Nilamadhab Mishra

Histone deacetylase inhibitors modulate renal disease in the MRL-lpr/lpr mouse

Studies in human systemic lupus erythematosus (SLE) suggest a possible role for histone deacetylases (HDACs) in skewed gene expression and disease pathogenesis. We used the MRL-lpr/lpr murine model of lupus to demonstrate that HDACs play a key role in the heightened levels of both Th1 and Th2 cytokine expression that contribute to disease. The availability of specific HDAC inhibitors (HDIs) such as trichostatin A (TSA) and suberonylanilide hydroxamic acid (SAHA) permits the study of the role of HDACs in gene regulation. Our results indicate that HDIs downregulate IL-12, IFN-gamma, IL-6, and IL-10 mRNA and protein levels in MRL-lpr/lpr splenocytes. This effect on gene transcription is associated with an increased accumulation of acetylated histones H3 and H4 in total cellular chromatin. To elucidate the in vivo effects of TSA on lupuslike disease, we treated MRL-lpr/lpr mice with TSA (0.5 mg/kg/d) for 5 weeks. Compared with vehicle-treated control mice, TSA-treated mice exhibited a significant reduction in proteinuria, glomerulonephritis, and spleen weight. Taken together, these findings suggest that increased expression of HDACs leading to an altered state of histone acetylation may be of pathologic significance in MRL-lpr/lpr mice. In addition, TSA or other HDIs may have therapeutic benefit in the treatment of SLE.

Increased Cellular Free Cholesterol in Macrophage-specific Abca1 Knock-out Mice Enhances Pro-inflammatory Response of Macrophages

Macrophage-specific Abca1 knock-out (Abca1–M/–M) mice were generated to determine the role of macrophage ABCA1 expression in plasma lipoprotein concentrations and the innate immune response of macrophages. Plasma lipid and lipoprotein concentrations in chow-fed Abca1–M/–M and wild-type (WT) mice were indistinguishable. Compared with WT macrophages, Abca1–M/–M macrophages had a >95% reduction in ABCA1 protein, failed to efflux lipid to apoA-I, and had a significant increase in free cholesterol (FC) and membrane lipid rafts without induction of endoplasmic reticulum stress. Lipopolysaccharide (LPS)-treated Abca1–M/–M macrophages exhibited enhanced expression of pro-inflammatory cytokines and increased activation of the NF-κB and MAPK pathways, which could be diminished by silencing MyD88 or by chemical inhibition of NF-κB or MAPK. In vivo LPS injection also resulted in a higher pro-inflammatory response in Abca1–M/–M mice compared with WT mice. Furthermore, cholesterol depletion of macrophages with methyl-β-cyclodextrin normalized FC content between the two genotypes and their response to LPS; cholesterol repletion of macrophages resulted in increased cellular FC accumulation and enhanced cellular response to LPS. Our results suggest that macrophage ABCA1 expression may protect against atherosclerosis by facilitating the net removal of excess lipid from macrophages and dampening pro-inflammatory MyD88-dependent signaling pathways by reduction of cell membrane FC and lipid raft content.

Organismal Differences in Post-translational Modifications in Histones H3 and H4

Post-translational modifications (PTMs) of histones play an important role in many cellular processes, notably gene regulation. Using a combination of mass spectrometric and immunobiochemical approaches, we show that the PTM profile of histone H3 differs significantly among the various model organisms examined. Unicellular eukaryotes, such as Saccharomyces cerevisiae (yeast) and Tetrahymena thermophila (Tet), for example, contain more activation than silencing marks as compared with mammalian cells (mouse and human), which are generally enriched in PTMs more often associated with gene silencing. Close examination reveals that many of the better-known modified lysines (Lys) can be either methylated or acetylated and that the overall modification patterns become more complex from unicellular eukaryotes to mammals. Additionally, novel species-specific H3 PTMs from wild-type asynchronously grown cells are also detected by mass spectrometry. Our results suggest that some PTMs are more conserved than previously thought, including H3K9me1 and H4K20me2 in yeast and H3K27me1, -me2, and -me3 in Tet. On histone H4, methylation at Lys-20 showed a similar pattern as H3 methylation at Lys-9, with mammals containing more methylation than the unicellular organisms. Additionally, modification profiles of H4 acetylation were very similar among the organisms examined.

Trichostatin A reverses skewed expression of CD154, interleukin-10, and interferon-γ gene and protein expression in lupus T cells

In systemic lupus erythematosus (SLE), T helper cells exhibit increased and prolonged expression of cell-surface CD40 ligand (CD154), spontaneously overproduce interleukin-10 (IL-10), but underproduce interferon-gamma (IFN-γ). We tested the hypothesis that the imbalance of these gene products reflects skewed expression of CD154, IL-10, and IFN-γ genes. Here, we demonstrate that the histone deacetylase inhibitor, trichostatin A, significantly down-regulated CD154 and IL-10 and up-regulated IFN-γ gene expression in SLE T cells. This reversal corrected the aberrant expression of these gene products, thereby enhancing IFN-γ production and inhibiting IL-10 and CD154 expression. That trichostatin A can simultaneously reverse the skewed expression of multiple genes implicated in the immunopathogenesis of SLE suggests that this pharmacologic agent may be a candidate for the treatment of this autoimmune disease.

Modulation of Renal Disease in MRL/ lpr Mice by Suberoylanilide Hydroxamic Acid

Epigenetic regulation of gene expression is involved in the development of many diseases. Histone acetylation is a posttranslational modification of the nucleosomal histone tails that is regulated by the balance of histone deacetylases and histone acetyltransferases. Alterations in the balance of histone acetylation have been shown to cause aberrant expression of genes that are a hallmark of many diseases, including systemic lupus erythematosus. In this study, we determined whether suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor: 1) inhibits inflammatory mediator production in vitro and 2) modulates lupus progression in vivo. Mesangial cells isolated from 10-wk-old MRL/lpr mice were stimulated with LPS/IFN-γ and incubated with SAHA. TNF-α, IL-6, NO, and inducible NO synthase expression were inhibited by SAHA. We then treated MRL/lpr mice with daily injections of SAHA from age 10 to 20 wk. The animals treated with SAHA had decreased spleen size and a concomitant decrease in CD4−CD8− (double-negative) T cells compared with controls. Serum autoantibody levels and glomerular IgG and C3 deposition in SAHA-treated mice were similar to controls. In contrast, proteinuria and pathologic renal disease were significantly inhibited in the mice receiving SAHA. These data indicate that SAHA blocks mesangial cell inflammatory mediator production in vitro and disease progression in vivo in MRL/lpr mice.

Proteomic and genomic approaches reveal critical functions of H3K9 methylation and heterochromatin protein-1γ in reprogramming to pluripotency

Reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) involves a marked reorganization of chromatin. To identify post-translational histone modifications that change in global abundance during this process, we have applied a quantitative mass-spectrometry-based approach. We found that iPSCs, compared with both the starting fibroblasts and a late reprogramming intermediate (pre-iPSCs), are enriched for histone modifications associated with active chromatin, and depleted for marks of transcriptional elongation and a subset of repressive modifications including H3K9me2/me3. Dissecting the contribution of H3K9 methylation to reprogramming, we show that the H3K9 methyltransferases Ehmt1, Ehmt2 and Setdb1 regulate global H3K9me2/me3 levels and that their depletion increases iPSC formation from both fibroblasts and pre-iPSCs. Similarly, we find that inhibition of heterochromatin protein-1γ (Cbx3), a protein known to recognize H3K9 methylation, enhances reprogramming. Genome-wide location analysis revealed that Cbx3 predominantly binds active genes in both pre-iPSCs and pluripotent cells but with a strikingly different distribution: in pre-iPSCs, but not in embryonic stem cells, Cbx3 associates with active transcriptional start sites, suggesting a developmentally regulated role for Cbx3 in transcriptional activation. Despite largely non-overlapping functions and the predominant association of Cbx3 with active transcription, the H3K9 methyltransferases and Cbx3 both inhibit reprogramming by repressing the pluripotency factor Nanog. Together, our findings demonstrate that Cbx3 and H3K9 methylation restrict late reprogramming events, and suggest that a marked change in global chromatin character constitutes an epigenetic roadblock for reprogramming.

The histone deacetylase inhibitor trichostatin A upregulates regulatory T cells and modulates autoimmunity in NZB/W F1 mice

We sought to determine if the histone deacetylase inhibitor (HDI), trichostatin A (TSA), would alter systemic lupus erythematosus (SLE) in NZB/W mice. Fourteen to sixteen-week-old female NZB/W F1 mice were given TSA (1.0mg/kg body weight (BW)) intraperitonealy (i.p.) daily, TSA (1.0mg/kg BW) i.p.+anti-CD25 (250mg/mouse) i.p. every third day, only anti-CD25 (250mg/mouse) i.p., DMSO or isotype IgG. Disease progression was assessed as they aged. Mice were sacrificed at 26 or 38 weeks of age, tissues collected and evaluated. At 36 weeks, TSA-treated animals had decreased anti-double stranded DNA (dsDNA) autoantibodies and decreased protein excretion compared to controls. Spleen size and the percentage of CD4+CD69+ cells were decreased, with an increase in CD4+CD25+ T cells in the TSA-treated mice. Real-time reverse transcription-polymerase chain reaction (RT-PCR) analysis of T cells showed a decrease in IL-6 production but an increase in TGF-beta1 and Foxp3 in the TSA-treated animals. Kidney analysis showed a decrease in IgG and C3 deposition, decrease in pathologic glomerular disease and renal MCP-1, MMP-9, and IL-6 mRNA expression. Anti-CD25-treated mice euthanized at 26 weeks of age showed decreased Foxp3+CD4+CD25+ T cells compared to TSA-treated mice. These data suggest TSA administration modulates lupus-like disease, in part, by increasing T regulatory cells.

SYSTEMIC LUPUS ERYTHEMATOSUS IN THE ELDERLY

Aging modifies the clinical presentation and course of autoimmune disorders, although the mechanisms by which this occurs remain to be determined. Current evidence cited above supports the general concept that there is a natural senescence of the immune system. This evidence would suggest that somehow senescence directly affects gene expression, resulting in biochemical abnormalities that culminate in T-cell immunodysfunctions. This may be a principal factor that attenuates the autoimmune response to self-antigen and, therefore, the disease course. The authors speculate that there is a disorder primary to the T cell in SLE that is expressed as abnormal immunologic responses to self-antigens, resulting in autoimmunity. Although understanding of this primary T cell disorder is still limited, clinicians now know that the T cell harbors abnormal signaling pathways that reflect defective biochemical functions and seem to be genetically regulated. This aberrant signaling would be anticipated to affect both principal T cell subsets. It may hinder the capacity of cells, such as CD8 T cells, to effectively down-regulate the response of autoreactive CD4 helper T cells to autoantigens. Loss of self-regulation would manifest itself as loss of tolerance, a fundamental component of autoimmunity. The future challenge is to understand how aberrant signaling leads to loss of tolerance. Given this underlying genetic susceptibility in an aged individual whose T cells also are undergoing natural senescence, the authors suggest that it is conceivable that a stress factor may tip the balance in the favor of clinical disease. One such factor may be unspecified environmental stimuli. Yet another consideration is an intercurrent illness, such as an infection. It remains to be determined, however, what these environmental stimuli are and how they impact on the immune system to trigger disease.

Histone Deacetylase 9 Represses Cholesterol Efflux and Alternatively Activated Macrophages in Atherosclerosis Development

Objective— Recent genome-wide association studies revealed that a genetic variant in the loci corresponding to histone deacetylase 9 (HDAC9) is associated with large vessel stroke. HDAC9 expression was upregulated in human atherosclerotic plaques in different arteries. The molecular mechanisms how HDAC9 might increase atherosclerosis is not clear. Approach and Results— In this study, we show that systemic and bone marrow cell deletion of HDAC9 decreased atherosclerosis in LDLr−/− (low density lipoprotein receptor) mice with minimal effect on plasma lipid concentrations. HDAC9 deletion resulted upregulation of lipid homeostatic genes, downregulation of inflammatory genes, and polarization toward an M2 phenotype via increased accumulation of total acetylated H3 and H3K9 at the promoters of ABCA1 (ATP-binding cassette transporter), ABCG1, and PPAR-&ggr; (peroxisome proliferator-activated receptor) in macrophages. Conclusions— We conclude that macrophage HDAC9 upregulation is atherogenic via suppression of cholesterol efflux and generation of alternatively activated macrophages in atherosclerosis.

Histone Deacetylase 9 Deficiency Protects against Effector T Cell-mediated Systemic Autoimmunity

Co-repressor histone deacetylase 9 (HDAC9) plays a key role in the development and differentiation of many types of cells, including regulatory T cells. However, the biological function of HDAC9 in T effector cells is unknown. Systemic autoimmune diseases like lupus, diabetes, and rheumatoid arthritis have dysfunctional effector T cells. To determine the role of HDAC9 in systemic autoimmunity, we created MRL/lpr mice with HDAC9 deficiency that have aberrant effector T cell function. HDAC9 deficiency led to decreased lympho-proliferation, inflammation, autoantibody production, and increased survival in MRL/lpr mice. HDAC9-deficient mice manifested Th2 polarization, decreased T effector follicular cells positive for inducible co-stimulator, and activated T cells in vivo compared with HDAC9-intact MRL/lpr mice. HDAC9 deficiency also resulted in increased GATA3 and roquin and decreased BCL6 gene expression. HDAC9 deficiency was associated with increased site-specific lysine histone acetylation at H3 (H3K9, H3K14, and H3K18) globally that was localized to IL-4, roquin, and peroxisome proliferator-activated receptor-γ promoters with increased gene expression, respectively. In kidney and spleen, HDAC9 deficiency decreased inflammation and cytokine and chemokine production due to peroxisome proliferator-activated receptor γ overexpression. These findings suggest that HDAC9 acts as an epigenetic switch in effector T cell-mediated systemic autoimmunity.