Natsumi Araya

Cytoplasmic destruction of p53 by the endoplasmic reticulum-resident ubiquitin ligase ‘Synoviolin'

Synoviolin, also called HRD1, is an E3 ubiquitin ligase and is implicated in endoplasmic reticulum ‐associated degradation. In mammals, Synoviolin plays crucial roles in various physiological and pathological processes, including embryogenesis and the pathogenesis of arthropathy. However, little is known about the molecular mechanisms of Synoviolin in these actions. To clarify these issues, we analyzed the profile of protein expression in synoviolin‐null cells. Here, we report that Synoviolin targets tumor suppressor gene p53 for ubiquitination. Synoviolin sequestrated and metabolized p53 in the cytoplasm and negatively regulated its cellular level and biological functions, including transcription, cell cycle regulation and apoptosis. Furthermore, these p53 regulatory functions of Synoviolin were irrelevant to other E3 ubiquitin ligases for p53, such as MDM2, Pirh2 and Cop1, which form autoregulatory feedback loops. Our results provide novel insights into p53 signaling mediated by Synoviolin.

Abnormally High Levels of Virus-Infected IFN-γ+CCR4+CD4+CD25+ T Cells in a Retrovirus-Associated Neuroinflammatory Disorder

Background Human T-lymphotropic virus type 1 (HTLV-1) is a human retrovirus associated with both HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), which is a chronic neuroinflammatory disease, and adult T-cell leukemia (ATL). The pathogenesis of HAM/TSP is known to be as follows: HTLV-1-infected T cells trigger a hyperimmune response leading to neuroinflammation. However, the HTLV-1-infected T cell subset that plays a major role in the accelerated immune response has not yet been identified. Principal Findings Here, we demonstrate that CD4+CD25+CCR4+ T cells are the predominant viral reservoir, and their levels are increased in HAM/TSP patients. While CCR4 is known to be selectively expressed on T helper type 2 (Th2), Th17, and regulatory T (Treg) cells in healthy individuals, we demonstrate that IFN-γ production is extraordinarily increased and IL-4, IL-10, IL-17, and Foxp3 expression is decreased in the CD4+CD25+CCR4+ T cells of HAM/TSP patients as compared to those in healthy individuals, and the alteration in function is specific to this cell subtype. Notably, the frequency of IFN-γ-producing CD4+CD25+CCR4+Foxp3− T cells is dramatically increased in HAM/TSP patients, and this was found to be correlated with disease activity and severity. Conclusions We have defined a unique T cell subset—IFN-γ+CCR4+CD4+CD25+ T cells—that is abnormally increased and functionally altered in this retrovirus-associated inflammatory disorder of the central nervous system.

HTLV-1 induces a Th1-like state in CD4 + CCR4 + T cells

Human T-lymphotropic virus type 1 (HTLV-1) is linked to multiple diseases, including the neuroinflammatory disease HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) and adult T cell leukemia/lymphoma. Evidence suggests that HTLV-1, via the viral protein Tax, exploits CD4+ T cell plasticity and induces transcriptional changes in infected T cells that cause suppressive CD4+CD25+CCR4+ Tregs to lose expression of the transcription factor FOXP3 and produce IFN-γ, thus promoting inflammation. We hypothesized that transformation of HTLV-1-infected CCR4+ T cells into Th1-like cells plays a key role in the pathogenesis of HAM/TSP. Here, using patient cells and cell lines, we demonstrated that Tax, in cooperation with specificity protein 1 (Sp1), boosts expression of the Th1 master regulator T box transcription factor (T-bet) and consequently promotes production of IFN-γ. Evaluation of CSF and spinal cord lesions of HAM/TSP patients revealed the presence of abundant CD4+CCR4+ T cells that coexpressed the Th1 marker CXCR3 and produced T-bet and IFN-γ. Finally, treatment of isolated PBMCs and CNS cells from HAM/TSP patients with an antibody that targets CCR4+ T cells and induces cytotoxicity in these cells reduced both viral load and IFN-γ production, which suggests that targeting CCR4+ T cells may be a viable treatment option for HAM/TSP.

Positive feedback loop via astrocytes causes chronic inflammation in virus-associated myelopathy

Human T-lymphotropic virus type 1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a rare neurodegenerative disease characterized by chronic inflammation in the spinal cord. We hypothesized that a positive feedback loop driven by chemokines may be responsible for the chronic inflammation in HAM/TSP. We aimed to determine the identity of these chemokines, where they are produced, and how they drive chronic inflammation in HAM/TSP. We found that patients with HAM/TSP have extraordinarily high levels of the chemokine CXCL10 (also known as IP-10) and an abundance of cells expressing the CXCL10-binding receptor CXCR3 in the cerebrospinal fluid. Histological analysis revealed that astrocytes are the main producers of CXCL10 in the spinal cords of patients with HAM/TSP. Co-culture of human astrocytoma cells with CD4+ T cells from patients with HAM/TSP revealed that astrocytes produce CXCL10 in response to IFN-γ secreted by CD4+ T cells. Chemotaxis assays results suggest that CXCL10 induces migration of peripheral blood mononuclear cells to the central nervous system and that anti-CXCL10 neutralizing antibody can disrupt this migration. In short, we inferred that human T-lymphotropic virus type 1-infected cells in the central nervous system produce IFN-γ that induces astrocytes to secrete CXCL10, which recruits more infected cells to the area via CXCR3, constituting a T helper type 1-centric positive feedback loop that results in chronic inflammation.

Human T-lymphotropic virus type 1 (HTLV-1) and regulatory T cells in HTLV-1-associated neuroinflammatory disease.

Human T-lymphotropic virus type 1 (HTLV-1) is a retrovirus that is the causative agent of adult T cell leukemia/lymphoma (ATL) and associated with multiorgan inflammatory disorders, including HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) and uveitis. HTLV-1-infected T cells have been hypothesized to contribute to the development of these disorders, although the precise mechanisms are not well understood. HTLV-1 primarily infects CD4+ T helper (Th) cells that play a central role in adaptive immune responses. Based on their functions, patterns of cytokine secretion, and expression of specific transcription factors and chemokine receptors, Th cells that are differentiated from naïve CD4+ T cells are classified into four major lineages: Th1, Th2, Th17, and T regulatory (Treg) cells. The CD4+CD25+CCR4+ T cell population, which consists primarily of suppressive T cell subsets, such as the Treg and Th2 subsets in healthy individuals, is the predominant viral reservoir of HTLV-1 in both ATL and HAM/TSP patients. Interestingly, CD4+CD25+CCR4+ T cells become Th1-like cells in HAM/TSP patients, as evidenced by their overproduction of IFN-γ, suggesting that HTLV-1 may intracellularly induce T cell plasticity from Treg to IFN-γ+ T cells. This review examines the recent research into the association between HTLV-1 and Treg cells that has greatly enhanced understanding of the pathogenic mechanisms underlying immune dysregulation in HTLV-1-associated neuroinflammatory disease.

CSF CXCL10, CXCL9, and Neopterin as Candidate Prognostic Biomarkers for HTLV-1-Associated Myelopathy/Tropical Spastic Paraparesis

Background Human T-lymphotropic virus type 1 (HTLV-1) -associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a rare chronic neuroinflammatory disease. Since the disease course of HAM/TSP varies among patients, there is a dire need for biomarkers capable of predicting the rate of disease progression. However, there have been no studies to date that have compared the prognostic values of multiple potential biomarkers for HAM/TSP. Methodology/Principal Findings Peripheral blood and cerebrospinal fluid (CSF) samples from HAM/TSP patients and HTLV-1-infected control subjects were obtained and tested retrospectively for several potential biomarkers, including chemokines and other cytokines, and nine optimal candidates were selected based on receiver operating characteristic (ROC) analysis. Next, we evaluated the relationship between these candidates and the rate of disease progression in HAM/TSP patients, beginning with a first cohort of 30 patients (Training Set) and proceeding to a second cohort of 23 patients (Test Set). We defined “deteriorating HAM/TSP” as distinctly worsening function (≥3 grades on Osames Motor Disability Score (OMDS)) over four years and “stable HAM/TSP” as unchanged or only slightly worsened function (1 grade on OMDS) over four years, and we compared the levels of the candidate biomarkers in patients divided into these two groups. The CSF levels of chemokine (C-X-C motif) ligand 10 (CXCL10), CXCL9, and neopterin were well-correlated with disease progression, better even than HTLV-1 proviral load in PBMCs. Importantly, these results were validated using the Test Set. Conclusions/Significance As the CSF levels of CXCL10, CXCL9, and neopterin were the most strongly correlated with rate of disease progression, they represent the most viable candidates for HAM/TSP prognostic biomarkers. The identification of effective prognostic biomarkers could lead to earlier detection of high-risk patients, more patient-specific treatment options, and more productive clinical trials.

Fucoidan therapy decreases the proviral load in patients with human T-lymphotropic virus type-1-associated neurological disease.

BACKGROUND Human T-lymphotropic virus type-1 (HTLV-1) is a human retrovirus that causes HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) and adult T-cell leukaemia (ATL). A higher viral load in individuals with HTLV-1 infection increases their risk of developing HAM/TSP and ATL. Moreover, the high proviral load is associated with the clinical progression of HAM/TSP. Reduction of the number of HTLV-1-infected cells is therefore crucial for preventing and treating HTLV-1-associated diseases. Recently, fucoidan, a complex sulphated polysaccharide derived from marine seaweed, has been demonstrated to exert inhibitory effects on HTLV-1 infection in vitro. In this study, we examined the in vivo effects of fucoidan on HTLV-1 infection. METHODS In this single-centre open-label trial, 13 patients with HAM/TSP were treated with 6 g fucoidan daily for 6-13 months. The HTLV-1 proviral DNA load and frequencies of HTLV-1-specific CD8(+) T-cells, natural killer cells, invariant natural killer T-cells and dendritic cells in the peripheral blood were analysed. Furthermore, the in vitro inhibitory effect of fucoidan on cell-to-cell HTLV-1 infection was examined by using luciferase reporter cell assays. RESULTS Fucoidan inhibited the cell-to-cell transmission of HTLV-1 in vitro. Furthermore, fucoidan therapy resulted in a 42.4% decrease in the HTLV-1 proviral load without affecting the host immune cells. During the treatment, no exacerbation was observed. Four patients with HAM/TSP developed diarrhoea, which improved immediately after stopping fucoidan administration. CONCLUSIONS Fucoidan is a new potential therapeutic agent for the prevention and treatment of HTLV-1-associated diseases.

The E3 ligase synoviolin controls body weight and mitochondrial biogenesis through negative regulation of PGC‐1β

Obesity is a major global public health problem, and understanding its pathogenesis is critical for identifying a cure. In this study, a gene knockout strategy was used in post‐neonatal mice to delete synoviolin (Syvn)1/Hrd1/Der3, an ER‐resident E3 ubiquitin ligase with known roles in homeostasis maintenance. Syvn1 deficiency resulted in weight loss and lower accumulation of white adipose tissue in otherwise wild‐type animals as well as in genetically obese (ob/ob and db/db) and adipose tissue‐specific knockout mice as compared to control animals. SYVN1 interacted with and ubiquitinated the thermogenic coactivator peroxisome proliferator‐activated receptor coactivator (PGC)‐1β, and Syvn1 mutants showed upregulation of PGC‐1β target genes and increase in mitochondrion number, respiration, and basal energy expenditure in adipose tissue relative to control animals. Moreover, the selective SYVN1 inhibitor LS‐102 abolished the negative regulation of PGC‐1β by SYVN1 and prevented weight gain in mice. Thus, SYVN1 is a novel post‐translational regulator of PGC‐1β and a potential therapeutic target in obesity treatment.

E3 ubiquitin ligase synoviolin is involved in liver fibrogenesis.

Background and Aim Chronic hepatic damage leads to liver fibrosis, which is characterized by the accumulation of collagen-rich extracellular matrix. However, the mechanism by which E3 ubiquitin ligase is involved in collagen synthesis in liver fibrosis is incompletely understood. This study aimed to explore the involvement of the E3 ubiquitin ligase synoviolin (Syno) in liver fibrosis. Methods The expression and localization of synoviolin in the liver were analyzed in CCl4-induced hepatic injury models and human cirrhosis tissues. The degree of liver fibrosis and the number of activated hepatic stellate cells (HSCs) was compared between wild type (wt) and Syno+/− mice in the chronic hepatic injury model. We compared the ratio of apoptosis in activated HSCs between wt and Syno+/− mice. We also analyzed the effect of synoviolin on collagen synthesis in the cell line from HSCs (LX-2) using siRNA-synoviolin and a mutant synoviolin in which E3 ligase activity was abolished. Furthermore, we compared collagen synthesis between wt and Syno−/− mice embryonic fibroblasts (MEF) using quantitative RT-PCR, western blotting, and collagen assay; then, we immunohistochemically analyzed the localization of collagen in Syno−/− MEF cells. Results In the hepatic injury model as well as in cirrhosis, synoviolin was upregulated in the activated HSCs, while Syno+/− mice developed significantly less liver fibrosis than in wt mice. The number of activated HSCs was decreased in Syno+/− mice, and some of these cells showed apoptosis. Furthermore, collagen expression in LX-2 cells was upregulated by synoviolin overexpression, while synoviolin knockdown led to reduced collagen expression. Moreover, in Syno−/− MEF cells, the amounts of intracellular and secreted mature collagen were significantly decreased, and procollagen was abnormally accumulated in the endoplasmic reticulum. Conclusion Our findings demonstrate the importance of the E3 ubiquitin ligase synoviolin in liver fibrosis.

Mogamulizumab, an Anti-CCR4 Antibody, Targets Human T-Lymphotropic Virus Type 1–infected CD8+ and CD4+ T Cells to Treat Associated Myelopathy

BACKGROUND Human T-lymphotropic virus type 1 (HTLV-1) can cause chronic spinal cord inflammation, known as HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Since CD4(+)CCR4(+) T cells are the main HTLV-1 reservoir, we evaluated the defucosylated humanized anti-CCR4 antibody mogamulizumab as a treatment for HAM/TSP. METHODS We assessed the effects of mogamulizumab on peripheral blood mononuclear cells from 11 patients with HAM/TSP. We also studied how CD8(+) T cells, namely CD8(+) CCR4(+) T cells and cytotoxic T lymphocytes, are involved in HTLV-1 infection and HAM/TSP pathogenesis and how they would be affected by mogamulizumab. RESULTS Mogamulizumab effectively reduced the HTLV-1 proviral load (56.4% mean reduction at a minimum effective concentration of 0.01 µg/mL), spontaneous proliferation, and production of proinflammatory cytokines, including interferon γ (IFN-γ). Like CD4(+)CCR4(+) T cells, CD8(+)CCR4(+) T cells from patients with HAM/TSP exhibited high proviral loads and spontaneous IFN-γ production, unlike their CCR4(-) counterparts. CD8(+)CCR4(+) T cells from patients with HAM/TSP contained more IFN-γ-expressing cells and fewer interleukin 4-expressing cells than those from healthy donors. Notably, Tax-specific cytotoxic T lymphocytes that may help control the HTLV-1 infection were overwhelmingly CCR4(-). CONCLUSIONS We determined that CD8(+)CCR4(+) T cells and CD4(+)CCR4(+) T cells are prime therapeutic targets for treating HAM/TSP and propose mogamulizumab as a new treatment.