Single-cell transcriptome landscape of circulating CD4+ T cell populations in autoimmune diseases.

CD4+ T cells are key mediators of various autoimmune diseases; however, their role in disease progression remains unclear due to cellular heterogeneity. Here, we evaluated CD4+ T cell subpopulations using decomposition-based transcriptome characterization and canonical clustering strategies. This approach identified 12 independent gene programs governing whole CD4+ T cell heterogeneity, which can explain the ambiguity of canonical clustering. In addition, we performed a meta-analysis using public single-cell datasets of over 1.8 million peripheral CD4+ T cells from 953 individuals by projecting cells onto the reference and cataloging cell frequency and qualitative alterations of the populations in 20 diseases. The analyses revealed that the 12 transcriptional programs were useful in characterizing each autoimmune disease and predicting its clinical status. Moreover, genetic variants associated with autoimmune diseases showed disease-specific enrichment within the 12 gene programs. The results collectively provide a landscape of single-cell transcriptomes of CD4+ T cell subpopulations involved in autoimmune disease.

Analysis of gut microbiome, host genetics, and plasma metabolites reveals gut microbiome-host interactions in the Japanese population.

Interaction between the gut microbiome and host plays a key role in human health. Here, we perform a metagenome shotgun-sequencing-based analysis of Japanese participants to reveal associations between the gut microbiome, host genetics, and plasma metabolome. A genome-wide association study (GWAS) for microbial species (n = 524) identifies associations between the PDE1C gene locus and Bacteroides intestinalis and between TGIF2 and TGIF2-RAB5IF gene loci and Bacteroides acidifiaciens. In a microbial gene ortholog GWAS, agaE and agaS, which are related to the metabolism of carbohydrates forming the blood group A antigen, are associated with blood group A in a manner depending on the secretor status determined by the East Asian-specific FUT2 variant. A microbiome-metabolome association analysis (n = 261) identifies associations between bile acids and microbial features such as bile acid metabolism gene orthologs including bai and 7ß-hydroxysteroid dehydrogenase. Our publicly available data will be a useful resource for understanding gut microbiome-host interactions in an underrepresented population.

RGMa collapses the neuronal actin barrier against disease-implicated protein and exacerbates ALS.

Repulsive guidance molecule A (RGMa) was originally identified as a neuronal growth cone-collapsing factor. Previous reports have demonstrated the multifunctional roles of RGMa mediated by neogenin1. However, the pathogenic involvement of RGMa in amyotrophic lateral sclerosis (ALS) remains unclear. Here, we demonstrated that RGMa concentration was elevated in the cerebrospinal fluid of both patients with ALS and transgenic mice overexpressing the mutant human superoxide dismutase1 (mSOD1 mice). Treatment with humanized anti-RGMa monoclonal antibody ameliorated the clinical symptoms in mSOD1 mice. Histochemical analysis revealed that the anti-RGMa antibody significantly decreased mutant SOD1 protein accumulation in the motor neurons of mSOD1 mice via inhibition of actin depolymerization. In vitro analysis revealed that the anti-RGMa antibody inhibited the cellular uptake of the mutant SOD1 protein, presumably by reinforcing the neuronal actin barrier. Collectively, these data suggest that RGMa leads to the collapse of the neuronal actin barrier and promotes aberrant protein deposition, resulting in exacerbation of the ALS pathology.

[Questionnaire for Kinki area Japanese Society of Neurology board members regarding transitional medicine for neurological disease patients].

In July 2020, The Special Committee for Measures Against Transition from Pediatric to Adult Health Care of the Japanese Society of Neurology was established to address transitional care for patients with childhood-onset neurological disorders. One of the measures used was a questionnaire regarding transitional medicine given to the 129 board members in the Kinki area. Of the 46 respondents, 42 answered that they would "generally examine such patients" or "judge on a case-by-case basis" for patients referred from a pediatric physician. Most of the responses noted "epilepsy" and "neuromuscular disease" as target conditions. Generally, doctors in an adult medical department do not form a relationship with the patient or their family members, different than pediatric department doctors. Furthermore, adult clinical departments typically do not have sufficient knowledge regarding treatment of diseases such as developmental disorders. The present support system for transitional medicine is not sufficient and there is no means for reimbursement. Several issues must be resolved to facilitate a smooth medical transition.

Autoimmune-mediated astrocytopathy.

Recently accumulating evidence identified the disease entity where astrocytes residing within the central nervous system (CNS) are the target of autoantibody-mediated autoimmunity. Aquaporin4 (AQP4) is the most common antigen to serve as astrocyte-targeted autoimmune responses. Here, in this review, the clinical and pathological aspects of AQP4-mediated astrocyte disease are discussed together with the pathogenic role of anti-AQP4 antibody. More recently, the mechanism of immune dysregulation resulting in the production of astrocyte-targeted autoantibody is also revealed, and the postulated hypothesis is discussed.

Reconstruction of the personal information from human genome reads in gut metagenome sequencing data.

Human DNA present in faecal samples can result in a small number of human reads in gut shotgun metagenomic sequencing data. However, it is presently unclear how much personal information can be reconstructed from such reads, and this has not been quantitatively evaluated. Such a quantitative evaluation is necessary to clarify the ethical concerns related to data sharing and to enable efficient use of human genetic information in stool samples, such as for research and forensics. Here we used genomic approaches to reconstruct personal information from the faecal metagenomes of 343 Japanese individuals with associated human genotype data. Genetic sex could be accurately predicted based on the sequencing depth of sex chromosomes for 97.3% of the samples. Individuals could be re-identified from the matched genotype data based on human reads recovered from the faecal metagenomic data with 93.3% sensitivity using a likelihood score-based method. This method also enabled us to predict the ancestries of 98.3% of the samples. Finally, we performed ultra-deep shotgun metagenomic sequencing of five faecal samples as well as whole-genome sequencing of blood samples. Using genotype-calling approaches, we demonstrated that the genotypes of both common and rare variants could be reconstructed from faecal samples. This included clinically relevant variants. Our approach can be used to quantify personal information contained within gut metagenome data.

Single-cell RNA-seq analysis identifies distinct myeloid cells in a case with encephalitis temporally associated with COVID-19 vaccination.

Recently accumulating evidence has highlighted the rare occurrence of COVID-19 vaccination-induced inflammation in the central nervous system. However, the precise information on immune dysregulation related to the COVID-19 vaccination-associated autoimmunity remains elusive. Here we report a case of encephalitis temporally associated with COVID-19 vaccination, where single-cell RNA sequencing (scRNA-seq) analysis was applied to elucidate the distinct immune signature in the peripheral immune system. Peripheral blood mononuclear cells (PBMCs) were analyzed using scRNA-seq to clarify the cellular components of the patients in the acute and remission phases of the disease. The data obtained were compared to those acquired from a healthy cohort. The scRNA-seq analysis identified a distinct myeloid cell population in PBMCs during the acute phase of encephalitis. This specific myeloid population was detected neither in the remission phase of the disease nor in the healthy cohort. Our findings illustrate induction of a unique myeloid subset in encephalitis temporally associated with COVID-19 vaccination. Further research into the dysregulated immune signature of COVID-19 vaccination-associated autoimmunity including the cerebrospinal fluid (CSF) cells of central nervous system (CNS) is warranted to clarify the pathogenic role of the myeloid subset observed in our study.

Identification of double-stranded DNA in the cerebrospinal fluid of patients with acute neuromyelitis optica spectrum disorder.

Neuromyelitis optica spectrum disorder (NMOSD) is an inflammatory disease of the central nervous system (CNS) characterized by severe myelitis and optic neuritis. Double-stranded DNA (dsDNA) is involved in the pathogenesis of various autoimmune diseases, such as systemic lupus erythematosus. However, its role in NMOSD remains unclear. In this study, the concentration of dsDNA in the cerebrospinal fluid (CSF) was quantified in 23 patients with NMOSD and 16 patients with other neurological diseases (ONDs). CSF dsDNA levels in patients with NMOSD (median: 0.03 ng/µL) were significantly higher than those in patients with ONDs (median: 0.01 ng/µl). CSF dsDNA levels showed no significant difference before and after treatment. Elevation of CSF dsDNA levels may suggest its essential role in the augmentation of CNS inflammation in patients with NMOSD.

Health-related quality of life in Japanese patients with multiple sclerosis.

OBJECTIVES: Neurological disabilities, especially physical issues, can adversely affect the daily lives of people with multiple sclerosis (MS) and negatively impact their health-related quality of life (HRQOL). On the other hand, physical and psychiatric symptoms are variable in people with MS, and QOL can be influenced by cultural and educational background. This study aimed to evaluate the association of HRQOL with disabilities, fatigue, and depression in Japanese subjects with MS. METHODS: Evaluation of HRQOL, fatigue, and depression was performed in 184 Japanese individuals with MS, using the Functional Assessment of MS (FAMS), Fatigue Severity Scale (FSS), and Beck Depression Inventory-Second Edition (BDI-II), respectively. RESULTS: Multiple linear regression analysis demonstrated negative correlations of the Expanded Disability Status Scale (EDSS) with scores on the FAMS subscales of mobility, symptoms, thinking and fatigue, total FAMS, and additional concerns. The FSS score had negative correlations with mobility, symptoms, emotional well-being, thinking and fatigue, total FAMS, and additional concerns. There were negative correlations between BDI-II scores and all items of FAMS. CONCLUSIONS: HRQOL had relatively close correlations with disabilities and fatigue, and depression had an especially close relationship with HRQOL.

Myasthenia gravis-specific aberrant neuromuscular gene expression by medullary thymic epithelial cells in thymoma.

Myasthenia gravis (MG) is a neurological disease caused by autoantibodies against neuromuscular-associated proteins. While MG frequently develops in thymoma patients, the etiologic factors for MG are not well understood. Here, by constructing a comprehensive atlas of thymoma using bulk and single-cell RNA-sequencing, we identify ectopic expression of neuromuscular molecules in MG-type thymoma. These molecules are found within a distinct subpopulation of medullary thymic epithelial cells (mTECs), which we name neuromuscular mTECs (nmTECs). MG-thymoma also exhibits microenvironments dedicated to autoantibody production, including ectopic germinal center formation, T follicular helper cell accumulation, and type 2 conventional dendritic cell migration. Cell-cell interaction analysis also predicts the interaction between nmTECs and T/B cells via CXCL12-CXCR4. The enrichment of nmTECs presenting neuromuscular molecules within MG-thymoma is further confirmed immunohistochemically and by cellular composition estimation from the MG-thymoma transcriptome. Altogether, this study suggests that nmTECs have a significant function in MG pathogenesis via ectopic expression of neuromuscular molecules.

[A case of IgG4-related disease exclusively affecting pia matter and cerebral parenchyma].

A 61-year-old man presented with slowly progressive cognitive decline. Brain MRI revealed left frontal lobe lesions with gadolinium enhancement along pia mater. Brain biopsy was performed and histopathological findings was compatible with the diagnosis of IgG4-related disease (IgG4-RD). Serum IgG4 level was within a normal range, and no other systemic organs were suggested to be involved by clinical symptoms or laboratory findings. Intravenous methylprednisolone therapy followed by oral prednisone induction markedly improved the cognitive functions and MRI findings detected at the initial diagnosis. Our case highlights the importance of including IgG4-RD as one of the differential diagnosis when encountering the patients suffering from isolated cranial lesions even in the absence of normal serum IgG4 level.

Exploring steroid tapering in patients with neuromyelitis optica spectrum disorder treated with satralizumab in SAkuraSky: A case series.

BACKGROUND: Neuromyelitis optica spectrum disorder (NMOSD) is a rare, chronic, autoimmune disease, characterized by astrocytopathic lesions in the central nervous system (Beekman et al., 2019; Fujihara et al., 2020). The main aim of NMOSD maintenance therapy is to reduce the frequency and severity of relapses and minimize future disability (Fujihara et al., 2020). Oral corticosteroids are used long-term to prevent relapses, but are associated with serious complications (Kessler et al., 2016; Kimbrough et al., 2012). In the SAkuraSky study, satralizumab reduced the risk of relapse in patients with NMOSD compared with placebo, with comparable rates of serious adverse events and infections between treatment arms (Yamamura et al., 2019). Here, we report on 16 patients who tapered their steroid dose during the openlabel extension (OLE) period of SAkuraSky. METHODS: SAkuraSky was a phase 3, multicenter, randomized, double-blind (DB), placebo-controlled study of satralizumab in combination with immunosuppressive therapies (ISTs) in patients with NMOSD. Patients were randomized 1:1 to receive 120 mg subcutaneous satralizumab or placebo in addition to a stable dose of their baseline IST. After completing the DB period or experiencing relapse, patients could enter the OLE period. In the OLE, all patients received satralizumab, and IST doses could be tapered at the investigator's discretion. We assessed the different steroid tapering patterns and their impact on relapse and safety. Patients were considered to have tapered their steroids if their steroid dose at the clinical cut-off date (CCOD: February 18, 2020) was lower than on the first day of the OLE. Annualized relapse rate (ARR) was calculated as the number of relapses divided by the total number of patientyears at risk. RESULTS: Overall, 36 patients receiving oral corticosteroids entered the OLE, of whom 16 tapered their steroid dose. The mean age (range) at baseline was 44.9 (16-73) years, all 16 were female, 14 (88%) were Japanese, and 15 (94%) were AQP4-IgG seropositive. None were receiving any additional ISTs. Patients tapered their steroids from a median of 10 (range: 5-25) mg/day at OLE baseline to 2.75 (0-15) mg/day at the CCOD. Three patients discontinued steroids entirely, and all three remained relapse free. One patient who remained relapse free had temporary increases in steroid dose. Three relapses were observed in two patients who tapered steroids during the OLE; all three relapses required treatment. One of the relapses occurred shortly after a drop in steroid dose from 25 to 10 mg/day. The ARR for steroid-tapered patients was numerically lower in the OLE period than the satralizumab group in the DB period. The safety profile of satralizumab was in line with the overall SAkuraSky population. Two serious infections were observed in steroid-tapered patients in the OLE, both in the same patient: one event (hepatitis E) occurred before the patient began tapering their steroid dose; and one event (influenza) occurred while the patient was tapering. CONCLUSION: During the OLE of SAkuraSky, 16 patients tapered steroids and the ARR did not increase from the DB period. Patient numbers limit interpretation.

Enhancement of Regnase-1 expression with stem loop-targeting antisense oligonucleotides alleviates inflammatory diseases.

Regnase-1 is an ribonuclease that plays essential roles in restricting inflammation through degrading messenger RNAs (mRNAs) involved in immune reactions via the recognition of stem-loop (SL) structures in the 3' untranslated regions (3'UTRs). Dysregulated expression of Regnase-1 is associated with the pathogenesis of inflammatory and autoimmune diseases in mice and humans. Here, we developed a therapeutic strategy to suppress inflammatory responses by blocking Regnase-1 self-regulation, which was mediated by the simultaneous use of two antisense phosphorodiamidate morpholino oligonucleotides (MOs) to alter the binding of Regnase-1 toward the SL structures in its 3'UTR. Regnase-1-targeting MOs not only enhanced Regnase-1 expression by stabilizing mRNAs but also effectively reduced the expression of multiple proinflammatory transcripts that were controlled by Regnase-1 in macrophages. Intratracheal administration of Regnase-1-targeting MOs ameliorated acute respiratory distress syndrome and chronic fibrosis through suppression of inflammatory cascades. In addition, intracranial treatment with Regnase-1-targeting MOs attenuated the development of experimental autoimmune encephalomyelitis by promoting the expansion of homeostatic microglia and regulatory T cell populations. Regnase-1 expression was inversely correlated with disease severity in patients with multiple sclerosis, and MOs targeting human Regnase-1 SL structures were effective in mitigating cytokine production in human immune cells. Collectively, MO-mediated disruption of the Regnase-1 self-regulation pathway is a potential therapeutic strategy to enhance Regnase-1 abundance, which, in turn, provides therapeutic benefits for treating inflammatory diseases by suppressing inflammation.

[Perspective on transition from pediatric to adult health care for patients with neurological disease: current situation and issues].

An improvement in efficacy treatment and development of the social support system has led to many patients with neurological disease being able to reach adulthood. Therefore health care for life from pediatrics to adulthood has become necessary. The Special Committee for Measures Against Transition from Pediatric to Adult Health Care of the Japanese Society of Neurology officially started to examine the current situation and issues of transition from pediatric to adult health care in July 2020. Pediatric neurologists and adult neurologists have an awareness of this issue of constructing a better transition from pediatric to adult health care. However, there are some tasks that need to be resolved in the medical system. We intend to improve the understanding of transition and assessment of medical service fees for transition in cooperation with the Japanese Society of Neurology and the Japanese Society of Child Neurology.

[The efficacy of eculizumab against post-thymectomy exacerbations in thymoma associated myasthenia gravis (MG)].

We report a 62-year-old woman with thymoma associated myasthenia gravis (MG). She had significant dysphagia and was treated with corticosteroids, intravenous immunoglobulin (IVIG), immunoadsorption plasmapheresis (IAPP), and immunosuppressive drugs, and the extended thymectomy. Her symptoms gradually improved, but 3 weeks after thymectomy, her bulbar symptoms recurred. Although she was treated with repeated IVIG and IAPP, her symptom remained. Finally, after starting eculizumab did her symptoms go into complete remission. This case suggests the efficacy of anti-complement therapy for postoperative exacerbation of MG.

Cell-Free DNA Derived From Neutrophils Triggers Type 1 Interferon Signature in Neuromyelitis Optica Spectrum Disorder.

BACKGROUND AND OBJECTIVES: Recently accumulating evidence suggests the pivotal role of type 1 interferon (IFN-1) signature in the pathogenesis of neuromyelitis optica spectrum disorder (NMOSD). However, the mechanism of the initial trigger that augments IFN-1 pathway in the peripheral immune system of NMOSD has yet to be elucidated. METHODS: Clinical samples were obtained from 32 patients with aquaporin-4 antibody-positive NMOSD and 23 healthy subjects. IFN-1 induction in peripheral blood mononuclear cells (PBMCs) by serum-derived cell-free DNA (cfDNA) was assessed in combination with blockades of DNA sensors in vitro. CfDNA fraction was analyzed for DNA methylation profiles by bisulfite sequencing, elucidating the cellular origin of cfDNA. The induction of neutrophil extracellular trap related cell death (NETosis) was further analyzed in NMOSD and control groups, and the efficacy of pharmacologic intervention of NETosis was assessed. RESULTS: Enhanced IFN-1 induction by cfDNA derived from NMOSD was observed in PBMCs with cofactor of LL37 antimicrobial peptide. DNase treatment, cGAS inhibitor, and Toll-like receptor 9 antagonist efficiently inhibited IFN-1 production. DNA methylation pattern of cfDNA in patients with NMOSD demonstrated that the predominant cellular source of cfDNA was neutrophils. Whole blood transcriptome analysis also revealed neutrophil activation in NMOSD. In addition, enhanced NETosis induction was observed with NMOSD-derived sera, and efficient pharmacologic inhibition of NETosis with dipyridamole was observed. DISCUSSION: Our study highlights the previously unrevealed role of cfDNA predominantly released by neutrophil in the induction of IFN-1 signature in NMOSD and further indicate a novel pharmacologic target in NMOSD.