Impaired Chromatin Remodelling at STAT1-Regulated Promoters Leads to Global Unresponsiveness of Toxoplasma gondii-Infected Macrophages to IFN-γ

Intracellular pathogens including the apicomplexan and opportunistic parasite Toxoplasma gondii profoundly modify their host cells in order to establish infection. We have shown previously that intracellular T. gondii inhibit up-regulation of regulatory and effector functions in murine macrophages (MΦ) stimulated with interferon (IFN)-γ, which is the cytokine crucial for controlling the parasites' replication. Using genome-wide transcriptome analysis we show herein that infection with T. gondii leads to global unresponsiveness of murine macrophages to IFN-γ. More than 61% and 89% of the transcripts, which were induced or repressed by IFN-γ in non-infected MΦ, respectively, were not altered after stimulation of T. gondii-infected cells with IFN-γ. These genes are involved in a variety of biological processes, which are mostly but not exclusively related to immune responses. Analyses of the underlying mechanisms revealed that IFN-γ-triggered nuclear translocation of STAT1 still occurred in Toxoplasma-infected MΦ. However, STAT1 bound aberrantly to oligonucleotides containing the IFN-γ-responsive gamma-activated site (GAS) consensus sequence. Conversely, IFN-γ did not induce formation of active GAS-STAT1 complexes in nuclear extracts from infected MΦ. Mass spectrometry of protein complexes bound to GAS oligonucleotides showed that T. gondii-infected MΦ are unable to recruit non-muscle actin to IFN-γ-responsive DNA sequences, which appeared to be independent of stimulation with IFN-γ and of STAT1 binding. IFN-γ-induced recruitment of BRG-1 and acetylation of core histones at the IFN-γ-regulated CIITA promoter IV, but not β-actin was diminished by >90% in Toxoplasma-infected MΦ as compared to non-infected control cells. Remarkably, treatment with histone deacetylase inhibitors restored the ability of infected macrophages to express the IFN-γ regulated genes H2-A/E and CIITA. Taken together, these results indicate that Toxoplasma-infected MΦ are unable to respond to IFN-γ due to disturbed chromatin remodelling, but can be rescued using histone deacetylase inhibitors

Conditional expression of liver-enriched transcriptional activator protein augments Acholeplasma laidlawii-induced granulysin gene expression in a human monocytic cell line, THP-1

The antimicrobial protein granulysin is considered to play an important role in the defence mechanism against bacterial infection. We previously reported that Acholeplasma laidlawii-induced transactivation of the granulysin promoter in a human monocytic cell line, THP-1, is regulated by activator protein-1 and CCAAT/enhancer binding protein-β (C/EBPβ), but not by nuclear factor-κB. Moreover, liver-enriched transcriptional inhibitory protein (LIP), a C/EBPβ isoform, was strongly induced in A. laidlawii-stimulated THP-1 cells. However, the level of liver-enriched transcriptional activator protein (LAP), another C/EBPβ isoform, was essentially constant. Accordingly, we speculated that LIP would down-regulate A. laidlawii-induced granulysin gene expression in THP-1 cells. In the present study, we examined whether LAP augments A. laidlawii-induced granulysin gene expression using conditional LAP-expressing THP-1 cells in a tetracycline-controlled expression system. Our results indicated that conditional expression of LAP augmented A. laidlawii-induced expression of granulysin mRNA. In addition, the granulysin protein was observed in A. laidlawii-stimulated, LAP-expressing THP-1 cells. Our results suggest that the expression of LAP plays a critical role in the expression of the granulysin gene in macrophages