Inductive and suppressive networks regulate TLR9-dependent gene expression in vivo

Bacterial DNA expressing unmethylated CpG motifs binds to TLR9, thereby stimulating a broadly protective, innate immune response. Although CpG-mediated signal transduction has been studied, the scope of TLR9-dependent gene expression is incompletely understood. To resolve these issues, mice were treated with immunostimulatory CpG oligonucleotides (ODN) and splenic mRNA levels monitored from 30 min through 3 days by microarray. Through the unique application of bioinformatic analysis to these experimental data, this study is the first to describe the complex regulatory networks responsible for TLR9-mediated gene expression. Current results are the first to establish that CpG-induced stimulation of the innate immune system proceeds in multiple waves over time, and gene up-regulation is mediated by a small number of temporally activated “major inducers” and “minor inducers”. An additional study of TNF knockout mice supports the conclusion that the regulatory networks identified by our bioinformatic analysis accurately identified CpG ODN-driven gene–gene interactions in vivo. Equally important, this work identifies the counter-regulatory mechanisms embedded within the signaling cascade that suppresses the proinflammatory response triggered in vivo by CpG DNA stimulation. Identifying these network interactions provides novel and global insights into the regulation of TLR9-mediated gene activation, improves our understanding of TLR-mediated host defense, and facilitates the development of interventions designed to optimize the nature and duration of the ensuing response

Factors Influencing the Differentiation of Human Monocytic Myeloid-Derived Suppressor Cells Into Inflammatory Macrophages

Monocytic myeloid-derived suppressor cells (mMDSC) accumulate within tumors where they create an immunosuppressive milieu that inhibits the activity of cytotoxic T and NK cells thereby allowing cancers to evade immune elimination. The toll-like receptors 7/8 agonist R848 induces human mMDSC to mature into inflammatory macrophage (MACinflam). This work demonstrates that TNFα, IL-6, and IL-10 produced by maturing mMDSC are critical to the generation of MACinflam. Neutralizing any one of these cytokines significantly inhibits R848-dependent mMDSC differentiation. mMDSC cultured in pro-inflammatory cytokine IFNγ or the combination of TNFα plus IL-6 differentiate into MACinflam more efficiently than those treated with R848. These mMDSC-derived macrophages exert anti-tumor activity by killing cancer cells. RNA-Seq analysis of the genes expressed when mMDSC differentiate into MACinflam indicates that TNFα and the transcription factors NF-κB and STAT4 are major hubs regulating this process. These findings support the clinical evaluation of R848, IFNγ, and/or TNFα plus IL-6 for intratumoral therapy of established cancers

CpG Oligonucleotides as Cancer Vaccine Adjuvants

Adjuvants improve host responsiveness to co-delivered vaccines through a variety of mechanisms. Agents that trigger cells expressing Toll-like receptors (TLR) activate an innate immune response that enhances the induction of vaccine-specific immunity. When administered in combination with vaccines designed to prevent or slow tumor growth, TLR agonists have significantly improved the generation of cytotoxic T lymphocytes. Unfortunately, vaccines containing TLR agonists have rarely been able to eliminate large established tumors when administered systemically. To improve efficacy, attention has focused on delivering TLR agonists intra-tumorally with the intent of altering the tumor microenvironment. Agonists targeting TLRs 7/8 or 9 can reduce the frequency of Tregs while causing immunosuppressive MDSC in the tumor bed to differentiate into tumoricidal macrophages thereby enhancing tumor elimination. This work reviews pre-clinical and clinical studies concerning the utility of TLR 7/8/9 agonists as adjuvants for tumor vaccines

image_1.jpeg

<p>Monocytic myeloid-derived suppressor cells (mMDSC) accumulate within tumors where they create an immunosuppressive milieu that inhibits the activity of cytotoxic T and NK cells thereby allowing cancers to evade immune elimination. The toll-like receptors 7/8 agonist R848 induces human mMDSC to mature into inflammatory macrophage (MAC_inflam). This work demonstrates that TNFα, IL-6, and IL-10 produced by maturing mMDSC are critical to the generation of MAC_inflam. Neutralizing any one of these cytokines significantly inhibits R848-dependent mMDSC differentiation. mMDSC cultured in pro-inflammatory cytokine IFNγ or the combination of TNFα plus IL-6 differentiate into MAC_inflam more efficiently than those treated with R848. These mMDSC-derived macrophages exert anti-tumor activity by killing cancer cells. RNA-Seq analysis of the genes expressed when mMDSC differentiate into MAC_inflam indicates that TNFα and the transcription factors NF-κB and STAT4 are major hubs regulating this process. These findings support the clinical evaluation of R848, IFNγ, and/or TNFα plus IL-6 for intratumoral therapy of established cancers.</p

Activation of type I interferon-dependent genes characterizes the “core response” induced by CpG DNA

Synthetic ODNs expressing CpG motifs trigger an innate immune response via TLR9. pDCs are major effectors of this response. Two structurally distinct classes of CpG ODNs have been identified that differentially activate pDCs. “K” ODNs trigger the production of TNF-α and IL-6, whereas “D” ODNs preferentially induce the secretion of IFN-α. As K and D ODNs have distinct therapeutic effects, knowledge of their shared and sequence-specific activity is of considerable importance. This work uses the CAL-1 human pDC line to analyze the effect of CpG stimulation on gene expression. Genes up-regulated by both K and D ODNs (n=92) were largely dependent on type I IFN signaling and characterized functionally by antiviral activity. K ODNs induced a short-term increase in IFN-α/β production and uniquely up-regulated genes that supported antibacterial responses. In contrast, D ODNs triggered a persistent increase in IFN-α/β production and uniquely up-regulated genes associated with metabolic functions. Thus, the core functionality of human pDCs mediated by TLR9 ligation rests on a type I IFN response that differs from the response induced by the structural elements unique to specific classes of ODNs