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The stimulation of TLRs by pathogen-derived molecules leads to the production of proinflammatory cytokines. Because uncontrolled inflammation can be life threatening, TLR regulation is important; however, few studies have identified the signaling pathways that contribute to the modulation of TLR expression. In this study, we examined the relationship between activation and the transcriptional regulation of TLR9. We demonstrate that infection of primary human epithelial cells, B cells, and plasmacytoid dendritic cells with dsDNA viruses induces a regulatory temporary negative-feedback loop that blocks TLR9 transcription and function. TLR9 transcriptional downregulation was dependent on TLR9 signaling and was not induced by TLR5 or other NF-κB activators, such as TNF-α. Engagement of the TLR9 receptor induced the recruitment of a suppressive complex, consisting of NF-κBp65 and HDAC3, to an NF-κB cis element on the TLR9 promoter. Knockdown of HDAC3 blocked the transient suppression in which TLR9 function was restored. These results provide a framework for understanding the complex pathways involved in transcriptional regulation of TLR9, immune induction, and inflammation against viruses., (Copyright © 2014 by The American Association of Immunologists, Inc.)

EBV infects most of the human population and is associated with a number of human diseases including cancers. Moreover, evasion of the immune system and chronic infection is an essential step for EBV-associated diseases. In this paper, we show that EBV can alter the regulation and expression of TLRs, the key effector molecules of the innate immune response. EBV infection of human primary B cells resulted in the inhibition of TLR9 functionality. Stimulation of TLR9 on primary B cells led to the production of IL-6, TNF-α, and IgG, which was inhibited in cells infected with EBV. The virus exerts its inhibitory function by decreasing TLR9 mRNA and protein levels. This event was observed at early time points after EBV infection of primary cells, as well as in an immortalized lymphoblastoid cell line. We determined that the EBV oncoprotein latent membrane protein 1 (LMP1) is a strong inhibitor of TLR9 transcription. Overexpression of LMP1 in B cells reduced TLR9 promoter activity, mRNA, and protein levels. LMP1 mutants altered in activating the NF-κB pathway prevented TLR9 promoter deregulation. Blocking the NF-κB pathway recovered TLR9 promoter activity. Mutating the NF-κB cis element on the TLR9 promoter restored luciferase transcription in the presence of LMP1. Finally, deletion of the LMP1 gene in the EBV genome abolished the ability of the virus to induce TLR9 downregulation. Our study describes a mechanism used by EBV to suppress the host immune response by deregulating the TLR9 transcript through LMP1-mediated NF-κB activation.

Human metapneumoviruses (HMPVs) are recently identified Paramyxoviridae that contribute to respiratory tract infections in children. No effective treatments or vaccines are available. Successful defense against virus infection relies on early detection by germ line-encoded pattern recognition receptors and activation of cytokine and type I IFN genes. Recently, the RNA helicase retinoic acid-inducible gene I (RIG-I) has been shown to sense HMPV. In this study, we investigated the abilities of two prototype strains of HMPV (A1 [NL\1\00] and B1 [NL\1\99]) to activate RIG-I and induce type I IFNs. Despite the abilities of both HMPV-A1 and HMPV-B1 to infect and replicate in cell lines and primary cells, only the HMPV-A1 strain triggered RIG-I to induce IFNA/B gene transcription. The failure of the HMPV-B1 strain to elicit type I IFN production was dependent on the B1 phosphoprotein, which specifically prevented RIG-I-mediated sensing of HMPV viral 5' triphosphate RNA. In contrast to most cell types, plasmacytoid dendritic cells displayed a unique ability to sense both HMPV-A1 and HMPV-B1 and in this case sensing was via TLR7 rather than RIG-I. Collectively, these data reveal differential mechanisms of sensing for two closely related viruses, which operate in cell type-specific manners.

TLR9 is critical in parasite recognition and host resistance to experimental infection with Trypanosoma cruzi. However, no information is available regarding nucleotide sequences and cellular events involved on T. cruzi recognition by TLR9. In silico wide analysis associated with in vitro screening of synthetic oligonucleotides demonstrates that the retrotransposon VIPER elements and mucin-like glycoprotein (TcMUC) genes in the T. cruzi genome are highly enriched for CpG motifs that are immunostimulatory for mouse and human TLR9, respectively. Importantly, infection with T. cruzi triggers high levels of luciferase activity under NF-kappaB-dependent transcription in HEK cells cotransfected with human TLR9, but not in control (cotransfected with human MD2/TLR4) HEK cells. Further, we observed translocation of TLR9 to the lysosomes during invasion/uptake of T. cruzi parasites by dendritic cells. Consistently, potent proinflammatory activity was observed when highly unmethylated T. cruzi genomic DNA was delivered to the endo-lysosomal compartment of host cells expressing TLR9. Thus, together our results indicate that the unmethylated CpG motifs found in the T. cruzi genome are likely to be main parasite targets and probably become available to TLR9 when parasites are destroyed in the lysosome-fused vacuoles during parasite invasion/uptake by phagocytes.