Patrick Gordon

UBE2L3 Polymorphism Amplifies NF-κB Activation and Promotes Plasma Cell Development, Linking Linear Ubiquitination to Multiple Autoimmune Diseases

UBE2L3 is associated with increased susceptibility to numerous autoimmune diseases, but the underlying mechanism is unexplained. By using data from a genome-wide association study of systemic lupus erythematosus (SLE), we observed a single risk haplotype spanning UBE2L3, consistently aligned across multiple autoimmune diseases, associated with increased UBE2L3 expression in B cells and monocytes. rs140490 in the UBE2L3 promoter region showed the strongest association. UBE2L3 is an E2 ubiquitin-conjugating enzyme, specially adapted to function with HECT and RING-in-between-RING (RBR) E3 ligases, including HOIL-1 and HOIP, components of the linear ubiquitin chain assembly complex (LUBAC). Our data demonstrate that UBE2L3 is the preferred E2 conjugating enzyme for LUBAC in vivo, and UBE2L3 is essential for LUBAC-mediated activation of NF-κB. By accurately quantifying NF-κB translocation in primary human cells from healthy individuals stratified by rs140490 genotype, we observed that the autoimmune disease risk UBE2L3 genotype was correlated with basal NF-κB activation in unstimulated B cells and monocytes and regulated the sensitivity of NF-κB to CD40 stimulation in B cells and TNF stimulation in monocytes. The UBE2L3 risk allele correlated with increased circulating plasmablast and plasma cell numbers in SLE individuals, consistent with substantially elevated UBE2L3 protein levels in plasmablasts and plasma cells. These results identify key immunological consequences of the UBE2L3 autoimmune risk haplotype and highlight an important role for UBE2L3 in plasmablast and plasma cell development.

Soft tissue and subcutaneous calcification in connective tissue diseases

Purpose of reviewCalcinosis is a recognized manifestation of many connective-tissue diseases, especially juvenile dermatomyositis (JDM) and systemic sclerosis; however, little is known about the pathogenesis and the treatment of this condition. The purpose of this review is to discuss the most recently published data about calcinosis in connective-tissue diseases with emphasis on the pathogenesis and its treatment. Recent findingsCalcinosis is more common in patients with sustained disease-activity and longer disease duration. JDM patients with anti-p140 antibodies and tumour necrosis factor (TNF) &agr;-308AA allele are at an increased risk. Low levels of the calcium-regulating proteins, fetuin-A and osteopontin, have been found in the serum of patients with JDM. Macrophages and cytokines interleukin (IL) 6, IL-1&bgr; and TNF&agr; isolated from the calcific tissues in JDM have also been implicated in the pathologic process. Raised tissue expression of advanced glycation end products and their receptor has been noted in patients with systemic sclerosis and systemic lupus erythematosus with calcinosis. SummaryMany agents have been used for treatment of calcinosis but none has been accepted as a standard therapy. Case studies have shown that aggressive treatment of the underling inflammatory condition with intravenous immunoglobulin, anti TNF agents, thalidomide and haematopoietic stem cell transplantation has also led to improvement of the calcinosis. Aggressive management of the underlying inflammatory condition should help in treating as well as decreasing the incidence of calcinosis. Some case studies have focused on agents such as warfarin, bisphosphonates, diltiazem and others, which are primarily aimed at treating the process of calcinosis with varying success.

Atherosclerosis and autoimmunity

Novel risk factors for the progression of atherosclerosis such as C-reactive protein (CRP) and adhesion molecules have stimulated much recent interest in the role of inflammation in athero-sclerotic disease. There is also evidence emerging that autoimmunity may have a role in the pathogenesis of atherosclerosis. In this article we explore the evidence for the role of autoimmunity in human atherosclerosis, both in the general population and in the context of the antiphospholipid syndrome. In particular we will focus on several autoantigens, review the evidence for their role in the process of atherosclerosis and the nature of the immune responses.

2017 European League Against Rheumatism/American College of Rheumatology classification criteria for adult and juvenile idiopathic inflammatory myopathies and their major subgroups

Objective To develop and validate new classification criteria for adult and juvenile idiopathic inflammatory myopathies (IIM) and their major subgroups. Methods Candidate variables were assembled from published criteria and expert opinion using consensus methodology. Data were collected from 47 rheumatology, dermatology, neurology and paediatric clinics worldwide. Several statistical methods were used to derive the classification criteria. Results Based on data from 976 IIM patients (74% adults; 26% children) and 624 non-IIM patients with mimicking conditions (82% adults; 18% children), new criteria were derived. Each item is assigned a weighted score. The total score corresponds to a probability of having IIM. Subclassification is performed using a classification tree. A probability cut-off of 55%, corresponding to a score of 5.5 (6.7 with muscle biopsy) ‘probable IIM’, had best sensitivity/specificity (87%/82% without biopsies, 93%/88% with biopsies) and is recommended as a minimum to classify a patient as having IIM. A probability of ≥90%, corresponding to a score of ≥7.5 (≥8.7 with muscle biopsy), corresponds to ‘definite IIM’. A probability of <50%, corresponding to a score of <5.3 (<6.5 with muscle biopsy), rules out IIM, leaving a probability of ≥50 to <55% as ‘possible IIM’. Conclusions The European League Against Rheumatism/American College of Rheumatology (EULAR/ACR) classification criteria for IIM have been endorsed by international rheumatology, dermatology, neurology and paediatric groups. They employ easily accessible and operationally defined elements, and have been partially validated. They allow classification of ‘definite’, ‘probable’ and ‘possible’ IIM, in addition to the major subgroups of IIM, including juvenile IIM. They generally perform better than existing criteria.

T-regs in autoimmune hepatitis-systemic lupus erythematosus/mixed connective tissue disease overlap syndrome are functionally defective and display a Th1 cytokine profile

Autoimmune hepatitis (AIH), a severe hepatopathy characterized by hypergammaglobulinaemia, autoantibodies and interface hepatitis, is occasionally associated with systemic autoimmune manifestations [systemic lupus erythematosus (SLE); mixed connective tissue disease (MCTD)]. In both AIH and SLE/MCTD numerical and/or functional impairment of regulatory T-cells (T-regs) is believed to favour autoimmunity. To investigate whether immune-tolerance breakdown profiles differ in patients with AIH and SLE/MCTD, isolated AIH or systemic autoimmunity, we studied phenotypic and functional features of T-regs in 10 patients with AIH-SLE/MCTD, 22 with AIH, 12 with SLE and 20 healthy subjects. Compared to health, CD4(pos)CD25(pos) cells were decreased in number and expressed high levels of the CD127 activation marker in all three disease groups; in AIH-SLE/MCTD and in SLE they displayed low levels of FOXP3. In AIH-SLE/MCTD, they also contained a high proportion of IFNγ positive cells, indicating a Th1 profile. Similarly, in AIH-SLE/MCTD, CD4(pos)CD25(pos)CD25(high) T-regs were reduced in number and contained an increased proportion of activated CD127(pos) and IFNγ(pos) cells. Such skewing towards a Th1 profile was also present at effector level, as a high frequency of IFNγ-producing cells was observed within AIH-SLE/MCTD CD4(pos)CD25(neg) responder cells. Impairment in suppressor function both of CD4(pos)CD25(pos) cells and CD4(pos)CD25(pos)CD127(neg) T-regs was observed in all three autoimmune conditions, but while addition of CD4(pos)CD25(pos)CD127(neg) T-regs decreased CD4(pos)CD25(neg) responder cell proliferation in healthy subjects and partially in AIH patients, it had no effect in AIH-SLE/MCTD and SLE patients. In conclusion, in AIH-SLE/MCTD T-regs display a distinctive phenotypic and functional signature, characterized by marked activation, elevated IFNγ production and by a profound impairment of suppressive function, suggesting that multiple autoimmune manifestations may derive from a complex defect of immune-regulation.

Rituximab in the treatment of inflammatory myopathies: a review.

Several uncontrolled studies have encouraged the use of rituximab (RTX) in patients with myositis. Unfortunately, the first placebo-phase trial to assess the efficacy of RTX in refractory myositis did not show a significant difference between the two treatment groups, and doubts have been expressed about its study design. In this review we present an up-to-date overview of the reported experiences of RTX therapy in myositis. A PubMed search was performed to find all the available cases of refractory myositis patients treated with RTX up to July 2015. The following terms were assessed: inflammatory myopathies OR anti-synthetase syndrome OR polymyositis OR dermatomyositis AND RTX. A total of 48 studies were included. We identified 458 patients with myositis treated with RTX. We found a rate of response to RTX of 78.3%. RTX can play a role in the management of patients with myositis, at least in those with positive myositis-specific autoantibodies.

Second-line agents in myositis: 1-year factorial trial of additional immunosuppression in patients who have partially responded to steroids

Objective. Ciclosporin and MTX are used in idiopathic inflammatory myopathies (DM and PM) when patients incompletely respond to glucocorticoids. Their effectiveness is unproved in randomized controlled trials (RCTs). We evaluated their benefits in a placebo-controlled factorial RCT. Methods. A 56-week multicentre factorial-design double-blind placebo-controlled RCT compared steroids alone, MTX (15–25 mg weekly) plus steroids, ciclosporin (1–5 mg/kg/day) plus steroids and all three treatments. It enrolled adults with myositis (by Bohan and Peter criteria) with active disease receiving corticosteroids. Results. A total of 359 patients were screened and 58 randomized. Of the latter, 37 patients completed 12 months of treatment, 7 were lost to follow-up and 14 discontinued treatment. Patients completing 12 months of treatment showed significant improvement (P < 0.001 on paired t-tests) in manual muscle testing (14% change), walking time (22% change) and function (9% change). Intention to treat and completer analyses indicated that ciclosporin monotherapy, MTX monotherapy and ciclosporin/MTX combination therapy showed no significant treatment effects in comparison with placebo. Conclusion. Neither MTX nor ciclosporin (by themselves or in combination) improved clinical features in myositis patients who had incompletely responded to glucocorticoids. Trial Registration: International Standard Randomized Controlled Trial Number Register; http://www.controlled-trials.com/; ISRCTN40085050

Improved monitoring of clinical response in Systemic Lupus Erythematosus by longitudinal trend in soluble vascular cell adhesion molecule-1

BackgroundTo determine whether optimal use of serial measurements of serum levels of soluble cell adhesion molecules (CAM) can improve monitoring of disease activity in SLE.MethodsSerum levels of soluble CAM and conventional SLE biomarkers were measured in serial samples (n = 80) from 21 SLE patients during and after flare and correlated in longitudinal analysis with disease activity determined by ECLAM score. Blood samples from a second cohort of 34 SLE patients were subject to flow cytometry to correlate serum biomarkers with B cell subsets.ResultsBy adjusting for the baseline level (at the first visit), delta soluble vascular cell adhesion molecule-1 (sVCAM-1) showed stronger correlation with changes in ECLAM score and improved sensitivity and specificity for identifying SLE responders versus non-responders compared to conventional SLE biomarkers including anti-dsDNA antibody titre and complement C3. Multiple regression analysis identified delta sVCAM-1 as the best marker of SLE clinical response. sVCAM-1 levels were significantly correlated with CD95+CD27+ activated memory B cells, CD95+ plasmablasts and circulating plasma cell numbers in SLE patients.ConclusionSubtracting a baseline level of sVCAM-1 for each individual substantially improved its utility as a biomarker. Delta sVCAM-1 was superior to conventional SLE biomarkers for monitoring changes in disease activity. This suggests that serial monitoring of serum sVCAM-1 trends should be considered in SLE patients to document responses to treatment. We hypothesise that the correlation between activated B cell subsets and circulating plasma cell numbers with soluble VCAM-1 serum levels in SLE may relate to the important role of VCAM-1 in B lymphocyte survival and maturation in bone marrow and secondary lymphoid tissues.

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

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 × 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 Communitys Seventh Framework Programme.

Systemic lupus erythematosus in pregnancy

When a woman is known to have systemic lupus erythematosis, planning and preparation can improve the outcome of pregnancy and reduce the severity of some of the potential problems. All treatment options have risks but these risks have to be balanced against the risks of no treatment or, ‘too late’ treatment. In pregnancy, close fetal surveillance is important. Once there are definite signs of fetal harm this cannot always be corrected and late management decisions can only institute rescue. Urgent premature delivery is a frequent phenomenon. Proactive medical care of the mother, together with accurate information about the state of the baby, may reduce the frequency of sudden or early delivery thereby assisting neonatal management. Currently, some risks such as neonatal lupus with congenital heart block, retain a high mortality. The construction of an agreed detailed prospective management plan, understood by all those involved, can lead to increased satisfaction with the care process and a better outcome.When a woman is known to have systemic lupus erythematosis, planning and preparation can improve the outcome of pregnancy and reduce the severity of some of the potential problems. All treatment options have risks but these risks have to be balanced against the risks of no treatment or, ‘too late’ treatment. In pregnancy, close fetal surveillance is important. Once there are definite signs of fetal harm this cannot always be corrected and late management decisions can only institute rescue. Urgent premature delivery is a frequent phenomenon. Proactive medical care of the mother, together with accurate information about the state of the baby, may reduce the frequency of sudden or early delivery thereby assisting neonatal management. Currently, some risks such as neonatal lupus with congenital heart block, retain a high mortality. The construction of an agreed detailed prospective management plan, understood by all those involved, can lead to increased satisfaction with the care process and a better outcome.When a woman is known to have systemic lupus erythematosis, planning and preparation can improve the outcome of pregnancy and reduce the severity of some of the potential problems. All treatment options have risks but these risks have to be balanced against the risks of no treatment or, ‘too late’ treatment. In pregnancy, close fetal surveillance is important. Once there are definite signs of fetal harm this cannot always be corrected and late management decisions can only institute rescue. Urgent premature delivery is a frequent phenomenon. Proactive medical care of the mother, together with accurate information about the state of the baby, may reduce the frequency of sudden or early delivery thereby assisting neonatal management. Currently, some risks such as neonatal lupus with congenital heart block, retain a high mortality. The construction of an agreed detailed prospective management plan, understood by all those involved, can lead to increased satisfaction with the care process and a better outcome.