FRI0271 Dimethyl fumarate inhibits ube2l3 mediated tlr7 signalling and autoreactive b cell development in sle. (12th June 2018)

• Record Type: Journal Article
• Authors: Mauro, D.; Tsang, V.; Lucey-Clayton, I.; Rivellese, F.; Pagani, S.; Alam, F.; Pontarini, E.; Nerviani, A.; Pakozdi, A.; Rajakariah, R.; Pyne, D.; Vyse, T.J.; Pitzalis, C.; Lewis, M.J.
• Is Part Of: Annals of the rheumatic diseases. Volume 77(2018)Supplement 2
• Page Start: 674
• Page End: 674
• Publication Date: 2018-06-12
• Subjects: Rheumatism -- Periodicals; 616.723005
• DOI: 10.1136/annrheumdis-2018-eular.6960
• Languages: English
• ISSNs: 0003-4967

Abstract

Abstract : Background: Genetic studies have identified a single UBE2L3 risk haplotype which is associated with SLE and multiple autoimmune diseases, and leads to increased expression of UBE2L3 in eQTL studies. The E2 ubiquitin-conjugating enzyme UBE2L3 regulates NF-κB activation through regulation of the Linear Ubiquitination Chain Assembly Complex (LUBAC). Thus UBE2L3 regulates CD40-driven B cell activation. The UBE2L3 risk allele correlates with circulating plasmablast and plasma cell expansion in SLE individuals. Objectives: To determine the effect of UBE2L3 and linear ubiquitination on TLR7 signalling, and test the effect of Dimethyl Fumarate (DMF), which has recently been shown to inhibit UBE2L3, on B cell and plasmablast differentiation in SLE. Methods: Confocal microscopy, immunoprecipitation and western blot were used to assess linear ubiquitin chain accumulation in TLR7-HEK293 cells after TLR stimulation by Resiquimod. The effect of UBE2L3 and LUBAC on TLR7 signalling was dissected by the use pf UBE2L3/LUBAC overexpression, dominant negative mutants and shRNA silencing, measuring NF-kB reporter activity, IkappaBalpha phosphorylation, gene expression by qPCR and IL-8 secretion. DMF was administered in vitro to human B cells isolated from SLE patients (n=15) and controls and cultured with Resiquimod and/or IFNα for 5–7 days. B cell viability/proliferation, plasmablast differentiation were analysed by 10-colour flow cytometry. Supernatants were assayed for immunoglobulin secretion and autoantibody production. Results: TLR7 stimulation led to intracellular accumulation of linear ubiquitin chain comparable to TNFα. UBE2L3 and LUBAC co-overexpression enhanced TLR7 driven NF-kB activation and led to increased NF-κB target mRNA expression and increased secretion of IL-8. The effect was specific to UBE2L3 compared to other E2 enzymes. Dominant negative mutant UBE2L3(C86S) or HOIP(C885S) or UBE2L3/HOIP shRNA suppressed the response to TLR7 stimulation. DMF showed a dose-dependent inhibition of TLR7-mediated NF-kB activation. In primary SLE and healthy B cells, DMF suppressed proliferation of switched and unswitched CD27 +memory B cells and blocked plasmablast differentiation. DMF profoundly inhibited immunoglobulin secretion and anti-nuclear autoantibodies production in response to TLR7 and IFNa stimulus. Conclusions: Our data demonstrate that linear ubiquitination and UBE2L3 regulate TLR7 activation of NF-κB. UBE2L3 silencing or pharmacological antagonism of UBE2L3 by DMF suppressed the response to TLR7 activation. Excessive TLR7 signalling has been linked to SLE development and enhanced B cell autoreactivity. Thus our data identify a novel mechanism by which the UBE2L3 risk haplotype contributes to SLE susceptibility. DMF suppressed plasmablast differentiation and inhibited TLR7 and IFNa induced autoantibody production. These results support a role for repositioning DMF (currently used to treat multiple sclerosis) in the treatment of SLE. Disclosure of Interest: None declared