PU.1 and ICSBP control constitutive and IFN-gamma-regulated Tlr9 gene expression in mouse macrophages.
Macrophages are activated by unmethylated CpG-containing DNA (CpG DNA) via TLR9. IFN-gamma and LPS can synergize with CpG DNA to enhance proinflammatory responses in murine macrophages. Here, we show that LPS and IFN-gamma up-regulated Tlr9 mRNA in murine bone marrow-derived macrophages (BMM). The ability of LPS and IFN-gamma to induce Tlr9 mRNA expression in BMM was dependent on the presence of the growth factor, CSF-1, which is constitutively present in vivo. However, there were clear differences in mechanisms of Tlr9 mRNA induction. LPS stimulation rapidly removed the CSF-1 receptor (CSF-1R) from the cell surface, thereby blocking CSF-1-mediated transcriptional repression and indirectly inducing Tlr9 mRNA expression. By contrast, IFN-gamma activated the Tlr9 promoter directly and only marginally affected cell surface CSF-1R expression. An approximately 100-bp proximal promoter of the murine Tlr9 gene was sufficient to confer basal and IFN-gamma-inducible expression in RAW264.7 cells. A composite IFN regulatory factor (IRF)/PU.1 site upon the major transcription start site was identified. Mutation of the binding sites for PU.1 or IRF impaired basal promoter activity, but only the IRF-binding site was required for IFN-gamma induction. The mRNA expression of the IRF family member IFN consensus-binding protein [(ICSBP)/IRF8] was coregulated with Tlr9 in macrophages, and constitutive and IFN-gamma-inducible Tlr9 mRNA expression was reduced in ICSBP-deficient BMM. This study therefore characterizes the regulation of mouse Tlr9 expression and defines a molecular mechanism by which IFN-gamma amplifies mouse macrophage responses to CpG DNA.
Schroder
Kate
K
Special Research Centre for Functional and Applied Genomics, Institute for Molecular Bioscience, University of Queensland, St. Lucia, Brisbane 4072, Australia.
Lichtinger
Monika
M
Irvine
Katharine M
KM
Brion
Kristian
K
Trieu
Angela
A
Ross
Ian L
IL
Ravasi
Timothy
T
Stacey
Katryn J
KJ
Rehli
Michael
M
Hume
David A
DA
Sweet
Matthew J
MJ
D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D018899:CpG Islands; D005786:Gene Expression Regulation; D050835:Interferon Regulatory Factors / Q000502:physiology*; D007371:Interferon-gamma / Q000494:pharmacology / Q000502:physiology*; D008070:Lipopolysaccharides / Q000494:pharmacology; D016173:Macrophage Colony-Stimulating Factor / Q000378:metabolism; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011518:Proto-Oncogene Proteins / Q000502:physiology*; D016186:Receptor, Macrophage Colony-Stimulating Factor / Q000378:metabolism; D011994:Recombinant Proteins; D020218:Response Elements; D051217:Toll-Like Receptor 9 / Q000096:biosynthesis* / Q000235:genetics; D015534:Trans-Activators / Q000502:physiology*; D000099185:Interferon Regulatory Factor-8; D000099232:Proto-Oncogene Protein Spi-1
D050835:Interferon Regulatory Factors; D007371:Interferon-gamma; D008070:Lipopolysaccharides; D016173:Macrophage Colony-Stimulating Factor; D011518:Proto-Oncogene Proteins; D016186:Receptor, Macrophage Colony-Stimulating Factor; D011994:Recombinant Proteins; D051217:Toll-Like Receptor 9; D015534:Trans-Activators; D000099185:Interferon Regulatory Factor-8; D000099232:Proto-Oncogene Protein Spi-1; C495370:Tlr9 protein, mouse