A toolbox for class I HDACs reveals isoform specific roles in gene regulation and protein acetylation.

The class I histone deacetylases are essential regulators of cell fate decisions in health and disease. While pan- and class-specific HDAC inhibitors are available, these drugs do not allow a comprehensive understanding of individual HDAC function, or the therapeutic potential of isoform-specific targeting. To systematically compare the impact of individual catalytic functions of HDAC1, HDAC2 and HDAC3, we generated human HAP1 cell lines expressing catalytically inactive HDAC enzymes. Using this genetic toolbox we compare the effect of individual HDAC inhibition with the effects of class I specific inhibitors on cell viability, protein acetylation and gene expression. Individual inactivation of HDAC1 or HDAC2 has only mild effects on cell viability, while HDAC3 inactivation or loss results in DNA damage and apoptosis. Inactivation of HDAC1/HDAC2 led to increased acetylation of components of the COREST co-repressor complex, reduced deacetylase activity associated with this complex and derepression of neuronal genes. HDAC3 controls the acetylation of nuclear hormone receptor associated proteins and the expression of nuclear hormone receptor regulated genes. Acetylation of specific histone acetyltransferases and HDACs is sensitive to inactivation of HDAC1/HDAC2. Over a wide range of assays, we determined that in particular HDAC1 or HDAC2 catalytic inactivation mimics class I specific HDAC inhibitors. Importantly, we further demonstrate that catalytic inactivation of HDAC1 or HDAC2 sensitizes cells to specific cancer drugs. In summary, our systematic study revealed isoform-specific roles of HDAC1/2/3 catalytic functions. We suggest that targeted genetic inactivation of particular isoforms effectively mimics pharmacological HDAC inhibition allowing the identification of relevant HDACs as targets for therapeutic intervention.

Interleukins (from IL-1 to IL-38), interferons, transforming growth factor ß, and TNF-α: Receptors, functions, and roles in diseases.

There have been extensive developments on cellular and molecular mechanisms of immune regulation in allergy, asthma, autoimmune diseases, tumor development, organ transplantation, and chronic infections during the last few years. Better understanding the functions, reciprocal regulation, and counterbalance of subsets of immune and inflammatory cells that interact through interleukins, interferons, TNF-α, and TGF-ß offer opportunities for immune interventions and novel treatment modalities in the era of development of biological immune response modifiers particularly targeting these molecules or their receptors. More than 60 cytokines have been designated as interleukins since the initial discoveries of monocyte and lymphocyte interleukins (called IL-1 and IL-2, respectively). Studies of transgenic or gene-deficient mice with altered expression of these cytokines or their receptors and analyses of mutations and polymorphisms in human genes that encode these products have provided essential information about their functions. Here we review recent developments on IL-1 to IL-38, TNF-α, TGF-ß, and interferons. We highlight recent advances during the last few years in this area and extensively discuss their cellular sources, targets, receptors, signaling pathways, and roles in immune regulation in patients with allergy and asthma and other inflammatory diseases.

Development of a Cellular Model Mimicking Specific HDAC Inhibitors.

Class I histone deacetylases (HDACs) are important regulators of cellular functions in health and disease. HDAC1, HDAC2, HDAC3, and HDAC8 are promising targets for the treatment of cancer, neurological, and immunological disorders. These enzymes have both catalytic and non-catalytic functions in the regulation of gene expression. We here describe the generation of a genetic toolbox by the CRISPR/Cas9 methodology in nearly haploid human tumor cells. This novel model system allows to discriminate between catalytic and structural functions of class I HDAC enzymes and to mimic the treatment with specific HDAC inhibitors.

Targeting foam cell formation in inflammatory brain diseases by the histone modifier MS-275.

OBJECTIVE: To assess class I-histone deacetylase (HDAC) inhibition on formation of lipid-accumulating, disease-promoting phagocytes upon myelin load in vitro, relevant for neuroinflammatory disorders like multiple sclerosis (MS) and cerebral X-linked adrenoleukodystrophy (X-ALD). METHODS: Immunohistochemistry on postmortem brain tissue of acute MS (n = 6) and cerebral ALD (n = 4) cases to analyze activation and foam cell state of phagocytes. RNA-Seq of in vitro differentiated healthy macrophages (n = 8) after sustained myelin-loading to assess the metabolic shift associated with foam cell formation. RNA-Seq analysis of genes linked to lipid degradation and export in MS-275-treated human HAP1 cells and RT-qPCR analysis of HAP1 cells knocked out for individual members of class I HDACs or the corresponding enzymatically inactive knock-in mutants. Investigation of intracellular lipid/myelin content after MS-275 treatment of myelin-laden human foam cells. Analysis of disease characteristic very long-chain fatty acid (VLCFA) metabolism and inflammatory state in MS-275-treated X-ALD macrophages. RESULTS: Enlarged foam cells coincided with a pro-inflammatory, lesion-promoting phenotype in postmortem tissue of MS and cerebral ALD patients. Healthy in vitro myelin laden foam cells upregulated genes linked to LXRα/PPARγ pathways and mimicked a program associated with tissue repair. Treating these cells with MS-275, amplified this gene transcription program and significantly reduced lipid and cholesterol accumulation and, thus, foam cell formation. In macrophages derived from X-ALD patients, MS-275 improved the disease-associated alterations of VLCFA metabolism and reduced the pro-inflammatory status of these cells. INTERPRETATION: These findings identify class I-HDAC inhibition as a potential novel strategy to prevent disease promoting foam cell formation in CNS inflammation.

Identification of Broad-Spectrum Antiviral Compounds by Targeting Viral Entry.

Viruses are a major threat to human health and economic well-being. In recent years Ebola, Zika, influenza, and chikungunya virus epidemics have raised awareness that infections can spread rapidly before vaccines or specific antagonists can be made available. Broad-spectrum antivirals are drugs with the potential to inhibit infection by viruses from different groups or families, which may be deployed during outbreaks when specific diagnostics, vaccines or directly acting antivirals are not available. While pathogen-directed approaches are generally effective against a few closely related viruses, targeting cellular pathways used by multiple viral agents can have broad-spectrum efficacy. Virus entry, particularly clathrin-mediated endocytosis, constitutes an attractive target as it is used by many viruses. Using a phenotypic screening strategy where the inhibitory activity of small molecules was sequentially tested against different viruses, we identified 12 compounds with broad-spectrum activity, and found a subset blocking viral internalisation and/or fusion. Importantly, we show that compounds identified with this approach can reduce viral replication in a mouse model of Zika infection. This work provides proof of concept that it is possible to identify broad-spectrum inhibitors by iterative phenotypic screenings, and that inhibition of host-pathways critical for viral life cycles can be an effective antiviral strategy.