Yumi Tsuchida

Polygenic burdens on cell-specific pathways underlie the risk of rheumatoid arthritis

Recent evidence suggests that a substantial portion of complex disease risk alleles modify gene expression in a cell-specific manner. To identify candidate causal genes and biological pathways of immune-related complex diseases, we conducted expression quantitative trait loci (eQTL) analysis on five subsets of immune cells (CD4+ T cells, CD8+ T cells, B cells, natural killer (NK) cells and monocytes) and unfractionated peripheral blood from 105 healthy Japanese volunteers. We developed a three-step analytical pipeline comprising (i) prediction of individual gene expression using our eQTL database and public epigenomic data, (ii) gene-level association analysis and (iii) prediction of cell-specific pathway activity by integrating the direction of eQTL effects. By applying this pipeline to rheumatoid arthritis data sets, we identified candidate causal genes and a cytokine pathway (upregulation of tumor necrosis factor (TNF) in CD4+ T cells). Our approach is an efficient way to characterize the polygenic contributions and potential biological mechanisms of complex diseases.

Immunophenotyping of rheumatoid arthritis reveals a linkage between HLA-DRB1 genotype, CXCR4 expression on memory CD4+ T cells, and disease activity

Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease that leads to destructive arthritis. Although the HLA class II locus is the strongest genetic risk factor for rheumatoid arthritis, the relationship between HLA class II alleles and lymphocyte activation remains unclear. We performed immunophenotyping of peripheral blood mononuclear cells on 91 HLA-DRB1-genotyped RA patients and 110 healthy donors. The frequency of memory CXCR4+CD4+ T cells, and not Th1 and Th17 cells, was significantly associated with disease severity by multiple linear regression analysis. RA patients with one or more susceptible HLA-DR haplotypes (shared epitope: SE) displayed a significantly higher frequency of memory CXCR4+CD4+ T cells. Moreover, the frequency of memory CXCR4+CD4+ T cells significantly correlated with the expression level of HLA-DR on B cells, which was elevated in RA patients with SE. In vitro analysis and transcriptomic pathway analysis suggested that the interaction between HLA-DR and T cell receptors is an important regulator of memory CXCR4+CD4+ T cells. Clinically, a higher frequency of memory CXCR4+CD4+ T cells predicted a better response to CTLA4-Ig. Memory CXCR4+CD4+ T cells may serve as a powerful biomarker for unraveling the linkage between HLA-DRB1 genotype and disease activity in RA.

Characteristics of granulomatosis with polyangiitis patients in Japan

Abstract Objectives. Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is a disease with significant ethnic differences. Reports on characteristics of Japanese granulomatosis with polyangiitis (GPA) patients are limited, and this study was undertaken to determine the characteristics of Japanese GPA patients. Methods. This was a retrospective chart study of 24 Japanese GPA patients. GPA was defined according to the European Medicines Agency algorithm. Results. The percentage of MPO-ANCA-positive patients was 33.3%, higher than the percentages reported in studies from Western countries. MPO-ANCA-positive GPA patients differed from PR3-ANCA-positive GPA patients in organs involved at diagnosis with MPO-ANCA-positive patients having nose and sinus involvement less frequently compared to PR3-ANCA-positive patients. Interstitial lung infiltrates were more common among MPO-ANCA-positive GPA patients compared to PR3-ANCA-positive GPA patients. Conclusion. Among Japanese GPA patients, the proportion of MPO-ANCA-positive patients is higher compared to reports from Western countries, and those patients are often different from the classical picture of GPA.

TGF-β3 Inhibits Antibody Production by Human B Cells

TGF-β is a pleotropic cytokine involved in various biological processes. Of the three isoforms of TGF-β, TGF-β1 has long been recognized as an important inhibitory cytokine in the immune system and has been reported to inhibit B cell function in both mice and humans. Recently, it has been suggested that TGF-β3 may play an important role in the regulation of immune system in mice. Murine CD4+CD25-LAG3+ regulatory T cells suppress B cell function through the production of TGF-β3, and it has been reported that TGF-β3 is therapeutic in a mouse model of systemic lupus erythematosus. The effect of TGF-β3 on human B cells has not been reported, and we herein examined the effect of TGF-β3 on human B cells. TGF-β3 suppressed B cell survival, proliferation, differentiation into plasmablasts, and antibody secretion. Although the suppression of human B cells by TGF-β1 has long been recognized, the precise mechanism for the suppression of B cell function by TGF-β1 remains elusive; therefore, we examined the effect of TGF-β1 and β3 on pathways important in B cell activation and differentiation. TGF-β1 and TGF-β3 inhibited some of the key molecules of the cell cycle, as well as transcription factors important in B cell differentiation into antibody secreting cells such as IRF4, Blimp-1, and XBP1. TGF-β1 and β3 also inhibited B cell receptor signaling. Our results suggest that TGF-β3 modifying therapy might be therapeutic in autoimmune diseases with B cell dysregulation in humans.

A Case of Polymyxin b-Immobilized Fiber Column Treatment for Rapidly Progressive Interstitial Pneumonia Associated with Clinically Amyopathic Dermatomyositis

We report a case of rapidly progressive interstitial pneumonia associated with clinically amyopathic dermatomyositis who responded to single course of polymyxin b-immobilized fiber column treatment. Initial treatment with pulsed corticosteroids and cyclophosphamide, intravenous immunoglobulin, and cyclosporine seemed to suppress the activity of interstitial lung disease temporarily, but signs of relapse were detected such as elevation of serum KL-6 level and progressing pulmonary shadows in chest computed tomography scan. After polymyxin b-immobilized fiber column treatment, the areas of pulmonary shadows drastically decreased. Gradually, arterial partial pressure of oxygen/fraction of inspired oxygen (PaO2/FiO2) ratio recovered, and serum ferritin level and KL-6 level decreased. These findings indicate that polymyxin b-immobilized fiber column treatment could be promising in combination with conventional therapy for rapidly progressive interstitial pneumonia associated with clinically amyopathic dermatomyositis, especially at the early phase of relapse.

Macrophage activation syndrome associated with tocilizumab treatment in adult-onset Still’s disease

We read with interest the article by Bannai et al. published in the March 2016 issue titled ‘‘Successful tocilizumab therapy in seven patients with refractory adult-onset Still’s disease [1].’’ In this article, the authors describe seven patients with adult-onset Still’s disease (AOSD) who were treated with tocilizumab (TCZ), including a patient who developed macrophage activation syndrome (MAS) following TCZ treatment, and they argue for the need to monitor patients for signs of MAS when administering TCZ to patients with active AOSD. At our institution, there was also a patient with AOSD who developed signs of MAS after administration of TCZ for active AOSD. Development of MAS after the administration of TCZ for AOSD may be more common than previously thought, and rheumatologists need to be careful when initiating TCZ for active AOSD. The patient was a Japanese female with a previous history of Grave’s disease that had been treated with radioiodine ablation and surgical therapy. She developed spiking fever, arthralgia, and salmon colored rash, and she was diagnosed with AOSD when she was 19 years old. She was initially treated at another hospital with high-dose steroids, intravenous cyclophosphamide, cyclosporine (CyA), and leukocytapheresis (LCAP), and her symptoms resolved. Half a year later, while being treated with betamethasone 1.5 mg/ day and cyclosporine, she experienced a relapse. LCAP was performed again but was ineffective. Betamethasone was increased to 3 mg/day and methotrexate was started; however, her fever persisted, and she was referred to our hospital for further therapy. Upon referral to our hospital, she was taking betamethasone 3 mg/day, methotrexate 15 mg/week, and cyclosporine 100 mg/day, and she still had spiking fever, rash, and arthralgia. C-reactive protein level (CRP) was 20.21 mg/dl, and serum ferritin was 24,165 ng/ml. Her steroids were further increased to prednisolone (PSL) 40 mg/day, and the dosage of CyA was also increased. After the fever resolved and CRP decreased to 4.91 mg/dl, TCZ was administered at 8mg/kg. Within a day, she developed a fever. Her platelet levels decreased from 2.8 10/mL to 1.6 10/mL in two days, and ferritin increased to 14,254 ng/mL. AST increased to 91 IU/L, and fibrinogen decreased to 270 mg/dl. Her laboratory data met the PRINTO diagnostic criteria for MAS [2]. CMV antigenemia was negative, and there were no signs suggesting an infectious cause for MAS. She was treated with pulse methylprednisolone therapy followed by PSL 65 mg/day. CyA was increased to 350 mg/day to obtain a trough level of 135 ng/ml. After her fever, rash, and arthralgia resolved and CRP levels normalized, tocilizumab was again administered. There were no signs of MAS, and TCZ was continued every two weeks along with steroids, methotrexate, and cyclosporine, and her steroids were successfully tapered. Half a year later, her steroids had been tapered to PSL 15 mg/day and her disease was still stable, and tocilizumab treatment was changed from 8 mg/kg every two weeks to 8mg/kg every four weeks with no signs of relapse. Three years later, while taking PSL 15 mg/day, CyA 300 mg/day, and MTX 7.5 mg/week in addition to tocilizumab, she expressed wishes to have a child. Although she experienced minor flares that required short-term increase in steroids, methotrexate successfully was tapered and discontinued. Now, five years after initiation of TCZ treatment, she is being treated with tocilizumab 8 mg/kg every four weeks, PSL 6 mg/day, and CyA 200 mg/day and is in remission. Although we cannot decisively conclude if TCZ administration caused MAS in this patient, the case reported by Bannai et al. [1] and this case suggest that MAS might be induced when TCZ is administered to active AOSD patients. It has been suggested that various inflammatory cytokines in addition to IL-6, including IL-1, IL-18, and IFN-g, play important roles in the development of MAS [3], and it is possible that blocking of a single cytokine by tocilizumab may lead to increased levels of other inflammatory cytokines, perhaps through a negative feedback loop, and thus MAS. There have been many reports of TCZ therapy successfully initiated in highly active AOSD with higher CRP and/or ferritin levels compared to our patient [4–7]. Traditional disease activity parameters such as CRP and ferritin are not able to predict the development of MAS following TCZ therapy, and other disease parameters that can predict this phenomenon need to be investigated. In the meantime, rheumatologists need to be careful in monitoring for signs of MAS when administering TCZ for active AOSD.

Increased serum concentrations of IL-1 beta, IL-21 and Th17 cells in overweight patients with rheumatoid arthritis

BackgroundsObesity is associated with worse disease activity and drug responses in patients with rheumatoid arthritis (RA). However, the immunological mechanisms responsible for the relationship between RA and obesity have not yet been clarified in detail. This study aimed to elucidate the immunological mechanisms contributing to the pathogenesis of RA in overweight patients.MethodsThe frequencies of CD4+ T cell, B cell and monocyte subsets were analyzed in RA (n = 81) and healthy donors (n = 99) by flow cytometry, and were compared between three groups (body mass index (BMI) <20,  ≥20 to 25, >25). Serum cytokines were measured using multiplex ELISA. Gene expression was analyzed by quantitative PCR. Clinical information was extracted from medical records.ResultsThe frequencies of T helper (Th)17 (CD4+CD45RA-CXCR5-CXCR3-CCR6+) cells and plasmablasts (PB) were significantly increased in patients with RA with BMI >25. Significant correlation was observed between BMI and Th17 cells in patients with RA. No significant differences in cell frequencies between the three BMI groups were observed in the healthy donors. Serum interleukin (IL)-1β and IL-21 significantly correlated with BMI in RA patients. Gene expression patterns in Th17 cells from overweight patients with RA showed the characteristics of pathogenic Th17 cells.ConclusionsQuantitative and qualitative changes in Th17 cells were characteristic in overweight patients with RA.

HLA-DRB1 Shared Epitope Alleles and Disease Activity Are Correlated with Reduced T Cell Receptor Repertoire Diversity in CD4+ T Cells in Rheumatoid Arthritis

Objective. Shared epitope (SE) alleles are the most significant genetic susceptibility locus in rheumatoid arthritis (RA); however, their target populations in CD4+ T cells are not well elucidated. We analyzed the association between SE alleles and the T cell receptor (TCR) repertoire diversity of naive and memory CD4+ T cells using next-generation sequencing (NGS). Methods. The TCR beta chains in naive and memory CD4+ T cells from the peripheral blood of 22 patients with RA and 18 age- and sex-matched healthy donors (HD) were analyzed by NGS. The Renyi entropy was used to evaluate TCR repertoire diversity and its correlations with SE alleles and other variables were examined. Serum cytokine levels were measured by multiplex ELISA. Results. The TCR repertoire diversity in memory CD4+ T cells was reduced in SE allele-positive patients with RA compared with HD, and showed a significant negative correlation with the SE allele dosage in RA. The TCR repertoire diversity of naive and memory T cells was also negatively correlated with disease activity, and the SE allele dosage and disease activity were independently associated with reduced TCR repertoire diversity. TCR repertoire diversity showed a significant positive correlation with the serum interleukin 2 levels. Conclusion. SE alleles and disease activity were negatively correlated with the TCR repertoire diversity of CD4+ T cells in RA. Considering the pivotal role of CD4+ T cells in RA, restoring the altered TCR repertoire diversity will provide a potential RA therapeutic target.

Transcriptome analysis of peripheral blood from patients with rheumatoid arthritis: a systematic review

In the era of precision medicine, transcriptome analysis of whole gene expression is an essential technology. While DNA microarray has a limited dynamic range and a problem of background hybridization, RNA sequencing (RNA-seq) has a broader dynamic range and a lower background signal that increase the sensitivity and reproducibility. While transcriptome analyses in rheumatoid arthritis (RA) have generally focused on whole peripheral blood mononuclear cells (PBMC), analyses of detailed cell subsets have an increased need for understanding the pathophysiology of disease because the involvement of CD4+ T cells in the pathogenesis of RA has been established. Transcriptome analysis of detailed CD4+ T cell subsets or neutrophils shed new light on the pathophysiology of RA. There are several analyses about the effect of biological treatment. Many studies report the association between type I interferon signature gene expression and response to therapy.