The role of the Epstein Barr Virus in B cell tolerance loss in rheumatoid arthritis disease.

PhD ThesesAlthough many efforts have been done in understanding the aetiology of rheumatoid arthritis (RA), the Epstein-Barr Virus (EBV) remains one of the strongest candidates to be investigated as possible environmental factors in inducing RA. Of relevance, a high percentage of RA patients are characterised by a strong immune response to EBV, with high blood DNAviral load and circulating cross-reactive (auto)antibodies reacting against self-citrullinated antigens (ACPA) and viral proteins. Due to its lymphotropic behaviour, EBV might be able to rescue an autoreactive B cell phenotype and thus eludes the immune system checkpoints. Also, the virus and the infected B cells have been proposed as a possible source of citrullinated epitopes triggering the disease. To obtain long-standing EBV-infected B cells, I setup an in vitro co-culture system whereby RA fibroblasts-like synoviocytes (RA-FLS) obtained from synovial fluid and synovial tissue of patient were cultured with CD19+B cells for 28 days. RA-FLS were used as feeding environment for the CD19+B cells obtained from ACPA+ RA patients and healthy donor. CpG was added to induce plasmacells differentiation to test the presence of ACPA antibodies. B cells were then recovered, and analysed trough fluorescence activated cell sorting (Facs). Finally, molecular biology analysis has been performed to detect and quantify specific EBV-related gene such as the BamH1-W repeat region. Also, RA-FLS were recovered in order to investigate any possible modification induced by the CD19+B cells in the system. Preliminary data suggested that the EBV+ CD19+B cells population has a higher proliferation rate when incubated with RA-FLS, although this is not exclusive of RA patient since such proliferation was detected also in healthy donor. Nevertheless, this might reflect an in vivo mechanism in 6 which the EBV+ CD19+B cells from the peripheral compartment might find a preferential proliferating niche in the synovium, due to the RA-FLS. Furthermore, I am proposing a new method to obtain naturally EBV+ RACD19+ B cells in order to develop a better tool to study the role of the virus in RA pathogenesis

Rituximab versus tocilizumab in rheumatoid arthritis: synovial biopsy-based biomarker analysis of the phase 4 R4RA randomized trial

Patients with rheumatoid arthritis (RA) receive highly targeted biologic therapies without previous knowledge of target expression levels in the diseased tissue. Approximately 40% of patients do not respond to individual biologic therapies and 5–20% are refractory to all. In a biopsy-based, precision-medicine, randomized clinical trial in RA (R4RA; n = 164), patients with low/absent synovial B cell molecular signature had a lower response to rituximab (anti-CD20 monoclonal antibody) compared with that to tocilizumab (anti-IL6R monoclonal antibody) although the exact mechanisms of response/nonresponse remain to be established. Here, in-depth histological/molecular analyses of R4RA synovial biopsies identify humoral immune response gene signatures associated with response to rituximab and tocilizumab, and a stromal/fibroblast signature in patients refractory to all medications. Post-treatment changes in synovial gene expression and cell infiltration highlighted divergent effects of rituximab and tocilizumab relating to differing response/nonresponse mechanisms. Using ten-by-tenfold nested cross-validation, we developed machine learning algorithms predictive of response to rituximab (area under the curve (AUC) = 0.74), tocilizumab (AUC = 0.68) and, notably, multidrug resistance (AUC = 0.69). This study supports the notion that disease endotypes, driven by diverse molecular pathology pathways in the diseased tissue, determine diverse clinical and treatment–response phenotypes. It also highlights the importance of integration of molecular pathology signatures into clinical algorithms to optimize the future use of existing medications and inform the development of new drugs for refractory patients

Rituximab versus tocilizumab in rheumatoid arthritis: synovial biopsy-based biomarker analysis of the phase 4 r4ra randomized trial

Patients with rheumatoid arthritis (RA) receive highly targeted biologic therapies without previous knowledge of target expression levels in the diseased tissue. Approximately 40% of patients do not respond to individual biologic therapies and 5–20% are refractory to all. In a biopsy-based, precision-medicine, randomized clinical trial in RA (R4RA; n = 164), patients with low/absent synovial B cell molecular signature had a lower response to rituximab (anti-CD20 monoclonal antibody) compared with that to tocilizumab (anti-IL6R monoclonal antibody) although the exact mechanisms of response/nonresponse remain to be established. Here, in-depth histological/molecular analyses of R4RA synovial biopsies identify humoral immune response gene signatures associated with response to rituximab and tocilizumab, and a stromal/fibroblast signature in patients refractory to all medications. Post-treatment changes in synovial gene expression and cell infiltration highlighted divergent effects of rituximab and tocilizumab relating to differing response/nonresponse mechanisms. Using ten-by-tenfold nested cross-validation, we developed machine learning algorithms predictive of response to rituximab (area under the curve (AUC) = 0.74), tocilizumab (AUC = 0.68) and, notably, multidrug resistance (AUC = 0.69). This study supports the notion that disease endotypes, driven by diverse molecular pathology pathways in the diseased tissue, determine diverse clinical and treatment–response phenotypes. It also highlights the importance of integration of molecular pathology signatures into clinical algorithms to optimize the future use of existing medications and inform the development of new drugs for refractory patients

Riociguat treatment in patients with chronic thromboembolic pulmonary hypertension: Final safety data from the EXPERT registry

Objective: The soluble guanylate cyclase stimulator riociguat is approved for the treatment of adult patients with pulmonary arterial hypertension (PAH) and inoperable or persistent/recurrent chronic thromboembolic pulmonary hypertension (CTEPH) following Phase

GLI ANTICORPI ANTI PROTEINE CITRULLINATE: RUOLO NELLA PRODUZIONE DI RANTES DA PARTE DI SINOVIOCITI UMANI

Fra le malattie autoimmuni infiammatorie croniche, l’artrite reumatoide (AR) riveste un ruolo di primo piano colpendo tra lo 0,5 e l’1% degli adulti nel mondo sviluppato. L’hallmark patologico della malattia è un progressivo danno articolare a livello delle piccole e grandi articolazioni, più comune in mani e piedi per le prime, alle ginocchia per le seconde. Suddetto danno è causato da un infiltrato infiammatorio localizzato a livello della membrana sinoviale, il rivestimento di connettivo che ricopre internamente le capsule articolari; l’infiltrato tende ad espandersi nelle strutture limitrofe, provocando erosioni dell’osso. L’eziologia e la patogenesi di tale malattia non sono ancora completamente note ma recenti scoperte denotano nitidamente i ruoli sia della componente genetica che di quella ambientale. L’AR è, quindi, una malattia eterogenea, che può variare da forme lievi con sintomi lentamente progressivi, a forme caratterizzate dall’alternarsi di fasi di remissione e riacutizzazione, fino a una malattia caratterizzata da infiammazione sistemica con impegno extra-articolare e grave danno erosivo delle articolazioni. Negli ultimi anni sono state numerose le ipotesi riguardanti il ruolo peculiare che una popolazione di anticorpi caratteristica dell’artrite reumatoide, gli anticorpi anti peptidi/proteine citrullinate (ACPA), potrebbe avere nell’onset della malattia. Lo scopo di questo lavoro di tesi è di analizzare il ruolo degli ACPA nella produzione di citochine e chemochine da parte dei sinoviociti, anche chiamati sinoviociti fibroblasto simili o sinoviociti di tipo fibroblastico (FLS). Fra i vari attori cellulari che possono prendere parte in RA, i FLS sembrano giocare un ruolo fondamentale, contribuendo anche con la produzione di mediatori dell'infiammazione ed enzimi proteolitici che degradano la matrice extracellulare (MMP). Sono stati selezionati alcuni pazienti con AR nel cui siero sono presenti ACPA a titolo elevato. Gli ACPA sono stati in seguito isolati dal siero mediante cromatografia di affinità su peptidi citrullinati immobilizzati su fase solida. La specificità degli anticorpi purificati è stata poi confermata mediante test ELISA. Da frammenti di tessuto sinoviale prelevato durante sinoviectomie, in seguito, sono tate approntate colture di (FLS). Tutti gli esperimenti sono stati effettuati al 3°-5° passaggio delle cellule in coltura. Gli ACPA sono stati incubati a tempi e concentrazioni differenti su sinoviociti prelevati da pazienti con AR oppure con osteoartrosi (OA). Si è poi proceduto a valutare il pattern di citochine prodotte nel supernatante di coltura. In esperimenti preliminari è stato utilizzato un multiarray specifico per citochine umane ed è stato osservato un notevole aumento di produzione di CCL5/RANTES a seguito dell’incubazione dei FLS con APCA. Ulteriori conferme di questo risultato iniziale sono arrivate mediante ELISA “classico”, basato su anticorpi monoclonali e policlonali specifici per CCL5/RANTES. Il risultato finale ha mostrato che gli ACPA inducono produzione di CCL5/RANTES da parte dei FLS; tale produzione è particolarmente elevata quando i FLS derivano da pazienti con AR. In parallelo al dosaggio della proteina nel surnatante, è stato dosato mediante RT-PCR l’mRNA codificante CCL5/RANTES ed è stato dimostrato un aumento dopo stimolo con ACPA. Attualmente sono in corso esperimenti atti a valutare il legame degli ACPA ai FLS e i meccanismi coinvolti nelle cascate di fosforilazione

Data_Sheet_1_Follicular dendritic cell differentiation is associated with distinct synovial pathotype signatures in rheumatoid arthritis.PDF

Follicular dendritic cells (FDCs) fundamentally contribute to the formation of synovial ectopic lymphoid-like structures in rheumatoid arthritis (RA) which is associated with poor clinical prognosis. Despite this critical role, regulation of FDC development in the RA synovium and its correlation with synovial pathotype differentiation remained largely unknown. Here, we demonstrate that CNA.42+ FDCs distinctively express the pericyte/fibroblast-associated markers PDGFR-β, NG2, and Thy-1 in the synovial perivascular space but not in established follicles. In addition, synovial RNA-Seq analysis revealed that expression of the perivascular FDC markers was strongly correlated with PDGF-BB and fibroid synovitis, whereas TNF-α/LT-β was significantly associated with lymphoid synovitis and expression of CR1, CR2, and FcγRIIB characteristic of mature FDCs in lymphoid follicles. Moreover, PDGF-BB induced CNA.42+ FDC differentiation and CXCL13 secretion from NG2+ synovial pericytes, and together with TNF-α/LT-β conversely regulated early and late FDC differentiation genes in unsorted RA synovial fibroblasts (RASF) and this was confirmed in flow sorted stromal cell subsets. Furthermore, RASF TNF-αR expression was upregulated by TNF-α/LT-β and PDGF-BB; and TNF-α/LT-β-activated RASF retained ICs and induced B cell activation in in vitro germinal center reactions typical of FDCs. Additionally, FDCs trapped peptidyl citrulline, and strongly correlated with IL-6 expression, and plasma cell, B cell, and T cell infiltration of the RA synovium. Moreover, synovial FDCs were significantly associated with RA disease activity and radiographic features of tissue damage. To the best of our knowledge, this is the first report describing the reciprocal interaction between PDGF-BB and TNF-α/LT-β in synovial FDC development and evolution of RA histological pathotypes. Selective targeting of this interplay could inhibit FDC differentiation and potentially ameliorate RA in clinically severe and drug-resistant patients.</p

Axl and MerTK regulate synovial inflammation and are modulated by IL-6 inhibition in rheumatoid arthritis

International audienceThe TAM tyrosine kinases, Axl and MerTK, play an important role in rheumatoid arthritis (RA). Here, using a unique synovial tissue bioresource of patients with RA matched for disease stage and treatment exposure, we assessed how Axl and MerTK relate to synovial histopathology and disease activity, and their topographical expression and longitudinal modulation by targeted treatments. We show that in treatment-naive patients, high AXL levels are associated with pauci-immune histology and low disease activity and inversely correlate with the expression levels of pro-inflammatory genes. We define the location of Axl/MerTK in rheumatoid synovium using immunohistochemistry/fluorescence and digital spatial profiling and show that Axl is preferentially expressed in the lining layer. Moreover, its ectodomain, released in the synovial fluid, is associated with synovial histopathology. We also show that Toll-like-receptor 4-stimulated synovial fibroblasts from patients with RA modulate MerTK shedding by macrophages. Lastly, Axl/MerTK synovial expression is influenced by disease stage and therapeutic intervention, notably by IL-6 inhibition. These findings suggest that Axl/MerTK are a dynamic axis modulated by synovial cellular features, disease stage and treatment

Rituximab versus tocilizumab in rheumatoid arthritis: synovial biopsy-based biomarker analysis of the phase 4 R4RA randomized trial.

Patients with rheumatoid arthritis (RA) receive highly targeted biologic therapies without previous knowledge of target expression levels in the diseased tissue. Approximately 40% of patients do not respond to individual biologic therapies and 5-20% are refractory to all. In a biopsy-based, precision-medicine, randomized clinical trial in RA (R4RA; n = 164), patients with low/absent synovial B cell molecular signature had a lower response to rituximab (anti-CD20 monoclonal antibody) compared with that to tocilizumab (anti-IL6R monoclonal antibody) although the exact mechanisms of response/nonresponse remain to be established. Here, in-depth histological/molecular analyses of R4RA synovial biopsies identify humoral immune response gene signatures associated with response to rituximab and tocilizumab, and a stromal/fibroblast signature in patients refractory to all medications. Post-treatment changes in synovial gene expression and cell infiltration highlighted divergent effects of rituximab and tocilizumab relating to differing response/nonresponse mechanisms. Using ten-by-tenfold nested cross-validation, we developed machine learning algorithms predictive of response to rituximab (area under the curve (AUC) = 0.74), tocilizumab (AUC = 0.68) and, notably, multidrug resistance (AUC = 0.69). This study supports the notion that disease endotypes, driven by diverse molecular pathology pathways in the diseased tissue, determine diverse clinical and treatment-response phenotypes. It also highlights the importance of integration of molecular pathology signatures into clinical algorithms to optimize the future use of existing medications and inform the development of new drugs for refractory patients

Rituximab versus tocilizumab in rheumatoid arthritis: synovial biopsy-based biomarker analysis of the phase 4 R4RA randomized trial

Patients with rheumatoid arthritis (RA) receive highly targeted biologic therapies without previous knowledge of target expression levels in the diseased tissue. Approximately 40% of patients do not respond to individual biologic therapies and 5–20% are refractory to all. In a biopsy-based, precision-medicine, randomized clinical trial in RA (R4RA; n = 164), patients with low/absent synovial B cell molecular signature had a lower response to rituximab (anti-CD20 monoclonal antibody) compared with that to tocilizumab (anti-IL6R monoclonal antibody) although the exact mechanisms of response/nonresponse remain to be established. Here, in-depth histological/molecular analyses of R4RA synovial biopsies identify humoral immune response gene signatures associated with response to rituximab and tocilizumab, and a stromal/fibroblast signature in patients refractory to all medications. Post-treatment changes in synovial gene expression and cell infiltration highlighted divergent effects of rituximab and tocilizumab relating to differing response/nonresponse mechanisms. Using ten-by-tenfold nested cross-validation, we developed machine learning algorithms predictive of response to rituximab (area under the curve (AUC) = 0.74), tocilizumab (AUC = 0.68) and, notably, multidrug resistance (AUC = 0.69). This study supports the notion that disease endotypes, driven by diverse molecular pathology pathways in the diseased tissue, determine diverse clinical and treatment–response phenotypes. It also highlights the importance of integration of molecular pathology signatures into clinical algorithms to optimize the future use of existing medications and inform the development of new drugs for refractory patients

Unique expansion of IL-21+ Tfh and Tph cells under control of ICOS identifies Sjögren's syndrome with ectopic germinal centres and MALT lymphoma.

OBJECTIVES To explore the relevance of T-follicular-helper (Tfh) and pathogenic peripheral-helper T-cells (Tph) in promoting ectopic lymphoid structures (ELS) and B-cell mucosa-associated lymphoid tissue (MALT) lymphomas (MALT-L) in Sjögren's syndrome (SS) patients. METHODS Salivary gland (SG) biopsies with matched peripheral blood were collected from four centres across the European Union. Transcriptomic (microarray and quantitative PCR) analysis, FACS T-cell immunophenotyping with intracellular cytokine detection, multicolor immune-fluorescence microscopy and hybridisation were performed to characterise lesional and circulating Tfh and Tph-cells. SG-organ cultures were used to investigate functionally the blockade of T-cell costimulatory pathways on key proinflammatory cytokine production. RESULTS Transcriptomic analysis in SG identified Tfh-signature, interleukin-21 (IL-21) and the inducible T-cell co-stimulator (ICOS) costimulatory pathway as the most upregulated genes in ELS+SS patients, with parotid MALT-L displaying a 400-folds increase in IL-21 mRNA. Peripheral CD4CXC-motif chemokine receptor 5 (CXCR5)programmed cell death protein 1 (PD1)ICOS Tfh-like cells were significantly expanded in ELS+SS patients, were the main producers of IL-21, and closely correlated with circulating IgG and reduced complement C4. In the SG, lesional CD4CD45ROICOSPD1 cells selectively infiltrated ELS+ tissues and were aberrantly expanded in parotid MALT-L. In ELS+SG and MALT-L parotids, conventional CXCR5CD4PD1ICOSFoxp3 Tfh-cells and a uniquely expanded population of CXCR5CD4PD1ICOSFoxp3 Tph-cells displayed frequent IL-21/interferon-γ double-production but poor IL-17 expression. Finally, ICOS blockade in SG-organ cultures significantly reduced the production of IL-21 and inflammatory cytokines IL-6, IL-8 and tumour necrosis factor-α (TNF-α). CONCLUSIONS Overall, these findings highlight Tfh and Tph-cells, IL-21 and the ICOS costimulatory pathway as key pathogenic players in SS immunopathology and exploitable therapeutic targets in SS