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PU.1 and ICSBP control constitutive and IFN-gamma-regulated Tlr9 gene expression in mouse macrophages
Kate Schroder1, Monika Lichtinger, Katharine M Irvine
1Special Research Centre for Functional and Applied Genomics, Institute for Molecular Bioscience, University of Queensland, St. Lucia, Brisbane 4072, Australia.
Abstract:
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.
Insights
Interferon-gamma (IFN-γ) and lipopolysaccharide (LPS) boost macrophage responses to CpG DNA by upregulating Toll-like receptor 9 (TLR9) mRNA. IFN-γ directly activates the TLR9 promoter, enhancing CpG DNA responses in macrophages.
Area of Science:
- Immunology
- Molecular Biology
- Cell Biology
Background:
- Macrophages are key immune cells activated by unmethylated CpG-containing DNA (CpG DNA) through Toll-like receptor 9 (TLR9).
- Interferon-gamma (IFN-γ) and lipopolysaccharide (LPS) can synergize with CpG DNA to enhance pro-inflammatory responses in murine macrophages.
- Understanding the regulation of TLR9 expression is crucial for modulating macrophage-mediated immune responses.
Purpose of the Study:
- To investigate the mechanisms by which LPS and IFN-γ upregulate Toll-like receptor 9 (TLR9) mRNA expression in murine macrophages.
- To identify the regulatory elements and transcription factors involved in the IFN-γ-mediated induction of Tlr9 gene expression.
- To elucidate how these regulatory pathways influence macrophage responses to CpG DNA.
Main Methods:
- Murine bone marrow-derived macrophages (BMM) and RAW264.7 cells were used to study Tlr9 mRNA expression.
- Quantitative real-time PCR was employed to measure Tlr9 mRNA levels.
- Reporter gene assays with truncated Tlr9 promoter constructs were performed.
- Site-directed mutagenesis was used to identify critical transcription factor binding sites.
- The role of CSF-1 receptor (CSF-1R) and IFN consensus-binding protein (ICSBP/IRF8) was assessed.
Main Results:
- Both LPS and IFN-γ upregulate Tlr9 mRNA in BMM, dependent on the growth factor CSF-1.
- LPS indirectly induces Tlr9 mRNA by removing CSF-1R from the cell surface, blocking repression.
- IFN-γ directly activates the Tlr9 promoter via an IRF-binding site, with minimal effect on CSF-1R.
- A proximal 100-bp promoter region conferred basal and IFN-γ-inducible expression.
- ICSBP/IRF8 deficiency reduced basal and IFN-γ-inducible Tlr9 mRNA expression.
Conclusions:
- LPS and IFN-γ differentially regulate Tlr9 mRNA expression in macrophages through distinct mechanisms.
- IFN-γ amplifies macrophage responses to CpG DNA by directly inducing Tlr9 expression via IRF transcription factors.
- This study defines a molecular mechanism for IFN-γ-mediated enhancement of TLR9-dependent immune responses.
