Pathological role of activated mTOR in CXCR3+ memory B cells of rheumatoid arthritis.

OBJECTIVES: B cells play an important pathological role in RA. In this study, we investigated the role of metabolic regulator mTOR in B cells and its relevance to the pathology of RA. METHODS: Peripheral blood mononuclear cells were isolated from 31 normal subjects and 86 RA patients and the gated B cells were assessed for mTOR phosphorylation and chemokine receptor expression. In vitro studies on peripheral blood B cells isolated from the control and RA patients investigated the molecular mechanisms. RESULTS: Higher concentrations of CXCL10 (CXCR3 ligands) and lower percentages of CXCR3+ memory B cells were present in the peripheral blood of RA patients relative to the control. RA patients with high CXCL10 concentrations had smaller percentage of CXCR3+ memory B cells and high disease activity. One-year treatment with TNF inhibitors increased the percentage of CXCR3+ memory B cells and reduced serum CXCL10 concentrations. mTOR phosphorylation in B cells was further enhanced in RA patients, compared with the control, and was selectively enhanced in CXCR3+ memory B cells. mTOR phosphorylation in CXCR3+ memory B cells correlated with disease activity. In vitro, mTOR phosphorylation in B cells enhanced IL-6 production and increased RANKL expression. CONCLUSION: mTOR activation in CXCR3+ memory B cells of RA patients is associated with disease activity, mediated through IL-6 production and RANKL expression. The obtained results also suggest that TNF inhibitors mediate an impact on the association between CXCL10 and mTOR activated CXCR3+ memory B cells.

In vitro pharmacological effects of peficitinib on lymphocyte activation: a potential treatment for systemic sclerosis with JAK inhibitors.

OBJECTIVES: Peficitinib, a novel Janus kinase (JAK) inhibitor, demonstrated promising results in treating RA in phase 3 clinical trials. This in vitro study was undertaken to characterize the pharmacological properties of peficitinib and investigate the involvement of JAK and signal transducer and activator of transcription (STAT) pathways in the pathological processes of SSc, which is also an autoimmune disease. METHODS: Phosphorylation levels of STAT molecules were assessed in peripheral blood mononuclear cells collected from patients with RA or SSc and healthy subjects, and in skin specimens obtained from 19 patients with SSc. In vitro inhibition of STAT phosphorylation and cytokine/chemokine production by peficitinib, tofacitinib and baricitinib were also characterized. RESULTS: Higher spontaneous STAT1 or STAT3 phosphorylation was observed in peripheral T-cells and monocytes from patients with RA and SSc compared with healthy subjects. In skin sections from patients with SSc, phosphorylated STAT3-positive cells were found in almost all cases, irrespective of disease subtype or patient characteristics. Conversely, phosphorylated STAT1-positive cells were observed only in samples from untreated patients with diffuse disease of short duration. Peficitinib inhibited STAT phosphorylation induced by various cytokines, with comparable efficacy to tofacitinib and baricitinib. Peficitinib also suppressed cytokine and chemokine production by peripheral blood mononuclear cells and skin fibroblasts. CONCLUSION: Our results suggest that JAK/STAT pathways are constitutively activated in SSc and RA, and that the JAK inhibitor may represent a novel therapeutic option for SSc.

Type I and II interferons commit to abnormal expression of chemokine receptor on B cells in patients with systemic lupus erythematosus.

Memory B cells are increased in systemic lupus erythematosus (SLE) cases, but the qualitative abnormalities and induction mechanism of these cells are unclear. Here, we subclassified B cells by their chemokine receptor expression and investigated their induction mechanism. The peripheral blood of patients with SLE showed higher levels of CXCR5- and CXCR3+ B cells. CXCR5-CXCR3+ B cell levels were elevated in patients with active SLE, which decreased with improving disease conditions. Interferon (IFN)-γ stimulation increased CXCR3 expression, whereas IFN-ß stimulation reduced CXCR5 expression in B cells. Furthermore, CXCR5-CXCR3+ B cells were induced by a combination of IFN-ß and IFN-γ stimulation. Renal tissue examination of patients with active lupus nephritis confirmed the presence of CD19+CXCR3+ B cells. Collectively, the results revealed qualitative abnormalities accompanying reduced CXCR5 expression via type I IFN and enhanced CXCR3 expression via type II IFN in SLE, suggesting their involvement in B cell infiltration into tissues and inflammatory pathogenesis.

Expansion of T follicular helper-T helper 1 like cells through epigenetic regulation by signal transducer and activator of transcription factors.

OBJECTIVES: T follicular helper (Tfh) cells are critical in the development and progression of systemic lupus erythematosus (SLE). To assess the characteristics and mechanisms of differentiation of Tfh cells, we investigated the phenotype of T helper cells in patients with SLE and underlying epigenetic modifications by cytokine-induced signal transducer and activators of transcription (STAT) family factors. METHODS: Peripheral blood mononuclear cells from patients and healthy donors were analysed by flow cytometry. CD4+ T cells were isolated and cultured under various stimulations. Expression of characteristic markers and phosphorylation of STATs were analysed by flow cytometry and quantitative PCR. Histone modifications were analysed by chromatin immunoprecipitation (ChIP)-PCR. RESULTS: Differentiation of CD4+CXCR5+CXCR3+Bcl-6+T-bet+IL-21+IFN-γ+Tfh-Th1-like cells was induced by interleukin (IL)-12-induced activation of STAT1 and STAT4 simultaneously. The loci of Bcl-6 and T-bet at STAT binding sites were marked by bivalent histone modifications. After IL-12 stimulation, both STAT1 and STAT4 directly bound on BCL6 and TBX21 gene loci accompanied by suppression of repressive histone mark trimethylated histone 3 lysine 27. Levels of serum IL-12 and interferon (IFN)-γ, expression of IL-12 receptors and proportion of CXCR5+CXCR3+ activated Tfh-Th1-like cells were increased in patients with SLE. Furthermore, the level of pSTAT1, pSTAT4 and T-bet were higher in activated Tfh-Th1-like cells than non-Tfh-Th1 cells. CONCLUSION: Our findings suggest that IL-12-mediated co-activation of STAT1 and STAT4 alters histone modification, resulting in differentiation of Tfh-Th1-like cells that are characteristically expanded in patients with SLE. This could be one of the underlying mechanisms responsible for expansion of Tfh-Th1-like cells and potentially helpful towards development of cell-specific treatment for SLE.

Preclinical Characterization of ASP2713, a Novel Igß and FcγRIIB Cross-Linking Antibody, for Prediction of Human Pharmacokinetics and Clinically Effective Dose.

Previously, we reported the fundamental pharmacological characteristics of a novel Igß and Fc gamma receptor IIB cross-linking antibody, ASP2713, as a new treatment option for systemic lupus erythematosus. The aims of the present study were to investigate ASP2713's characteristics with regard to pharmacological effect, pharmacokinetics (PK), and receptor occupancy, and to predict its human PK and clinically effective dose. The relationship between the concentration and receptor occupancy of ASP2713 for Igß of B cell receptors was examined using whole blood B cells. Calculated EC50 values in cynomolgus monkeys and healthy volunteers were 0.35 and 0.058 µg/mL, respectively. Dose-dependent inhibition of anti-tetanus toxoid (TTx) antibody production, PK, and receptor occupancy of ASP2713 in TTx-sensitized cynomolgus monkeys suggested a minimally effective dose of 1 mg/kg by single intravenous (IV) administration. Scaling-up of monkey PK parameters to humans by allometric scaling predicted a clinically effective dose of 0.4 mg/kg IV administration at 4-week intervals to maintain a trough concentration in humans which achieved the same receptor occupancy expected at the effective dose in monkeys. This study aids in understanding the characteristics of ASP2713 and can be used as a basis for clinical dose setting.

Kdm5a promotes B cell activation in systemic lupus erythematosus via downregulation of A20 by histone modification.

Systemic lupus erythematosus (SLE) is a classic autoimmune connective tissue disease, which leads to multiple organ system injury. Tumor necrosis factor-induced protein 3 (TNFAIP3), generally called A20, has been documented to go together with the development of SLE. However, the role and mechanism of A20 in the progression of SLE are still unrevealed. In our study, A20 was downregulated in B cells from SLE patients and B cell responsiveness was significantly elevated in SLE patients. Overexpression of A20 restrained the proliferation and induced the apoptosis of B cells. Additionally, trimethylation of histone H3 Lysine 4 (H3K4me3) was decreased in the A20 promoter of SLE B cells. Lysine demethylase 5 A (Kdm5a) was significantly increased in B cells from SLE patients and negatively correlated with A20 expression. Further, Kdm5a knockdown increased the H3K4me3 level and A20 expression. More importantly, Kdm5a promoted the proliferation and inhibited the apoptosis of B cells in SLE via downregulation of A20. In general, Kdm5a promoted the proliferation and inhibited the apoptosis of B cells in SLE via downregulation of A20 by decreasing H3K4me3 enrichment level in the A20 promoter, suggesting a novel mechanism underlying SLE progression, and providing a promising therapeutic target for SLE. AVAILABILITY OF DATA AND MATERIALS: All data generated or analyzed during this study are included in this published article and its additional files.

Discovery of a novel Igß and FcγRIIB cross-linking antibody, ASP2713, and its potential application in the treatment of systemic lupus erythematosus.

B cell-targeted therapies have evolved as established therapies for systemic lupus erythematosus (SLE); however, existing approaches still do not thoroughly satisfy clinical requirements due to limited efficacy against memory B cells, autoantibody-producing plasmablasts and disease heterogeneity. To provide a new treatment option for SLE, we created a novel anti-Igß antibody with enhanced affinity for Fc gamma receptor (FcγR) IIB called ASP2713. ASP2713 cross-reacted with both human and cynomolgus monkey Igß and showed increased binding affinity for human and monkey FcγRIIB compared to native human IgG1. This binding property allows dominant B cell binding and induction of intrinsic negative feedback signals. In human B cells, ASP2713 significantly and concentration-dependently induced FcγRIIB ITIM phosphorylation, while suppressing proliferation under B cell receptor stimulation. This pharmacological effect was also confirmed in in vitro B cell proliferation and antibody production assays using peripheral B cells isolated from patients with SLE. In a cynomolgus monkey tetanus toxoid-induced antibody production model, ASP2713 almost completely inhibited the increase in antigen-specific antibodies with superior efficacy to rituximab. Additionally, ASP2713 significantly suppressed recall antibody production in response to secondary tetanus toxoid immunization, indicating the memory B cell- and plasmablast-targeting potential of ASP2713. Our results suggest that ASP2713 may have therapeutic potential as a treatment for SLE, where B cells play a pathogenic role.

Janus Kinase Inhibitor Baricitinib Modulates Human Innate and Adaptive Immune System.

The purpose of this study was to elucidate the mechanism of action of baricitinib on Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling, which involves in human innate and adaptive immune system. The effects of baricitinib were evaluated using human monocyte-derived dendritic cells (MoDCs), plasmacytoid dendritic cells (pDCs), B cells, and T cells. Baricitinib concentration-dependently suppressed the expression of CD80/CD86 on MoDCs and the production of type-I interferon (IFN) by pDCs. Baricitinib also suppressed the differentiation of human B cells into plasmablasts by B cell receptor and type-I IFN stimuli and inhibited the production of interleukin (IL)-6 from B cells. Human CD4+ T cells proliferated after T cell receptor stimulation with anti-CD3 and anti-CD28 antibody; however, such proliferation was suppressed by baricitinib in a concentration-dependent manner. In addition, baricitinib inhibited Th1 differentiation after IL-12 stimulation and Th17 differentiation by TGF-ß1, IL-6, IL-1ß, and IL-23 stimulation. Tofacitinib showed similar effects in these experiments. In naive CD4+ T cells, IFN-α and IFN-γ induced phosphorylation of STAT1, which was inhibited by baricitinib and tofacitinib. Furthermore, IL-6-induced phosphorylation of STAT1 and STAT3 was also inhibited by JAK inhibitors. In conclusion, the results indicated that baricitinib suppresses the differentiation of plasmablasts, Th1 and Th17 cells, as well as innate immunity, such as the T cell stimulatory capacity of dendritic cells. Thus, JAK inhibitors can be potentially clinically effective not only in rheumatoid arthritis but other immune-related diseases.

Sequential regulation of gibberellin, brassinosteroid, and jasmonic acid biosynthesis occurs in rice coleoptiles to control the transcript levels of anti-microbial thionin genes.

Transcripts of thionin genes encoding antimicrobial peptides were present at a high level in rice coleoptiles just after germination, and decreased to an undetectable level after about 3 d, but this decline was suppressed by co-treatment with gibberellic acid (GA(3)) and brassinolide (BL). The temporal expression patterns of key enzyme genes for the biosyntheses of gibberellins (GAs) and brassinosteroids (BRs) were correlated with the fluctuation of thionin mRNAs. Jasmonic acid (JA) replaced the effect of GA3 and BL, and its change in endogenous level was parallel to that of the thionin genes. These results strongly suggest that thionin gene expression was positively regulated by JA, whose endogenous level was synergistically regulated by GAs and BRs. In contrast, thionin gene expression in etiolated seedlings remained high while the endogenous level of JA was low, suggesting the presence of another signaling pathway in the dark to maintain the thionin level.