Nicholas J. Bernard

Rheumatoid arthritis: Prevotella copri associated with new-onset untreated RA.

By parallel genetic sequencing the intestinal microbiome, researchers have discovered a high prevalence of the bacteria Prevotella copri in patients with rheumatoid arthritis (RA). “It has been suspected that RA was linked to infections or perhaps even diet, but this is the first finding of an association with a specific microbe,” says Professor Dan Littman about his collaborative study now published in eLife. Previous work using mouse models of arthritis identified that intestinal microbiota can affect inflammation in the joints. Littman explains, “we sought to find out if something similar was going on in human disease.” By overpopulating the intestines of mice with segmented filamentous bacteria, researchers have previously induced intestinal type 17 T helper cells that Littman says “might circulate and contribute to systemic autoimmune diseases”. For this reason his team wanted to characterize the intestinal microbiome of patients with new onset untreated RA (NORA). The researchers performed parallel sequencing of bacterial DNA from human faecal samples and detected P. copri in 75% (33 of 44) of patients with NORA and only in 21.4% (6 of 28) of healthy individuals (with a threshold for detection of >5% abundance). Unexpectedly, they RHEUMATOID ARTHRITIS

Rheumatoid arthritis: Changes in ACPA Fc glycosylation patterns prior to RA onset.

knowledge of the effect of antibody Fc glycosylation comes from monoclonal antibody or mouse studies. For this reason, the researchers compared Fc glycosylation patterns of total serum IgG1 and ACPAIgG1 in order to determine the time frame of Fc glycosylation relative to the onset of RA. They took serum samples from patients with arthralgia (n = 183) at various intervals following initial medical consultation with a rheumatologist (baseline). After a median time of 12 months (IQR 6–24), 105 patients went on to develop arthritis, (RA = 48, UA = 57). Total serum IgG and ACPA were isolated by antigen affinity chromatography and trypsin digests were analysed by mass spectrometry for Fc glycosylation, including galactosylation, sialylation and fucosylation. RHEUMATOID ARTHRITIS

Osteoarthritis: Repositioning verapamil—for Wnt of an OA treatment

Verapamil a class IV antiarrhythmic agent approved in 1982 by the FDA for the treatment of complications of cardiovascular disease, such as angina and hypertension, was found to be the most powerful dose-dependent activator of FRZB expression in human OA chondrocytes. Seven other calciumchannel blockers were also tested, but none had an effect on FRZB expression or Wnt/β-catenin signalling. OSTEOARTHRITIS

Inflammation: A TLR5-TNF positive feedback loop in rheumatoid arthritis.

The researchers isolated peripheral blood mononuclear cells from patients with RA treated with DMARDs or with TNF inhibitors plus or minus DMARDs. Flagellin treatment of monocytes in vitro had an NF-κB-dependent, dosedependent, positive effect on chemotaxis, and anti-TLR5 antibody treatment inhibited chemotaxis of these cells. Monocytes from patients treated with TNF inhibitors had lower TLR5 expression than monocytes from patients who were treated with DMARDs without TNF inhibitors, suggesting that TNF and TLR5 pathways synergize INFLAMMATION

Connective tissue diseases: Inhibiting cathepsin S to treat SLE and lupus nephritis

(approximately 1.31 mg/day, stable plasma concentration of 400–600 ng/ml) protected MRL-Faslpr mice (n = 15), which spontaneously develop SLE and lupus nephritis, from severe disease. The researchers found significantly less IL-10 and TNF in plasma, a reduced percentage of activated dendritic cells and CD4+ T cells in the spleen, and disrupted spatial organisation of germinal centre formation in treated mice compared with control mice. The RO5461111-treated mice also developed fewer glomerular IgG deposits and had fewer circulating anti-dsDNA IgG antibodies than control mice. CONNECTIVE TISSUE DISEASES

Inflammation: New classification criteria for autoinflammatory periodic fevers.

mevalonate kinase deficiency (MKD; n = 74), TNF-receptor-associated periodic fever syndrome (TRAPS; n = 86) or cryopyrin-associated periodic syndromes (CAPS; n = 67). Gold-standard was defined as confirmatory genetic results of two MEFV mutations (≥1 in exon 10) for FMF, two MVK mutations (excluding variants with an unknown role in pathogenesis) for MKD, heterozygous TNFRSF1A mutations (excluding low-penetrance or unknown mutations) for TRAPS, and heterozygous NLRP3 mutations (excluding functional polymorphisms and variants with either low-penetrance or an unknown role in pathogenesis) for CAPS. Patients with chronic (nonperiodic) disease were excluded. To establish the classification criteria, the researchers compared the four goldstandard groups with a control group of patients with periodic fever, apthosis, pharyngitis and adenitis (PFAPA; n = 199), a non-monogenic disease. The groups were then split randomly into two sets, a training set (n = 412), to establish the classification criteria, and a validation set (n = 305). Univariate and multivariate analysis of clinical variables between the four diseases in the training set enabled the formulation of weighted classification scores totalling 100–110 points for each disease. For example, variables included absence of chest pain (11) and presence of painful lymph nodes (13) for MKD (cut-off ≥42), absence of vomiting (14) and presence of perorbital oedema (21) in TRAPS (cut-off ≥43), absence of adominal pain (15) and presence of urticarial rash (25) for CAPS (cut-off ≥52), and the absence of urticarial rash (15) and presence of abdominal pain (9) in FMF (cut-off ≥60). All criteria had high sensitivity and specificity in the gold-standard groups. Importantly, the criteria also performed well when applied to the group of patients who had been excluded from the testing set owing to nonconfirmatory genetic results (specificity: FMF, 87%; TRAPS, 84%; CAPS, 95%; MKD, 89%; sensitivity: FMF, 68%; TRAPS, 59%; CAPS, 70%; MKD, 53%). However, in their paper, the authors admit these data are overstated as selection from the registry probably enriched the dataset for patients already suspected of having an autoinflammatory disease. The true test of the new criteria will, therefore, be in daily practice. “Many patients have mutations in more than one gene,” says Gattorno on the difficulty of diagnosing periodic fevers, “so nonconfirmatory genetic tests can overestimate the importance of genetic variants in patients with an unclear inflammatory phenotype. This problem will be exacerbated by next-generation sequencing. Studies to correlate and validate data from molecular and clinical analysis, like ours, will be critical for classification and understanding the effect of genetic variants.” In reference to developing the criteria beyond provisional status, Gattorno adds, “We are working to establish a consensus among clinicians and geneticists to identify definitive classification criteria by combining clinical criteria and results from genetic analysis.” Hoffman’s assessment of the provisional criteria is that the benefit to experts might be limited but, he says, “This scorebased approach may help physicians who are not familiar with these diagnoses.” He predicts, “An approach like this could be adapted to develop a user-friendly web or computer-based diagnostic algorithm for use by patients or physicians.”

Bone: Fighting osteoporosis black and blue

The researchers also administered the compound orally to ovariectomized mice and to mice with RANKL-induced osteoporosis. Femurs were scanned by microcomputed tomography to measure bone density parameters, including trabecular number, thickness, separation and spacing. Delphinidin treatment (figure, right panel) prevented cancellous bone degradation (figure, middle panel) in both mouse models. In response to Niida’s publication, Mone Zaidi, an osteoporosis expert at the Icahn School of Medicine at Mount Sinai (NY, USA) notes the “renewed interest in BONE

Connective tissue diseases: TLR4 and fibronectin—persistent fibrogenesis in SSc

Researchers in the USA are close to understanding the mechanism of chronic fibrosis associated with systemic sclerosis (SSc). John Varga and colleagues identified fibronectin extra domain A (FnEDA) as an endogenous damageassociated ligand for Toll-like receptor 4 (TLR4) and showed that skin fibroblasts in patients with SSc are chronically exposed to higher than normal levels of FnEDA. Varga says this chronic activation of fibroblasts, which involves transforming growth factor-β (TGF-β), “establishes a vicious cycle of persistent fibrogenesis.” In explanation of how the project began, Varga says “fibrosis is a major cause of morbidity and mortality in SSc. While we don’t know what triggers fibrosis, we had even less understanding of why it persists, even when the initial injury is removed.” FnEDA, which is transiently expressed in extracellular matrix during tissue repair, is a multifunctional, alternative splice variant of fibronectin. In Science Translational Medicine, the researchers, led by Swati Bhattacharyya, showed that FnEDA is 5-fold more abundant in serum from patients with diffuse cutaneous SSc (n = 46, 20.2 ± 1.6 μg/ml) than from healthy individuals (n = 16, 4.5 ± 0.8 μg/ml). With quantitative PCR and immunofluorescence they showed an increase in FnEDA expression in skin from biopsy samples from SSc patients, and in vitro, FnEDA RNA and protein production by foreskin fibroblasts was upregulated by SMADdependent TGF-β stimulation. With TLR4-defective C3HeJ mice, siRNA knockdown or a small molecule inhibitor of TLR4, the researchers then showed that TLR4 is essential for FnEDAinduced collagen production by fibroblasts. Finally, TLR4 inhibition attenuated muscle necrosis, dermal thickening, cutaneous fibrosis and COL1A1 and ACAT2 expression in the skin of bleomycin-treated mice. Varga predicts “these studies might open new doors for innovative treatments and explain chronic innate immunity in SSc.” He wants to “identify patients who have the most active TLR signalling and develop novel drugs that selectively block fibroblast–FnEDA cross-talk, and thereby dampen chronic fibroblast activation.”

Connective tissue diseases: A mechanism of B cell hyperactivity in SLE

Researchers have identified a mechanism for B cell hyperactivity in patients with systemic lupus erythematosus (SLE). Published in Science Translational Medicine, the data show that microRNAs (miRNAs) miR-7, miR-21 and miR-22 enhance B-cell receptor signalling by suppressing the expression of PTEN, a process that could contribute to the characteristic production of autoantibodies in SLE. Phosphatidylinositol 3,4,5-trisphosphate 3-phosphatase and dual-specificity protein phosphatase PTEN is a known tumour suppressor, but deficiency of the protein has also been shown to cause an SLE-like disease in mice. “We relied on the mouse models to point us in the right direction,” comments Peter Lipsky, corresponding author of the study. By fluorescence-activated cell sorting, his team showed that B-cell expression (except memory B cells) of PTEN was lower in patients with SLE than in healthy donors. Importantly, the researchers also identified an inverse correlation between the level of PTEN expression and SLE disease activity.

Connective tissue diseases: How do autoreactive B cells survive in SLE—autophagy?

Data now published in Annals of the Rheumatic Diseases indicate “autophagy is abnormal in SLE in both T‐cell and B‐cell compartments, but is more significantly different in B cells,” says the lead researcher of the study, Timothy Vyse of King’s College London. The researchers found that autophagy is required for differentiation of plasmablasts and that more autophagosomes are formed in naive B cells from patients with systemic lupus erythematosus (SLE) and in a mouse model of SLE, compared with healthy individuals and control mice, respectively. Although autoreactive B cells are characteristic of SLE, a role for B‐cell autophagy in the pathogenesis of SLE had not previously been studied. Autophagy is an ATG‐family‐protein‐ regulated intracellular mechanism for the vesicular catabolism and recycling of harmful or superfluous cell contents and, therefore, its regulation is important for cell survival and apoptosis. Programmed cell death of autoreactive cells at early stages of ontogeny prevents autoimmunity, indicating that autophagy might be involved in the control of tolerance checkpoints that prevent autoreactive B‐cell and T‐cell survival. The researchers found that B‐cell autophagy was activated in the early stages of disease in an SLE mouse model (NZB/W F1 mice) and also in patients with SLE. Patients with SLE were excluded from the study if they were being, or had been, treated with rituximab (≤1 year) or methylprednisolone (≤1 month). Using multispectral‐imaging flow cytometry and a tracer dye (CytoID, Enzo Life Sciences) specific for an ATG family member (LC3) the researchers found that, compared with healthy individuals, B cells from patients with SLE (n = 43) had more autophagosomes; disease activity (SELENA‐SLEDAI) correlated positively with autophagosome density. Many pharmaceuticals inhibit autophagy and could, therefore, be used as novel treatments for SLE. One difficulty in translating these exciting new findings to the bedside, however, is that autophagy is a physiological mechanism required for the viability and proliferation of all cells, not just autoreactive cells. Vyse speculates “the potential therapeutic role for inhibition of autophagy in lymphocytes in SLE and other autoimmune diseases should be explored,” but he agrees that targeting autoreactive cells would be preferential.