Stage-specific GATA3 induction promotes ILC2 development after lineage commitment.

Group 2 innate lymphoid cells (ILC2s) are a subset of innate lymphocytes that produce type 2 cytokines, including IL-4, IL-5, and IL-13. GATA3 is a critical transcription factor for ILC2 development at multiple stages. However, when and how GATA3 is induced to the levels required for ILC2 development remains unclear. Herein, we identify ILC2-specific GATA3-related tandem super-enhancers (G3SE) that induce high GATA3 in ILC2-committed precursors. G3SE-deficient mice exhibit ILC2 deficiency in the bone marrow, lung, liver, and small intestine with minimal impact on other ILC lineages or Th2 cells. Single-cell RNA-sequencing and subsequent flow cytometry analysis show that GATA3 induction mechanism, which is required for entering the ILC2 stage, is lost in IL-17RB+PD-1- late ILC2-committed precursor stage in G3SE-deficient mice. Cnot6l, part of the CCR4-NOT deadenylase complex, is a possible GATA3 target during ILC2 development. Our findings implicate a stage-specific regulatory mechanism for GATA3 expression during ILC2 development.

A distal enhancer of GATA3 regulates Th2 differentiation and allergic inflammation.

Asthma is a widespread airway disorder where GATA3-dependent Type-2 helper T (Th2) cells and group 2 innate lymphoid cells (ILC2s) play vital roles. Asthma-associated single nucleotide polymorphisms (SNPs) are enriched in a region located 926-970 kb downstream from GATA3 in the 10p14 (hG900). However, it is unknown how hG900 affects the pathogenesis of allergic airway inflammation. To investigate the roles of the asthma-associated GATA3 enhancer region in experimental allergic airway inflammation, we first examined the correlation between GATA3 expression and the activation of the hG900 region was analyzed by flow cytometry and ChIP-qPCR. We found that The activation of enhancers in the hG900 region was strongly correlated to the levels of GATA3 in human peripheral T cell subsets. We next generated mice lacking the mG900 region (mG900KO mice) were generated by the CRISPR-Cas9 system, and the development and function of helper T cells and ILCs in mG900KO mice were analyzed in steady-state conditions and allergic airway inflammation induced by papain or house dust mite (HDM). The deletion of the mG900 did not affect the development of lymphocytes in steady-state conditions or allergic airway inflammation induced by papain. However, mG900KO mice exhibited reduced allergic inflammation and Th2 differentiation in the HDM-induced allergic airway inflammation. The analysis of the chromatin conformation around Gata3 by circular chromosome conformation capture coupled to high-throughput sequencing (4C-seq) revealed that the mG900 region interacted with the transcription start site of Gata3 with an influencing chromatin conformation in Th2 cells. These findings indicate that the mG900 region plays a pivotal role in Th2 differentiation and thus enhances allergic airway inflammation.

Calcium pyrophosphate crystal arthritis associated with immune checkpoint inhibitor successfully treated with intra-articular glucocorticoid: A case report.

Immune checkpoint inhibitors (ICIs) sometimes induce immune-related adverse events (irAEs), and arthritis is one of the irAE symptoms. Recently, crystal-induced arthritis, such as calcium pyrophosphate (CPP) crystal deposition disease and gout, has been reported to occur after ICI administration. However, the distinction between ICI-associated crystal arthritis and ICI-induced non-crystal arthritis is difficult because their symptoms are similar. Besides, optimal treatment for ICI-associated crystal arthritis has not been established. Here, we report a patient who developed CPP crystal arthritis twice after pembrolizumab (ICI) administration and was successfully treated with intra-articular glucocorticoid injection. He suffered arthritis and acute interstitial nephritis simultaneously after ICI administration. Musculoskeletal ultrasound of his affected joint suggests that his arthritis was crystal-induced arthritis, and arthrocentesis detected CPP crystal in synovial fluid. Thus, we diagnosed his arthritis as ICI-associated cystal arthritis. Therefore, our case encourages the use of musculoskeletal ultrasound in patients with arthritis after treatment with ICI because it may distinguish between ICI-associated crystal arthritis and ICI-induced non-crystal arthritis. Besides, ICI-associated crystal arthritis could be treatable by intra-articular glucocorticoid injection.

Suppression of Type I Interferon Signaling in Myeloid Cells by Autoantibodies in Severe COVID-19 Patients.

PURPOSE: Auto-antibodies (auto-abs) to type I interferons (IFNs) have been identified in patients with life-threatening coronavirus disease 2019 (COVID-19), suggesting that the presence of auto-abs may be a risk factor for disease severity. We therefore investigated the mechanism underlying COVID-19 exacerbation induced by auto-abs to type I IFNs. METHODS: We evaluated plasma from 123 patients with COVID-19 to measure auto-abs to type I IFNs. We performed single-cell RNA sequencing (scRNA-seq) of peripheral blood mononuclear cells from the patients with auto-abs and conducted epitope mapping of the auto-abs. RESULTS: Three of 19 severe and 4 of 42 critical COVID-19 patients had neutralizing auto-abs to type I IFNs. Patients with auto-abs to type I IFNs showed no characteristic clinical features. scRNA-seq from 38 patients with COVID-19 revealed that IFN signaling in conventional dendritic cells and canonical monocytes was attenuated, and SARS-CoV-2-specific BCR repertoires were decreased in patients with auto-abs. Furthermore, auto-abs to IFN-α2 from COVID-19 patients with auto-abs recognized characteristic epitopes of IFN-α2, which binds to the receptor. CONCLUSION: Auto-abs to type I IFN found in COVID-19 patients inhibited IFN signaling in dendritic cells and monocytes by blocking the binding of type I IFN to its receptor. The failure to properly induce production of an antibody to SARS-CoV-2 may be a causative factor of COVID-19 severity.

Polyclonally Derived Alloantigen-Specific T Regulatory Cells Exhibit Target-Specific Suppression and Capture MHC Class II from Dendritic Cells.

Foxp3+ T regulatory (Treg) cells prevent allograft rejection and graft-versus-host disease. Although polyclonal Tregs have been used both in animal models and in humans, the fine specificity of their suppressive function is poorly defined. We have generated mouse recipient-derived alloantigen-specific Tregs in vitro and explored the fine specificity of their suppressive function and their mechanism of action in vitro and in vivo. In vitro, when alloantigen and peptide Ag were both presented on the same dendritic cell, both responses were suppressed by iTregs specific either for the alloantigen or for the peptide Ag. In vivo, iTreg suppression was limited to the cognate Ag, and no bystander suppression was observed when both allo-antigen and peptide Ag were present on the same dendritic cell. In vitro, alloantigen-specific Tregs captured cognate MHC but failed to capture noncognate MHC. Our results demonstrate that a polyclonal population of iTregs generated from naive T cells can mediate highly specific function in vivo and support the view that Treg therapy, even with unselected polyclonal populations, is likely to be target antigen-specific and that bystander responses to self-antigens or to infectious agents are unlikely.

Mechanochemical synthesis of fluoride-ion conducting glass and glass-ceramic in ZrF4-BaF2 binary system.

Fluoride glasses in the binary system ZrF4-BaF2 were prepared via mechanochemical treatment. The glass-forming region of the ZrF4-BaF2 system obtained using the mechanochemical method was wider than that obtained using the conventional melt-quenching method. The glass-ceramic 60ZrF4·40BaF2 (mol%) sample was obtained by heat treatment and shows a higher conductivity of 1.2 × 10-6 S cm-1 at 200 °C than the pristine glass. This study revealed that mechanochemical treatment was effective for the synthesis of fluoride glasses.

Development of a Kidney Prognostic Score in a Japanese Cohort of Patients With Antineutrophil Cytoplasmic Autoantibody Vasculitis.

Introduction: Glomerulonephritis is frequent in antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) and crucial to disease outcomes. We conducted a detailed assessment of renal pathology in Japanese patients with AAV, and developed a new score that would predict renal outcome. Methods: Two hundred twenty-one patients who were diagnosed with AAV and underwent a kidney biopsy were enrolled. Data on glomerular, tubular, interstitial, and vascular lesions from kidney biopsies were analyzed; the 3 established classification and prognostic scoring systems (Berden Classification, Mayo Clinic/RPS Chronicity Score [MCCS], and ANCA Renal Risk Score [ARRS]) were validated. Further, we developed a new prognostic score by including variables relevant for Japanese patients with ANCA-glomerulonephritis. Results: Median follow-up was 60 months (interquartile range: 6-60). End-stage kidney disease (ESKD) risk prediction by the MCCS and the ARRS was confirmed. Moreover, our analysis identified 4 items with significant ESKD risk prediction capacity, namely percentage of cellular, fibrocellular, and fibrous crescents; and sclerotic glomeruli. Based on our findings, we created a score evaluating the percentage of these lesions to total glomeruli, the Percentage of ANCA Crescentic Score (PACS). The area under the receiver operating characteristic (ROC) curve evaluating PACS was 0.783. The PACS had a comparable performance as the ARRS in predicting ESKD. The optimal PACS cut-off for ESKD risk over 60 months was 43%. In addition, the percentage of cellular crescents and presence of interstitial inflammation were independent predictors of kidney function recovery. Conclusion: We developed a new score predicting renal prognosis using histopathological data of Japanese patients with ANCA-glomerulonephritis. Studies are needed to validate our results in international cohorts.

Physiological and immunological barriers in the lung.

The lungs serve as the primary organ for respiration, facilitating the vital exchange of gases with the bloodstream. Given their perpetual exposure to external particulates and pathogens, they possess intricate protective barriers. Cellular adhesion in the lungs is robustly maintained through tight junctions, adherens junctions, and desmosomes. Furthermore, the pulmonary system features a mucociliary clearance mechanism that synthesizes mucus and transports it to the outside. This mucus is enriched with chemical barriers like antimicrobial proteins and immunoglobulin A (IgA). Additionally, a complex immunological network comprising epithelial cells, neural cells, and immune cells plays a pivotal role in pulmonary defense. A comprehensive understanding of these protective systems offers valuable insights into potential pathologies and their therapeutic interventions.

Single-cell RNA sequencing of submandibular gland reveals collagen type XV-positive fibroblasts as a disease-characterizing cell population of IgG4-related disease.

OBJECTIVES: IgG4-related disease (IgG4-RD) is a systemic autoimmune disease with an unknown etiology, affecting single/multiple organ(s). Pathological findings include the infiltration of IgG4-producing plasma cells, obliterative phlebitis, and storiform fibrosis. Although immunological studies have shed light on the dysregulation of lymphocytes in IgG4-RD pathogenesis, the role of non-immune cells remains unclear. This study aimed to investigate the demographics and characteristics of non-immune cells in IgG4-RD and explore potential biomarkers derived from non-immune cells in the sera. METHODS: We conducted single-cell RNA sequence (scRNA-seq) on non-immune cells isolated from submandibular glands of IgG4-RD patients. We focused on fibroblasts expressing collagen type XV and confirmed the presence of those fibroblasts using immunohistochemistry. Additionally, we measured the levels of collagen type XV in the sera of IgG4-RD patients. RESULTS: The scRNA-seq analysis revealed several distinct clusters consisting of fibroblasts, endothelial cells, ductal cells, and muscle cells. Differential gene expression analysis showed upregulation of COL15A1 in IgG4-RD fibroblasts compared to control subjects. Notably, COL15A1-positive fibroblasts exhibited a distinct transcriptome compared to COL15A1-negative counterparts. Immunohistochemical analysis confirmed a significant presence of collagen type XV-positive fibroblasts in IgG4-RD patients. Furthermore, immune-suppressive therapy in active IgG4-RD patients resulted in decreased serum levels of collagen type XV. CONCLUSIONS: Our findings suggest that collagen type XV-producing fibroblasts may represent a disease-characterizing non-immune cell population in IgG4-RD and hold potential as a disease-monitoring marker.

Immunogenicity and influence on disease activity of recombinant zoster vaccine in patients with rheumatoid arthritis treated with DMARDs.

OBJECTIVES: This study aimed to determine the immunogenicity and the influence on disease activity of an adjuvanted recombinant varicella-zoster virus (VZV) subunit vaccine (RZV) in patients with rheumatoid arthritis (RA) treated with disease-modifying antirheumatic drugs (DMARDs). METHODS: This prospective longitudinal study enrolled 53 patients with RA (aged ≥50 years) treated with DMARDs (conventional synthetic (cs)DMARDs 20, biological (b)DMARDs 23 and targeted synthetic (ts)DMARDs 10) and 10 control individuals. The participants received two intramuscular RZV 2 months apart. VZV-specific CD4+ T cell responses (cell-mediated immunity; CMI) and IgG antibody responses (humoral immunity; HI) were assessed at 0 and 3 months after the first RZV administration using flow cytometry and enzyme immunoassay, respectively. Disease activity (Disease Activity Score 28-C reactive protein and Clinical Disease Activity Index), flares and adverse events were monitored for 6 months after the first vaccination. RESULTS: VZV-specific CMI and HI significantly increased in the three DMARDs-treated patients with RA after RZV administration compared with the corresponding prevaccination values (p<0.001-0.014), and the magnitudes and fold-increases of those responses were not significantly different among the three DMARDs-treated patients with RA. Furthermore, the vaccine response rates of CMI and HI were not significantly different between csDMARDs-treated patients and b-DMARDs or ts-DMARDs-treated patients. Meanwhile, no significant increases in disease activity indices or adverse events were observed in these patients during the 6-month follow-up period after the first vaccination. RZV-induced RA flares occurred in two patients (3.8%) but were mild and controllable. CONCLUSION: RZV is robustly immunogenic and has a clinically acceptable safety profile in elderly patients with RA receiving DMARDs.

Short-term and long-term outcomes of patients with anti-melanoma differentiation-associated gene 5 antibody-positive dermatomyositis.

OBJECTIVES: Anti-melanoma differentiation-associated gene 5 (MDA5) antibody-positive dermatomyositis (MDA5-DM) is a subtype of dermatomyositis characterized by frequent interstitial lung disease and reduced muscle involvement. This study aimed to determine the short-term and long-term outcomes of patients with MDA5-DM. METHODS: Information on baseline characteristics, treatments, and short-term and long-term outcomes of patients with MDA5-DM including survival, relapse, and the titer of anti-MDA5 antibody, was retrospectively collected. Descriptive statistics regarding clinical outcomes were calculated, and a comparison of clinical parameters between patients with and without relapse was performed. The short-term survival according to the use of Janus kinase inhibitors (JAKi) was also assessed. RESULTS: A total of 154 patients with MDA5-DM were included in the study. Forty patients (25.9%) died during the remission induction phase, with respiratory failure being the most common cause of mortality. Among the 114 patients who survived the remission induction phase, the 5-year cumulative survival and relapse-free survival rates were 96.8% and 77.4%, respectively, and 7.9% of patients achieved complete drug-free remission. Fifty-four patients achieved normalization of anti-MDA5 antibody titers and only two of them relapsed after normalization. In the severe patients, the 6-month survival rate became significantly higher after the emergence of the JAKi treatment compared with before its existence (p= 0.03). CONCLUSIONS: Although relapse often occurs, the long-term survival of MDA5-DM patients who survived the remission induction phase is generally favorable. The status of the anti-MDA5 antibody is associated with relapse. JAKi may improve the survival of refractory patients with severe MDA5-DM.

Eosinophils Contribute to Oral Tolerance via Induction of RORγt-Positive Antigen-Presenting Cells and RORγt-Positive Regulatory T Cells.

Oral tolerance has been defined as the specific suppression of immune responses to an antigen by prior oral administration of the antigen. It has been thought to serve to suppress food allergy. Previous studies have shown that dendritic cells (DCs) and regulatory T cells (Tregs) are involved in the induction of oral tolerance. However, the detailed mechanisms of Treg induction in oral tolerance remain largely unknown. Eosinophils have been recognized as effector cells in allergic diseases, but in recent years, the diverse functions of tissue-resident eosinophils have been reported. Eosinophils in the intestine have been reported to induce Tregs by releasing TGF-ß, but the role of eosinophils in oral tolerance is still controversial. In this study, we analyzed the roles of eosinophils in oral tolerance using eosinophil-deficient ΔdblGATA mice (mice lacking a high-affinity GATA-binding site in the GATA1 promoter). ΔdblGATA mice showed impaired antigen-induced oral tolerance compared to wild-type mice. The induction of RORγt+ Tregs in mesenteric lymph nodes (MLNs) by oral tolerance induction was impaired in ΔdblGATA mice compared to wild-type mice. An increase in RORγt+ antigen-presenting cells (APCs), which are involved in RORγt+ Treg differentiation, in the intestine and MLNs was not seen in ΔdblGATA mice. Notably, the expansion of group 3 innate lymphoid cells (ILC3s), a subset of RORγt+ APCs, by oral tolerance induction was seen in wild-type mice but not ΔdblGATA mice. These results suggest that eosinophils are crucial in the induction of oral tolerance, possibly via the induction of RORγt+ APCs and RORγt+ Tregs.

Reduced-dose versus high-dose glucocorticoids added to rituximab on remission induction in ANCA-associated vasculitis: predefined 2-year follow-up study.

OBJECTIVES: The LoVAS trial reported non-inferiority in remission induction rates between the reduced-dose and conventional high-dose glucocorticoid regimens plus rituximab for antineutrophil cytoplasm antibody (ANCA)-associated vasculitis at 6 months; however, maintenance glucocorticoid requirements and long-term outcomes are unknown. METHODS: A total of 140 patients with new-onset ANCA-associated vasculitis without severe glomerulonephritis or alveolar haemorrhage were randomised to receive reduced-dose prednisolone (0.5 mg/kg/day) plus rituximab (375 mg/m2/week×4) or high-dose prednisolone (1 mg/kg/day) plus rituximab. After achieving remission, patients received the rituximab maintenance therapy (1 g/6 months). RESULTS: A total of 134 patients were analysed. Among patients who achieved remission with the protocolised treatments, the majority of patients in the reduced-dose group (89.7%) and 15.5% in the high-dose group discontinued prednisolone (median time to withdrawal, 150 and 375 days, respectively). During 24-month trial period, two patients in the reduced-dose group (2.8%) died, while five patients in the high-dose group (7.6%) died (p=0.225). Relapse occurred in nine patients in the reduced-dose group (13.0%) (two major and seven minor) and five in the high-dose group (7.6%) (two major and three minor) (p=0.311). Serious adverse events (SAEs) were less frequent in the reduced-dose group (36 events in 19 patients, 27.5%) than in the high-dose group (54 events in 30 patients, 46.2%) (p=0.025). CONCLUSION: At 24 months, frequencies of relapse did not differ between the groups, and SAEs were less frequent in the reduced-dose group due to the lower event rate in the 6-month induction phase. The bias to myeloperoxidase-ANCA positivity (85.8%) in the trial population should be noted. TRIAL REGISTRATION NUMBER: NCT02198248.

Enhanced efficacy of the novel recombinant clone VasSF in a mouse model of antineutrophil cytoplasmic antibody-associated vasculitis.

Based on the efficacy of intravenous immunoglobulin (IVIg) for the treatment of antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV), we developed a recombinant single-chain-fragment variable clone, VasSF, therapeutic against AAV in a mouse model (SCG/Kj mice). VasSF is thought to bind to vasculitis-associated apolipoprotein A-II (APOA2) as a target molecule. VasSF is a promising new drug against AAV, but difficulties in the yield and purification of VasSF remain unresolved. We produced monomers of new VasSF molecules by modifying the plasmid structure for VasSF expression and simplifying the purification method using high-performance liquid chromatography. We compared the therapeutic effects between 5-day continuous administration of the monomers, as in IVIg treatment, and single shots of 5-day-equivalent doses. We also evaluated the life-prolonging effect of the single-shot treatment. Two-dimensional western blots were used to examine the binding of VasSF to APOA2. Our improved manufacturing method resulted in a 100-fold higher yield of VasSF than in our previous study. Monomerization of VasSF stabilized its efficacy. Single shots of a small amount (1/80 000 of IVIg) produced sufficient therapeutic effects, including decreased glomerular crescent formation, a decreasing trend of serum ANCA against myeloperoxidase (MPO-ANCA), decreases in multiple proinflammatory cytokines, and a trend toward prolonged survival. Two-dimensional western blots confirmed the binding of VasSF to APOA2. The newly produced pure VasSF monomers are stable and therapeutic for AAV with a single low-dose injection, possibly by removing vasculitis-associated APOA2. Thus, the new VasSF described herein is a promising drug against AAV.

Cytotoxicity, Differentiation, and Biocompatibility of Root-End Filling: A Comprehensive Study.

Assessing the biocompatibility of endodontic root-end filling materials through cell line responses is both essential and of utmost importance. This study aimed to the cytotoxicity of the type of cell death through apoptosis and autophagy, and odontoblast cell-like differentiation effects of MTA, zinc oxide-eugenol, and two experimental Portland cements modified with bismuth (Portland Bi) and barium (Portland Ba) on primary cell cultures. Material and methods: The cells corresponded to human periodontal ligament and gingival fibroblasts (HPLF, HGF), human pulp cells (HPC), and human squamous carcinoma cells from three different patients (HSC-2, -3, -4). The cements were inoculcated in different concentrations for cytotoxicity evaluation, DNA fragmentation in electrophoresis, apoptosis caspase activation, and autophagy antigen reaction, odontoblast-like cells were differentiated and tested for mineral deposition. The data were subject to a non-parametric test. Results: All cements caused a dose-dependent reduction in cell viability. Contact with zinc oxide-eugenol induced neither DNA fragmentation nor apoptotic caspase-3 activation and autophagy inhibitors (3-methyladenine, bafilomycin). Portland Bi accelerated significantly (p < 0.05) the differentiation of odontoblast-like cells. Within the limitation of this study, it was concluded that Portland cement with bismuth exhibits cytocompatibility and promotes odontoblast-like cell differentiation. This research contributes valuable insights into biocompatibility, suggesting its potential use in endodontic repair and biomimetic remineralization.

IL-21 is required for the maintenance and pathogenesis of murine Vγ4+ IL-17-producing γδT cells.

Murine IL-17-producing γδT (γδT17) cells are divided into two subsets: natural γδT17 (nγδT17) cells, whose development is restricted to the fetal thymus, and inducible γδT17 cells, which require antigen exposure for their IL-17 production and are presumed to develop from Rorc + Il17a - CCR9 + immature γδT17 cells in the adult thymus and whose T cell receptor (TCR) is biased toward Vγ4. Although IL-23 is known to be involved in developing γδT17 cells, the roles of other cytokines, such as IL-21, which is involved in developing Th17 cells like IL-23, in the development, maintenance, and pathophysiology of γδT17 cells remain unknown. Here, we show that IL-21 is dispensable for the fetal thymic development of nγδT17 cells but is required for the peripheral maintenance of Vγ4+nγδT17 cells. Upon stimulation with γδTCR, IL-1 plus IL-21 induces the proliferation of Vγ4+nγδT17 cells via STAT3 as effectively as IL-1 plus IL-23. Using bone marrow chimeric mice, we demonstrated that immature γδT17 cells are produced de novo in the adult mice from donor adult bone marrow cells and that IL-21 is dispensable for their development. Instead, IL-21 is required to expand newly induced Vγ4+γδT17 cells in the periphery upon immunization. Finally, using adoptive transfer experiments of γδT17 cells, we found that IL-21 receptors on γδT17 cells are involved in maintaining Vγ4+γδT17 cells, subsequent infiltration of Th17 cells into the spinal cord, and exacerbation of experimental autoimmune encephalomyelitis. Collectively, IL-21 plays a vital role in the maintenance and pathogenesis of Vγ4+γδT17 cells.