Background and objectives Genome-wide association studies have identified a strong association between a single risk haplotype of the UBE2L3 gene and systemic lupus erythematosus (SLE). UBE2L3 is an E2 ubiquitin-conjugating enzyme that critically regulates ubiquitination by the linear ubiquitin chain assembly complex (LUBAC) with consequent effects on NF-kB signalling and inflammatory responses. Herein, we dissect the role of UBE2L3 in regulating NF-kB signalling in human B-cells and monocytes and demonstrate the risk haplotype drives plasmablast and plasma cell expansion in patients with SLE.
Materials and methods UBE2L3 genotype data from GWAS in SLE was imputed using 1000 Genomes reference data. UBE2L3 function and signalling pathways were studied in vitro in HEK293 cells, or ex vivo using B cells and monocytes from healthy individuals or SLE patients (NF-kB translocation by Imagestream, multicolour flow cytometry of B cell subsets) stratified by UBE2L3 genotype. Parameters of SLE disease activity were collected and correlated with flow cytometry results.
Results Data from SLE GWAS, imputed to 1000 Genomes level identified rs140490 as the most strongly associated UBE2L3 SNP, located at -270bp of the promoter region (p = 8.6 × 10–14; OR 1.30, 95% CI: 1.21–1.39). Microarray/western blot studies found that the rs140490 risk allele correlated with increased UBE2L3 expression in human B cells and monocytes. Overexpression of UBE2L3 in combination with LUBAC in HEK293-NF-kB reporter cell lines led to marked upregulation of NF-kB activity, which was abolished by a dominant-negative mutant UBE2L3[C86S]. RNAi blockade of UBE2L3 antagonised TNF signalling by inhibiting IκBα processing. UBE2L3 expression was 3–4-fold elevated in peripheral blood plasmablasts and plasma cells (p < 0.0001), with increased UBE2L3 expression in plasma cells from SLE patients compared to controls (p = 0.01). The T/T genotype at rs140490 was associated with a significant expansion in plasmablast and plasma cell populations in SLE patients (both p < 0.001), and showed a trend to correlation with increased SLEDAI scores (p = 0.06). Imagestream analysis demonstrated that rs140490 genotype correlated with both basal NF-kB activation in healthy individuals, as well as the sensitivity of NF-kB to CD40 stimulation in B cells and TNF stimulation in monocytes.
Conclusions The UBE2L3 risk haplotype exerts a critical rate-limiting effect on TNF and CD40 signalling through regulation of LUBAC and NF-kB activation. This is the first demonstration that a complex trait variant at UBE2L3 regulates both basal NF-kB activation and sensitivity of NF-kB to stimulation in ex vivo human cells, resulting in accelerated B cell differentiation in SLE.