Niche-Selective Inhibition of Pathogenic Th17 Cells by Targeting Metabolic Redundancy.

Targeting glycolysis has been considered therapeutically intractable owing to its essential housekeeping role. However, the context-dependent requirement for individual glycolytic steps has not been fully explored. We show that CRISPR-mediated targeting of glycolysis in T cells in mice results in global loss of Th17 cells, whereas deficiency of the glycolytic enzyme glucose phosphate isomerase (Gpi1) selectively eliminates inflammatory encephalitogenic and colitogenic Th17 cells, without substantially affecting homeostatic microbiota-specific Th17 cells. In homeostatic Th17 cells, partial blockade of glycolysis upon Gpi1 inactivation was compensated by pentose phosphate pathway flux and increased mitochondrial respiration. In contrast, inflammatory Th17 cells experience a hypoxic microenvironment known to limit mitochondrial respiration, which is incompatible with loss of Gpi1. Our study suggests that inhibiting glycolysis by targeting Gpi1 could be an effective therapeutic strategy with minimum toxicity for Th17-mediated autoimmune diseases, and, more generally, that metabolic redundancies can be exploited for selective targeting of disease processes.

The histone chaperone CAF-1 cooperates with the DNA methyltransferases to maintain Cd4 silencing in cytotoxic T cells.

The transcriptional repression of alternative lineage genes is critical for cell fate commitment. Mechanisms by which locus-specific gene silencing is initiated and heritably maintained during cell division are not clearly understood. To study the maintenance of silent gene states, we investigated how the Cd4 gene is stably repressed in CD8+ T cells. Through CRISPR and shRNA screening, we identified the histone chaperone CAF-1 as a critical component for Cd4 repression. We found that the large subunit of CAF-1, Chaf1a, requires the N-terminal KER domain to associate with the histone deacetylases HDAC1/2 and the histone demethylase LSD1, enzymes that also participate in Cd4 silencing. When CAF-1 was lacking, Cd4 derepression was markedly enhanced in the absence of the de novo DNA methyltransferase Dnmt3a but not the maintenance DNA methyltransferase Dnmt1. In contrast to Dnmt1, Dnmt3a deficiency did not significantly alter levels of DNA methylation at the Cd4 locus. Instead, Dnmt3a deficiency sensitized CD8+ T cells to Cd4 derepression mediated by compromised functions of histone-modifying factors, including the enzymes associated with CAF-1. Thus, we propose that the heritable silencing of the Cd4 gene in CD8+ T cells exploits cooperative functions among the DNA methyltransferases, CAF-1, and histone-modifying enzymes.

Author Correction: c-MAF-dependent regulatory T cells mediate immunological tolerance to a gut pathobiont.

In this Letter, the 'Competing interests' statement should have stated: 'D.R.L. consults for and has equity in Vedanta Biosciences.' The original Letter has not been corrected.

c-MAF-dependent regulatory T cells mediate immunological tolerance to a gut pathobiont.

Both microbial and host genetic factors contribute to the pathogenesis of autoimmune diseases. There is accumulating evidence that microbial species that potentiate chronic inflammation, as in inflammatory bowel disease, often also colonize healthy individuals. These microorganisms, including the Helicobacter species, can induce pathogenic T cells and are collectively referred to as pathobionts. However, how such T cells are constrained in healthy individuals is not yet understood. Here we report that host tolerance to a potentially pathogenic bacterium, Helicobacter hepaticus, is mediated by the induction of RORγt+FOXP3+ regulatory T (iTreg) cells that selectively restrain pro-inflammatory T helper 17 (TH17) cells and whose function is dependent on the transcription factor c-MAF. Whereas colonization of wild-type mice by H. hepaticus promoted differentiation of RORγt-expressing microorganism-specific iTreg cells in the large intestine, in disease-susceptible IL-10-deficient mice, there was instead expansion of colitogenic TH17 cells. Inactivation of c-MAF in the Treg cell compartment impaired differentiation and function, including IL-10 production, of bacteria-specific iTreg cells, and resulted in the accumulation of H. hepaticus-specific inflammatory TH17 cells and spontaneous colitis. By contrast, RORγt inactivation in Treg cells had only a minor effect on the bacteria-specific Treg and TH17 cell balance, and did not result in inflammation. Our results suggest that pathobiont-dependent inflammatory bowel disease is driven by microbiota-reactive T cells that have escaped this c-MAF-dependent mechanism of iTreg-TH17 homeostasis.

Feasibility of telephone-delivered therapy for common mental health difficulties embedded in pediatric epilepsy clinics.

BACKGROUND: Mental and physical health treatment should be delivered together for children and young people with epilepsy. Training healthcare professionals (HCPs) in epilepsy services to deliver mental health interventions is an important way to facilitate integrated care. OBJECTIVE: To determine the feasibility of remotely delivered assessment and psychological treatment for mental health difficulties delivered by HCPs in pediatric epilepsy clinics with limited formal training in psychological interventions. We hypothesized that it would be (i) feasible to train HCPs to deliver the psychological intervention and (ii) that participants receiving the psychological therapy would report reductions in symptoms of mental health difficulties including anxiety, depression, and behavior difficulties and improve quality of life. METHODS: Thirty-four children and young people with epilepsy who had impairing symptoms of a common mental health difficulty (anxiety, depression, disruptive behavior, and/or trauma) were allocated to receive 6 months of a modular cognitive behavioral intervention delivered by a HCP with limited formal psychological therapy experience. Thirteen HCPs were trained in delivery of the intervention. Healthcare professional competence was assessed in a two-stage process. Parent-reported measures of mental health symptoms and quality of life were completed at baseline and following the intervention. Paired t-tests were used to analyze changes in symptoms over time. RESULTS: All thirteen HCPs who participated in the training were considered competent in therapeutic delivery by the end of the training period. Twenty-three patients completed pre- and post-intervention measures and were included in the analysis. There were statistically significant improvements in: symptoms of mental health problems (p = 0.01; Cohen's d = 0.62), total impact of mental health problems (p = 0.03; Cohen's d = 0.52), anxiety and depression symptoms (p = 0.02; Cohen's d = 0.57) and quality of life (p = 0.01; Cohen's d = 0.57). CONCLUSION: A modular cognitive behavioral treatment delivered over the telephone by HCPs with limited experience of psychological therapy was feasible and effective in treating mental health problems in children and young people with epilepsy. Health-related Quality of Life also improved over the duration of treatment. A randomized controlled trial (RCT) is needed to demonstrate efficacy of the intervention.

Exit site infection and peritonitis due to Serratia species in patients receiving peritoneal dialysis: Epidemiology and clinical outcomes.

AIM: To study the epidemiology and clinical outcomes of catheter-related infections of Serratia species in peritoneal dialysis (PD) patients. METHODS: We retrospectively reviewed the patient characteristics, antibiotics susceptibility/resistance patterns and treatment outcomes of exit site infection (ESI) and peritonitis due to Serratia in PD patients during the period of 2004 to 2017. RESULTS: One hundred and sixty-one patients had Serratia ESI, of which 10 (6.2%) progressed to tunnel tract involvement and 11 (6.8%) developed PD peritonitis. Nineteen (11.8%) patients with Serratia ESI failed to respond to medical treatment and required catheter removal. Fifty-six (34.8%) patients had repeat Serratia ESI, which occurred at 12.9 ± 13.6 months after the previous episode. Twenty-two patients had Serratia peritonitis, which accounted for 1% of peritonitis during the study period. Ten (45.5%) patients responded to medical treatment while 12 (54.5%) patients required catheter removal. Nine patients (36.4%) failed to resume PD and were converted to long-term haemodialysis. Two patients had repeat peritonitis at 2 months and 3 years, respectively, after the initial episode. Serratia species in PD patients showed high rates of resistance to ampicillin, and first- and second-generation cephalosporins, but were generally susceptible to aminoglycosides, carboxy-/ureido-penicillins and carbapenems. CONCLUSION: Our results suggest that Serratia ESI show low risk of progression to peritonitis and favourable response to medical therapy, while Serratia peritonitis was associated with high rates of catheter removal and peritoneal failure.

Clinical characteristics and outcomes of peritoneal dialysis (PD)-related peritonitis after colonoscopy.

Colonoscopy is known to be associated with peritonitis in peritoneal dialysis (PD) patients. Antibiotic prophylaxis is recommended before colonoscopy. This study aims to investigate the clinical characteristics and outcomes of patients with PD-related peritonitis after colonoscopy. PD patients who were followed up in Pamela Youde Nethersole Eastern Hospital, with colonoscopy done from 1 January 2009 to 31 December 2019, were included for record review retrospectively. During this period, 74 patients underwent 115 colonoscopies. Fourteen patients (12.2%) developed PD-related peritonitis within 1 week after colonoscopy. There was no statistically significant difference in mean age, PD vintage, PD modality and history of PD-related peritonitis between patients with or without colonoscopy-related peritonitis. Polypectomy was more common in patients who developed peritonitis (78.6%) compared to those without peritonitis (35.6%) (p = 0.006). Ten of the 14 PD patients who had colonoscopy-related peritonitis responded to medical treatment while 4 patients required PD catheter removal. Two patients converted to maintenance haemodialysis and two died. Only 33% of Gram-negative bacteria isolated were sensitive to intravenous cefuroxime which was given as prophylactic antibiotic before colonoscopy. In conclusion, the overall risk of PD patients developing peritonitis post colonoscopy was 12.2%. Polypectomy was associated with higher risk of colonoscopy-related peritonitis. Large-scale study is needed to delineate effective antibiotic prophylaxis for colonoscopy-related peritonitis.

CD4 expression in effector T cells depends on DNA demethylation over a developmentally established stimulus-responsive element.

The epigenetic patterns that are established during early thymic development might determine mature T cell physiology and function, but the molecular basis and topography of the genetic elements involved are not fully known. Here we show, using the Cd4 locus as a paradigm for early developmental programming, that DNA demethylation during thymic development licenses a novel stimulus-responsive element that is critical for the maintenance of Cd4 gene expression in effector T cells. We document the importance of maintaining high CD4 expression during parasitic infection and show that by driving transcription, this stimulus-responsive element allows for the maintenance of histone H3K4me3 levels during T cell replication, which is critical for preventing de novo DNA methylation at the Cd4 promoter. A failure to undergo epigenetic programming during development leads to gene silencing during effector T cell replication. Our study thus provides evidence of early developmental events shaping the functional fitness of mature effector T cells.

Aberrant activation of hedgehog signaling pathway in ovarian cancers: effect on prognosis, cell invasion and differentiation.

Aberrant activation of hedgehog (HH) pathway has been implicated in the development of human malignancies. This study aimed at investigating the role of HH molecules in human ovarian carcinogenesis. The expression profiles of HH molecules were examined in ovarian tumor samples and ovarian cancer cell lines and the in vitro effects of HH molecules on cell proliferation, apoptosis, migration, invasion and cell differentiation as well as related downstream target genes were assessed. Overexpression of Patched and Gli1 protein in ovarian cancers correlated with poor survival of the patients (P = 0.008; P = 0.004). Significantly elevated expression of Sonic hedgehog messenger RNA was observed in ovarian cancers compared with normal tissues and benign ovarian tumors and such differential expression was specific to histological types (P < 0.05). Ectopic Gli1 overexpression in ovarian cancer cells conferred increased cell proliferation, cell mobility, invasiveness and change in differentiation in association with increased expression of E-cadherin, vimentin, Bcl-2, caspases as well as beta1 integrin, membrane type 1 matrix metalloproteinase (MT1-MMP) and vascular endothelial growth factor (VEGF). Treatment with 3-keto-N-(aminoethyl-aminocaproyl-dihydrocinnamoyl)-cyclopamine induced cancer cell apoptosis, suppressed cell growth, mobility and invasiveness and induced cancer cell dedifferentiation with decreased expression of E-cadherin, cytokeratin 7, Snail, calretinin, vimentin, Bcl-2, caspases, beta1 integrin, MT1-MMP and VEGF. Our data suggested that abnormal HH signaling activation plays important roles in the development and progression of ovarian cancers. Gli1 expression is an independent prognostic marker. Inhibition of the HH pathway molecules might be a valid therapeutic strategy for ovarian cancers.

Aberrant activation of hedgehog signaling pathway contributes to endometrial carcinogenesis through beta-catenin.

The hedgehog and Wnt signaling pathways play important roles in human cancers with possible interaction. This study aimed at analysis and correlation of the expression of Gli1, a transcriptional factor and target gene of hedgehog signaling pathway, with clinicopathological parameters and expression of beta-catenin, an important member of the Wnt pathway, in normal, hyperplastic and malignant endometrium. Immunohistochemical study on 15 normal endometrium, 14 simple and complex hyperplasia without atypia, 37 atypical complex hyperplasia and 80 endometrial cancers showed significant Gli1 overexpression and beta-catenin nuclear immunoreactivity in endometrial cancers and atypical endometrial hyperplasia when compared with normal endometrium (P<0.05). Overexpression of Gli1 in endometrial cancers correlated with well-differentiated histological grade (P<0.001), non-myometrial invasion (P=0.004) and superficial myometrial invasion (P=0.041). beta-Catenin nuclear immunoreactivity was also associated with well-differentiated histology (P=0.013). Gli1 overexpression positively correlated with beta-catenin nuclear immunoreactivity in atypical complex hyperplasia (P=0.013) and endometrial carcinoma (P=0.017). Similar Gli1 and beta-catenin protein expression pattern was observed in normal and endometrial cancer cell lines by western blotting. We further showed a complex formation between Gli1 and beta-catenin protein in endometrial cancer cell lines in an immunoprecipitation study. Ectopic overexpression of Gli1 into endometrial cancer cells led to reduced expression of beta-catenin in cell cytoplasm and increased expression of beta-catenin in the nuclei. In summary, overexpression of Gli1 was an early event in endometrial carcinogenesis. Aberrant activation of hedgehog pathway may play important roles in endometrial cancer through beta-catenin nuclear accumulation.

Stage-specific epigenetic regulation of CD4 expression by coordinated enhancer elements during T cell development.

The inheritance of gene expression patterns is dependent on epigenetic regulation, but the establishment and maintenance of epigenetic landscapes during T cell differentiation are incompletely understood. Here we show that two stage-specific Cd4 cis-elements, the previously characterized enhancer E4p and a novel enhancer E4m, coordinately promote Cd4 transcription in mature thymic MHC-II-specific T cells, in part through the canonical Wnt pathway. Specifically, E4p licenses E4m to orchestrate DNA demethylation by TET1 and TET3, which in turn poises the Cd4 locus for transcription in peripheral T cells. Cd4 locus demethylation is important for subsequent Cd4 transcription in activated peripheral T cells wherein these cis-elements become dispensable. By contrast, in developing thymocytes the loss of TET1/3 does not affect Cd4 transcription, highlighting an uncoupled event between transcription and epigenetic modifications. Together our findings reveal an important function for thymic cis-elements in governing gene expression in the periphery via a heritable epigenetic mechanism.

Constitutive turnover of histone H2A.Z at yeast promoters requires the preinitiation complex.

The assembly of the preinitiation complex (PIC) occurs upstream of the +1 nucleosome which, in yeast, obstructs the transcription start site and is frequently assembled with the histone variant H2A.Z. To understand the contribution of the transcription machinery in the disassembly of the +1 H2A.Z nucleosome, conditional mutants were used to block PIC assembly. A quantitative ChIP-seq approach, which allows detection of global occupancy change, was employed to measure H2A.Z occupancy. Blocking PIC assembly resulted in promoter-specific H2A.Z accumulation, indicating that the PIC is required to evict H2A.Z. By contrast, H2A.Z eviction was unaffected upon depletion of INO80, a remodeler previously reported to displace nucleosomal H2A.Z. Robust PIC-dependent H2A.Z eviction was observed at active and infrequently transcribed genes, indicating that constitutive H2A.Z turnover is a general phenomenon. Finally, sites with strong H2A.Z turnover precisely mark transcript starts, providing a new metric for identifying cryptic and alternative sites of initiation.

Tyrosine kinase B receptor and BDNF expression in ovarian cancers - Effect on cell migration, angiogenesis and clinical outcome.

In this report, we demonstrated that overexpression of tropomyosin-related kinase B (TrkB) was associated with shorter survival in ovarian cancer patients. Brain-derived neurotrophic factor (BDNF), the TrkB ligand, induced activation (phosphorylation) of TrkB in a dose dependent manner. Besides demonstrating the effect of BDNF/TrkB pathway in enhancing cancer cell migration and invasion but inhibiting apoptosis, we also report for the first time that exogenous hepatocyte growth factor induced TrkB expression at both mRNA and protein levels as well as phosphorylation. Our findings suggest that BDNF/TrkB pathway is important in ovarian carcinogenesis and TrkB may be a potential therapeutic target for ovarian cancer.