Anne-Laure Chanson

Histone deacetylase inhibitors impair innate immune responses to Toll-like receptor agonists and to infection

Regulated by histone acetyltransferases and deacetylases (HDACs), histone acetylation is a key epigenetic mechanism controlling chromatin structure, DNA accessibility, and gene expression. HDAC inhibitors induce growth arrest, differentiation, and apoptosis of tumor cells and are used as anticancer agents. Here we describe the effects of HDAC inhibitors on microbial sensing by macrophages and dendritic cells in vitro and host defenses against infection in vivo. HDAC inhibitors down-regulated the expression of numerous host defense genes, including pattern recognition receptors, kinases, transcription regulators, cytokines, chemokines, growth factors, and costimulatory molecules as assessed by genome-wide microarray analyses or innate immune responses of macrophages and dendritic cells stimulated with Toll-like receptor agonists. HDAC inhibitors induced the expression of Mi-2β and enhanced the DNA-binding activity of the Mi-2/NuRD complex that acts as a transcriptional repressor of macrophage cytokine production. In vivo, HDAC inhibitors increased the susceptibility to bacterial and fungal infections but conferred protection against toxic and septic shock. Thus, these data identify an essential role for HDAC inhibitors in the regulation of the expression of innate immune genes and host defenses against microbial pathogens.

Protection from lethal Gram-negative bacterial sepsis by targeting Toll-like receptor 4

Toll-like receptor 4 (TLR4), the signal-transducing molecule of the LPS receptor complex, plays a fundamental role in the sensing of LPS from Gram-negative bacteria. Activation of TLR4 signaling pathways by LPS is a critical upstream event in the pathogenesis of Gram-negative sepsis, making TLR4 an attractive target for novel antisepsis therapy. To validate the concept of TLR4-targeted treatment strategies in Gram-negative sepsis, we first showed that TLR4−/− and myeloid differentiation primary response gene 88 (MyD88)−/− mice were fully resistant to Escherichia coli–induced septic shock, whereas TLR2−/− and wild-type mice rapidly died of fulminant sepsis. Neutralizing anti-TLR4 antibodies were then generated using a soluble chimeric fusion protein composed of the N-terminal domain of mouse TLR4 (amino acids 1–334) and the Fc portion of human IgG1. Anti-TLR4 antibodies inhibited intracellular signaling, markedly reduced cytokine production, and protected mice from lethal endotoxic shock and E. coli sepsis when administered in a prophylactic and therapeutic manner up to 13 h after the onset of bacterial sepsis. These experimental data provide strong support for the concept of TLR4-targeted therapy for Gram-negative sepsis.

Macrophage migration inhibitory factor promotes innate immune responses by suppressing glucocorticoid‐induced expression of mitogen‐activated protein kinase phosphatase‐1

The pro‐inflammatory cytokine macrophage migration inhibitory factor (MIF) acts as a physiological counter‐regulator of the immuno‐suppressive effects of glucocorticoids. However, the mechanisms whereby MIF exerts its counter‐balancing effect remain largely unknown. Here we report that MAPK phosphatase 1 (MKP‐1), an archetypal member of dual specificity phosphatase that inactivates MAPK activity in response to pro‐inflammatory stimuli, is a critical target of MIF‐glucocorticoid crosstalk. Recombinant MIF counter‐regulated in a dose‐dependent fashion dexamethasone inhibition of TNF and IL‐8 production by RAW 264.7 macrophages and U‐937 promonocytes stimulated with lipoplysaccharides (LPS) or with LPS plus phorbol 12‐myristate 13‐acetate. Stimulation of RAW 264.7 macrophages with dexamethasone or dexamethasone plus LPS led to a robust up‐regulation of MKP‐1 mRNA and protein expressions that were counter‐regulated by addition of recombinant MIF. Antisense MIF macrophages expressing reduced levels of endogenous MIF produced higher amount of MKP‐1 and lower amount of TNF after exposure to dexamethasone and dexamethasone plus LPS, indicating that endogenous MIF acts in an autocrine fashion to override glucocorticoid‐induced MKP‐1 expression and inhibition of cytokine production. Taken together, these data identify MKP‐1 as a molecular target of MIF‐glucocorticoid crosstalk and provide a molecular basis for the control of macrophage responses by a pair of physiological regulators of innate immunity.

Histone Deacetylase Inhibitors Impair Antibacterial Defenses of Macrophages

Histone deacetylases (HDACs) control gene expression by deacetylating histones and nonhistone proteins. HDAC inhibitors (HDACi) are powerful anticancer drugs that exert anti-inflammatory and immunomodulatory activities. We recently reported a proof-of-concept study demonstrating that HDACi increase susceptibility to bacterial infections in vivo. Yet, still little is known about the effects of HDACi on antimicrobial innate immune defenses. Here we show that HDACi belonging to different chemical classes inhibit at multiple levels the response of macrophages to bacterial infection. HDACi reduce the phagocytosis and the killing of Escherichia coli and Staphylococcus aureus by macrophages. In line with these findings, HDACi decrease the expression of phagocytic receptors and inhibit bacteria-induced production of reactive oxygen and nitrogen species by macrophages. Consistently, HDACi impair the expression of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunits and inducible nitric oxide synthase. These data indicate that HDACi have a strong impact on critical antimicrobial defense mechanisms in macrophages.

A functional microsatellite of the macrophage migration inhibitory factor gene associated with meningococcal disease

Macrophage migration inhibitory factor (MIF) is an abundantly expressed proinflammatory cytokine playing a critical role in innate immunity and sepsis and other inflammatory diseases. We examined whether functional MIF gene polymorphisms (–794 CATT5–8 microsatellite and –173 G/C SNP) were associated with the occurrence and outcome of meningococcal disease in children. The CATT5 allele was associated with the probability of death predicted by the Pediatric Index of Mortality 2 (P=0.001), which increased in correlation with the CATT5 copy number (P=0.04). The CATT5 allele, but not the —173 G/C alleles, was also associated with the actual mortality from meningoccal sepsis [OR 2.72 (1.2‐6.4), P=0.02]. A family‐based association test (i.e., transmission disequilibrium test) performed in 240 trios with 1 afflicted offspring indicated that CATT5 was a protective allele (P=0.02) for the occurrence of meningococcal disease. At baseline and after stimulation with Neisseria meningitidis in THP‐1 monocytic cells or in a whole‐blood assay, CATT5 was found to be a low‐expression MIF allele (P= 0.005 and P=0.04 for transcriptional activity; P=0.09 and P=0.09 for MIF production). Taken together, these data suggest that polymorphisms of the MIF gene affecting MIF expression are associated with the occurrence, severity, and outcome of meningococcal disease in children.—Renner, P., Roger, T., Bochud, P.‐Y., Sprong, T., Sweep, F. C. G. J., Bochud, M., Faust, S. N., Haralambous, E., Betts, H., Chanson, A.‐L., Reymond, M. K., Mermel, E., Erard, V., van Deuren, M., Read, R. C., Levin, M., Calandra, T. A functional microsatellite of the macrophage migration inhibitory factor gene associated with meningococcal disease. FASEB J. 26, 907–916 (2012). www.fasebj.org

Macrophage Migration Inhibitory Factor Deficiency Is Associated With Impaired Killing of Gram-Negative Bacteria by Macrophages and Increased Susceptibility to Klebsiella pneumoniae Sepsis

The cytokine macrophage migration inhibitory factor (MIF) is an important component of the early proinflammatory response of the innate immune system. However, the antimicrobial defense mechanisms mediated by MIF remain fairly mysterious. In the present study, we examined whether MIF controls bacterial uptake and clearance by professional phagocytes, using wild-type and MIF-deficient macrophages. MIF deficiency did not affect bacterial phagocytosis, but it strongly impaired the killing of gram-negative bacteria by macrophages and host defenses against gram-negative bacterial infection, as shown by increased mortality in a Klebsiella pneumonia model. Consistent with MIFs regulatory role of Toll-like 4 expression in macrophages, MIF-deficient cells stimulated with lipopolysaccharide or Escherichia coli exhibited reduced nuclear factor κB activity and tumor necrosis factor (TNF) production. Addition of recombinant MIF or TNF corrected the killing defect of MIF-deficient macrophages. Together, these data show that MIF is a key mediator of host responses against gram-negative bacteria, acting in part via a modulation of bacterial killing by macrophages.

Regulation of constitutive and microbial pathogen-induced human macrophage migration inhibitory factor (MIF) gene expression

The cytokine macrophage migration inhibitory factor (MIF) is an important regulator of innate immunity, inflammation and oncogenesis. However, four decades after its identification, the molecular mechanism(s) regulating the expression of the MIF gene remain largely unknown. Analyses of human monocytic (THP‐1), epithelial (HeLa and A549) and keratinocytic (HaCat) cells transfected with wild‐type, truncated and mutated MIF promoter reporter constructs, and electrophoretic mobility shift assay, chromatin immunoprecipitation, and siRNA inhibition indicated that the transcription factors specificity protein (Sp)1 and cAMP response element‐binding protein (CREB) are critical positive regulators of constitutive human MIF gene expression. Albeit located in a cytosine guanine dinucleotide island, the MIF gene was found to be hypomethylated, an observation consistent with high baseline transcriptional activity. Moreover, stimulation of THP‐1 cells and of peripheral blood mononuclear cells with microbial products up‐regulated phosphorylated Sp1 nuclear content, Sp1 DNA‐binding activity, MIF promoter activity and MIF mRNA levels in a MEK1/2‐, Sp1‐dependent manner. Taken together with previous observations of an important role for MIF in pro‐inflammatory macrophage responses, these present findings suggest a key role for Sp1 and CREB in transcriptional regulation of MIF gene expression and MIF‐dependent host antimicrobial innate immune defense.

Histone deacetylase inhibitors repress macrophage migration inhibitory factor (MIF) expression by targeting MIF gene transcription through a local chromatin deacetylation.

The cytokine macrophage migration inhibitory factor plays a central role in inflammation, cell proliferation and tumorigenesis. Moreover, macrophage migration inhibitory factor levels correlate with tumor aggressiveness and metastatic potential. Histone deacetylase inhibitors are potent antitumor agents recently introduced in the clinic. Therefore, we hypothesized that macrophage migration inhibitory factor would represent a target of histone deacetylase inhibitors. Confirming our hypothesis, we report that histone deacetylase inhibitors of various chemical classes strongly inhibited macrophage migration inhibitory factor expression in a broad range of cell lines, in primary cells and in vivo. Nuclear run on, transient transfection with macrophage migration inhibitory factor promoter reporter constructs and transduction with macrophage migration inhibitory factor expressing adenovirus demonstrated that trichostatin A (a prototypical histone deacetylase inhibitor) inhibited endogenous, but not episomal, MIF gene transcription. Interestingly, trichostatin A induced a local and specific deacetylation of macrophage migration inhibitory factor promoter-associated H3 and H4 histones which did not affect chromatin accessibility but was associated with an impaired recruitment of RNA polymerase II and Sp1 and CREB transcription factors required for basal MIF gene transcription. Altogether, this study describes a new molecular mechanism by which histone deacetylase inhibitors inhibit MIF gene expression, and suggests that macrophage migration inhibitory factor inhibition by histone deacetylase inhibitors may contribute to the antitumorigenic effects of histone deacetylase inhibitors.

MIF Deficiency is Associated with Impaired Killing of Gram-negative Bacteria by Macrophages and Increased Susceptibility to Klebsiella pneumoniae Sepsis

The cytokine macrophage migration inhibitory factor (MIF) is an important component of the early proinflammatory response of the innate immune system. However, the antimicrobial defense mechanisms mediated by MIF remain fairly mysterious. In the present study, we examined whether MIF controls bacterial uptake and clearance by professional phagocytes, using wild-type and MIF-deficient macrophages. MIF deficiency did not affect bacterial phagocytosis, but it strongly impaired the killing of gram-negative bacteria by macrophages and host defenses against gram-negative bacterial infection, as shown by increased mortality in a Klebsiella pneumonia model. Consistent with MIFs regulatory role of Toll-like 4 expression in macrophages, MIF-deficient cells stimulated with lipopolysaccharide or Escherichia coli exhibited reduced nuclear factor κB activity and tumor necrosis factor (TNF) production. Addition of recombinant MIF or TNF corrected the killing defect of MIF-deficient macrophages. Together, these data show that MIF is a key mediator of host responses against gram-negative bacteria, acting in part via a modulation of bacterial killing by macrophages.

Migration Inhibitory Factor Deficiency Is Associated With Impaired Killing of Gram-Negative Bacteria by Macrophages and Increased Susceptibility to Klebsiella pneumoniae Sepsis

The cytokine macrophage migration inhibitory factor (MIF) is an important component of the early proinflammatory response of the innate immune system. However, the antimicrobial defense mechanisms mediated by MIF remain fairly mysterious. In the present study, we examined whether MIF controls bacterial uptake and clearance by professional phagocytes, using wild-type and MIF-deficient macrophages. MIF deficiency did not affect bacterial phagocytosis, but it strongly impaired the killing of gram-negative bacteria by macrophages and host defenses against gram-negative bacterial infection, as shown by increased mortality in a Klebsiella pneumonia model. Consistent with MIFs regulatory role of Toll-like 4 expression in macrophages, MIF-deficient cells stimulated with lipopolysaccharide or Escherichia coli exhibited reduced nuclear factor κB activity and tumor necrosis factor (TNF) production. Addition of recombinant MIF or TNF corrected the killing defect of MIF-deficient macrophages. Together, these data show that MIF is a key mediator of host responses against gram-negative bacteria, acting in part via a modulation of bacterial killing by macrophages.