Innate immunity to intraphagosomal pathogens is mediated by interferon regulatory factor 8 (IRF-8) that stimulates the expression of macrophage-specific Nramp1 through antagonizing repression by c-Myc.

Macrophages are a central arm of innate immune defense against intracellular pathogens. They internalize microbes into phagosomes where the invaders are being killed by oxygen and nitrogen reactive species. Despite this battery of antimicrobial molecules, some are able to thrive within the phagosome thus termed intraphagosomal pathogens among which are Salmonella, Leishmania, and Mycobacteria. In mice, a single dominant gene termed Nramp1/Slc11a1 controls innate resistance to such pathogens. This gene is expressed exclusively in myeloid cells. Previously, we have shown that the restricted expression of Nramp1 is regulated by a myeloid cell-specific transcription factor termed IRF-8/ICSBP. It is demonstrated here that the induction of Nramp1 expression in activated macrophages is accompanied by a promoter shift from a repression state elicited by c-Myc to an activation state elicited by the induction of IRF-8 in activated macrophages. This transition from repression to activation is facilitated by a competitive protein-protein interaction with the transcription factor Miz-1. To show that IRF-8 is directly involved in the elimination of intraphagosomal pathogens through the regulation of Nramp1 gene expression, we bred wild type as well as IRF-8 and Nramp1 null mouse strains and examined macrophages derived from bone marrow and peritoneum. Our results clearly show that the absence of IRF-8 and Nramp1 leads to the same phenotype; defective killing of intraphagosomal Salmonella enterica serovar typhimurium and Mycobacterium bovis. Thus, interplay between repression and activation state of the Nramp1 promoter mediated by IRF-8 provides the molecular basis by which macrophages resist intraphagosomal pathogens at early stage after infection.

Crohn's disease and SLC11A1 promoter polymorphism.

Crohn's disease (CD) is a chronic multifactorial inflammatory disease. The prevalence of CD in Ashkenazi Jews is higher than in Sephardic Jews. SLC11A1, also known as Nramp1, is a divalent cation antiporter essential for the elimination of intraphagosomal pathogens. SLC11A1 has seven alleles in the promoter region and previous studies have suggested an association between CD and SLC11A1. The aim of this study was to check for a possible association between SLC11A1 promoter alleles and CD in Ashkenazi Jewish patients. DNA samples from healthy Ashkenazi donors and Ashkenazi CD patients were obtained and analyzed for SLC11A1 promoter polymorphism by PCR and DNA sequencing. One hundred thirty-one samples from healthy donors and 131 samples from CD patients were analyzed. Four alleles were identified: approximately 70% of the samples carried allele 3; approximately 30%, allele 2; approximately 1%, allele 1; and <1%, allele 5. There was no difference in allele frequencies between healthy donors and CD patients. No correlation was found between mutations in NOD2/CARD15 and the phenotype of CD. We conclude that the difference in SLC11A1 promoter polymorphism plays no role in CD in Ashkenazi Jews.

Identification of IRF-8 and IRF-1 target genes in activated macrophages.

Interferon regulatory factor 1 (IRF-1) and IRF-8, also known as interferon consensus sequence binding protein (ICSBP), are important regulators of macrophage differentiation and function. These factors exert their activities through the formation of heterocomplexes. As such, they are coactivators of various interferon-inducible genes in macrophages. To gain better insights into the involvement of these two transcription factors in the onset of the innate immune response and to identify their regulatory network in activated macrophages, DNA microarray was employed. Changes in the expression profile were analyzed in peritoneal macrophages from wild type mice and compared to IRF-1 and IRF-8 null mice, before and following 4 h exposure to IFN-gamma and LPS. The expression pattern of 265 genes was significantly changed (up/down) in peritoneal macrophages extracted from wild type mice following treatment with IFN-gamma and LPS, while no changes in the expression levels of these genes were observed in samples of the same cell-type from both IRF-1 and IRF-8 null mice. Among these putative target genes, numerous genes are involved in macrophage activity during inflammation. The expression profile of 10 of them was further examined by quantitative RT-PCR. In addition, the promoter regions of three of the identified genes were analyzed by reporter gene assay for the ability to respond to IRF-1 and IRF-8. Together, our results suggest that both IRF-1 and IRF-8 are involved in the transcriptional regulation of these genes. We therefore suggest a broader role for IRF-1 and IRF-8 in macrophages differentiation and maturation, being important inflammatory mediators.

Nramp1-mediated innate resistance to intraphagosomal pathogens is regulated by IRF-8, PU.1, and Miz-1.

Natural resistance-associated macrophage protein 1 (Nramp1) is a proton/divalent cation antiporter exclusively expressed in monocyte/macrophage cells with a unique role in innate resistance to intraphagosomal pathogens. In humans, it is linked to several infectious diseases, including leprosy, pulmonary tuberculosis, visceral leishmaniasis, meningococcal meningitis, and human immunodeficiency virus as well as to autoimmune diseases such as rheumatoid arthritis and Crohn's disease. Here we demonstrate that the restricted expression of Nramp1 is mediated by the macrophage-specific transcription factor IRF-8. This factor exerts its activity via protein-protein interaction, which facilitates its binding to target DNA. Using yeast two-hybrid screen we identified Myc Interacting Zinc finger protein 1 (Miz-1) as new interacting partner. This interaction is restricted to immune cells and takes place on the promoter Nramp1 in association with PU.1, a transcription factor essential for myelopoiesis. Consistent with these data, IRF-8 knockout mice are sensitive to a repertoire of intracellular pathogens. Accordingly, IRF-8-/- mice express low levels of Nramp1 that can not be induced any further. Thus, our results explain in molecular terms the role of IRF-8 in conferring innate resistance to intracellular pathogens and point to its possible involvement in autoimmune diseases.

Characterization of the murine Nramp1 promoter: requirements for transactivation by Miz-1.

Murine Nramp1 encodes a divalent cation transporter that is expressed in late endosomes/lysosomes of macrophages, and the transported cations facilitate intracellular pathogen growth control. The Nramp1 promoter is TATA box-deficient, has two initiator elements, and is repressed by c-Myc, in accordance with the notion that genes that deplete the iron content of the cell cytosol antagonize cell growth. Repression via c-Myc occurs at the initiator elements, whereas a c-Myc-interacting protein (Miz-1) stimulates transcription. Here we demonstrate that a non-canonical E box (CAACTG) inhibits basal promoter activity and activation by Miz-1. A consensus Sp1-binding site or GC box is also necessary for Miz-1-dependent transactivation, but not repression. Repression occurs by c-Myc competing with p300/CBP for binding Miz-1. Our results show that an Sp1 site mutant inhibits coactivation by p300 and that the murine Nramp1 promoter is preferentially expressed within macrophages (relative to a beta-actin control) compared with non-macrophage cells. The effect of the Sp1 site mutation on promoter function shows cell-type specificity: stimulation in COS-1 and inhibition in RAW264.7 cells. Miz-1-directed RNA interference confirms a stimulatory role for Miz-1 in Nramp1 promoter function. c-Myc, Miz-1, and Sp1 were identified as binding to the Nramp1 core promoter in control cells and following acute stimulation with interferon-gamma and lipopolysaccharide. These results provide a description of sites that modulate the activity of the initiator-binding protein Miz-1 and indicate a stimulatory role for GC box-binding factors in macrophages and a inhibitory role for E box elements in proliferating cells.