Session Type: ACR Poster Session A
Session Time: 9:00AM-11:00AM
A single risk haplotype spanning UBE2L3 is strongly associated with SLE and multiple autoimmune diseases. The E2 ubiquitin-conjugating enzyme UBE2L3 regulates TNFα and CD40 induced NF-kB activation via regulation of the Linear Ubiquitination Chain Assembly Complex (LUBAC). We have shown that the UBE2L3 risk allele correlates with plasmablast and plasma cell expansion in SLE individuals by regulating NF-κB activation in B cells. Dimethyl Fumarate (DMF), a current oral therapy for multiple sclerosis and psoriasis, has been recently identified as a UBE2L3 inhibitor. Given the known role of excessive TLR7 signalling in SLE, we aimed to determine the effect of UBE2L3 and linear ubiquitination on TLR7 signalling and to explore whether UBE2L3-mediated plasmablast differentiation can be inhibited by DMF.
Transient overexpression and shRNA silencing were used in TLR7-HEK293 NF-kB reporter cell line to investigate the effect of UBE2L3/LUBAC on NF-kB activation, gene expression by qPCR and IL-8 secretion by ELISA. Western blot was used to measure linear ubiquitin chain polymerisation, confirmed by confocal microscopy. DMF was administered to CD19+ peripheral blood human B cells cultured in the presence of resiquimod, IFNα, or both for up to 7 days. B cell viability, activation, differentiation and proliferation were analysed by flow cytometry. Immunoglobulin secretion was quantified by ELISA and ANA detected by Hep2 immunofluorescence assay.
Overexpression of catalytically active LUBAC components increased TLR7-induced NF-kB activation by reporter assay. NF-kB activation, NF-κB target gene expression by qPCR and IL-8 secretion were further enhanced by co-overexpression of UBE2L3 with LUBAC, basally and after TLR7 stimulation, but was abolished by dominant negative mutant UBE2L3(C86S) or HOIP(C885S), and in UBE2L3 silenced cells. TLR7 engagement triggered accumulation of linear ubiquitin chains comparable to TNFα. DMF exerted a dose-dependent suppression of NF-kB activation following TLR7 engagement. DMF reduced human primary B cell survival, and suppressed proliferation of switched and unswitched CD27+ memory B cells. DMF inhibited TLR7 stimulated immunoglobulin secretion by in vitro differentiated B cells. Co-stimulation of B cells with TLR7 ligand resiquimod and IFNa induced anti-nuclear autoantibody production which was suppressed by DMF.
Our results establish that linear ubiquitination and UBE2L3 regulate TLR7 activation of NF-κB. Silencing UBE2L3/LUBAC, use of dominant negative mutant UBE2L3 or HOIP, or use of the UBE2L3 inhibitor DMF suppressed TLR7 driven NF-κB activation. Excessive TLR7 signalling has been linked to SLE development by impaired checkpoint permissiveness in autoreactive B cells, leading to autoantibody production in the context of type I interferon. DMF demonstrated potent suppression of CD27+ memory B cell differentiation and inhibited TLR7 and IFNa induced autoantibody production. These results suggest that there may be a role for repositioning DMF in the treatment of SLE and autoantibody driven autoimmune diseases.
To cite this abstract in AMA style:Mauro D, Tsang V, Clayton-Lucey IA, Pagani S, Alam F, Pontarini E, Nerviani A, Pakozdi A, Cove-Smith A, Rajakariar R, Pyne D, Vyse TJ, Pitzalis C, Lewis MJ. Regulation of TLR7 Signalling By UBE2L3 Dependent Linear Ubiquitination Explains Dimethyl Fumarate Suppression of Autoreactive B Cell Development in SLE [abstract]. Arthritis Rheumatol. 2017; 69 (suppl 10). https://acrabstracts.org/abstract/regulation-of-tlr7-signalling-by-ube2l3-dependent-linear-ubiquitination-explains-dimethyl-fumarate-suppression-of-autoreactive-b-cell-development-in-sle/. Accessed September 22, 2023.
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ACR Meeting Abstracts - https://acrabstracts.org/abstract/regulation-of-tlr7-signalling-by-ube2l3-dependent-linear-ubiquitination-explains-dimethyl-fumarate-suppression-of-autoreactive-b-cell-development-in-sle/