Histone Deacetylase Inhibitors Enhance CD4 T Cell Susceptibility to NK Cell Killing but Reduce NK Cell Function

In the search for a cure for HIV-1 infection, histone deacetylase inhibitors (HDACi) are being investigated as activators of latently infected CD4 T cells to promote their targeting by cytotoxic T-lymphocytes (CTL). However, HDACi may also inhibit CTL function, suggesting different immunotherapy approaches may need to be explored. Here, we study the impact of different HDACi on both Natural Killer (NK) and CTL targeting of HIV-1 infected cells. We found HDACi down-regulated HLA class I expression independently of HIV-1 Nef which, without significantly compromising CTL function, led to enhanced targeting by NK cells. HDACi-treated HIV-1-infected CD4 T cells were also more effectively cleared than untreated controls during NK co-culture. However, HDACi impaired NK function, reducing degranulation and killing capacity. Depending on the HDACi and dose, this impairment could counteract the benefit gained by treating infected target cells. These data suggest that following HDACi-induced HLA class I down-regulation NK cells kill HIV-1-infected cells, although HDACi-mediated NK cell inhibition may negate this effect. Our data emphasize the importance of studying the effects of potential interventions on both targets and effectors

DNA Methylation Status of the Methylenetetrahydrofolate Reductase Gene is associated with Depressive Symptoms in Japanese Workers: A Cross-Sectional Study

Abstract Background: Polymorphisms in methylenetetrahydrofolate reductase (MTHFR) gene, that is considered to be the most important genetic determinant of blood homocysteine concentration, are associated with various diseases, including psychiatric disorders. However, the epigenetic factors influencing on the transcription and expression of this gene are unclear. The current study aims to detect the relationship between epigenetic factor-DNA methylation status, on the human MTHFR gene and depressive symptoms in Japanese workers. Methods: 774 DNA samples were extracted from saliva samples collected from subjects recruited for a mental health study, and an Illumina Human Methylation 450K Microarray Assay was used to examine DNA methylation profile across the human MTHFR gene. Depressive symptoms were determined by K6 questionnaire. Four independent DNA pools were created based on K6 scores, and the methylation levels were compared among the pools. Results: The DNA methylation level was lower in subjects with higher degrees of depression for both the entire gene (p=2.10×1

Mucosa-associated invariant T cells link intestinal immunity with antibacterial immune defects in alcoholic liver disease

Background/aims: Intestinal permeability with systemic distribution of bacterial products are central in the immunopathogenesis of alcoholic liver disease (ALD), yet links with intestinal immunity remain elusive. Mucosa-associated invariant T cells (MAIT) are found in liver, blood and intestinal mucosa and are a key component of antibacterial host defences. Their role in ALD is unknown. Methods/design: We analysed frequency, phenotype, transcriptional regulation and function of blood MAIT cells in severe alcoholic hepatitis (SAH), alcohol-related cirrhosis (ARC) and healthy controls (HC). We also examined direct impact of ethanol, bacterial products from faecal extracts and antigenic hyperstimulation on MAIT cell functionality. Presence of MAIT cells in colon and liver was assessed by quantitative PCR and immunohistochemistry/gene expression respectively. Results: In ARC and SAH, blood MAIT cells were dramatically depleted, hyperactivated and displayed defective antibacterial cytokine/cytotoxic responses. These correlated with suppression of lineage-specific transcription factors and hyperexpression of homing receptors in the liver with intrahepatic preservation of MAIT cells in ALD. These alterations were stronger in SAH, where surrogate markers of bacterial infection and microbial translocation were higher than ARC. Ethanol exposure in vitro, in vivo alcohol withdrawal and treatment with Escherichia coli had no effect on MAIT cell frequencies, whereas exposure to faecal bacteria/antigens induced functional impairments comparable with blood MAIT cells from ALD and significant MAIT cell depletion, which was not observed in other T cell compartments. Conclusions: In ALD, the antibacterial potency of MAIT cells is compromised as a consequence of contact with microbial products and microbiota, suggesting that the ‘leaky’ gut observed in ALD drives MAIT cell dysfunction and susceptibility to infection in these patients

Analysis of folylpoly-γ-glutamate synthetase gene expression in human B-precursor ALL and T-lineage ALL cells

BACKGROUND: Expression of folylpoly-γ-glutamate synthetase (FPGS) gene is two- to three-fold higher in B-precursor ALL (Bp- ALL) than in T-lineage ALL (T-ALL) and correlates with intracellular accumulation of methotrexate (MTX) polyglutamates and lymphoblast sensitivity to MTX. In this report, we investigated the molecular regulatory mechanisms directing FPGS gene expression in Bp-ALL and T-ALL cells. METHODS: To determine FPGS transcription rate in Bp-ALL and T-ALL we used nuclear run-on assays. 5'-RACE was used to uncover potential regulatory regions involved in the lineage differences. We developed a luciferase reporter gene assay to investigate FPGS promoter/enhancer activity. To further characterize the FPGS proximal promoter, we determined the role of the putative transcription binding sites NFY and E-box on FPGS expression using luciferase reporter gene assays with substitution mutants and EMSA. RESULTS: FPGS transcription initiation rate was 1.6-fold higher in NALM6 vs. CCRF-CEM cells indicating that differences in transcription rate led to the observed lineage differences in FPGS expression between Bp-ALL and T-ALL blasts. Two major transcripts encoding the mitochondrial/cytosolic and cytosolic isoforms were detected in Bp-ALL (NALM6 and REH) whereas in T-ALL (CCRF-CEM) cells only the mitochondrial/cytosolic transcript was detected. In all DNA fragments examined for promoter/enhancer activity, we measured significantly lower luciferase activity in NALM6 vs. CCRF-CEM cells, suggesting the need for additional yet unidentified regulatory elements in Bp-ALL. Finally, we determined that the putative transcription factor binding site NFY, but not E-box, plays a role in FPGS transcription in both Bp- and T-lineage. CONCLUSION: We demonstrated that the minimal FPGS promoter region previously described in CCRF-CEM is not sufficient to effectively drive FPGS transcription in NALM6 cells, suggesting that different regulatory elements are required for FPGS gene expression in Bp-cells. Our data indicate that the control of FPGS expression in human hematopoietic cells is complex and involves lineage-specific differences in regulatory elements, transcription initiation rates, and mRNA processing. Understanding the lineage-specific mechanisms of FPGS expression should lead to improved therapeutic strategies aimed at overcoming MTX resistance or inducing apoptosis in leukemic cells

Mycobacterium tuberculosis Rv3802c Encodes a Phospholipase/Thioesterase and Is Inhibited by the Antimycobacterial Agent Tetrahydrolipstatin

The cell wall of M. tuberculosis is central to its success as a pathogen. Mycolic acids are key components of this cell wall. The genes involved in joining the α and mero mycolates are located in a cluster, beginning with Rv3799c and extending at least until Rv3804c. The role of each enzyme encoded by these five genes is fairly well understood, except for Rv3802c. Rv3802 is one of seven putative cutinases encoded by the genome of M. tuberculosis. In phytopathogens, cutinases hydrolyze the waxy layer of plants, cutin. In a strictly mammalian pathogen, such as M. tuberculosis, it is likely that these proteins perform a different function. Of the seven, we chose to focus on Rv3802c because of its location in a mycolic acid synthesis gene cluster, its putative essentiality, its ubiquitous presence in actinomycetes, and its conservation in the minimal genome of Mycobacterium leprae. We expressed Rv3802 in Escherichia coli and purified the enzymatically active form. We probed its activities and inhibitors characterizing those relevant to its possible role in mycolic acid biosynthesis. In addition to its reported phospholipase A activity, Rv3802 has significant thioesterase activity, and it is inhibited by tetrahydrolipstatin (THL). THL is a described anti-tuberculous compound with an unknown mechanism, but it reportedly targets cell wall synthesis. Taken together, these data circumstantially support a role for Rv3802 in mycolic acid synthesis and, as the cell wall is integral to M. tuberculosis pathogenesis, identification of a novel cell wall enzyme and its inhibition has therapeutic and diagnostic implications

Absence of Membrane Phosphatidylcholine Does Not Affect Virulence and Stress Tolerance Phenotypes in the Opportunistic Pathogen Pseudomonas aeruginosa

During growth in presence of choline, both laboratory and clinical Pseudomonas aeruginosa strains synthesize phosphatidylcholine (PC), and PC makes up ∼4% of the total membrane phospholipid content. In all the strains tested, PC synthesis occurred only when choline is provided exogenously. Mutants defective in synthesis of PC were generated in the strain backgrounds PAO1 and PA14. Minimum inhibitory concentration studies testing sensitivity of PC-deficient strains towards various antibiotics and cationic antimicrobial peptides revealed no differences as compared to wild-type strains. Mutants incapable of synthesizing PC were also found to be unaffected in motility and biofilm formation on abiotic surfaces, colonization of biotic surfaces and virulence in a mouse infection model. A global phenotypic microarray was further used to identify conditions wherein membrane PC may play a role of in P. aeruginosa. No culture conditions were identified wherein wild-type and PC-deficient mutants showed phenotypic differences. Membrane PC may serve a highly specific role during P. aeruginosa interactions with its eukaryotic hosts based on all the clinical strains tested retaining the ability to synthesize it during availability of choline

MAIT cells launch a rapid, robust and distinct hyperinflammatory response to bacterial superantigens and quickly acquire an anergic phenotype that impedes their cognate antimicrobial function: Defining a novel mechanism of superantigen-induced immunopathology and immunosuppression

Superantigens (SAgs) are potent exotoxins secreted by Staphylococcus aureus and Streptococcus pyogenes. They target a large fraction of T cell pools to set in motion a "cytokine storm" with severe and sometimes life-threatening consequences typically encountered in toxic shock syndrome (TSS). Given the rapidity with which TSS develops, designing timely and truly targeted therapies for this syndrome requires identification of key mediators of the cytokine storm's initial wave. Equally important, early host responses to SAgs can be accompanied or followed by a state of immunosuppression, which in turn jeopardizes the host's ability to combat and clear infections. Unlike in mouse models, the mechanisms underlying SAg-associated immunosuppression in humans are ill-defined. In this work, we have identified a population of innate-like T cells, called mucosa-associated invariant T (MAIT) cells, as the most powerful source of pro-inflammatory cytokines after exposure to SAgs. We have utilized primary human peripheral blood and hepatic mononuclear cells, mouse MAIT hybridoma lines, HLA-DR4-transgenic mice, MAIThighHLA-DR4+ bone marrow chimeras, and humanized NOD-scid IL-2Rγnull mice to demonstrate for the first time that: i) mouse and human MAIT cells are hyperresponsive to SAgs, typified by staphylococcal enterotoxin B (SEB); ii) the human MAIT cell response to SEB is rapid and far greater in magnitude than that launched by unfractionated conventional T, invariant natural killer T (iNKT) or γδ T cells, and is characterized by production of interferon (IFN)-γ, tumor necrosis factor (TNF)-α and interleukin (IL)-2, but not IL-17A; iii) high-affinity MHC class II interaction with SAgs, but not MHC-related protein 1 (MR1) participation, is required for MAIT cell activation; iv) MAIT cell responses to SEB can occur in a T cell receptor (TCR) Vβ-specific manner but are largely contributed by IL-12 and IL-18; v) as MAIT cells are primed by SAgs, they also begin to develop a molecular signature consistent with exhaustion and failure to participate in antimicrobial defense. Accordingly, they upregulate lymphocyte-activation gene 3 (LAG-3), T cell immunoglobulin and mucin-3 (TIM-3), and/or programmed cell death-1 (PD-1), and acquire an anergic phenotype that interferes with their cognate function against Klebsiella pneumoniae and Escherichia coli; vi) MAIT cell hyperactivation and anergy co-utilize a signaling pathway that is governed by p38 and MEK1/2. Collectively, our findings demonstrate a pathogenic, rather than protective, role for MAIT cells during infection. Furthermore, we propose a novel mechanism of SAg-associated immunosuppression in humans. MAIT cells may therefore provide an attractive therapeutic target for the management of both early and late phases of severe SAg-mediated illnesses

Treatment of enterohemorrhagic Escherichia coli (EHEC) infection and hemolytic uremic syndrome (HUS)

Verotoxigenic Escherichia coli (VTEC) are a specialized group of E. coli that can cause severe colonic disease and renal failure. Their pathogenicity derives from virulence factors that enable the bacteria to colonize the colon and deliver extremely powerful toxins known as verotoxins (VT) or Shiga toxins (Stx) to the systemic circulation. The recent devastating E. coli O104:H4 epidemic in Europe has shown how helpless medical professionals are in terms of offering effective therapies. By examining the sources and distribution of these bacteria, and how they cause disease, we will be in a better position to prevent and treat the inevitable future cases of sporadic disease and victims of common source outbreaks. Due to the complexity of pathogenesis, it is likely a multitargeted approach is warranted. Developments in terms of these treatments are discussed

Drugs and drug-like molecules can modulate the function of mucosal-associated invariant T cells

The major-histocompatibility-complex-(MHC)-class-I-related molecule MR1 can present activating and non-activating vitamin-B-based ligands to mucosal-associated invariant T cells (MAIT cells). Whether MR1 binds other ligands is unknown. Here we identified a range of small organic molecules, drugs, drug metabolites and drug-like molecules, including salicylates and diclofenac, as MR1-binding ligands. Some of these ligands inhibited MAIT cells ex vivo and in vivo, while others, including diclofenac metabolites, were agonists. Crystal structures of a T cell antigen receptor (TCR) from a MAIT cell in complex with MR1 bound to the non-stimulatory and stimulatory compounds showed distinct ligand orientations and contacts within MR1, which highlighted the versatility of the MR1 binding pocket. The findings demonstrated that MR1 was able to capture chemically diverse structures, spanning mono- and bicyclic compounds, that either inhibited or activated MAIT cells. This indicated that drugs and drug-like molecules can modulate MAIT cell function in mammals

High prevalence of obesity, central obesity and abnormal glucose tolerance in the middle-aged Finnish population

<p>Abstract</p> <p>Background</p> <p>There is a worldwide increase in the prevalence of obesity and disturbances in glucose metabolism. The aim of this study was to assess the current prevalence of obesity, central obesity and abnormal glucose tolerance in Finnish population, and to investigate the associations between body mass index (BMI), waist circumference and abnormal glucose tolerance.</p> <p>Methods</p> <p>A cross-sectional population-based survey was conducted in Finland during October 2004 and January 2005. A total of 4500 randomly selected individuals aged 45–74 years were invited to a health examination that included an oral glucose tolerance test. The participation rate was 62% in men and 67% in women.</p> <p>Results</p> <p>The prevalence of obesity was 23.5% (95% Confidence Interval (CI) 21.1–25.9) in men, and 28.0% (95% CI 25.5–30.5) in women. The overall prevalence of abnormal glucose tolerance (including type 2 diabetes, impaired glucose tolerance, or impaired fasting glucose) was 42.0% (95% CI 39.2–44.8) in men and 33.4% (95% CI 30.9–36.0) in women. The prevalence of previously unknown, screen-detected type 2 diabetes was 9.3% (95% CI 7.7–11.0) in men and 7.3% (95% CI 5.9–8.7) in women. Central obesity was associated with abnormal glucose tolerance within each of the three BMI categories normal (< 25 kg/m²), overweight (25–29 kg/m²), and obese (≥ 30 kg/m²).</p> <p>Conclusion</p> <p>In a population-based random sample of Finnish population, prevalences of obesity, central obesity and abnormal glucose tolerance were found to be high. A remarkably high number of previously undetected cases of type 2 diabetes was detected. Waist circumference is a predictor of abnormal glucose tolerance in all categories of obesity.</p