How to translate genetic findings into clinical applications in spondyloarthritis?

Spondyloarthritis (SpA) is characterized by a strong genetic predisposition evidenced by the identification of up to 50 susceptibility loci, in addition to HLA-B27, the major genetic factor associated with the disease. These loci have not only deepened our understanding of disease pathogenesis but also offer the potential to improve disease management. Diagnostic delay is a major issue in SpA. HLA-B27 testing is widely used as diagnostic biomarker in SpA but its predictive value is limited. Several attempts have been made to develop more sophisticated polygenic risk score (PRS). However, these scores currently offer very little improvement as compared to HLA-B27 and are still difficult to implement in clinical routine. Genetics might also help to predict disease outcome including treatment response. Several genetic variants have been reported to be associated with radiographic damage or with poor response to TNF blockers, unfortunately with lack of coherence across studies. Large-scale studies should be conducted to obtain more robust findings. Genetic and genomic evidence in complex diseases can be further used to support the identification of new drug targets and to repurpose existing drugs. Although not fully driven by genetics, development of IL-17 blockers has been facilitated by the discovery of the association between IL23R variants and SpA. Development of recent approaches combining GWAS findings with functional genomics will help to prioritize new drug targets in the future. Although very promising, translational genetics in SpA remains challenging and will require a multidisciplinary approach that integrates genetics, genomics, immunology, and clinical research

Genetics and Functional Genomics of Spondyloarthritis

Spondyloarthritis (SpA) is a chronic inflammatory disorder with high heritability but with complex genetics. It encompasses several entities that share common clinical features. Most of the genetic studies in SpA have been restricted to ankylosing spondylitis (AS), the prototypical form of SpA. However, there is growing evidence of shared genetic background between all the SpA subtypes and also with some other immune-mediated diseases. The most important part of SpA heritability comes from the HLA-B27 allele in the major histocompatibility complex (MHC) that explains around 25% of the attributable heredity. Several other loci outside of the MHC have been shown to be involved in the disease. However, all these non-MHC loci explain only a small additional fraction of disease predisposition. Thus, a substantial fraction of SpA genetic basis remains poorly understood. Gene expression profiling is a complementary approach to elucidate the underlying mechanisms and pathways that drive the disease. Several expression profiling studies have been undertaken in SpA. However, results have been quite disappointing with little overlap between the studies largely due to the small sample sizes, resulting in limited power to discover small effects. In this review, we summarize current knowledge on genetic findings concerning SpA and we describe strategic approaches for identification of additional variants, with a focus on rare variants in familial forms. We also provide an overview of gene expression studies in SpA and discuss the possibilities offered by high-throughput RNA sequencing technologies, in particular in sorted cells. Finally, issues in establishing molecular mechanisms underlying genetic association hits and potential translational applications will be addressed

Evidence for Altered Cytoskeleton Mobilization Pathway in Splenic Dendritic Cells (DC) from HLA-B27/human b2 microglobulin Transgenic Rats (B27-rats)

Background: Although the association of the MHC class I allele HLA-B27 with Spondyloarthropathy (SpA) has been known for almost 35 years different hypotheses on its relation to disease mechanism still exist in parallel. Several lines of rats transgenic for HLA-B27 and human β2-microglobulin develop an inflammatory disease that strikingly resembles human SpA. It is hypothesized that disease in HLA-B27-transgenic rats arises as a consequence of interaction between antigen-presenting cells expressing high levels of HLA-B27 and peripheral T lymphocytes, and may result from a rupture of tolerance towards gut bacteria. Methods: We used 2D PAGE and iTRAQ to compare the protein expression profile of HLA-B27 dendritic cells (DCs) to that of healthy HLA-B7 expressing and nontransgenic (NTG) rat DCs. MHC II surface expression and apoptotic sensitivity were quantified using flow cytometry. Results: Three protein sets from the proteome analysis were indicative for aberrant cellular processes. First, all proteins involved in protein processing and MHC I assembly were upregulated in B27 DCs, illustrating the higher pressure on the ER due to misfolding of the HLA-B27 heavy chain. Second, all proteins directly influencing actin-dynamics were downregulated. We showed earlier that this not only influences motility, but also plays an important role in deficient immunological synapse formation. Third, the key thiol protease Cathepsin S involved in MHC II synthesis was downregulated, which led us to quantify RT1-B and RT1-D surface expression. Downregulation concerned both CD4+ and CD4- OX62+ HLA-B27 DC subpopulations and maturation enlarged differences in both population bias and expression intensity. Deficient actin dynamics could also contribute to this lower MHC II surface expression. Study of sensitivity to MHC class II-mediated apoptosis by antibody stimulation showed that compared to NTG, both B7 and B27 CD4+ DC were more prone to apoptosis but did not mutually differ. In contrast, overnight culturing resulted in a higher cell death in B27 than in control CD4- DC, even without antibody stimulation. Interestingly, decreased actin dynamics could also be involved in DC apoptosis. Conclusions: We have demonstrated that DCs are a very vulnerable cell type in HLA-B27 rats. Deficient cytoskeletal dynamics could immobilize matured DC in the tissue or induce aberrant migration patterns upon activation. On top of that abnormal intracellular trafficking and membrane organization together with a reduced expression of MHC class II molecules makes them aberrant in T-cell communication by deficient immunological synapse formation. Especially the reduced motility and viability of the tolerigenic CD4- DC could play an important role in initiating a systemic auto-immune response

Complexe Majeur d'Histocompatibilité et génomique fonctionnelle dans les spondylarthrites

La SpA est un rhumatisme inflammatoire chronique fréquent, dont la prévalence est de 0,3% en France. Les mécanismes pathologiques qui en sont à l origine demeurent largement incertains. Néanmoins, l héritabilité de la maladie est élevée, impliquant de multiples facteurs génétiques, dont la région du complexe majeur d histocompatibilité (CMH) et plus particulièrement l allèle HLA-B27 qui y exerce un rôle prédominant. L objectif de ce travail était d identifier de nouvelles cibles moléculaires, en vue d améliorer la compréhension de la physiopathologie de la SpA, par des approches génétiques et de génomiques fonctionnelles.La première partie de mon travail a consisté en l identification de polymorphismes du CMH associés à la SpA et distinct de HLA-B27. Les études d association portant sur les données génétiques de 3 cohortes indépendantes nous ont permis d identifié 5 variants associés à la SpA indépendamment du HLA-B27. Les deux polymorphismes situés à proximité des gènes MICA et MAPK14 semblent particulièrement intéressants pour leur implication potentielle dans la pathogénèse de la SpA. En marge de cette étude, nous avons entrepris de déterminer la prévalence du HLA-B27 dans une cohorte française représentative de la population générale, qui était de 6,9% chez les témoins et de 74,2% chez les sujets atteints de SpA.Les études fonctionnelles conduites sur des cellules dendritiques dérivées de monocytes (MD-DCs) ont permis d identifier un défaut de réponse proliférative des LT CD4+ stimulés par les MD-DCs de patients atteints de SpA, ainsi qu une signature transcriptomique de 81 gènes caractéristique des MD-DCs de ces patients. Parmi les gènes validés, la surexpression d ADAMTS15, de F13A1 et de SELL pourrait jouer un rôle dans l inflammation liée à la pathologie, alors que la sous-régulation de CITED2 paraitrait corrélée à une dérégulation de la voie Wnt. Enfin, nos investigations sur les MD-DCS nous ont amené à identifier une corrélation entre l haplotype d ERAP1 prédisposant à la SpA, et un niveau accru d expression de ce gène ainsi que de la protéine ERAP1.Spondyloarthritis (SpA) is a frequent chronic inflammatory rheumatic disorder, with a prevalence of 0.3% in France. Pathological mechanisms leading to SpA remain largely uncertain. Nevertheless, the heritability of this disorder is high, likely involving multiple genetic factors, among which the major histocompatibility complex (MHC) region and particularly the HLA-B27 allele which plays a prominent role. The objective of this work was to achieve a better understanding of SpA physiopathology via genetic and transcriptomic approaches. The first part of my work consisted in identification of MHC polymorphisms associated with SpA, distinct of HLA-B27. Association studies based on the genetic data of 3 independent cohorts have allowed to identify 5 SNPs associated to SpA, independently of HLA-B27. Two polymorphisms localized next to MICA and MAPK14 genes seem particularly interesting for their implication in SpA pathogenesis. In parallel of this study, we characterized HLA-B27 prevalence in a French cohort corresponding to 6.9% in healthy controls and 74.2% in SpA patients. Functional studies on monocyte-derived dendritic cells (MD-DCs) revealed altered capacity to stimulate allogeneic CD4+ T cell responses by MD-DCs from SpA patients and a transcriptomic signature of 81 genes differentially expressed in those cells, as compared to those from healthy controls. Among validated genes, ADAMTS15, F13A1 and SELL could play a role in SpA inflammation, whereas CITED2 seemed to be correlated to Wnt pathway. Finally, a strong correlation between ERAP1 SpA-susceptibility haplotype and an increased expression of this gene and the ERAP1 protein has been identified.PARIS5-Bibliotheque electronique (751069902) / SudocSudocFranceF

Transgenic rat OX62(+) DCs exhibit multiple cellular deficiencies and the tolerigenic CD4(-) subset suffers reduced viability

Background: Although the association of the MHC class I allele HLA-B27 with Spondyloarthropathy (SpA) has been known for almost 35 years different hypotheses on its relation to disease mechanism still exist in parallel. Several lines of rats transgenic for HLA-B27 and human β2-microglobulin develop an inflammatory disease that strikingly resembles human SpA. It is hypothesized that disease in HLA-B27- transgenic rats arises as a consequence of interaction between antigen-presenting cells expressing high levels of HLA-B27 and peripheral T lymphocytes, and may result from a rupture of tolerance towards gut bacteria. Methods: We used 2D PAGE and iTRAQ to compare the protein expression profile of HLA-B27 dendritic cells (DCs) to that of healthy HLAB7 expressing and nontransgenic (NTG) rat DCs. MHC II surface expression and apoptotic sensitivity were quantified using flow cytometry. Results: Three protein sets from the proteome analysis were indicative for aberrant cellular processes. First, all proteins involved in protein processing and MHC I assembly were upregulated in B27 DCs, illustrating the higher pressure on the ER due to misfolding of the HLA-B27 heavy chain. Second, all proteins directly influencing actin-dynamics were downregulated. We showed earlier that this not only influences motility, but also plays an important role in deficient immunological synapse formation. Third, the key thiol protease Cathepsin S involved in MHC II synthesis was downregulated, which led us to quantify RT1-B and RT1-D surface expression. Downregulation concerned both CD4+ and CD4- OX62+ HLA-B27 DC subpopulations and maturation enlarged differences in both population bias and expression intensity. Deficient actin dynamics could also contribute to this lower MHC II surface expression. Study of sensitivity to MHC class II-mediated apoptosis by antibody stimulation showed that compared to NTG, both B7 and B27 CD4+ DC were more prone to apoptosis but did not mutually differ. In contrast, overnight culturing resulted in a higher cell death in B27 than in control CD4- DC, even without antibody stimulation. Interestingly, decreased actin dynamics could also be involved in DC apoptosis. Conclusions: We have demonstrated that DCs are a very vulnerable cell type in HLA-B27 rats. Deficient cytoskeletal dynamics could immobilize matured DC in the tissue or induce aberrant migration patterns upon activation. On top of that abnormal intracellular trafficking and membrane organization together with a reduced expression of MHC class II molecules makes them aberrant in T-cell communication by deficient immunological synapse formation. Especially the reduced motility and viability of the tolerigenic CD4- DC could play an important role in initiating a systemic auto-immune response

Inhibition of anti-tuberculosis T-lymphocyte function with tumour necrosis factor antagonists

Reactivation of latent Mycobacterium tuberculosis (Mtb) infection is a major complication of anti-tumour necrosis factor (TNF)-α treatment, but its mechanism is not fully understood. We evaluated the effect of the TNF antagonists infliximab (Ifx), adalimumab (Ada) and etanercept (Eta) on anti-mycobacterial immune responses in two conditions: with ex vivo studies from patients treated with TNF antagonists and with the in vitro addition of TNF antagonists to cells stimulated with mycobacterial antigens. In both cases, we analysed the response of CD4(+ )T lymphocytes to purified protein derivative (PPD) and to culture filtrate protein (CFP)-10, an antigen restricted to Mtb. The tests performed were lymphoproliferation and immediate production of interferon (IFN)-γ. In the 68 patients with inflammatory diseases (rheumatoid arthritis, spondylarthropathy or Crohn's disease), including 31 patients with a previous or latent tuberculosis (TB), 14 weeks of anti-TNF-α treatment had no effect on the proliferation of CD4(+ )T lymphocytes. In contrast, the number of IFN-γ-releasing CD4(+ )T lymphocytes decreased for PPD (p < 0.005) and CFP-10 (p < 0.01) in patients with previous TB and for PPD (p < 0.05) in other patients (all vaccinated with Bacille Calmette-Guérin). Treatments with Ifx and with Eta affected IFN-γ release to a similar extent. In vitro addition of TNF antagonists to CD4(+ )T lymphocytes stimulated with mycobacterial antigens inhibited their proliferation and their expression of membrane-bound TNF (mTNF). These effects occurred late in cultures, suggesting a direct effect of TNF antagonists on activated mTNF(+ )CD4(+ )T lymphocytes, and Ifx and Ada were more efficient than Eta. Therefore, TNF antagonists have a dual action on anti-mycobacterial CD4(+ )T lymphocytes. Administered in vivo, they decrease the frequency of the subpopulation of memory CD4(+ )T lymphocytes rapidly releasing IFN-γ upon challenge with mycobacterial antigens. Added in vitro, they inhibit the activation of CD4(+ )T lymphocytes by mycobacterial antigens. Such a dual effect may explain the increased incidence of TB in patients treated with TNF antagonists as well as possible differences between TNF antagonists for the incidence and the clinical presentation of TB reactivation

Genomewide association study of acute anterior uveitis identifies new susceptibility loci

Acknowledgments The authors thank all participating subjects with AS and healthy individuals who provided the DNA and clinical information necessary for this study. We would like to acknowledge the contributions of Anna Deminger, Sahlgrenska Academy at University of Gothenburg, and Urban Hellman, Umeå University, for their assistance in case recruitment and assessment and handling biological samples Funding Information: The survey was conducted by NatCen and the genomewide scan data were analyzed and deposited by the Wellcome Trust Sanger Institute. Information on how to access the data can be found on the Understanding Society website https: www. understandingsociety.ac.uk/ . We acknowledge and thank the TCRA AS Group for their support in recruiting patients for the study. M.A.B. is funded by a National Health and Medical Research Council (Australia) Senior Principal Research Fellowship, and support for this study was received from a National Health and Medical Research Council (Australia) program Grant (566938) and project Grant (569829), and from the Australian Cancer Research Foundation and Rebecca Cooper Medical Research Foundation. We are also very grateful for the invaluable support received from the National Ankylosing Spondylitis Society (UK) and Spondyloarthritis Association of America in case recruitment. Additional financial and technical support for patient recruitment was provided by the National Institute for Health Research Oxford Musculoskeletal Biomedical Research Unit and NIHR Thames Valley Comprehensive Local Research and an unrestricted educational grant from Abbott Laboratories. The authors acknowledge the sharing of data and samples by the BSRBR-AS Register in Aberdeen. Chief Investigator, Prof Gary Macfarlane and Dr Gareth Jones, Deputy Chief Investigator, created the BSRBR-AS study, which was commissioned by the British Society for Rheumatology, funded in part by Abbvie, Pfizer, and UCB. We are grateful to every patient, past and present staff of the BSRBR-AS register team, and to all clinical staff who recruited patients, followed them up and entered data – details here: https://www.abdn.ac.uk/iahs/research/ epidemiology/spondyloarthritis.php#panel1011. Funding was also received from the Swedish Research Council and The Swedish state under the agreement between the Swedish government and the county councils, the ALF agreement. The Irish data was derived from participants in ASRI – The Ankylosing Spondylitis Registry of Ireland, which is funded by unrestricted grants from Abbvie and Pfizer. Funding bodies involved played no role in the study design, performance, or preparation of this manuscript. Funding Information: X.F.H. was funded by the National Natural Science Foundation of China (31771390). The TASC study was funded by the National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS) grants P01-052915, R01-AR046208. Funding was also received from the University of Texas Health Science Center at Houston CTSA grant UL1RR02418, Cedars-Sinai GCRC grant MO1-RR00425, Intramural Research Program, NIAMS/NIH, and Rebecca Cooper Foundation (Australia). This study was funded, in part, by Arthritis Research UK (Grants 19536 and 18797), by the Wellcome Trust (Grant number 076113), and by the Oxford Comprehensive Biomedical Research Centre ankylosing spondylitis chronic disease cohort (Theme Code: A91202). The New Zealand data was derived from participants in the Spondyloarthritis Genetics and the Environment Study (SAGE) and was funded by The Health Research Council, New Zealand. H.X. was funded by the National Natural Science Foundation of China Grant 81020108029 and 30872339. French sample collection was performed by the Groupe Française d’Etude Génétique des Spondylarthrites, coordinated by Professor Maxime Breban, and funded by the Agence Nationale de Recherche GEMISA grant reference ANR-10-MIDI-0002. We acknowledge the Understanding Society: The UK Household Longitudinal Study. This is led by the Institute for Social and Economic Research at the University of Essex and funded by the Economic and Social Research Council. Publisher Copyright: © 2020 Association for Research in Vision and Ophthalmology Inc.. All rights reserved.Peer reviewedPublisher PD

Major histocompatibility complex associations of ankylosing spondylitis are complex and involve further epistasis with ERAP1

Ankylosing spondylitis (AS) is a common, highly heritable, inflammatory arthritis for which HLA-B*27 is the major genetic risk factor, although its role in the aetiology of AS remains elusive. To better understand the genetic basis of the MHC susceptibility loci, we genotyped 7,264 MHC SNPs in 22,647 AS cases and controls of European descent. We impute SNPs, classical HLA alleles and amino-acid residues within HLA proteins, and tested these for association to AS status. Here we show that in addition to effects due to HLA-B*27 alleles, several other HLA-B alleles also affect susceptibility. After controlling for the associated haplotypes in HLA-B, we observe independent associations with variants in the HLA-A, HLA-DPB1 and HLA-DRB1 loci. We also demonstrate that the ERAP1 SNP rs30187 association is not restricted only to carriers of HLA-B*27 but also found in HLA-B*40:01 carriers independently of HLA-B*27 genotype

Caractéristiques et devenir des patients traités par anti-TNF alpha pour une spondylarthrite

PARIS7-Xavier Bichat (751182101) / SudocSudocFranceF

La spondylarthrite du rat HLA-B27 (Rôle des cellules présentatrices d'antigène)

Les spondylarthropathies (SpA) sont fortement asssociées à la molécule HLA-B27, dont le rôle dans le déclenchement de la maladie reste encore inconnu. Ce travail a pour but d'étudier le dysfonctionnement des cellules dendritiques (DC) dans le modèle du rat transgénique pour le HLA-B27. Nous avons confirmé l'existence d'un déficit fonctionnel de stimulation des lymphocytes T (LT) par les DC de rat transgénique pour le HLA-B27. Cette anomalie n'est pas une conséquence de l'état inflammatoire chronique du rat malade. Elle résulte au moins en partie d'une diminution de la formation de conjugués indépendants d'antigène entre les DC B27 et les LT naïfs. Nous avons recherché les mécanismes moléculaires qui pourraient être responsables de cette perturbation, en comparant les DC B27 aux DC de rats non transgéniques (ntg) et de rats transgéniques pour le HLA-B7.The spondylarthropathies (SpA) are a group of inflammatoryrheumatic disorders strongly associated with the HLA-B27 molecule. The mechanism of this association remains unknown. In this work, we further investigated the functional defect of dendritic cells (DCs) in HLA-B27 transgenic rat model. We have confirmed that spenic DCs form the disease-prone 33-3 line have a striking defect in their capacity to stimulate the T cell proliferative response in vitro. This defect is not a consequence of the chronic inflammatory process in these rats, but result in part to a decrease antigen-independent conjugate formation, between 33-3 DC and naive T cells. We investigated the molecular mechanism responsible for this defect by comparing DCs from HLA-B27 transgenic rats with DCs from nontransgenic and from HLA-B7 transgenic rats.PARIS5-BU Méd.Cochin (751142101) / SudocSudocFranceF