Dataset Information

Stat1 acetylation inhibits inducible nitric oxide synthase expression in interferon-gamma-treated RAW264.7 murine macrophages.

ABSTRACT:

Background

We hypothesized that acetylation of the Stat1 regulates interferon-gamma (IFN-gamma) mediated macrophage expression of inducible nitric oxide synthase (iNOS).

Methods

RAW 264.7 iNOS expression was induced with IFN-gamma. Deacetylase inhibitors trichostatin A (TSA) or valproic acid (VPA) were added. Stat1 and iNOS mRNA and protein were measured. Acetylated Stat1 was determined by immunoprecipitation. Chromatin immunoprecipitation assessed in vivo binding of Stat1 to the iNOS promoter.

Results

IFN-gamma significantly increased nitrite, iNOS protein and iNOS mRNA, and iNOS promoter activation. (P < .01 vs control for nitrite, protein, and mRNA). TSA-mediated acetylation decreased these to levels that were not different from controls. IFN-gamma increased acetylated Stat1 by 5-fold (P < .02 vs control); TSA + IFN-gamma caused an additional 4-fold increase in acetylated Stat1 (P < .05 vs IFN alone). Stat1 binding to the iNOS promoter increased 8-fold with IFN-gamma (P < .01 vs control). In TSA + IFN-gamma, Stat1 binding was not different from controls. Although less potent than TSA, VPA also significantly decreased nitrite, iNOS protein, iNOS mRNA, Stat1 acetylation, and Stat1 binding.

Conclusions

Acetylation of Stat1 protein correlates with decreased Stat1 binding to the iNOS promoter with resultant inhibition of IFN-gamma-mediated iNOS expression. Acetylation of the Stat1 protein may downregulate iNOS expression in proinflammatory states.

SUBMITTER: Guo L

PROVIDER: S-EPMC2034510 | biostudies-literature | 2007 Aug

REPOSITORIES: biostudies-literature

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Publications

Stat1 acetylation inhibits inducible nitric oxide synthase expression in interferon-gamma-treated RAW264.7 murine macrophages.

Guo Lucie L Guo Hongtao H Gao Chengjiang C Mi Zhiyong Z Russell William B WB Kuo Paul C PC

Surgery 20070801 2

<h4>Background</h4>We hypothesized that acetylation of the Stat1 regulates interferon-gamma (IFN-gamma) mediated macrophage expression of inducible nitric oxide synthase (iNOS).<h4>Methods</h4>RAW 264.7 iNOS expression was induced with IFN-gamma. Deacetylase inhibitors trichostatin A (TSA) or valproic acid (VPA) were added. Stat1 and iNOS mRNA and protein were measured. Acetylated Stat1 was determined by immunoprecipitation. Chromatin immunoprecipitation assessed in vivo binding of Stat1 to the ...[more]

PMID: 17689680

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Project description:BackgroundMultiple sclerosis (MS) is characterized by neuroinflammation and demyelination orchestrated by activated neuroglial cells, CNS infiltrating leukocytes, and their reciprocal interactions through inflammatory signals. An inflammatory stimulus triggers inducible nitric oxide synthase (NOS2), a pro-inflammatory marker of microglia/macrophages (MG/Mφ) to catalyze sustained nitric oxide production. NOS2 during neuroinflammation, has been associated with MS disease pathology; however, studies dissecting its role in demyelination are limited. We studied the role of NOS2 in a recombinant β-coronavirus-MHV-RSA59 induced neuroinflammation, an experimental animal model mimicking the pathological hallmarks of MS: neuroinflammatory demyelination and axonal degeneration.ObjectiveUnderstanding the role of NOS2 in murine-β-coronavirus-MHV-RSA59 demyelination.MethodsBrain and spinal cords from mock and RSA59 infected 4-5-week-old MHV-free C57BL/6 mice (WT) and NOS2-/- mice were harvested at different disease phases post infection (p.i.) (day 5/6-acute, day 9/10-acute-adaptive and day 30-chronic phase) and compared for pathological outcomes.ResultsNOS2 was upregulated at the acute phase of RSA59-induced disease in WT mice and its deficiency resulted in severe disease and reduced survival at the acute-adaptive transition phase. Low survival in NOS2-/- mice was attributed to (i) high neuroinflammation resulting from increased accumulation of macrophages and neutrophils and (ii) Iba1 + phagocytic MG/Mφ mediated-early demyelination as observed at this phase. The phagocytic phenotype of CNS MG/Mφ was confirmed by significantly higher mRNA transcripts of phagocyte markers-CD206, TREM2, and Arg1 and double immunolabelling of Iba1 with MBP and PLP. Further, NOS2 deficiency led to exacerbated demyelination at the chronic phase as well.ConclusionTaken together the results imply that the immune system failed to control the disease progression in the absence of NOS2. Thus, our observations highlight a protective role of NOS2 in murine-β-coronavirus induced demyelination.

Project description:Interferon gamma (IFN-γ) restricts the intracellular replication of many pathogens, but the mechanism by which IFN-γ confers cell-intrinsic pathogen resistance remains unclear. For example, intracellular replication of the bacterial pathogen Legionella pneumophila in macrophages is potently curtailed by IFN-γ. However, consistent with prior studies, no individual genetic deficiency that we tested completely abolished IFN-γ-mediated control. Intriguingly, we observed that the glycolysis inhibitor 2-deoxyglucose (2DG) partially rescued L. pneumophila replication in IFN-γ-treated macrophages. 2DG inhibits glycolysis and triggers the unfolded protein response, but unexpectedly, it appears these effects are not responsible for perturbing the antimicrobial activity of IFN-γ. Instead, we found that 2DG rescues bacterial replication by inhibiting the expression of two key antimicrobial factors, inducible nitric oxide synthase (iNOS) and immune-responsive gene 1 (IRG1). Using immortalized and primary macrophages deficient in iNOS and IRG1, we confirmed that loss of both iNOS and IRG1, but not individual deficiency in either gene, partially reduced IFN-γ-mediated restriction of L. pneumophila Further, using a combinatorial CRISPR/Cas9 mutagenesis approach, we found that mutation of iNOS and IRG1 in combination with four other genes (CASP11, IRGM1, IRGM3, and NOX2) resulted in a total loss of L. pneumophila restriction by IFN-γ in primary bone marrow macrophages. Our study defines a complete set of cell-intrinsic factors required for IFN-γ-mediated restriction of an intracellular bacterial pathogen and highlights the combinatorial strategy used by hosts to block bacterial replication in macrophages.IMPORTANCELegionella pneumophila is one example among many species of pathogenic bacteria that replicate within mammalian macrophages during infection. The immune signaling factor interferon gamma (IFN-γ) blocks L. pneumophila replication in macrophages and is an essential component of the immune response to L. pneumophila and other intracellular pathogens. However, to date, no study has identified the exact molecular factors induced by IFN-γ that are required for its activity. We generated macrophages lacking different combinations of IFN-γ-induced genes in an attempt to find a genetic background in which there is a complete loss of IFN-γ-mediated restriction of L. pneumophila We identified six genes that comprise the totality of the IFN-γ-dependent restriction of L. pneumophila replication in macrophages. Our results clarify the molecular basis underlying the potent effects of IFN-γ and highlight how redundancy downstream of IFN-γ is key to prevent exploitation of macrophages by pathogens.

Dataset Information

Stat1 acetylation inhibits inducible nitric oxide synthase expression in interferon-gamma-treated RAW264.7 murine macrophages.

Background

Methods

Results

Conclusions

Publications

Stat1 acetylation inhibits inducible nitric oxide synthase expression in interferon-gamma-treated RAW264.7 murine macrophages.

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