Arian Laurence

EZH2 is crucial for both differentiation of regulatory T cells and T effector cell expansion

The roles of EZH2 in various subsets of CD4+ T cells are controversial and its mechanisms of action are incompletely understood. FOXP3-positive Treg cells are a critical helper T cell subset, and dysregulation of Treg generation or function results in systemic autoimmunity. FOXP3 associates with EZH2 to mediate gene repression and suppressive function. Herein, we demonstrate that deletion of Ezh2 in CD4 T cells resulted in reduced numbers of Treg cells in vivo and differentiation in vitro and an increased proportion of memory CD4 T cells in part due to exaggerated production of effector cytokines. Furthermore, we found that both Ezh2-deficient Treg cells and T effector cells were functionally impaired in vivo: Tregs failed to constrain autoimmune colitis and T effector cells neither provided a protective response to T. gondii infection nor mediated autoimmune colitis. The dichotomous function of EZH2 in regulating differentiation and senescence in effector and regulatory T cells helps to explain the apparent existing contradictions in literature.

Reduced expression of transcriptional intermediary factor 1 gamma promotes metastasis and indicates poor prognosis of hepatocellular carcinoma

Transcriptional intermediary factor 1 gamma (TIF1γ) may play either a potential tumor‐suppressor or ‐promoter role in cancer. Here we report on a critical role of TIF1γ in the progression of hepatocellular carcinoma (HCC). Reduced expression of TIF1γ was detected in HCC, especially in advanced HCC tissues, compared to adjacent noncancerous tissues. HCC patients with low TIF1γ expression had shorter overall survival times and higher recurrence rates than those with high TIF1γ expression. Reduced TIF1γ expression was an independent and significant risk factor for recurrence and survival after curative resection. In HCC cells, TIF1γ played a dual role: It promoted tumor growth in early‐stage HCC, but not in advanced‐stage HCC, whereas it inhibited invasion and metastasis in both early‐ and advanced‐stage HCC. Mechanistically, we confirmed that TIF1γ inhibited transforming growth factor‐β/ Drosophila mothers against decapentaplegic protein (TGF‐β/Smad) signaling through monoubiquitination of Smad4 and suppressed the formation of Smad2/3/4 complex in HCC cells. TGF‐β‐inducing cytostasis and metastasis were both inhibited by TIF1γ in HCC. We further proved that TIF1γ suppressed cyotstasis‐related TGF‐β/Smad downstream c‐myc down‐regulation, as well as p21/cip1 and p15/ink4b up‐regulation in early‐stage HCC. Meanwhile, TGF‐β inducible epithelial‐mesenchymal transition and TGF‐β/Smad downstream metastatic cascades, including phosphatase and tensin homolog deleted on chromosome ten down‐regulation, chemokine (CXC motif) receptor 4 and matrix metalloproteinase 1 induction, and epidermal growth factor receptor– and protein kinase B–signaling transactivation, were inhibited by TIF1γ. In addition, we found that the down‐regulation of TIF1γ in HCC was caused by hypermethylation of CpG islands in the TIF1γ promoter, and demonstrated that the combination of TIF1γ and phosphorylated Smad2 was a more powerful predictor of poor prognosis. Conclusion: TIF1γ regulates tumor growth and metastasis through inhibition of TGF‐β/Smad signaling and may serve as a novel prognostic biomarker in HCC. (Hepatology 2014;60:1620–1636)

Targeting JAK/STAT signalling in inflammatory skin diseases with small molecule inhibitors

For most inflammatory skin diseases topical glucocorticosteroids and traditional oral immunosuppressive drugs remain the principle treatment choices, but this has started to change. A deeper understanding in individual disease pathogenesis, basic immune mechanisms and molecular signalling pathways, together with advances in pharmaceutical drug development, allow us to interfere more precisely with disease‐related factors. Some examples of inflammation‐controlling interventions include antibodies neutralizing disease‐associated cytokines, and small molecules targeting intracellular pathways relevant to cytokine production or cytokine signalling. So far, this is best established for psoriasis, an inflammatory skin disease dominated by Th17 cytokines. In this review, we focus on chronic inflammatory skin diseases where cytokines using type I/II cytokine receptors play a dominant role in disease pathogenesis and where novel treatments with inhibitors of the JAK/STAT pathway are already under clinical investigation. To better understand the rationale of using JAK/STAT inhibitors in the discussed skin diseases, we give an overview of important genetic and immunological associations with the JAK/STAT pathway and summarize the stage of clinical development of small molecular inhibitors. JAK/STAT inhibitors will presumably find wide application in dermatology, since they can be applied not only systematically but also topically for the treatment of inflammatory skin diseases.

Global analysis of DNA methylation in hepatocellular carcinoma by a liquid hybridization capture-based bisulfite sequencing approach.

BackgroundEpigenetic alterations, such as aberrant DNA methylation of promoter and enhancer regions, which lead to atypical gene expression, have been associated with carcinogenesis. In hepatocellular carcinoma (HCC), genome-wide analysis of methylation has only recently been used. For a better understanding of hepatocarcinogenesis, we applied an even higher resolution analysis of the promoter methylome to identify previously unknown regions and genes differentially methylated in HCC.ResultsOptimized liquid hybridization capture-based bisulfite sequencing (LHC-BS) was developed to quantitatively analyze 1.86 million CpG sites in individual samples from eight pairs of HCC and adjacent tissues. By linking the differentially methylated regions (DMRs) in promoters to the differentially expressed genes (DEGs), we identified 12 DMR-associated genes. We further utilized Illumina MiSeq combining the bisulfite sequencing PCR approach to validate the 12 candidate genes. Analysis of an additional 78 HCC pairs on the Illumina MiSeq platform confirmed that 7 genes showed either promoter hyper-methylation (SMAD6, IFITM1, LRRC4, CHST4, and TBX15) or hypo-methylation (CCL20 and NQO1) in HCC.ConclusionsNovel methylome profiling provides a cost-efficient approach to identifying candidate genes in human HCC that may contribute to hepatocarcinogenesis. Our work provides further information critical for understanding the epigenetic processes underlying tumorigenesis and development of HCC.

Biallelic interferon regulatory factor 8 mutation: A complex immunodeficiency syndrome with dendritic cell deficiency, monocytopenia, and immune dysregulation

Background: The homozygous K108E mutation of interferon regulatory factor 8 (IRF8) is reported to cause dendritic cell (DC) and monocyte deficiency. However, more widespread immune dysfunction is predicted from the multiple roles ascribed to IRF8 in immune cell development and function. Objective: We sought to describe the effect on hematopoiesis and immunity of the compound heterozygous R83C/R291Q mutation of IRF8, which is present in a patient with recurrent viral infection, granuloproliferation, and intracerebral calcification. Methods: Variant IRF8 alleles were identified by means of exome sequencing, and their function was tested by using reporter assays. The cellular phenotype was studied in detail by using flow cytometry, functional immunologic assay transcriptional profiling, and antigen receptor profiling. Results: Both mutations affected conserved residues, and R291Q is orthologous to R294, which is mutated in the BXH2 IRF8‐deficient mouse. R83C showed reduced nuclear translocation, and neither mutant was able to regulate the Ets/IRF composite element or interferon‐stimulated response element, whereas R291Q retained BATF/JUN interactions. DC deficiency and monocytopenia were observed in blood, dermis, and lung lavage fluid. Granulocytes were consistently increased, dysplastic, and hypofunctional. Natural killer cell development and maturation were arrested. TH1, TH17, and CD8+ memory T‐cell differentiation was significantly reduced, and T cells did not express CXCR3. B‐cell development was impaired, with fewer memory cells, reduced class‐switching, and lower frequency and complexity of somatic hypermutation. Cell‐specific gene expression was widely disturbed in interferon‐ and IRF8‐regulated transcripts. Conclusions: This analysis defines the clinical features of human biallelic IRF8 deficiency, revealing a complex immunodeficiency syndrome caused by DC and monocyte deficiency combined with widespread immune dysregulation.

PD-1 Inhibitory Receptor Downregulates Asparaginyl Endopeptidase and Maintains Foxp3 Transcription Factor Stability in Induced Regulatory T Cells

SUMMARY CD4+ T cell differentiation into multiple T helper (Th) cell lineages is critical for optimal adaptive immune responses. This report identifies an intrinsic mechanism by which programmed death‐1 receptor (PD‐1) signaling imparted regulatory phenotype to Foxp3+ Th1 cells (denoted as Tbet+iTregPDL1 cells) and inducible regulatory T (iTreg) cells. Tbet+iTregPDL1 cells prevented inflammation in murine models of experimental colitis and experimental graft versus host disease (GvHD). Programmed death ligand‐1 (PDL‐1) binding to PD‐1 imparted regulatory function to Tbet+iTregPDL1 cells and iTreg cells by specifically downregulating endo‐lysosomal protease asparaginyl endopeptidase (AEP). AEP regulated Foxp3 stability and blocking AEP imparted regulatory function in Tbet+iTreg cells. Also, Aep−/− iTreg cells significantly inhibited GvHD and maintained Foxp3 expression. PD‐1‐mediated Foxp3 maintenance in Tbet+ Th1 cells occurred both in tumor infiltrating lymphocytes (TILs) and during chronic viral infection. Collectively, this report has identified an intrinsic function for PD‐1 in maintaining Foxp3 through proteolytic pathway. Graphical Abstract Figure. No caption available. HighlightsAsparaginyl endopeptidase (AEP) is expressed in induced regulatory T cellsAEP cleaves Foxp3 and Aep−/− mice have elevated numbers of peripheral Treg cellsAEP deficiency increases Treg cell frequency and numbers in GvHD and melanomaPD‐1 signaling maintains Foxp3 protein expression by inhibiting AEP &NA; Th1 cells are known for their enhanced stability, so mechanisms that mediate their flexibility are poorly studied. Here, Stathopoulou et al. demonstrate that plasticity of Th1 cells to Tbet+iTreg cells is mediated by PD‐1 signaling via asparaginyl endopeptidase (AEP). AEP inhibition enhanced iTreg cells in GvHD and tumor models.

Loss of 11βHSD1 enhances glycolysis, facilitates intrahepatic metastasis, and indicates poor prognosis in hepatocellular carcinoma.

11Beta-hydroxysteroid dehydrogenase type 1 (11βHSD1), converting glucocorticoids from hormonally inactive cortisone to active cortisol, plays an essential role in glucose homeostasis. Accumulating evidence suggests that enhanced glycolytic activity is closely associated with postoperative recurrence and prognosis of hepatocellular carcinoma (HCC). Whether 11βHSD1 contributes to HCC metastasis and recurrence remains unclear. Here we found that expression of 11βHSD1 in human HCC (310 pairs) was frequently decreased compared to the adjacent non-neoplastic liver tissues (ANT), which correlated well with the intrahepatic-metastatic index, serum glycemia, and other malignant clinicopathological characteristics of HCC and predicted poor prognosis. Knockdown of 11βHSD1 in BEL-7402 cells drastically reduced the pH of culture medium and induced cell death. Meanwhile, overexpression of 11βHSD1 in SMMC-7721 HCC cells resulted in repression of cell migration, invasion, angiogenesis, and proliferation in vitro. When transferred into BALB/c nude mice, 11βHSD1 overexpression resulted in decreased intrahepatic metastasis, angiogenesis, and tumor size. F-18-2-fluoro-2-deoxyglucose accumulation assay measured by positron emission tomography elucidated that 11βHSD1 reduced glucose uptake and glycolysis in SMMC-7721 cells in vitro, and intrahepatic metastasis foci and subcutaneous tumor growth in vivo. We showed that 11βHSD1 repressed cell metastasis, angiogenesis and proliferation of HCC by causing disruption of glycolysis via the HIF-1α and c-MYC pathways. In conclusion, 11βHSD1 inhibits the intrahepatic metastasis of HCC via restriction of tumor glycolysis activity and may serve as a prognostic biomarker for patients.

Effect of Huaier granule on recurrence after curative resection of HCC: a multicentre, randomised clinical trial

Objective There is little evidence that adjuvant therapy after radical surgical resection of hepatocellular carcinoma (HCC) improves recurrence-free survival (RFS) or overall survival (OS). We conducted a multicentre, randomised, controlled, phase IV trial evaluating the benefit of an aqueous extract of Trametes robinophila Murr (Huaier granule) to address this unmet need. Design and results A total of 1044 patients were randomised in 2:1 ratio to receive either Huaier or no further treatment (controls) for a maximum of 96 weeks. The primary endpoint was RFS. Secondary endpoints included OS and tumour extrahepatic recurrence rate (ERR). The Huaier (n=686) and control groups (n=316) had a mean RFS of 75.5 weeks and 68.5 weeks, respectively (HR 0.67; 95% CI 0.55 to 0.81). The difference in the RFS rate between Huaier and control groups was 62.39% and 49.05% (95% CI 6.74 to 19.94; p=0.0001); this led to an OS rate in the Huaier and control groups of 95.19% and 91.46%, respectively (95% CI 0.26 to 7.21; p=0.0207). The tumour ERR between Huaier and control groups was 8.60% and 13.61% (95% CI −12.59 to −2.50; p=0.0018), respectively. Conclusions This is the first nationwide multicentre study, involving 39 centres and 1044 patients, to prove the effectiveness of Huaier granule as adjuvant therapy for HCC after curative liver resection. It demonstrated a significant prolongation of RFS and reduced extrahepatic recurrence in Huaier group. Trial registration NCT01770431; Post-results.

Tbet is a critical modulator of FoxP3 expression in autoimmune graft-versus-host disease

CD4+ T-helper subsets drive autoimmune chronic graft-versus-host disease, a major complication after allogeneic bone marrow transplantation. However, it remains unclear how specific T-helper subsets contribute to chronic graft-versus-host disease. T-helper type 1 cells are one of the major disease-mediating T-cell subsets and require interferon-γ signaling and Tbet expression for their function. Regulatory T cells on the other hand can inhibit T-helper type 1 cell-mediated responses. Using an established murine model that isolates the autoimmune component of graft-versus-host disease, we hypothesized that T-helper type 1 cells would restrict FoxP3-driven regulatory T cells. Upon transfer into immune-deficient syngeneic hosts, alloreactive Tbx21−/−CD4+ T cells led to marked increases in FoxP3+ cells and reduced clinical evidence of autoimmunity. To evaluate whether peripheral induction contributed to regulatory T-cell predominance, we adoptively transferred Tbx21−/− T cells that consisted of fate mapping for FoxP3: recipients of flow-purified effector cells that were Foxp3− and Tbx21−/− had enhanced T-regulatory-cell predominance during autoimmune graft-versus-host disease. These data directly demonstrated that peripheral T-regulatory-cell induction was inhibited by Tbet. Finally, Tbx21−/− T-regulatory cells cross-regulated autoimmune wild-type T-effector-cell cytokine production in vivo. The Tbet pathway therefore directly impairs T-regulatory-cell reconstitution and is consequently a feasible target in efforts to prevent autoimmune graft-versus-host disease.