Cytotoxic Tph subset with low B-cell helper functions and its involvement in systemic lupus erythematosus.

T peripheral helper (Tph) cells are thought to contribute to extra-follicular B cell activation and play a pathogenic role in autoimmune diseases. However, the role of Tph subsets is not fully elucidated. Here, we investigate the immunological functions of Tph subsets and their involvement in systemic lupus erythematosus (SLE). We have defined four Tph subsets (Tph1: CXCR3+CCR6-, Tph2: CXCR3-CCR6-, Tph17: CXCR3-CCR6+, and Tph1-17: CXCR3+CCR6+) and performed RNA sequencing after cell sorting. Tph1 and Tph17 subsets express substantial levels of IL21, indicating B cell helper functions. However, Tph2 and Tph1-17 subsets express low IL21. Interestingly, we have found Tph2 subset express high levels of CX3CR1, GZMB, PRF1, GLNY, S1PR5, TBX21, EOMES, ZNF863, and RUNX3, indicating a feature of CD4+ cytotoxic T lymphocytes. In SLE patients, the frequency of Tph1 and Tph2 subsets are significantly increased and positively correlated with SLE disease activity indexes. Tph1 cells expansion has been observed in patients with cutaneous and musculoskeletal manifestations. On the other hand, Tph2 cell expansion has been found in patients with lupus nephritis in addition to the above manifestations. Our findings imply that Tph1 and Tph2 subsets exert distinct immunological functions and are contributed to the complexity of clinical manifestations in SLE.

Effects of interleukin-6 signal inhibition on Treg subpopulations and association of Tregs with clinical outcomes in rheumatoid arthritis.

OBJECTIVES: Anti-IL-6 receptor antibodies are clinically efficacious in the management of rheumatoid arthritis (RA) with an associated increase in regulatory T cells (Tregs); however, the role of functional Treg subsets has yet to be clarified. This study aimed to evaluate how functional Treg subsets are altered by IL-6 receptor blockade and to analyze the relationship between these Treg subsets and the clinical outcome of RA. METHODS: We collected frozen peripheral blood mononuclear cells (PBMCs) from 40 patients with RA who started tocilizumab (TCZ) with or without MTX and 11 healthy controls (HCs). We fractionated Tregs with flow cytometry based on markers of phenotype and function and measured the proportions of detailed Treg subsets sequentially from baseline to week 52. RESULTS: The proportions of resting Tregs (rTregs) and rTregs+activated Tregs (aTregs) were significantly lower in RA patients at baseline than in HCs. The proportions of all those CD127low Tregs, rTregs, aTregs, and rTregs+aTregs were significantly increased with TCZ treatment. In patients treated with TCZ without MTX, rTreg were increased. Patients with an increase in the proportion of rTregs at week 12 had significantly less arthritis flares during the observation period. CONCLUSIONS: Blocking IL-6 receptor with TCZ increased the proportion of rTregs, a functional Treg subpopulation. Patients with an early increase in rTregs showed a favorable treatment course, and this increase in rTregs may reflect molecular remission induced by IL-6 signal inhibition.

Th17/IL-17A axis is critical for pulmonary arterial hypertension (PAH) in systemic sclerosis (SSc): SSc patients with high levels of serum IL-17A exhibit reduced lung functions and increased prevalence of PAH.

BACKGROUND: It is thought that systemic sclerosis (SSc) might be a T helper 17 (Th17) cell-driven autoimmune disease. Noticeably, pulmonary arterial hypertension (PAH) is a leading cause of death in patients with SSc. Here, we investigated the association between serum Th17-related cytokines and prevalence of PAH in SSc patients. METHODS: This study included 72 SSc patients and 51 healthy controls (HC). We determined clinical manifestations, immunophenotypes including Th subsets in peripheral blood lymphocytes, and the serum levels of interleukin (IL)-17A, IL-17A/F, IL-17B. IL-17C, IL-17D. IL-1ß, IL-6, IL-21, IL-22, and IL-23. RESULTS: The frequency of Th17 cells was significantly increased in SSc patients compared to HC and was positively correlated with the modified Rodnan skin scores. Furthermore, the serum levels of IL-17A, IL-17D, IL-1ß, and IL-6 were significantly increased in SSc patients compared to HC. SSc patients with detected IL-17A showed high levels of IL-17A/F, IL-1ß, IL-6, and IL-22, and high frequency of Th17 cells. Interestingly, these patients exhibited the reduced lung functions and increased prevalence of PAH significantly compared to patients with undetected IL-17A. Similarly, SSc patients with detected IL-17A and high IL-6 (≥1.2 pg/mL) exhibited the decreased lung functions and increased prevalence of PAH compared to patients with undetected IL-17A and low IL-6. CONCLUSION: We found that SSc patients with high levels of serum IL-17A or both IL-17A and IL-6 show reduced lung functions and high prevalence of PAH. Consequently, it is highly probable that Th17/IL-17A axis is critical for the prevalence of PAH in SSc patients.

Significant association of CX3CR1+CD8 T cells with aging and distinct clinical features in Sjögren's syndrome and IgG4-related disease.

OBJECTIVES: Recent studies have implicated cytotoxic CD4 and CD8 T cells in primary Sjögren's syndrome (pSS) and IgG4-related disease (IgG4-RD), but their association with immune aging and organ-specific clinical features remain unclear. CX3CR1 is expressed on cytotoxic CD4 and CD8 T cells. The aim of this study was to determine associations of peripheral CX3CR1+CD4 and CX3CR1+CD8 T cells with aging and clinical features. METHODS: Whole blood samples were freshly obtained from consecutive patients with active, treatment-naïve pSS (n=57), IgG4-RD (n=54), and healthy individuals (n=40) and analysed by flow cytometry for CX3CR1+CD4 and CX3CR1+CD8 proportions. Associations of those T cells with aging and clinical features were determined. RESULTS: CX3CR1+CD4 and CX3CR1+CD8 T cells selectively expressed perforin and granzyme B. Proportions of CX3CR1+CD4 and CX3CR1+CD8 T cells were significantly higher in pSS and IgG4-RD than in healthy individuals. Higher proportions of CX3CR1+CD8 T cells were associated with aging in pSS and IgG4-RD but not in healthy individuals. Sex differences were not associated with proportions of CX3CR1+CD8 T cells. Furthermore, patients with pSS with interstitial lung disease showed higher proportions of CX3CR1+CD8 T cells than those without interstitial lung disease. IgG4-RD patients with retroperitoneal fibrosis and/or aortitis exhibited higher proportions of CX3CR1+CD8 T cells compared with those with Mikulicz's disease. Moreover, proportions of CX3CR1+CD8 T cells were decreased following glucocorticoid treatment in paralleled with clinical improvements in IgG4-RD. CONCLUSIONS: CX3CR1+CD8 T cells might be involved in immune aging and distinct clinical phenotypes of patients with pSS or IgG4-RD.

T follicular helper cells and T peripheral helper cells in rheumatic and musculoskeletal diseases.

Recent technological progress has greatly advanced our understanding of human immunology. In particular, the discovery of human T follicular helper (Tfh) and T peripheral helper (Tph) cells has significantly advanced our understanding of human adaptive immune system. Tfh and Tph cells share similar molecular characteristics and both play critical roles in B cell differentiation and maturation. However, they differ in their functional properties, such as chemokine receptor expression and cytokine production. As a result, Tfh cells are mainly involved in B cell differentiation and maturation in germinal centres of secondary lymphoid tissues, while Tph cells are involved in B cell differentiation and tissue damage in peripheral inflammatory lesions. Importantly, the involvement of Tfh and Tph cells in the pathogenesis of rheumatic and musculoskeletal diseases has become clear. In rheumatoid arthritis and systemic lupus erythematosus, Tph cell infiltration is predominant in peripheral inflammatory lesions, whereas Tfh cell infiltration is predominant in the affected lesions of IgG4-related disease. Therefore, the contribution of Tfh and Tph cells to the development of rheumatic and musculoskeletal diseases varies depending on each disease. In this review, we provide an overview of human Tfh and Tph cells and summarise the latest findings on these novel T cell subsets in various rheumatic and musculoskeletal diseases.

Anti-human-TIGIT agonistic antibody ameliorates autoimmune diseases by inhibiting Tfh and Tph cells and enhancing Treg cells.

T cells play important roles in autoimmune diseases, but it remains unclear how to optimally manipulate them. We focused on the T cell immunoreceptor with Ig and ITIM domains (TIGIT), a coinhibitory molecule that regulates and is expressed in T cells. In autoimmune diseases, the association between TIGIT-expressing cells and pathogenesis and the function of human-TIGIT (hu-TIGIT) signalling modification have not been fully elucidated. Here we generated anti-hu-TIGIT agonistic monoclonal antibodies (mAbs) and generated hu-TIGIT knock-in mice to accurately evaluate the efficacy of mAb function. Our mAb suppressed the activation of CD4+ T cells, especially follicular helper T and peripheral helper T cells that highly expressed TIGIT, and enhanced the suppressive function of naïve regulatory T cells. These results indicate that our mAb has advantages in restoring the imbalance of T cells that are activated in autoimmune diseases and suggest potential clinical applications for anti-hu-TIGIT agonistic mAbs as therapeutic agents.

Sodium benzoate attenuates 2,8-dihydroxyadenine nephropathy by inhibiting monocyte/macrophage TNF-α expression.

Sodium benzoate (SB), a known D-amino acid oxidase (DAO) enzyme inhibitor, has an anti-inflammatory effect, although its role in renal damage has not been explored. 2,8-dihydroxyadenine crystal induced chronic kidney disease, in which TNF-α is involved in the pathogenesis, was established by oral adenine administration in C57BL/6JJcl mice (AdCKD) with or without SB to investigate its renal protective effects. SB significantly attenuated AdCKD by decreasing serum creatinine and urea nitrogen levels, and kidney interstitial fibrosis and tubular atrophy scores. The survival of AdCKD mice improved 2.6-fold by SB administration. SB significantly decreased the number of infiltrating macrophages observed in the positive F4/80 immunohistochemistry area and reduced the expression of macrophage markers and inflammatory genes, including TNF-α, in the kidneys of AdCKD. Human THP-1 cells stimulated with either lipopolysaccharide or TNF-α showed increased expression of inflammatory genes, although this was significantly reduced by SB, confirming the anti-inflammatory effects of SB. SB exhibited renal protective effects in AdCKD in DAO enzyme deficient mice, suggesting that anti-inflammatory effect of SB was independent of DAO enzyme activity. Moreover, binding to motif DNA sequence, protein level, and mRNA level of NF-κB RelB were significantly inhibited by SB in AdCKD kidneys and lipopolysaccharide treated THP-1 cells, respectively. We report that anti-inflammatory property of SB is independent of DAO enzymatic activity and is associated with down regulated NF-κB RelB as well as its downstream inflammatory genes such as TNF-α in AdCKD.

Distinct gene signatures of monocytes and B cells in patients with giant cell arteritis: a longitudinal transcriptome analysis.

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.

Distinct impact of malignancy and allergy on the clinical and immunological features of IgG4-related disease.

OBJECTIVES: To clarify the clinical and immunological characteristics of IgG4-RD based on the underlying diseases. METHODS: Consecutive patients with IgG4-RD treated at Keio University Hospital between 2010 and 2021 were divided according to the presence of malignancy or allergy into three groups. The clinical characteristics and 56 immune cell subsets in the peripheral blood were compared among the groups. RESULTS: Among 123 patients, 18 (14.6%) had malignancy including 4 with allergy (malignancy group), 57 (46.3%) had allergy alone (allergy group), and 48 (39.0%) had neither (idiopathic group). In the malignancy group, the patients were older (70.1 vs. 54.4 vs. 64.9 years, p<0.001), male-dominant (83.3 vs. 42.1 vs. 54.2%, p=0.008), and had smoking habits (77.8 vs. 42.1 vs. 43.8%, p=0.02). They also had significant involvement of the aorta/large vessels (33.3 vs. 7.0 vs. 20.8%, p=0.02), while the patients in the allergy group tended to have orbital/lacrimal gland involvement. Remission and relapse rates were not different between the groups; however, overall survival was significantly poorer in the malignancy group (p=0.02). Comprehensive immunophenotyping of the peripheral blood revealed that the increase in CXCR5+CD2-double negative T cells and the decrease in naive CD8 T cells were characteristic of the malignancy group. CONCLUSIONS: The clinical and immunological phenotypes of IgG4-RD differ among those with underlying diseases.

Disease-specific expansion of CD29+IL-17RA+ T effector cells possessing multiple signalling pathways in spondyloarthritis.

OBJECTIVES: T cells adhere to enthesis fibrocartilage via integrins and intrinsically require IL-17RA-mediated signals to maintain their effector function. We analysed CD29+IL-17RA+ T cells in inflamed lesions and peripheral blood in patients with SpA and investigated their association with disease activity and therapeutic response. METHODS: Transcriptome analysis of synovial fluid T cells from PsA was performed using publicly available bulk cell RNA sequencing data. Blood samples were obtained from healthy controls (n = 37), RA (n = 12), IgG4-related disease (IgG4-RD; n = 12), large vessel vasculitis (LVV; n = 12) and SpA (n = 28) and were analysed by flow cytometry. RESULTS: T cells in the inflamed joints of PsA showed CD29 and IL-17RA expression. CD29+IL-17RA+ T cells showed enriched CXCR3+CD45RA+ effector cells and activation of spleen tyrosine kinase (Syk), nuclear factor κB (NF-κB) and Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathways. The proportion of peripheral blood CD29+IL-17RA+ T cells was significantly increased in patients with SpA compared with patients with RA, IgG4-RD or LVV and in healthy controls. Based on the ASDAS-CRP scores, the proportion of CD29+IL-17RA+ T cells was positively correlated with disease activity in treatment-naïve patients with active SpA. Anti-IL-17 but not anti-TNF monoclonal antibodies reduced CD29+IL-17RA+ T cells. CONCLUSIONS: CD29+IL-17RA+ T effector cells with enhanced Syk, NF-κB and JAK-STAT pathways were specifically increased in SpA and were correlated with disease activity, implicating a role of this newly identified T cell population in the pathogenesis. Anti-IL-17 monoclonal antibodies may be effective for patients by reducing this pathogenic T cell population.

Differences in the strength of inhibition of interleukin-6 signalling by subcutaneous sarilumab and tocilizumab in rheumatoid arthritis patients.

OBJECTIVES: To assess differences in the strength of inhibition of IL-6/STAT3 signalling induced by subcutaneously (sc) administered tocilizumab (TCZ) and sarilumab (SAR). METHODS: Data were collected on patients with rheumatoid arthritis (RA) who achieved low disease activity (Clinical Disease Activity Index [CDAI]≤10) following treatment with weekly or bi-weekly administration of 162 mg sc of TCZ (TCZ qw group, n=8; TCZ q2w group, n=8), bi-weekly doses of 200 mg sc of SAR (SAR q2w group, n=7), or MTX (n=8) as a control. The clinical characteristics of each group were collected, and the serum concentrations of IL-6 and soluble IL-6 receptor (sIL-6R) were measured using ELISA. Whole blood samples from each group were stimulated with 100 ng/ml of IL-6. The proportion of phosphorylated (p)STAT3-positive CD4+ T cells was measured using phosflow cytometric analysis. RESULTS: The proportion of pSTAT3-positive CD4+ T cells following stimulation with 100 ng/ml of recombinant human IL-6 was significantly different among the groups (median 1.8% [0.9-3.0] vs. 7.7% [2.9-8.0] vs. 12.5% [11.4-16.6] vs. 71.5% [68.0-78.5] for the TCZ qw, SAR q2w, TCZ q2w, and MTX control groups, respectively; p<0.01 for all comparisons). CONCLUSIONS: SAR 200 mg q2w showed significantly stronger inhibition of IL-6/STAT3 signalling than TCZ sc q2w but weaker inhibition than TCZ sc qw. The results of this study may be useful for adjusting the IL-6 blockade treatment for patients with RA.

Serum cytokines and bone metabolic markers in patients with rheumatoid arthritis treated with biological disease modifying anti-rheumatic drugs.

INTRODUCTION: /objectives Several biological disease-modifying anti-rheumatic drugs (bDMARDs) have been widely used for the management of rheumatoid arthritis (RA). These drugs target different molecules important for the pathophysiology of RA; however, only a few studies have compared the effects of these biological drugs on cytokines and bone metabolic markers. The main aim of this study is to clarify the effects of bDMARDs with different modes of action on the cytokine and bone metabolic marker levels in patients with RA. METHODS: Patients with RA who were initiated on infliximab, tocilizumab, or abatacept as the first bDMARD were prospectively enrolled in this study. Serum cytokine and bone metabolic marker levels were measured longitudinally, and changes in their levels were compared. RESULTS: A total of 174 patients were enrolled in this study, with 55, 70, and 49 patients in the infliximab, tocilizumab, and abatacept groups, respectively. At six months, despite the similar clinical effectiveness of the three drugs, changes in the cytokine and bone metabolic marker levels were distinct; interferon-γ and tumor necrosis factor-α levels were significantly increased with infliximab, interleukin-6 levels were increased with tocilizumab, and interleukin-1ß and interleukin-8 levels were increased with abatacept treatment. Bone-specific alkaline phosphatase and osteocalcin levels increased more significantly with tocilizumab than with infliximab, while osteopontin and osteonectin levels decreased with infliximab treatment. CONCLUSIONS: bDMARDs with different modes of action exert different effects on the cytokine and bone metabolic marker levels in patients with RA.

Clinical and immunological effects of hydroxychloroquine in patients with active rheumatoid arthritis despite antirheumatic treatment.

OBJECTIVE: To investigate the efficacy and safety of hydroxychloroquine (HCQ) in patients with rheumatoid arthritis (RA). METHODS: Patients with active RA, despite conventional synthetic disease-modifying antirheumatic drugs (csDMARDs), were recruited. HCQ was administered for 24 weeks, in addition to prior treatment. The primary endpoint was the proportion of American College of Rheumatology (ACR) 20 achievement at week 24, compared to that of a propensity score matched historical control group. RESULTS: Sixty patients were enrolled and administered HCQ. We also identified 276 patients as candidates for the historical control group. Propensity score matching yielded 46 patients in each group. The proportion of ACR20 achievements at week 24 was significantly higher in the HCQ group than that in the control group (54.4% vs. 28.3%, P = 0.007). The proportion of ACR50 and ACR70 achievement at week 24 were also higher in the HCQ group than those in the control group (ACR50, 30.4% vs. 4.3%, P = 0.006; ACR70, 17.4% vs. 0%, P = 0.005). Neither hydroxychloroquine retinopathy nor any new safety signal was observed during the study. CONCLUSION: The addition of HCQ to csDMARDs was effective, with no new safety signal in patients with RA.

Role of interferons (IFNs) in the differentiation of T peripheral helper (Tph) cells.

Interleukin (IL)-21-producing T peripheral helper (Tph) cells are thought to contribute to extra-follicular B cell activation and play a pathogenic role in autoimmune diseases. In this study, we investigated the relationship between Tph cells and interferons (IFNs) in several autoimmune diseases because our previous study demonstrated that type I IFNs promote the differentiation of IL-21-producing Tph-like cells. The frequency of Tph cells in the blood as well as serum IFN-α2a and IFN-λ1 were markedly elevated in patients with active systemic lupus erythematosus (SLE) compared to other autoimmune diseases or healthy controls. Notably, the frequency of Tph cells was positively correlated with the SLE disease activity index, serum IFN-α and serum IFN-λ1 in SLE patients. Additionally, we found that type III IFNs (IFN-λ1, IFN-λ2 and IFN-λ3) promote the differentiation of programmed cell death-1 (PD-1)+ CXCR5 -CD4+ T cells and enhance the secretion of IL-21, IFN-γ and CXCL13. IFN-λ1, like IFN-α, up-regulated the mRNA expression of IL21, IFNG, CXCL13, CD244, SLAMF7, GZMB, PRF1, CCR5 and PRDM1, whereas it down-regulated that of CXCR5 and BCL6, reflecting a Tph-related gene expression pattern. IFN-α in combination with IFN-λ1, IFN-λ2 or IFN-λ3 significantly increased the differentiation of PD-1+CXCR5- Tph-like cells and the secretion of Tph-related cytokines as compared with each IFN alone, suggesting a cooperative interaction. From these findings, it is highly probable that type III IFNs in addition to type I IFNs play a key role in the differentiation of Tph cells and that high levels of IFN-α and IFN-λ1 trigger the differentiation and expansion of Tph cells in SLE.

Role of interferons (IFNs) in the differentiation of T peripheral helper (Tph) cells.

T follicular helper (Tfh) cells and T peripheral helper (Tph) cells produce interleukin (IL)-21 and are thought to contribute to follicular and extra-follicular B-cell activation, respectively, in autoimmune diseases. It is known that programmed cell death-1 (PD-1)-positive CXCR5+ Tfh-like cells are differentiated from human naive CD4+ T cells by IL-12 plus transforming growth factor (TGF)-ß. However, it remains unclear what cytokines are required for Tph differentiation. In this study, we found that interferon (IFN)-α and IFN-ß reduce the frequency of Tfh-like cells under the IL-12 plus TGF-ß condition, whereas they promote generation of PD-1+CXCR5-CD4+ T cells and secretion of IL-21, IFN-γ and CXCL13. Intracellular cytokine staining and T-cell-B-cell co-culture studies indicated that IFN-α promotes generation of IL-21+IFN-γ +CXCR5-CD4+ T cells thereby enhancing B-cell helper function. By IFN-α treatment, the mRNA levels of IL21, IFNG, CXCL13, CD244, SLAMF7, GZMB and PRDM1 were significantly up-regulated but BCL6 mRNA expression was down-regulated, suggesting a Tph-related gene expression pattern. On the other hand, IL-2-neutralization increased mRNA levels of IL21, CXCL13 and CXCR5, retained BCL6, but showed no clear effect on IFNG or PRDM1. RNA sequencing analyses revealed that PD-1hiCXCR5-CD4+ T cells prepared from in vitro culture show a Tph-related gene expression pattern similar with that of PD-1hiCXCR5- Tph cells obtained from the blood of patients with systemic lupus erythematosus. From our findings, it is highly probable that type I IFNs play a key role in differentiation of Tph cells and trigger Tph cell expansion in autoimmune diseases.

Interleukin-1 pathway in active large vessel vasculitis patients with a poor prognosis: a longitudinal transcriptome analysis.

OBJECTIVES: Large vessel vasculitis (LVV) is characterised by a high relapse rate. Because accurate assessment of the LVV disease status can be difficult, an accurate prognostic marker for initial risk stratification is required. We conducted a comprehensive longitudinal investigation of next-generation RNA-sequencing data for patients with LVV to explore useful biomarkers associated with clinical characteristics. METHODS: Key molecular pathways relevant to LVV pathogenesis were identified by examining the whole blood RNA from patients with LVV and healthy controls (HCs). The data were examined by pathway analysis and weighted gene correlation network analysis (WGCNA) to identify functional gene sets that were differentially expressed between LVV patients and HCs, and associated with clinical features. We then compared the expression of the selected genes during week 0, week 6, remission and relapse. RESULTS: The whole-transcriptome gene expression data for 108 samples obtained from LVV patients (n = 27) and HCs (n = 12) were compared. The pathway analysis and WGCNA revealed that molecular pathway related to interleukin (IL)-1 was significantly upregulated in LVV patients compared with HCs, which correlated with the positron emission tomography vascular activity score, a disease-extent score based on the distribution of affected arteries. Further analysis revealed that the expression levels of genes in the IL-1 signalling pathway remained high after conventional treatment and were associated with disease relapse. CONCLUSION: Upregulation of the IL-1 signalling pathway was a characteristic of LVV patients and was associated with the extent of disease and a poor prognosis.

Peripheral TIGIT+ T Follicular Helper Cells That Produce High Levels of Interleukin-21 via OX40 Represent Disease Activity in IgG4-Related Disease.

Objectives: 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. Methods: 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. Results: 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. Conclusions: 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.

Identification of neutrophil ß2-integrin LFA-1 as a potential mechanistic biomarker in ANCA-associated vasculitis via microarray and validation analyses.

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.

Platelet CXCL4 mediates neutrophil extracellular traps formation in ANCA-associated vasculitis.

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.

Lymphadenopathy in IgG4-related disease: a phenotype of severe activity and poor prognosis, with eotaxin-3 as a new biomarker.

OBJECTIVE: To clarify relevant proteins and clinical characteristics of a phenotype of IgG4-related disease (IgG4-RD) with lymphadenopathy. METHODS: We enrolled patients newly diagnosed with IgG4-RD in our department between January 2000 and June 2018 and performed proteomic analysis to measure serum concentrations of 1305 proteins. We extracted proteins overexpressed in patients with IgG4-RD with lymphadenopathy by comparing between those with lymphadenopathy, those without lymphadenopathy and healthy controls. We further reviewed all the patients with IgG4-RD in our institution and investigated the characteristics and prognosis of the patients with IgG4-RD with lymphadenopathy. RESULTS: Eighty-five patients with IgG4-RD were enrolled, of which, 55% had lymphadenopathy. Proteomic analysis in 31 patients with IgG4-RD and 6 healthy controls revealed that eotaxin-3 was a potential serum biomarker in the patients with lymphadenopathy versus those without lymphadenopathy and healthy controls. A cohort of 85 patients with IgG4-RD demonstrated that patients with lymphadenopathy showed a significantly higher serum IgG4, IgG4:IgG ratio, IgG4-RD responder index and eosinophilia (P < 0.001 for all), irrelevant of the extent to which organ involvement developed. Patients with lymphadenopathy treated with glucocorticoid alone relapsed with significantly higher rates than those without lymphadenopathy (P = 0.03). CONCLUSION: Lymphadenopathy in IgG4-RD represents a phenotype associated with high disease activities, eosinophilia and relapsing disease. Eotaxin-3 is a novel biomarker related to IgG4-RD with lymphadenopathy.