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ObjectivesLittle is known about the immunology underlying variable treatment response in rheumatoid arthritis (RA). We performed large scale transcriptome analyses of peripheral blood immune cell subsets to identify immune cells that predict treatment resistance.MethodsWe isolated 18 peripheral blood immune cell subsets of 55 pre-treatment RA patients and 39 healthy controls, and performed RNA sequencing. Transcriptome changes in RA and treatment effects were systematically characterized. Association between immune cell gene modules and treatment resistance was evaluated. We validated predictive value of identified parameters for treatment resistance using quantitative polymerase chain reaction (qPCR) and mass cytometric analysis cohorts. We also characterized the identified population by synovial single cell RNA-seq analysis.ResultsImmune cells of RA patients were characterized by enhanced interferon and IL6-JAK-STAT3 signaling that demonstrate partial normalization after treatment. A gene expression module of plasmacytoid dendritic cells (pDC) reflecting the expansion of pre-dendritic cells (pre-DC) exhibited strongest association with treatment resistance. Type I interferon signaling was negatively correlated to pre-DC gene expression. qPCR and mass cytometric analysis in independent cohorts validated that the pre-DC associated gene expression and the proportion of pre-DC were significantly higher before treatment in treatment-resistant patients. A cluster of synovial DCs showed both features of pre-DC and proinflammatory conventional DC2s.ConclusionsAn increase in pre-DC in peripheral blood predicted RA treatment resistance. Pre-DC could have pathophysiological relevance to RA treatment response.Key messagesWhat is already known about this subject?Limited information is available about the immune cells that are associated with RA treatment resistance.What does this study add?RA treatment resistance can be predicted by an increase in pre-DC in peripheral blood prior to treatment.The expression of genes reflecting an increase in pre-DC is negatively correlated to the type I interferon signature, which is associated with good therapeutic response.Synovial pre-DC-like cells are proinflammatory cDC2s.How might this impact on clinical practice or future developments?Stratified treatment of RA is possible using pre-DC as a biomarker, and it might be possible to develop new therapies for treatment-resistant RA by targeting pre-DC.

Background Behçet’s syndrome (BS) is an immune-mediated disease characterized by recurrent oral ulcers, genital ulcers, uveitis, and skin symptoms. HLA-B51, as well as other genetic polymorphisms, has been reported to be associated with BS; however, the pathogenesis of BS and its relationship to genetic risk factors still remain unclear. To address these points, we performed immunophenotyping and transcriptome analysis of immune cells from BS patients and healthy donors. Methods ImmuNexUT is a comprehensive database consisting of RNA sequencing data and eQTL database of immune cell subsets from patients with immune-mediated diseases and healthy donors, and flow cytometry data and transcriptome data from 23 BS patients and 28 healthy donors from the ImmuNexUT study were utilized for this study. Differential gene expression analysis and weighted gene co-expression network analysis (WGCNA) were performed to identify genes associated with BS and clinical features of BS. eQTL database was used to assess the relationship between genetic risk factors of BS with those genes. Results The frequency of Th17 cells was increased in BS patients, and transcriptome analysis of Th17 cells suggested the activation of the NFκB pathway in Th17 cells of BS patients. Next, WGCNA was used to group genes into modules with similar expression patterns in each subset. Modules of antigen-presenting cells were associated with BS, and pathway analysis suggested the activation of antigen-presenting cells of BS patients. Further examination of genes in BS-associated modules indicated that the expression of YBX3, a member of a plasmacytoid dendritic cell (pDC) gene module associated with BS, is influenced by a BS risk polymorphism, rs2617170, in pDCs, suggesting that YBX3 may be a key molecule connecting genetic risk factors of BS with disease pathogenesis. Furthermore, pathway analysis of modules associated with HLA-B51 indicated that the association of IL-17-associated pathways in memory CD8+ T cells with HLA-B51; therefore, IL-17-producing CD8+ T cells, Tc17 cells, may play a critical role in BS. Conclusions Various cells including CD4+ T cells, CD8+ T cells, and antigen-presenting cells are important in the pathogenesis of BS. Tc17 cells and YBX3 may be potential therapeutic targets in BS.

Human Leukocyte Antigen (HLA) alleles regulate susceptibility to rheumatoid arthritis (RA) and immune-mediated diseases. This study aims to elucidate the impact of HLA alleles to T cell subsets.We performed genome-wide and HLA allele association analysis for T cell receptor (TCR) beta chain repertoire in 13 purified T cell subsets from the ImmuNexUT database, consisting of 407 donors with ten immune-mediated diseases and healthy controls.HLA class II alleles were associated with TRBV gene usage and the public clones of CD4 T cells, while HLA class I alleles were associated with CD8 T cells. RA-risk and immune-mediated diseases-risk HLA alleles were associated with TRBV gene usage of naive and effector CD4 T cell subsets and public clones accumulating in Th17. Clonal diversity was independent of HLA alleles and was correlated with transcriptome changes that reflect TCR signaling.This study revealed in vivo evidence that both HLA alleles and environmental factors shape naive and effector TCR repertoires in RA and immune-mediated diseases patients.

Objectives We evaluated flow-cytometric and transcriptome features of peripheral blood immune cells from early-phase (disease duration Methods Fifty Japanese patients with SSc (12 early SSc cases and 38 late SSc cases) and 50 age- and sex-matched healthy controls were enrolled. A comparison of flow-cytometric subset proportions and RNA-sequencing of 24 peripheral blood immune cell subsets was performed. We evaluated differentially expressed genes (DEGs), characterized the co-expressed gene modules, and estimated the composition of subpopulations by deconvolution based on single-cell RNA-sequencing data. As a disease control, idiopathic inflammatory myositis (IIM) patients were also evaluated. Results Analysing the data from early and late SSc, fraction II effector regulatory T cell (Fr. II eTreg) genes showed a remarkable differential gene expression, enriched for genes related to oxidative phosphorylation. Although the flow-cytometric proportion of Fr. II eTregs was not changed in early SSc, deconvolution indicated expansion of the activated subpopulation. Co-expressed gene modules of Fr. II eTregs demonstrated enrichment of the DEGs of early SSc and correlation with the proportion of the activated subpopulation. These results suggested that DEGs in Fr. II eTregs from patients with early SSc were closely associated with the increased proportion of the activated subpopulation. Similar dysregulation of Fr. II eTregs was also observed in data from patients with early IIM. Conclusions RNA-seq of immune cells indicated the dysregulation of Fr. II eTregs in early SSc with increased proportion of the activated subpopulation.

Objective Previous gene expression analyses seeking genes specific to antineutrophil cytoplasmic antibody-associated vasculitis (AAV) have been limited due to crude cell separation and the use of microarrays. This study aims to identify AAV-specific gene expression profiles in a way that overcomes those limitations. Methods Blood samples were collected from 26 AAV patients and 28 healthy controls (HCs). Neutrophils were isolated by negative selection, whereas 19 subsets of peripheral blood mononuclear cells were sorted by fluorescence assisted cell sorting. RNA-sequencing was then conducted for each sample, and iterative weighted gene correlation network analysis (iterativeWGCNA) and random forest were consecutively applied to identify the most influential gene module in distinguishing AAV from HCs. Correlations of the identified module with clinical parameters were evaluated, and the biological role was assessed with hub gene identification and pathway analysis. Particularly, the module's association with neutrophil extracellular trap formation, NETosis, was analyzed. Finally, the module's overlap with GWAS-identified autoimmune disease genes (GADGs) was assessed for validation. Results A neutrophil module (Neu_M20) was ranked top in the random forest analysis among 255 modules created by iterativeWGCNA. Neu_M20 correlated with disease activity and neutrophil counts but not with the presence of antineutrophil cytoplasmic antibody. The module comprised pro-inflammatory genes, including those related to NETosis, supported by experimental evidence. The genes in the module significantly overlapped GADGs. Conclusion We identified the distinct group of pro-inflammatory genes in neutrophils, which characterize AAV. Further investigations are warranted to confirm our findings as they could serve as novel therapeutic targets.

Background. The epidermal growth factors amphiregulin (AREG) and heparin-binding epidermal growth factor (HB-EGF) are implicated in the pathogenesis of several autoimmune diseases, but their clinical and pathological roles in idiopathic inflammatory myopathy (IIM) are unclear. Methods. Serum AREG and HB-EGF levels were measured by ELISA in patients with IIM (n = 37), systemic sclerosis (n = 17), and rheumatoid arthritis (n = 10), and for seven age- and sex-matched healthy controls (HCs). Associations between serum AREG or HB-EGF levels and the clinical parameters were analyzed. Results. Serum AREG levels in IIM patients were significantly elevated compared to those in HCs (median, 20.7 and 10.7 pg/mL, respectively, p = 0.025). In particular, serum AREG levels in IIM patients with interstitial lung disease (ILD) were higher than those of HCs (22.4 pg/mL, p = 0.027). The disease duration in patients with elevated serum AREG levels was significantly shorter compared to those who had normal serum AREG levels (7 and 21 months, respectively, p = 0.0012). Serum HB-EGF levels were significantly increased in IIM patients with elevated CK levels (136.2 pg/mL, p = 0.020) and patients with anti-Mi-2 antibody (183.7 pg/mL, p = 0.045) compared to those in HCs (74.9 pg/mL). Conclusion. These results suggested that AREG could be a promising biomarker associated with early-phase IIM-related ILD, and that HB-EGF expression was associated with muscle injury and regeneration in IIM.

Summary Genetic studies have revealed many variant loci that are associated with immune-mediated diseases. To elucidate the disease pathogenesis, it is essential to understand the function of these variants, especially under disease-associated conditions. Here, we performed a large-scale immune cell gene-expression analysis, together with whole-genome sequence analysis. Our dataset consists of 28 distinct immune cell subsets from 337 patients diagnosed with 10 categories of immune-mediated diseases and 79 healthy volunteers. Our dataset captured distinctive gene-expression profiles across immune cell types and diseases. Expression quantitative trait loci (eQTL) analysis revealed dynamic variations of eQTL effects in the context of immunological conditions, as well as cell types. These cell-type-specific and context-dependent eQTLs showed significant enrichment in immune disease-associated genetic variants, and they implicated the disease-relevant cell types, genes, and environment. This atlas deepens our understanding of the immunogenetic functions of disease-associated variants under in vivo disease conditions.

Objective Immunological disturbances have been reported in systemic sclerosis (SSc). This study assessed the transcriptome disturbances in immune cell subsets in SSc and characterized a disease-related gene network module and immune cell cluster at single cell resolution. Methods Twenty-one Japanese SSc patients were enrolled and compared with 13 age- and sex-matched healthy controls (HC). Nineteen peripheral blood immune cell subsets were sorted by flow cytometry and bulk RNA-seq analysis was performed for each. Differential expression and pathway analyses were conducted. Iterative weighted gene correlation network analysis (iWGCNA) of each subset revealed clustered co-expressed gene network modules. Random forest analysis prioritized a disease-related gene module. Single cell RNA-seq analysis of 878 monocytes was integrated with bulk RNA-seq analysis and with a public database for single cell RNA-seq analysis of SSc patients. Results Inflammatory pathway genes were differentially expressed in widespread immune cell subsets of SSc. An inflammatory gene module from CD16+ monocytes, which included KLF10, PLAUR, JUNB and JUND, showed the greatest discrimination between SSc and HC. One of the clusters of SSc monocytes identified by single-cell RNA-seq analysis characteristically expressed these inflammatory co-expressed genes and was similar to lung infiltrating FCN1hi monocytes expressing IL1B. Conclusions Our integrated analysis of bulk and single cell RNA-seq analysis identified an inflammatory gene module and a cluster of monocytes that are relevant to SSc pathophysiology. They could serve as candidate novel therapeutic targets in SSc.

We successfully fabricate large-sized (20-cm long) graded-index (GI) core polymer optical waveguides with high thermal durability, using the soft-lithography method. Furthermore, we demonstrate that the fabricated GI-core waveguide shows lower inter-channel crosstalk than the SI-core waveguides fabricated using the same method.

We present two methods for fabricating densely-aligned graded-index (GI) multiple-core polymer optical waveguides utilizing soft-lithography and dispensers. For on-board optical interconnects with polymer waveguides, much higher data rate and channel density have been required in the past few years. However, because of the high scattering loss of polymer waveguides, their inter-channel crosstalk is of great concern, when narrower pitch is required. Meanwhile, we have demonstrated low propagation loss and low inter-channel crosstalk of polymer optical waveguides with GI cores due to the optical confinement effect of the GI-core. Hence, the channel density could be increased for GI multiple-core waveguides. In this paper, we successfully fabricate a polymer waveguide with GI cores directly on a substrate utilizing the soft-lithography method or a dispenser. We experimentally confirm that near parabolic refractive index profiles are formed in the parallel cores with 50 μm x 30 μm size at 125-μm pitch in a length of 20 cm. We also use a copolymer for forming the GI profile, and then, confirm the high-temperature stability of the parabolic index profile compared to the one formed with a doped polymer. Finally, we discuss the crosstalk properties of the fabricated waveguides.

A multi-channel polymer optical waveguide with graded-index (GI) cores is successfully fabricated directly on a substrate by means of the soft-lithographic method. The great advantage in the inter-channel crosstalk of densely-aligned GI-core waveguides is demonstrated both theoretically and experimentally.