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Bacterial CpG-containing (CpG) DNA promotes survival of murine macrophages and triggers production of proinflammatory mediators. The CpG DNA-induced inflammatory response is mediated via TLR9, whereas a recent study reported that activation of the Akt prosurvival pathway occurs via DNA-dependent protein kinase (DNA-PK) and independently of TLR9. We show, in this study, that Akt activation and survival of murine bone marrow-derived macrophages (BMM) triggered by CpG-containing phosphodiester oligodeoxynucleotides or CpG-containing phosphorothioate oligodeoxynucleotides was completely dependent on TLR9. In addition, survival triggered by CpG-containing phosphodiester oligodeoxynucleotides was not compromised in BMM from SCID mice that express a catalytically inactive form of DNA-PK. CpG DNA-induced survival of BMM was inhibited by the PI3K inhibitor, LY294002, but not by the MEK1/2 inhibitor, PD98059. The effect of LY294002 was specific to survival, because treatment of BMM with LY294002 affected CpG DNA-induced TNF-α production only modestly. Therefore, CpG DNA activates macrophage survival via TLR9 and the PI3K-Akt pathway and independently of DNA-PK and MEK-ERK.

Bacterial LPS triggers dramatic changes in gene expression in macrophages. We show here that LPS regulated several members of the histone deacetylase (HDAC) family at the mRNA level in murine bone marrow-derived macrophages (BMM). LPS transiently repressed, then induced a number of HDACs (Hdac-4, 5, 7) in BMM, whereas Hdac-1 mRNA was induced more rapidly. Treatment of BMM with trichostatin A (TSA), an inhibitor of HDACs, enhanced LPS-induced expression of the Cox-2, Cxcl2, and Ifit2 genes. In the case of Cox-2, this effect was also apparent at the promoter level. Overexpression of Hdac-8 in RAW264 murine macrophages blocked the ability of LPS to induce Cox-2 mRNA. Another class of LPS-inducible genes, which included Ccl2, Ccl7, and Edn1, was suppressed by TSA, an effect most likely mediated by PU.1 degradation. Hence, HDACs act as potent and selective negative regulators of proinflammatory gene expression and act to prevent excessive inflammatory responses in macrophages.

We previously reported that bacterial products such as LPS and CpG DNA down-modulated cell surface levels of the Colony Stimulating Factor (CSF)-1 receptor (CSF-1R) on primary murine macrophages in an all-or-nothing manner. Here we show that the ability of bacterial products to down-modulate the CSF-1R rendered bone marrow-derived macrophages (BMM) unresponsive to CSF-1 as assessed by Akt and ERK1/2 phosphorylation. Using toll-like receptor (tlr)9 as a model CSF-1-repressed gene, we show that LPS induced tlr9 expression in BMM only when CSF-1 was present, suggesting that LPS relieves CSF-1-mediated inhibition to induce gene expression. Using cDNA microarrays, we identified a cluster of similarly CSF-1 repressed genes in BMM. By real time PCR we confirmed that the expression of a selection of these genes, including integral membrane protein 2B (itm2b), receptor activity-modifying protein 2 (ramp2) and macrophage-specific gene 1 (mpg-1), were repressed by CSF-1 and were induced by LPS only in the presence of CSF-1. This pattern of gene regulation was also apparent in thioglycollate-elicited peritoneal macrophages (TEPM). LPS also counteracted CSF-1 action to induce mRNA expression of a number of transcription factors including interferon consensus sequence binding protein 1 (Icsbp1), suggesting that this mechanism leads to transcriptional reprogramming in macrophages. Since the majority of in vitro studies on macrophage biology do not include CSF-1, these genes represent a set of previously uncharacterised LPS-inducible genes. This study identifies a new mechanism of macrophage activation, in which LPS (and other toll-like receptor agonists) regulate gene expression by switching off the CSF-1R signal. This finding also provides a biological relevance to the well-documented ability of macrophage activators to down-modulate surface expression of the CSF-1R.

Summary Soluble egg antigen (SEA) from the helminth Schistosoma mansoni promotes T helper type 2 (Th2) responses by modulating antigen-presenting cell function. The Jagged/Notch pathway has recently been implicated in driving Th2 development. We show here that SEA rapidly up-regulated mRNA and protein expression of the Notch ligand Jagged-1 in both murine bone marrow-derived macrophages (BMMs) and human monocyte-derived macrophages (HMDMs). Another potential Th2-promoting factor, interleukin (IL)-33, was not transcriptionally induced by SEA in BMMs. Up-regulation of Jagged-1 mRNA by SEA was also apparent in conventional dendritic cells (DCs), although the effect was less striking than in BMMs. Conversely, SEA-pulsed DCs, but not BMMs, promoted IL-4 production upon T-cell activation, suggesting that Jagged-1 induction alone is insufficient for instructing Th2 development. A comparison of the responses initiated in BMMs by SEA and the bacterial endotoxin lipopolysaccharide (LPS) revealed common activation of extracellular signal-regulated kinase-1/2 (ERK-1/2) and p38 phosphorylation, as well as induction of Jagged-1 mRNA. However, only LPS triggered IκB degradation, phosphorylation of c-Jun N-terminal kinase (Jnk) and signal transducer and activator of transcription 1 (Stat1) Tyr701, and IL-33 and IL-12p40 mRNA up-regulation. Inducible gene expression was modified by the presence of the macrophage growth factor colony-stimulating factor (CSF)-1, which inhibited Jagged-1 induction by SEA and LPS, but enhanced LPS-induced IL-12p40 expression. Unlike LPS, SEA robustly activated signalling in HEK293 cells expressing either Toll-like receptor 2 (TLR2) or TLR4/MD2. Pharmacological inhibition of the ERK-1/2 pathway impaired SEA- and LPS-inducible Jagged-1 expression in BMMs. Taken together, our data suggest that Jagged-1 is an ERK-dependent target of TLR signalling that has a macrophage-specific function in the response to SEA. © 2008 Blackwell Publishing Ltd.

Macrophages are activated by unmethylated CpG-containing DNA (CpG DNA) via TLR9. IFN-γ and LPS can synergize with CpG DNA to enhance proinflammatory responses in murine macrophages. Here, we show that LPS and IFN-γ up-regulated Tlr9 mRNA in murine bone marrow-derived macrophages (BMM). The ability of LPS and IFN-γ 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-γ activated the Tlr9 promoter directly and only marginally affected cell surface CSF-1R expression. An ∼100-bp proximal promoter of the murine Tlr9 gene was sufficient to confer basal and IFN-γ-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-γ 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-γ-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-γ amplifies mouse macrophage responses to CpG DNA.