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Neutrophils form neutrophil extracellular traps (NETs), which are involved in the pathogenesis of ANCA-associated vasculitis (AAV). Recent reports suggest that platelets stimulated via toll-like receptor (TLR) pathways can induce NETs formation. However, the mechanism underlying the involvement of platelets in NETs formation in AAV is unknown. We investigated the role of platelets in the pathogenesis of AAV. Platelets from AAV patients and healthy controls (HCs) were co-cultured with peripheral neutrophils, and NETs formation was visualized and quantified. The expression levels of TLRs on platelets were examined by flow cytometry. Platelets were treated with a TLR agonist, platelet-derived humoral factor, CXCL4 (platelet factor 4: PF4), and/or anti-CXCL4 antibody to investigate the effects of TLR–CXCL4 signaling on NETs formation. Platelets from AAV significantly upregulated NETs formation in vitro. Flow cytometric analysis revealed that the proportion of TLR9 positive platelets was significantly higher in AAV than HCs. CXCL4 released from TLR9 agonist-stimulated platelets was significantly enhanced in AAV, which subsequently increased NETs formation. Further, neutralizing anti-CXCL4 antibody significantly inhibited NETs formation enhanced by platelets from AAV. TLR9 signaling and CXCL4 release underlie the key role that platelets play in NETs formation in the pathogenesis of AAV.

Background Interstitial lung disease (ILD) is a serious complication of connective tissue diseases (CTDs). Although immune dysregulation triggered by genetic and environmental factors is thought to provoke inflammation and subsequent fibrosis, precise mechanisms of these processes remain unclear. Recent reports suggest that activation of aryl hydrocarbon receptor (AhR) signals by various ligands such as tryptophan derivatives can induce hyper-immune responses and are involved in autoimmunity. We investigated the effects of AhR signals on the process of lung fibrosis and changes in immunological features using a bleomycin (BLM)-induced lung fibrosis mouse model. Methods BLM was administered intratracheally to C57BL/6JJcl mice and either 5,11-dihydroindolo[3,2-b]carbazole-6-carboxaldehyde (FICZ), a natural AhR ligand, or vehicle was subsequently injected intraperitoneally on day 0, 1, and 2 from BLM administration. Mice were sacrificed at week 3, and lung fibrosis was quantified by the histological changes using the Ashcroft score and deposition of soluble collagen levels in the lung using Sircol assay. The population of immune cells infiltrated into the lungs was analyzed using flow cytometry. Results Both the Ashcroft score and soluble collagen level in FICZ-treated mice were significantly lower than those in the vehicle group. Moreover, the survival rate of FICZ-treated mice was significantly higher than that of control mice during the 3 weeks after treatment. Interestingly, flow cytometric analysis revealed that the number of CD4 + Foxp3 + regulatory T cells (Tregs) was significantly increased and CD4 + IFNγ + and γδ + IL-17A + T cells were decreased in the lungs of FICZ-treated mice, while the total number of T, B, and NK cells were unaffected by FICZ treatment. Conclusions Our findings suggest that stimulation of AhR signals attenuated lung fibrosis by increasing Tregs and suppressing inflammatory T cell subsets in a BLM-induced fibrosis model. AhR signaling pathways may therefore be useful therapeutic targets for connective tissue disease-associated ILD .

Sustained clinical remission (CR) without drug treatment has not been achieved in patients with rheumatoid arthritis (RA). This implies a substantial difference between CR and the healthy state, but it has yet to be quantified. We report a longitudinal monitoring of the drug response at multi-omics levels in the peripheral blood of patients with RA. Our data reveal that drug treatments alter the molecular profile closer to that of HCs at the transcriptome, serum proteome, and immunophenotype level. Patient follow-up suggests that the molecular profile after drug treatments is associated with long-term stable CR. In addition, we identify molecular signatures that are resistant to drug treatments. These signatures are associated with RA independently of known disease severity indexes and are largely explained by the imbalance of neutrophils, monocytes, and lymphocytes. This high-dimensional phenotyping provides a quantitative measure of molecular remission and illustrates a multi-omics approach to understanding drug response. Little information is available on molecular changes in response to treatment of rheumatoid arthritis (RA). Here the authors report a multi-omics study collecting patients' transcriptome, proteome, and immunophenotype data to help understand the impact of drug treatments on RA molecular phenotypes.

Background We reported that the production of BAFF (B cell-activating factor) and IL-6, both of which are involved in survival and differentiation of B cells, is dysregulated in monocytes of patients with primary Sjögren’s syndrome (pSS). In this study, we investigate the relationship between possible aberrations of pSS monocytes and clinical features of pSS patients and the contribution of monocytes to B cell activation, a mechanism involved in the pathogenesis of pSS. Methods Expression of BAFF-receptor (BR3) on peripheral monocytes from patients with pSS ( n  = 67) and healthy controls (HC: n  = 37) was analyzed by FACS. Peripheral monocytes were stimulated with BAFF, and IL-6 production by the cells was measured by ELISA. Peripheral B cells were cultured with BAFF-stimulated monocytes in the presence or absence of anti-IL-6 receptor antibody, and IgG production by the cells was measured by ELISA. Patients’ serological data were collected from their clinical records. Patients’ disease activity was quantified based on their EULAR Sjögren’s syndrome disease activity index (ESSDAI) scores. Results The proportion of peripheral BR3-positive monocytes (BR3 + /CD14 + ) was significantly increased in pSS patients compared to HC. Moreover, IL-6 production by BAFF-stimulated monocytes was remarkably higher than HC and was significantly correlated with BR3 + /CD14 + ratios of patients. In addition, BR3 expression on pSS monocytes was elevated in anti-Ro/SSA and/or anti-La/SSB positive compared to negative patients. Remarkably, BR3 expression on peripheral monocytes was positively and significantly correlated with patients’ serum IgG and IgM levels and ESSDAI scores. Moreover, the amount of IgG produced by B cells was markedly higher in pSS patients compared to HC when the cells were co-cultured with BAFF-stimulated autologous monocytes in vitro. Notably, addition of anti-IL-6 receptor antibody into the co-culture system led to inhibition of IgG production by B cells. Conclusions Our data suggest that elevated BR3 expression in monocytes is associated with clinical features in pSS patients and that enhanced production of IL-6 by BAFF-stimulated monocytes plays a part in the overproduction of IgG by B cells in pSS. These results suggest that BAFF signaling pathways through BR3 in monocytes are possible therapeutic targets for pSS.

Multiple studies suggest that interleukin (IL)-21 plays a pivotal role in the differentiation of B cells and activation of cytotoxic T cells and is involved in the pathogenesis of IgG4-related disease (IgG4-RD). T cell immunoreceptor with immunoglobulin and ITIM domain (TIGIT) is a new marker of T follicular helper (Tfh) cells, yet its significance remains unknown. The objective of this study was to investigate whether TIGIT expression could detect high IL-21-producing peripheral Tfh populations and their association with disease activity in IgG4-RD. TIGIT expression in peripheral CD4+T cell subsets was comprehensively analyzed by multi-color flow cytometry. Single cell mapping was performed by t-SNE method, and IL-21 production was compared in TIGIT+ and TIGIT-T cells. The effect of OX40 signal on cytokine expression was analyzed by RNA-sequencing. Clinical significance of TIGIT+ and TIGIT- peripheral T cells was analyzed in active patients with IgG4-RD, both at baseline and after 12 weeks of glucocorticoid treatment. Unbiased single cell mapping revealed two high IL-21-producing peripheral T cell populations; TIGIT+ Tfh and TIGIT-T helper cells. OX40 signal was associated with high IL-21 production in TIGIT+ Tfh and TIGIT-T helper cells. IL-21 production in Tfh cells correlated with the proportion of TIGIT+ cells in Tfh cells, serum IgG4 level, and scores of disease activity. Furthermore, the skewing toward peripheral TIGIT+ Tfh cells, particularly TIGIT+Tfh2 subset correlated with disease activity and was corrected by glucocorticoid treatment in IgG4-RD. OX40 is associated with high IL-21 production in peripheral TIGIT+ Tfh cells, and the increase in peripheral TIGIT+ Tfh cells reflects disease activity in IgG4-RD.

Background Large vessel vasculitis (LVV) is a type of vasculitis characterized by granulomatous inflammation of medium- and large-sized arteries. Clinical assessment of acute phase reactants has been conventionally used to diagnose and monitor diseases; however, accurate assessment of vascular disease activity status can be difficult. In this study, we investigated comprehensive immuno-phenotyping to explore useful biomarkers associated with clinical characteristics. Methods Consecutive patients with newly diagnosed LVV who visited our institution between May 2016 and May 2019 were enrolled. The number of circulating T cells, B cells, natural killer cells, dendritic cells, monocytes, and granulocytes was examined and chronologically followed. Baseline and time-course changes in immuno-phenotyping associated with disease activity were assessed. Results Comprehensive immuno-phenotyping data from 90 samples from each of 20 patients with LVV were compared with those from healthy controls (HCs). The number of helper T (Th), follicular helper T (Tfh), CD8 + T, CD14 ++ CD16 + monocytes, and neutrophils were higher in patients with giant cell arteritis (GCA) and/or Takayasu arteritis (TAK) than in HCs. Among them, the number of CD8 + T and CD8 + Tem were higher in patients with TAK than in GCA. Notably, memory CD4 + and CD8 + T cells in patients with TAK remained high even in the remission phase. Further analysis revealed that the number of Th1, Th17, and Tfh cells was associated with disease relapse in GCA and TAK and that the number of CD8 + T cells was associated with relapse in TAK. Th1, Th17, and Tfh cells decreased after treatment with biologic agents, while CD8 + T cells did not. Conclusions Our results from peripheral immuno-phenotyping analysis indicate that the numbers of Th and Tfh cells changed along with the disease condition in both GCA and TAK, while that of CD8 + T cells did not, especially in TAK. Treatment with biologic agents decreased the proportion of Th and Tfh cells, but not CD8 + T cells, in the patients. Chronological immuno-phenotyping data explained the difference in therapeutic response, such as reactivities against biologics, between GCA and TAK.

Background Leukocyte activation by anti-neutrophil cytoplasmic antibody (ANCA) and the subsequent leukocyte–endothelium interaction play a key role in the development of endothelial damage in ANCA-associated vasculitis (AAV). In contrast to that of leukocyte activation, the exact role of the leukocyte–endothelium interaction via integrin remains unclear. Here, we performed microarray and validation analyses to explore association between the expression levels of lymphocyte function-associated antigen-1 (LFA-1) and the clinical characteristics of patients with AAV. Methods We performed gene set enrichment analysis (GSEA) to identify the functional gene sets differentially expressed between patients with AAV and other types of vasculitis and the healthy controls (HCs). Flow cytometry was performed to validate the GSEA results. Treatment-naïve patients were monitored until 24 weeks of treatment. To examine the role of LFA-1 in the neutrophil–endothelium interaction, we performed a leukocyte adhesion and transmigration assay using peripheral blood and human umbilical vein endothelial cells (HUVECs). Results GSEA revealed that the molecular pathways involving integrin-related genes were significantly upregulated in patients with AAV compared to that in patients with other types of vasculitis and the HCs. Flow cytometry revealed that the percentage of neutrophils expressing LFA-1 was significantly higher in patients with AAV than in those with large-vessel vasculitis or polyarteritis nodosa and the HCs. LFA-1 levels in the neutrophils were higher in patients with MPO-ANCA-positive expression than in those with a positive PR3-ANCA expression and correlated with the peripheral eosinophil count, serum rheumatoid factor titre, serum C-reactive protein levels, and the vasculitis activity score of systemic and chest components. After 24 weeks of treatment, including prednisolone, cyclophosphamide, rituximab, azathioprine, methotrexate, and/or tacrolimus, neutrophil LFA-1 expression remained high in the non-responder patients, but decreased in the responder patients. The in vitro assay showed that leukocyte migration toward HUVECs was dependent on the interaction between LFA-1 and intercellular adhesion molecule-1 (ICAM1); the migration of leukocytes was inhibited by blocking the adhesion of LFA-1 to ICAM1. Conclusions The expression of LFA-1 in neutrophils is increased in patients with AAV. Neutrophil LFA-1 levels correlate with the clinical features of AAV. Inhibiting the adhesion of LFA-1 and ICAM1 impedes the neutrophil–endothelium interaction and may have a therapeutic role in AAV.

Background Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is an autoimmune disease that affects small- to medium-sized blood vessels. Despite treatments having been improved, patients often experience disease relapses. It remains unclear how the immune cells involve in the development of vasculitis and how they fluctuate over the course of treatment. In this study, we aimed to identify the immune subsets and serum cytokines associated with disease relapse by comprehensive immuno-phenotyping in AAV patients. Methods We reviewed consecutive patients ( n  = 29) from Keio University Hospital who had been newly diagnosed with AAV from January 2015 to February 2019 and chronologically followed until 52 weeks. Numbers of circulating T cells, B cells, monocytes, and granulocytes were analyzed by flow cytometry (FACS). Serum levels of cytokines were measured by electrochemiluminescence enzyme immunoassay. Clinical information was obtained from patients’ records and association with time-course changes in immuno-phenotypes and serum levels of cytokines were assessed. Results Comprehensive immuno-phenotyping data from 161 samples from 29 AAV patients at diagnosis; at weeks 4, 12, 24, and 52 of treatment; and at time of major relapse were examined. FACS analysis from patients with relapse revealed that CD14 ++ CD16 + intermediate monocytes and plasma cells concomitantly changed associated with disease relapse, which were independent from treatment regimen, ANCA status, or disease phenotype. In particular, the number of CD14 ++ CD16 + intermediate monocytes at relapse was significantly higher than that in remission or in healthy controls. Serum cytokine measurement revealed that changes of monocyte-derived proinflammatory cytokines such as IL-1β, IL-6, IL-8, and TNF-α were associated with disease status. Conclusions Chronological changes in CD14 ++ CD16 + intermediate monocyte counts can be a marker of disease relapse in AAV patients.

We explored effective therapeutic targets for systemic sclerosis (SSc) patients with high risk for pulmonary arterial hypertension (PAH) by plasma proteomics analysis. A total of fifty-seven patients with SSc were enrolled in the study and the prevalence of PAH was 19.3%. On the other hand, 75.4% of SSc patients showed the ratio of forced vital capacity percentage/diffusing capacity of the lungs for carbon monoxide percentage> 1.6 and met criteria for high risk of PAH. Identification of elevated plasma proteins in SSc patients with high risk of PAH, followed by upstream regulator analysis, predicted interleukin (IL)-17A as a major upstream molecule. Furthermore, we performed in vitro neutralization study using MT-6194, a bispecific antibody targeting both IL-17A and IL-6 receptor. We found that MT-6194 broadly suppressed the increased expression of downstream molecules of IL-17A including IL-17A-related cytokines/chemokines, IL-17A-driven NF B pathway and IL-6-driven JAK/STAT pathway which were shown to be increased in SSc patients with high risk of PAH by the proteomics. Consequently, it is revealed that IL-17A is a promising target for early intervention in SSc patients with high risk for PAH.

Background Giant cell arteritis (GCA) is a primary large-vessel vasculitis (LVV) of unknown origin. Its management is a challenge due to the late onset of disease symptoms and frequent relapse; therefore, clarifying the pathophysiology of GCA is essential to improving treatment. This study aimed to identify the transition of molecular signatures in immune cells relevant to GCA pathogenesis by analyzing longitudinal transcriptome data in patients. Methods We analyzed the whole blood transcriptome of treatment-naive patients with GCA, patients with Takayasu arteritis (TAK), age-matched, old healthy controls (HCs), and young HCs. Characteristic genes for GCA were identified, and the longitudinal transition of those genes was analyzed using cell-type identification by estimating relative subsets of RNA transcripts (CIBERSORT). Results Repeated measures analysis of variance revealed 739 differentially expressed genes among all patients and HCs. Of the 739 genes, 15 were characteristically upregulated and 36 were downregulated in patients with GCA compared to those with TAK and HCs. Pathway enrichment analysis showed that downregulated genes in GCA were associated with B cell activation. CIBERSORT analysis revealed that upregulation of “M0-macrophages” and downregulation of B cells were characteristic of GCA. Upregulation of “M0-macrophages” reflects the activation of monocytes in GCA toward M0-like phenotypes, which persisted under 6 weeks of treatment. Combined treatment with prednisolone and an interleukin-6 receptor antagonist normalized molecular profiles more efficiently than prednisolone monotherapy. Conclusions Gene signatures of monocyte activation and B cell inactivation were characteristic of GCA and associated with treatment response.