Date of Award
PhD (Doctor of Philosophy)
Professor Caroline Jefferies
Science Foundation of Ireland who funded this study under Grant 08/IN.1/B2091.
Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterised by the presence of antibodies that recognise and target both protein and nucleic acid autoantigens. In addition, elevated levels of IL-23 and type I IFN are a major hallmark of this disease, playing critical roles in disease pathogenesis. TRIM21, a member of the tripartite motif containing (TRIM) E3 ligase family of proteins, is a well characterised SLE-associated autoantigen. Polymorphisms in the TRIM21 gene have been associated with SLE susceptibility suggesting it plays a critical role in disease pathogenesis. Studies into the function of TRIM21 have unveiled a dual function for this protein in regulating immune responses. Initially TRIM21 sustains interferon regulatory factor 3 (IRF3) levels, thus promoting interferon (IFN) and cytokine production, whereas following TLR7/9 stimulation, TRIM21 ubiquitinates IRF3 leading to its degradation via the proteasome, thus negatively regulating IFN and proinflammatory cytokine production. Following TRIM21 gene disruption, the enhanced stability of IRF3 observed has been associated with enhanced type I IFN and also IL-23pl9 levels, cytokines that play pivotal roles in SLE pathogenesis. We therefore investigated and identified a role for IRF3 in regulating IL-23pl9 production, thus explaining the association of TRIM21 with IL-23pl9 expression. Results demonstrate that loss of IRF3 leads to abrogated production of IL-23pl9 in response to TLR stimulation, thus indicating that IRF3 is a novel positive regulator of IL-23pl9 production. We have also shown that IRF3 is stably bound to the human IL-23pl9 promoter in monocytes, an association which increases with TLR3 stimulation. Patients with SLE show increased levels of IRF3 bound to the IL-23pl9 promoter both endogenously and following TLR3 activation compared with control subjects. Enhanced IRF3 binding was associated with increased IL-23pl9 production in monocytes from patients with SLE, thus providing novel insights into the molecular pathogenesis of SLE and the potential role for TLR3 in driving this response.
Despite the vast number of studies characterising the function of TRIM21, very little is known about the regulation of this protein particularly at a promoter level. Using Matlnspector, we identified a putative estrogen response element
within the TRIM21 gene promoter, which ChIP analysis proved to be functional. Estrogen was seen to upregulate TRIM21 expression through an ERa-dependent mechanism, a pathway which we observed to be overactive in SLE patients. Treatment of resting monocytes with an ERa antagonist abrogated estrogeninduced TRIM21 expression and as a consequence decreased IL-23 expression. Thus these findings identify TRIM21 as a novel ERa-regulated gene and provide further insights into the link between estrogen and the molecular pathogenesis of SLE.
In addition to promoter analysis, investigation into 3' untranslated region of TRIM21 led us to identify a number of putative microRNAs binding sites that hold potential in the regulation of TRIM21 expression. Investigation into one such microRNA, miR-381, revealed a novel role for this microRNA in the negative regulation of TRIM21. Patient analysis demonstrated that levels of miR-381 were altered in SLE immune cells leading to decreased TRIM21 expression following TLR7 activation. As TRIM21 in response to TLR stimulation, ubiquitinates and degrades both IRF3 and IRF7, the reduced levels of TRIM21 observed contributes to the overproduction of IL-23 observed in these patients thus highlighting the pathogenic role dysregulated miRNA expression plays in SLE. Taken together, this work has identified TRIM21 as a novel estrogen receptor regulated gene whose activity is altered in SLE leading to enhanced production of IL-23, a cytokine associated with the pathogenesis of SLE. Together with the identification of miR-381 as a novel regulator of TRIM21 expression, our findings have not only contributed to the understanding behind the molecular pathogenesis of SLE but also underlined the therapeutic potential in manipulating TRIM21 activity or levels for the treatment of SLE.
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Smith S. Investigation into the Role and Regulation of Tripartite Motif-containing protein 21 (TRIM21) in Systemic Lupus Erythematosus [PhD Thesis]. Dublin: Royal College of Surgeons in Ireland; 2013.