified in the peripheral blood of our SLE patients. The WG and control cohorts showed similar levels of expression for LIGHT and its receptor indicating that these signalling pathways are not involved in its pathogenesis. III Studies we performed using P/I activated Jurkat cells demonstrated that there is good correlation between LIGHT mRNA upregulation and the appearance of soluble LIGHT protein in tissue culture supernatants. The analysis of soluble LIGHT levels using ELISA demonstrated that there was a significant elevation of soluble LIGHT in the SLE cohort also a large percentage of the CD patients were strongly positive. The WG patients showed a significant reduction in soluble LIGHT compared to the controls, which provides further proof that it is not involved in its pathogenesis. Overall, our research shows that LIGHT is upregulated in both CD and SLE and given its function in promoting a strong Th1 response it is likely to contribute in the pathogenesis of both diseases. We speculate that increased LIGHT activity in SLE may promote the development of glomerulonephritis one of the more serious clinical manifestations of the disease, which can ultimately lead to renal failure and death. CD patients have a high incidence of developing secondary autoimmune disease. We hypothesize that in addition to its immediate effects in CD pathogenesis, sustained levels of soluble LIGHT may also contribute to a breakdown of selftolerance and lead to autoimmune disease development. Further research into the expression and function of LIGHT will lead to a better understanding of the mechanisms causing these diseases. In the future, this should lead to the development of new therapies for these and related diseases.

DOI

https://doi.org/10.21427/D7KK6M

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