Boeltz, S

Gordon, P

Lehner, P

Lewis, M

Shields, A

Spector, T

Vyse, S

Vyse, T

Walczak, H

The Lancet

Queen Mary Research Online

Effect of UBE2L3 genotype on regulation of the linear ubiquitin chain assembly complex in systemic lupus erythematosusMyles Lewis, Simon Vyse, Adrian Shields, Sebastian Boeltz, Patrick Gordon, Timothy Spector, Paul Lehner, Henning Walczak, Timothy Vyse Centre for Experimental Medicine and Rheumatology, Queen Mary University of London, London, UK (M Lewis PhD, S Vyse MSc);  Department of Medical and Molecular Genetics, King's College Hospital, London, UK (A Shields PhD, S Boeltz MSc, T Vyse PhD);  Rheumatology Department, King's College Hospital, London, UK (P Gordon PhD); Department of Twin Research and Genetic Epidemiology, King's College London, London, UK (T Spector PhD); Cambridge Institute for Medical Research, University of Cambridge, Cambridge, UK (P Lehner PhD); and Centre for Cell Death, Cancer and Inflammation, University College London, London, UK (H Walczak PhD)Correspondence to: Dr Myles Lewis, Queen Mary University of London, Centre for Experimental Medicine and Rheumatology, Charterhouse Square, London EC1M 6BQ, UKmyles.lewis@qmul.ac.ukAbstract Background A single risk haplotype across UBE2L3 is strongly associated with systemic lupus erythematosus (SLE) and many other autoimmune diseases. UBE2L3 is an E2 ubiquitin-conjugating enzyme with specificity for RING-in-between-RING E3 ligases, including HOIL-1 and HOIP, components of the linear ubiquitin chain assembly complex (LUBAC), which has a pivotal role in inflammation, through crucial regulation of NF-κB. We aimed to determine whether UBE2L3 regulates LUBAC-mediated activation of NF-κB, and determine the effect of UBE2L3 genotype on NF-κB activation and B cell differentiation.Methods UBE2L3 genotype data from SLE genome-wide association studies was imputed by use of 1000 Genomes data. UBE2L3 function was studied in a HEK293-NF-κB reporter cell line with standard molecular biology techniques. p65 NF-κB translocation in ex- vivo B cells and monocytes from genotyped healthy individuals was quantified by imaging flow cytometry. B cell subsets from healthy individuals and patients with SLE, stratified by UBE2L3 genotype, were determined by multicolour flow cytometry.Findings  rs140490, located at –270 base pairs of the  UBE2L3 promoter, was identified as the most strongly associated single nucleotide polymorphism (p=8·6 x 10-14, odds ratio 1·30, 95%CI 1·21–1·39). The rs140490 risk allele increased  UBE2L3 expression in B cells and monocytes. Marked upregulation of NF-κB was observed with combined overexpression of  UBE2L3 and LUBAC, but abolished by dominant-negative mutant  UBE2L3[C86S], or  UBE2L3 silencing. The  rs140490 genotype correlated with basal NF-κB activation in ex-vivo human B cells and monocytes, as well as NF-κB sensitivity to CD40 or tumour necrosis factor (TNF) stimulation. UBE2L3 expression was 3–4 times higher in circulating plasmablasts and plasma cells than in other B cell subsets, with higher levels in patients with SLE than in controls. The rs140490 genotype correlated with increasing plasmablast and plasma cell differentiation in patients with SLE.Interpretation This study shows that NF-κB activation mediated by LUBAC is exquisitely sensitive to the expression level of UBE2L3. The UBE2L3 risk haplotype is correlated with TNF and CD40 induced NF-κB activation in primary human cells, and with plasmablast and plasma cell expansion in SLE, consistent with the dependence of these cells on NF-κB as a survival factor. Since UBE2L3 is highly expressed in plasma cells,  UBE2L3 could be a novel therapeutic target in SLE.Funding Arthritis Research UK, Wellcome Trust, George Koukis Foundation,  European Community's Seventh Framework Programme.Contributors MJL and TJV designed the study. SV, MJL, AMS, and SB performed the experiments and analysed data. HW and PJL provided expert assistance, reagents, and conceptual advice. MJL and PAG provided samples from patients with SLE. TDS provided twin samples and genotype data. MJL and TJV wrote the paper. All authors discussed the results and implications and commented on the abstract at all stages.Declaration of interests We declare no competing interests.

Effect of UBE2L3 genotype on regulation of the linear ubiquitin chain assembly complex in systemic lupus erythematosus

Effect of UBE2L3 genotype on regulation of the linear ubiquitin chain assembly complex in systemic lupus erythematosus

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