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.

Induced pluripotent stem cell-based assays recapture multiple properties of human astrocytes.

The majority of the population of glial cells in the central nervous system consists of astrocytes, and impairment of astrocytes causes various disorders. It is useful to assess the multiple astrocytic properties in order to understand their complex roles in the pathophysiology. Although we can differentiate human astrocytes from induced pluripotent stem cells (iPSCs), it remains unknown how we can analyse and reveal the multiple properties of astrocytes in complexed human disease conditions. For this purpose, we tested astrocytic differentiation protocols from feeder-free iPSCs based on the previous method with some modifications. Then, we set up extra- and intracellular assessments of iPSC-derived astrocytes by testing cytokine release, calcium influx, autophagy induction and migration. The results led us to analytic methods with conditions in which iPSC-derived astrocytes behave as in vivo. Finally, we applied these methods for modelling an astrocyte-related disease, Alexander disease. An analytic system using iPSC-derived astrocytes could be used to recapture complexities in human astrocyte diseases.

[Data-driven drug discovery for drug repurposing].

To improve the decreased efficiency of drug discovery and development, drug repurposing (also called drug repositioning) has been expected, that it is a strategy for identifying new medical indications for approved, investigational or suspended drugs. Particularly, according to the rapid expansion of medical and life science data and the remarkable technological progress of AI technology in recent years, the approach of computational drug repurposing has been attracted as one of the applications in data-driven drug discovery. Computational drug repurposing is a method of systematical and strategical research for identifying novel indication candidates and prioritizing the indication candidates based on the various profiles of drugs, genes, and diseases. In this review article, the typical data science techniques for data-driven drug repurposing, 1. drug-target interaction prediction, 2. transcriptomics-based approach by using differentially gene expression profiles, 3. natural language processing and word embedding, and their current status were summarized. We have also introduced a use case of data-driven drug repurposing for the PPARγ/α agonist Netoglitazone that we actually analyzed. In addition, as an excellent successful case of data-driven drug repurposing in recent years, we have also discussed a repurposing case reported by BenevolentAI in 2020, that Baricitinib has been identified as a potential intervention for COVID-19, based on immunomodulatory treatment by its mechanism of action as a JAK1 and JAK2 inhibition.

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.

Changes in the pattern of cytokine production from peripheral blood mononuclear cells in patients with rheumatoid arthritis treated with infliximab and their relation to plasma arginase activity.

AIM: The aim of this study was to quantify the production of T-cell cytokines from the peripheral blood mononuclear cells (PBMCs) of RA patients before and after treatment with anti-tumor necrosis factor (TNF)-α infliximab (IFX). METHOD: We stimulated the PBMCs of RA patients (n = 24) in vitro and quantified the cytokines in the culture supernatant using enzyme-linked immunosorbent assay. RESULTS: Unexpectedly, the cytokines tested, interferon (IFN)-γ, interleukin (IL)-4 and IL-17, were all found to have increased, rather than decreased, after the treatment. When the patients were divided into two groups according to the plasma activity of arginase, which is implicated in the immune-suppressive function of myeloid-derived suppressor cells, the substantial increase in the cytokine production ex vivo was only detected in the group in which the arginase activity was decreased after the treatment with IFX. In fact, although the ex vivo production of IL-21 increased along with the other cytokines, the plasma concentration of IL-21 decreased significantly after IFX treatment. CONCLUSION: It is important to exercise caution in interpreting ex vivo cytokine production data, in that they can be negatively influenced by the immune-suppressive mechanisms that prevent excessive inflammation. Thus, to analyze the T-cell response accurately, T-cell markers that are detectable in the serum or plasma need to be discovered. The concentrations of IFN-γ, IL-4 and IL-17 were all below detection limits, but that of IL-21 was detectable in the plasma and inversely correlated with the production of IL-21 ex vivo. This may indicate the involvement of Th17 response in the pathogenesis of RA.

A novel method predicting clinical response using only background clinical data in RA patients before treatment with infliximab.

OBJECTIVES: The aim of the present study was to generate a novel method for predicting the clinical response to infliximab (IFX), using a machine-learning algorithm with only clinical data obtained before the treatment in rheumatoid arthritis (RA) patients. METHODS: We obtained 32 variables out of the clinical data on the patients from two independent hospitals. Next, we selected both clinical parameters and machine-learning algorithms and decided the candidates of prediction method. These candidates were verified by clinical variables on different patients from two other hospitals. Finally, we decided the prediction method to achieve the highest score. RESULTS: The combination of multilayer perceptron algorithm (neural network) and nine clinical parameters shows the best accuracy performance. This method could predict the good or moderate response to IFX with 92% accuracy. The sensitivity of this method was 96.7%, while the specificity was 75%. CONCLUSIONS: We have developed a novel method for predicting the clinical response using only background clinical data in RA patients before treatment with IFX. Our method for predicting the response to IFX in RA patients may have advantages over the other previous methods in several points including easy usability, cost-effectiveness and accuracy.

Nationwide epidemiological survey of 169 patients with adult Still's disease in Japan.

OBJECTIVES: A nationwide survey was conducted to assess the number of patients, clinical aspects, treatment, and prognosis of adult Still's disease (ASD) in Japan. METHODS: A primary questionnaire was sent to randomly selected medical institutions in order to estimate the number of patients. We sent a secondary questionnaire to the same institutions to characterize the clinical manifestations and treatment of ASD. RESULTS: The estimated prevalence of ASD was 3.9 per 100,000. Analysis of 169 patients showed a mean age at onset of 46 years. The main clinical symptoms were fever, arthritis, and typical rash in agreement with previous surveys. Oral glucocorticoids were used to treat 96% of the patients, while methotrexate was used in 41% and biological agents were used in 16%. Lymphadenopathy and macrophage activation syndrome were significantly associated with increased risk of relapse (P < 0.05, each). Patients who achieved remission after tocilizumab therapy had significantly longer disease duration (6.2 years) than patients who did not (1.9 years) (p < 0.05). CONCLUSIONS: The 2010-2011 nationwide survey of ASD identified important changes in treatment and improvement of prognosis compared with previous surveys.

Combination of tumor necrosis factor α and interleukin-6 induces mouse osteoclast-like cells with bone resorption activity both in vitro and in vivo.

OBJECTIVE: To clarify the function of osteoclast-like multinuclear cells differentiated from bone marrow-derived macrophages (BMMs) by a combination of tumor necrosis factor α (TNFα) and interleukin-6 (IL-6), and to investigate the molecular mechanisms underlying the differentiation. METHODS: BMMs were stimulated by TNFα and/or IL-6. The cells were then compared with conventional osteoclasts differentiated in vitro by RANKL. An in vitro pit formation assay on dentine slices and an in vivo resorption assay of calvarial bones were performed. We also evaluated the activities and expression levels of NF-κB, c-Fos, and NF-ATc1, which are essential to the differentiation of conventional osteoclasts. Small interfering RNA was used to knock down c-Fos. The effects of genetic ablation of STAT-3 and pharmacologic inhibitors of NF-AT, JAK, and ERK were also studied. RESULTS: Osteoclast-like cell differentiation depended on TNFα and IL-6 and was not inhibited by osteoprotegerin. These differentiated cells were associated with both in vitro and in vivo bone resorption activity. TNFα and IL-6 had a synergistic effect on the activity and expression of c-Fos. Knockdown of c-Fos inhibited the expression of NF-ATc1 and the differentiation of osteoclast-like cells. All of these inhibitors blocked differentiation of the cells in vitro, but surprisingly, the conditional knockout of STAT-3 did not. Tofacitinib also inhibited the bone destruction caused by TNFα and IL-6 in vivo. CONCLUSION: Our results demonstrate that a combination of the inflammatory cytokines TNFα and IL-6 can induce osteoclast-like cells that have in vitro and in vivo bone-resorptive activity.

Marked induction of c-Maf protein during Th17 cell differentiation and its implication in memory Th cell development.

Until recently, effector T helper (Th) cells have been classified into two subsets, Th1 and Th2 cells. Since the discovery of Th17 cells, which produce IL-17, much attention has been given to Th17 cells, mainly because they have been implicated in the pathogenesis of various inflammatory diseases. We have performed transcriptome analysis combined with factor analysis and revealed that the expression level of c-Maf, which is considered to be important for Th2 differentiation, increases significantly during the course of Th17 differentiation. The IL-23 receptor (IL-23R), which is important for Th17 cells, is among putative transcriptional targets of c-Maf. Interestingly, the analysis of c-Maf transgenic Th cells revealed that the overexpression of c-Maf did not lead to the acceleration of the early stage of Th17 differentiation but rather to the expansion of memory phenotype cells, particularly with Th1 and Th17 traits. Consistently, mouse wild-type memory Th cells expressed higher mRNA levels of c-Maf, IL-23R, IL-17, and IFN-γ than control cells; in contrast, Maf(-/-) memory Th cells expressed lower mRNA levels of those molecules. Thus, we propose that c-Maf is important for the development of memory Th cells, particularly memory Th17 cells and Th1 cells.

Geranylgeranyl-pyrophosphate regulates secretion of pentraxin 3 and monocyte chemoattractant protein-1 from rheumatoid fibroblast-like synoviocytes in distinct manners.

OBJECTIVES: We previously reported that 10 mg/day of simvastatin significantly reduced clinical scores of rheumatoid arthritis (RA) in active RA patients with hypercholesterolemia. In this study, we have investigated the mechanism by which simvastatin inhibits the production of the mediators of inflammation, such as pentraxin 3 (PTX3) and monocyte chemoattractant protein-1 (MCP-1), from fibroblast-like synoviocytes (FLS) derived from patients with RA. METHODS: FLS from RA patients were cultured with 0-10 µM simvastatin for 24 h. ELISA and real-time PCR were used to quantitate the protein level and the mRNA level of PTX3 and MCP-1, respectively. RESULTS: Simvastatin both reduced the secretion of PTX3 and MCP-1 in FLS cultures and inhibited their mRNA expression in these cells. The effects of simvastatin were all completely reversed in the presence of mevalonic acid or geranylgeranylpyrophosphate, but not in the presence of farnesyl-pyrophosphate. The geranylgeranyl transferase inhibitor GGTI-298 and the Rho kinase inhibitor Y-27632 inhibited the production of PTX3 but not of MCP-1. CONCLUSIONS: Although simvastatin inhibited the production of PTX3 and MCP-1 in RA FLS, the mechanisms were quite different. It inhibits PTX3 production in a Rho-dependent manner but MCP-1 production in a Rho-independent manner. These results shed light on novel aspects of the anti-inflammatory mechanisms of simvastatin and may prove its important role in the treatment of rheumatic diseases.

Analysis of cytokine production patterns of peripheral blood mononuclear cells from a rheumatoid arthritis patient successfully treated with rituximab.

We had a rheumatoid arthritis (RA) patient resistant to multiple drugs and who developed panniculitis due to etanercept treatment, then responded fairly well to rituximab. Intracellular staining of cytokines in the peripheral blood mononuclear cells before and after rituximab administration revealed that the cytokine production, representative of T-helper (Th)1-, Th2-, and Th17-type responses, decreased abruptly after the treatment. Interestingly, this timing coincided with that of the manifestation of the beneficial effect. This relationship may provide useful insight into the mechanism of action of the drug and hence about the pathogenesis of RA.

Efficacy of tacrolimus in infliximab-refractory progressive rheumatoid arthritis.

We report a Japanese male patient with intractable rheumatoid arthritis (RA), in whom tacrolimus was effective ultimately. Five years before the admission he was diagnosed as RA, which was resistant to various disease-modifying anti-rheumatic drugs (DMARDs). Two years before, administration of infliximab was initiated although the medicine failed to control RA. In spite of the multiple joint replacement, the RA disease activity worsened. Tacrolimus (1.5 mg/day) was administered. Twenty-four weeks of tacrolimus treatment reduced the disease activity score for 28 joints-erythrocyte sedimentation rate from 7.44 to 3.65. Herein, we present a patient with RA, who was successfully treated by tacrolimus, and in whom infliximab was not effective. Tacrolimus may be one of the drugs for RA patients refractory to the conventional treatments including methotrexate or tumor necrosis factor inhibitors.

Vasculo-Behçet's disease with non-traumatic subcapsular hematoma of the kidney and aneurysmal dilatations of the celiac and superior mesenteric arteries.

We report a patient with vasculo-Behçet's disease treated successfully with a high dose of prednisolone. In 2002, the patient was diagnosed with vasculo-Behçet's disease. He was admitted to our hospital because of sudden-onset right lower back pain in June 2006. Upon admission, abdominal angiography revealed aneurysmal dilatations of the celiac and superior mesenteric arteries. He was treated promptly with high-dose prednisolone, after which the aneurysms displayed no further enlargement. As we believe this case to be quite rare, we report this case with a literature review in support of this characterization.

Comparative study of circadian oscillatory network models of Drosophila.

The circadian clock of Drosophila is a model pathway for research in biological clock mechanisms, both with traditional experimental approaches and with emerging systems biology approaches utilizing mathematical modeling and in silico computer simulation. Dynamic diurnal oscillations are achieved by the complex interaction of components as a system, and mathematical reconstruction has proven to be an invaluable means of understanding such systematic behavior. In this study, we implemented eight published models of the Drosophila circadian clock in Systems Biology Markup Language (SBML) for comparative systems biology studies using E-Cell Simulation Environment version 3, to examine the system-level requirements for the clock mechanism to be robust, by calculating the period and amplitude sensitivity coefficients with simulation experiments. While all models were generally robust as determined by the network topology of the oscillatory feedback loop structure, existing models place relatively strong emphasis on transcription regulation, although this is a limitation on robustness. We suggest that more comprehensive modeling including protein phosphorylation, polymerization, and nuclear transport with regard to amplitude sensitivity will be necessary for understanding the light entrainment and temperature compensation of circadian clocks.

High concentration simvastatin induces apoptosis in fibroblast-like synoviocytes from patients with rheumatoid arthritis.

OBJECTIVE: We previously reported that 10 mg/day of simvastatin significantly reduced clinical scores of rheumatoid arthritis (RA) in patients with active RA with hypercholesterolemia. We have also reported that a certain pharmacological concentration of simvastatin, i.e., 0.05-0.1 microM, inhibits the production of interleukin 6 (IL-6) and IL-8 and the cell proliferation induced by tumor necrosis factor-alpha (TNF-alpha) in fibroblast-like synoviocytes (FLS) derived from patients with RA in vitro. We investigated other effects of simvastatin on FLS from the standpoint of cell viability and apoptosis. METHODS: RA FLS were cultured with or without 0.05-50 microM simvastatin for 48 h. Cell viability was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Apoptosis was measured by flow cytometric analysis using propidium iodide and annexin-V. Caspase-3 and -9 activities were analyzed by colorimetric assays. RESULTS: High concentrations of simvastatin, i.e., 1.0-50 microM, reduced cell viability and induced prominent apoptosis in FLS in a dose-dependent manner. The apoptosis induced by simvastatin was caspase-3- and caspase-9-dependent. These effects were completely reversed in the presence of mevalonic acid or geranylgeranyl-pyrophosphate, but not in the presence of farnesyl-pyrophosphate. Further, a geranylgeranyl transferase inhibitor and a RhoA kinase inhibitor mimicked the effect of simvastatin. CONCLUSION: These data, together with our previous report, suggest that low (pharmacological range) and high concentrations of simvastatin affect FLS differently: (1) at a low concentration, it inhibits IL-6 and IL-8 production and the cell proliferation of FLS induced by TNF-alpha (2) at high concentrations, it induces apoptosis in FLS. Understanding this dose-dependent biphasic effect of simvastatin may prove important for its clinical applications in the treatment of RA.

A mathematical model for the Kai-protein-based chemical oscillator and clock gene expression rhythms in cyanobacteria.

In the cyanobacterium, Synechococcus elongatus, most promoters are regulated by a circadian clock under continuous light (LL) conditions. Nevertheless, the basic circadian oscillation is primarily generated by alternating KaiC phosphorylation/dephosphorylation reactions at the posttranslational level. Indeed, the KaiC phosphorylation cycle was recently reconstituted in vitro by incubating KaiA, KaiB, and KaiC proteins with ATP. However, the molecular dynamics of this chemical oscillation and the mechanism that drives the circadian transcription/translation rhythms remain unknown. In this report, the KaiC phosphorylation cycle and the gene regulatory network in the cyanobacterial circadian system have been modeled. The model reproduces the robust KaiC phosphorylation cycle in the absence of de novo gene expression as is observed in vitro, as well as its coupling to transcriptional/translational feedback in LL conditions in vivo. Moreover, the model is consistent with most previous experiments, including various combinations of genetic knockout or overexpression of kai genes. It also predicts that multiple KaiC phosphorylation states and dynamic Kai protein interactions may be required for the cyanobacterial circadian system.