Frances Humby

Ectopic Lymphoid Structures Support Ongoing Production of Class-Switched Autoantibodies in Rheumatoid Synovium

Background Follicular structures resembling germinal centres (GCs) that are characterized by follicular dendritic cell (FDC) networks have long been recognized in chronically inflamed tissues in autoimmune diseases, including the synovium of rheumatoid arthritis (RA). However, it is debated whether these ectopic structures promote autoimmunity and chronic inflammation driving the production of pathogenic autoantibodies. Anti-citrullinated protein/peptide antibodies (ACPA) are highly specific markers of RA, predict a poor prognosis, and have been suggested to be pathogenic. Therefore, the main study objectives were to determine whether ectopic lymphoid structures in RA synovium: (i) express activation-induced cytidine deaminase (AID), the enzyme required for somatic hypermutation and class-switch recombination (CSR) of Ig genes; (ii) support ongoing CSR and ACPA production; and (iii) remain functional in a RA/severe combined immunodeficiency (SCID) chimera model devoid of new immune cell influx into the synovium. Methods and Findings Using immunohistochemistry (IHC) and quantitative Taqman real-time PCR (QT-PCR) in synovial tissue from 55 patients with RA, we demonstrated that FDC+ structures invariably expressed AID with a distribution resembling secondary lymphoid organs. Further, AID+/CD21+ follicular structures were surrounded by ACPA+/CD138+ plasma cells, as demonstrated by immune reactivity to citrullinated fibrinogen. Moreover, we identified a novel subset of synovial AID+/CD20+ B cells outside GCs resembling interfollicular large B cells. In order to gain direct functional evidence that AID+ structures support CSR and in situ manufacturing of class-switched ACPA, 34 SCID mice were transplanted with RA synovium and humanely killed at 4 wk for harvesting of transplants and sera. Persistent expression of AID and Iγ-Cμ circular transcripts (identifying ongoing IgM-IgG class-switching) was observed in synovial grafts expressing FDCs/CD21L. Furthermore, synovial mRNA levels of AID were closely associated with circulating human IgG ACPA in mouse sera. Finally, the survival and proliferation of functional B cell niches was associated with persistent overexpression of genes regulating ectopic lymphoneogenesis. Conclusions Our demonstration that FDC+ follicular units invariably express AID and are surrounded by ACPA-producing plasma cells provides strong evidence that ectopic lymphoid structures in the RA synovium are functional and support autoantibody production. This concept is further confirmed by evidence of sustained AID expression, B cell proliferation, ongoing CSR, and production of human IgG ACPA from GC+ synovial tissue transplanted into SCID mice, independently of new B cell influx from the systemic circulation. These data identify AID as a potential therapeutic target in RA and suggest that survival of functional synovial B cell niches may profoundly influence chronic inflammation, autoimmunity, and response to B cell–depleting therapies.

Activation-induced cytidine deaminase expression in follicular dendritic cell networks and interfollicular large B cells supports functionality of ectopic lymphoid neogenesis in autoimmune sialoadenitis and MALT lymphoma in Sjögren's syndrome

Demonstration of ectopic germinal center-like structures (GC-LSs) in chronically inflamed tissues in patients with autoimmune disorders is a relatively common finding. However, to what extent ectopic lymphoid structures behave as true GC and are able to support class switch recombination (CSR) and somatic hypermutation (SHM) of the Ig genes is still debated. In addition, no information is available on whether CSR and SHM can take place in the absence of GCs at extrafollicular sites in an ectopic lymphoid tissue. In this study, we show that in salivary glands (SGs) of Sjögren’s syndrome (SS) activation-induced cytidine deaminase (AID), the enzyme responsible for CSR and SHM is invariably expressed within follicular dendritic cell (FDC) networks but is not detectable in SGs in the absence of ectopic GC-LSs, suggesting that FDC networks play an essential role in sustaining the Ag-driven B cell proliferation within SS-SGs. We also show that the recently described population of interfollicular large B cells selectively expresses AID outside ectopic GC in the T cell-rich areas of periductal aggregates. Finally, we report that AID retains its exclusive association with numerous, residual GCs in parotid SS-MALT lymphomas, whereas neoplastic marginal zone-like B cells are consistently AID negative. These results strongly support the notion that ectopic lymphoid structures in SS-SGs express the molecular machinery to support local autoantibody production and B cell expansion and may play a crucial role toward lymphomagenesis.

Secondary and ectopic lymphoid tissue responses in rheumatoid arthritis: from inflammation to autoimmunity and tissue damage/remodeling.

Summary:  Rheumatoid arthritis is a chronic systemic inflammatory disease primarily affecting the synovium of diarthrodial joints. Despite the currently unknown etiology, overwhelming evidence indicates that both innate and adaptive immunity play a central role in disease pathogenesis. In this review, we consider recent evidence examining the mechanisms of lymphoid tissue reactivity in rheumatoid arthritis with a focus on the dynamics controlling secondary and ectopic lymphoid tissue response. We then examine the cellular and molecular mechanisms regulating the biopathology of these processes with specific emphasis on cell trafficking, contribution to autoimmunity, and joint damage‐repair. We finally provide a brief overview of the most recent studies addressing the clinical relevance of synovial lymphoid tissue analysis as a diagnostic and prognostic tool as well as its response to current biological therapies.

Mature Antigen-Experienced T Helper Cells Synthesize and Secrete the B Cell Chemoattractant CXCL13 in the Inflammatory Environment of the Rheumatoid Joint

OBJECTIVE Synovial B cells play a critical role in rheumatoid arthritis (RA), being involved in autoantibody synthesis, T cell activation, and cytokine production. CXCL13 is a B cell chemoattractant that is instrumental in synovial B cell organization; the regulatory determinants of CXCL13 in inflammation are poorly characterized. This study was undertaken to investigate the functional involvement of synovial T cells in the ectopic expression of CXCL13 in RA. METHODS CXCL13 production and regulation were addressed using immunohistochemistry, in situ hybridization, quantitative polymerase chain reaction, multicolor flow cytometry, and enzyme-linked immunosorbent assay, by in situ-ex vivo analysis and in vitro functional assays with rheumatoid synovial tissue and primary cells. RESULTS CXCL13 messenger RNA and protein expression and spontaneous CXCL13 secretion were detected in RA synovial fluid T cells but were not detected (or were detected only occasionally) in peripheral blood T cells. Analysis of tissue expression confirmed cytoplasm localization of CXCL13 in T lymphocytes infiltrating B cell follicles and small perivascular aggregates. Multicolor characterizations in synovial fluid demonstrated CXCL13 expression in antigen-experienced T helper cells, frequently characterized by terminal differentiation and the lack of the follicular helper T cell markers CXCR5 and BCL6 protein. In vitro functional assays revealed the enhancing effect of T cell receptor-CD28 engagement on CXCL13 production and secretion in primary cells. CONCLUSION Our findings define a new functional property of synovial T cells, demonstrating their active involvement in the local production of B cell chemoattractants, and support a direct contribution of the adaptive immune system and antigen-dependent signals in the mechanisms of B cell localization in RA.

New learnings on the pathophysiology of RA from synovial biopsies.

Purpose of reviewTo critically appraise the literature related to the pathophysiology of rheumatoid arthritis (RA) focusing on the contribution of synovial tissue pathology (synovitis) in determining diverse clinical outcome/therapeutic response. Recent findingsRA synovitis is highly heterogeneous with diverse cellular and molecular signatures (pathotypes) emerging as potential taxonomic classifiers of disease phenotypes.The challenge is to understand mechanistically the sophisticated interplay between systemic disease ‘initiators’ and joint-specific ‘localizing/perpetuating’ factors leading to disparate coupling of inflammation/tissue-destructive pathways and disease outcome. Synovial tissue analysis has been instrumental in enhancing understanding of R0A pathogenesis and developing targeted DMARD-biologic therapies. The next step is to elucidate the relationship of different synovial pathotypes/molecular signatures with therapeutic response/resistance in randomized clinical trials in order to develop effective therapies for ‘resistant’ patients. The development of ultrasound-guided synovial biopsy as a rapid, safe and well tolerated procedure that enables synovial tissue collection from most joints/patients will facilitate such studies. SummaryRA is a heterogeneous clinical and pathobiological entity. Specific pathways within synovial tissues are emerging as associated with diverse clinical evolution and therapeutic response/resistance that, if confirmed in randomized clinical trials, may lead to the development of synovial tissue analysis as a potential clinical tool for patient stratification.

Immunohistological assessment of the synovial tissue in small joints in rheumatoid arthritis: validation of a minimally invasive ultrasound-guided synovial biopsy procedure.

The aim of the present study was to perform an immunohistological assessment of the synovial tissue from involved small joints in rheumatoid arthritis (RA) and to explore the reliability of a mini-invasive ultrasound (US)-guided technique of small joint synovial biopsy for the histopathological assessment. Synovial tissue collected during arthrotomic surgery of small joints in nine patients served as the gold standard for the validation of the histological assessment. Small hand-joint synovial biopsies from an additional nine patients with erosive RA were obtained by a mini-invasive US-guided procedure, performed percutaneously by the portal and rigid forceps technique. Using digital image analysis, the area fractions of synovial macrophages (CD68 cells), T cells (CD3 cells) and B cells (CD20 cells) were measured in all high-power fields of every sample at different cutting levels. The representative sample was defined as the minimal number of high-power fields whose mean area fraction would reflect the overall mean area fraction within a percentage mean difference of 10%. For each patient, a range of three to five large samples for surgical biopsies and a range of 8–12 samples for US-guided biopsies were collected and analysed. In arthrotomic samples, the analysis of a randomly selected tissue area of 2.5 mm2 was representative of the overall value for CD68, CD3 and CD20 cells. US-guided samples allowed histological evaluation in 100% of cases, with a mean valid area of 18.56 mm2 (range 7.29–38.28 mm2). The analysis of a cumulative area of 2.5 mm2 from eight randomly selected sections (from different samples or from different cutting levels) allowed to reduce the percentage mean difference to less than 10% for CD68, CD3 and CD20 cells. In conclusion, US-guided synovial biopsy represents a reliable tool for the assessment of the histopathological features of RA patients with a mini-invasive approach.

Epstein–Barr virus persistence and infection of autoreactive plasma cells in synovial lymphoid structures in rheumatoid arthritis

Objectives Rheumatoid arthritis (RA) is associated with an increased Epstein–Barr virus (EBV) blood DNA load, a robust immune response to EBV and cross-reactive circulating antibodies to viral and self-antigens. However, the role of EBV in RA pathogenesis remains elusive. Here, we investigated the relationship between synovial EBV infection, ectopic lymphoid structures (ELS) and immunity to citrullinated self and EBV proteins. Methods Latent and lytic EBV infection was investigated in 43 RA synovial tissues characterised for presence/absence of ELS and in 11 control osteoarthritis synovia using RT-PCR, in situ hybridisation and immunohistochemistry. Synovial production of anti-citrullinated protein (ACPA) and anti-citrullinated EBV peptide (VCP1/VCP2) antibodies was investigated in situ and in vivo in the severe combined immunodeficiency (SCID)/RA chimeric model. Results EBV dysregulation was observed exclusively in ELS+ RA but not osteoarthritis (OA) synovia, as revealed by presence of EBV latent (LMP2A, EBV-encoded small RNA (EBER)) transcripts, EBER+ cells and immunoreactivity for EBV latent (LMP1, LMP2A) and lytic (BFRF1) antigens in ELS-associated B cells and plasma cells, respectively. Importantly, a large proportion of ACPA-producing plasma cells surrounding synovial germinal centres were infected with EBV. Furthermore, ELS-containing RA synovia transplanted into SCID mice supported production of ACPA and anti-VCP1/VCP2 antibodies. Analysis of CD4+ and CD8+ T-cell localisation and granzyme B expression suggests that EBV persistence in ELS-containing synovia may be favoured by exclusion of CD8+ T cells from B-cell follicles and impaired CD8-mediated cytotoxicity. Conclusions We demonstrated active EBV infection within ELS in the RA synovium in association with local differentiation of ACPA-reactive B cells.

EULAR definition of arthralgia suspicious for progression to rheumatoid arthritis

Background During the transition to rheumatoid arthritis (RA) many patients pass through a phase characterised by the presence of symptoms without clinically apparent synovitis. These symptoms are not well-characterised. This taskforce aimed to define the clinical characteristics of patients with arthralgia who are considered at risk for RA by experts based on their clinical experience. Methods The taskforce consisted of 18 rheumatologists, 1 methodologist, 2 patients, 3 health professionals and 1 research fellow. The process had three phases. In phase I, a list of parameters considered characteristic for clinically suspect arthralgia (CSA) was derived; the most important parameters were selected by a three-phased Delphi approach. In phase II, the experts evaluated 50 existing patients on paper, classified them as CSA/no-CSA and indicated their level of confidence. A provisional set of parameters was derived. This was studied for validation in phase III, where all rheumatologists collected patients with and without CSA from their outpatient clinics. Results The comprehensive list consisted of 55 parameters, of which 16 were considered most important. A multivariable model based on the data from phase II identified seven relevant parameters: symptom duration <1 year, symptoms of metacarpophalangeal (MCP) joints, morning stiffness duration ≥60 min, most severe symptoms in early morning, first-degree relative with RA, difficulty with making a fist and positive squeeze test of MCP joints. In phase III, the combination of these parameters was accurate in identifying patients with arthralgia who were considered at risk of developing RA (area under the receiver operating characteristic curve 0.92, 95% CI 0.87 to 0.96). Test characteristics for different cut-off points were determined. Conclusions A set of clinical characteristics for patients with arthralgia who are at risk of progression to RA was established.

Synovial Tissue Heterogeneity and Peripheral Blood Biomarkers

Rheumatoid arthritis is characterized by multiple pathobiological processes and heterogeneous clinical phenotypes. Not surprisingly, the inflamed synovium harbors an equally complex pathology. This includes variability in infiltrating and resident cell populations, spatial arrangements, and cell–cell interactions, as well as gene expression profiles. Remarkable progress in our understanding of the many facets of tissue heterogeneity has been facilitated by the increasing availability of patients’ material and the development of advanced research technologies. The next challenge is to capitalize on the large amount of data generated to elucidate the specific pathogenic pathways disparately activated in different patients and/or different phases of the disease. When tissue pathology can be reliably explored through noninvasive circulating biomarkers, then the circle will be closed. We attempt to highlight key advances in the understanding of synovial tissue heterogeneity in rheumatoid arthritis and summarize novel perspectives in synovial biomarker discovery in relation to peripheral blood.

High expression levels of the B cell chemoattractant CXCL13 in rheumatoid synovium are a marker of severe disease

OBJECTIVE The B cell chemoattractant chemokine ligand 13 (CXCL13) is emerging as a new biochemical marker in RA. This study was undertaken to dissect the relationship between CXCL13 expression levels in the synovium and clinico-pathological variables relevant to RA pathogenesis and outcome. METHODS Synovial tissues from 71 RA patients were evaluated by immunohistochemistry. Thirty paired samples were used for comparative gene expression analysis by quantitative real-time PCR. CXCL13 levels were analysed in relation to cellular, molecular and clinical features of inflammation, lymphocyte activation and joint damage. RESULTS In patients with early disease (<12 months duration), CXCL13 expression correlated significantly with synovial markers of local disease activity and systemic inflammation. Such correlation was less evident in established RA. Notably, the association with lymphocyte infiltration and with expression of B/T cell-related activation and proliferation genes, such as activation-induced cytidine deaminase, IFN-γ and IL-2, remained highly significant independent of disease duration and local disease activity. Patients featuring the highest levels of CXCL13 were more frequently ACPA positive and IgG ACPA titres were increased in the high CXCL13 expression group. Furthermore, the frequency of erosive disease on radiographs was significantly higher in the upper tertile of CXCL13 expression (P = 0.01 with adjustment for disease duration and ACPA). Accordingly, synovial CXCL13 and the local receptor activator of nuclear factor κB ligand (RANKL)/osteoprotegerin (OPG) ratio significantly co-varied (ρ = 0.52, P < 0.01), independent of the level of local inflammation. CONCLUSION Synovial CXCL13 appears to be a marker of a more severe pattern of RA disease, characterized by increased lymphocyte activation and bone remodelling beyond the level of conventional markers of inflammation.