Hiromi Takaki

TLR3/TICAM-1 signaling in tumor cell RIP3-dependent necroptosis

The engagement of Toll-like receptor 3 (TLR3) leads to the oligomerization of the adaptor TICAM-1 (TRIF), which can induces either of three acute cellular responses, namely, cell survival coupled to Type I interferon production, or cell death, via apoptosis or necrosis. The specific response elicited by TLR3 determines the fate of affected cells, although the switching mechanism between the two cell death pathways in TLR3-stimulated cells remains molecularly unknown. Tumor necrosis factor α (TNFα)-mediated cell death can proceed via apoptosis or via a non-apoptotic pathway, termed necroptosis or programmed necrosis, which have been described in detail. Interestingly, death domain-containing kinases called receptor-interacting protein kinases (RIPs) are involved in the signaling pathways leading to these two cell death pathways. Formation of the RIP1/RIP3 complex (called necrosome) in the absence of caspase 8 activity is crucial for the induction of necroptosis in response to TNFα signaling. On the other hand, RIP1 is known to interact with the C-terminal domain of TICAM-1 and to modulate TLR3 signaling. In macrophages and perhaps tumor cell lines, RIP1/RIP3-mediated necroptotic cell death can ensue the administration of the TLR agonist polyI:C. If this involved the TLR3/TICAM-1 pathway, the innate sensing of viral dsRNA would be linked to cytopathic effects and to persistent inflammation, in turn favoring the release of damage-associated molecular patterns (DAMPs) in the microenvironment. Here, we review accumulating evidence pointing to the involvement of the TLR3/TICAM-1 axis in tumor cell necroptosis and the subsequent release of DAMPs.

Strain-to-strain difference of V protein of measles virus affects MDA5-mediated IFN-β-inducing potential.

Laboratory-adapted and vaccine strains of measles virus (MV) induce type I interferon (IFN) in infected cells to a far greater extent than wild-type strains. We investigated the mechanisms for this differential type I IFN production in cells infected with representative MV strains. The overexpression of the wild-type V protein suppressed melanoma differentiation-associated gene 5 (MDA5)-induced IFN-β promoter activity, while this was not seen in A549 cells expressing CD150 transfected with the V protein of the vaccine strain. The V proteins of the wild-type also suppressed poly I:C-induced IFN regulatory factor 3 (IRF-3) dimerization. The V proteins of the wild-type and vaccine strain did not affect retinoic acid-inducible gene 1 (RIG-I)- or toll-IL-1R homology domain-containing adaptor molecule 1 (TICAM-1)-induced IFN-β promoter activation. We identified an amino acid substitution of the cysteine residue at position 272 (which is conserved among paramyxoviruses) to an arginine residue in the V protein of the vaccine strain. Only the V protein possessing the 272C residue binds to MDA5. The mutation introduced into the wild-type V protein (C272R) was unable to suppress MDA5-induced IRF-3 nuclear translocation and IFN-β promoter activation as seen in the V proteins of the vaccine strain, whereas the mutation introduced in the vaccine strain V protein (R272C) was able to inhibit MDA5-induced IRF-3 and IFN-β promoter activation. The other 6 residues of the vaccine strain V sequence inconsistent with the authentic sequence of the wild-type V protein barely affected the IRF-3 nuclear translocation. These data suggested that the structural difference of laboratory-adapted [corrected] MV V protein hampers MDA5 blockade and acts as a nidus for the spread/amplification of type I IFN induction. Ultimately, measles vaccine strains have two modes of IFN-β-induction for their attenuation: V protein mutation and production of defective interference (DI) RNA.

PolyI:C–Induced, TLR3/RIP3-Dependent Necroptosis Backs Up Immune Effector–Mediated Tumor Elimination In Vivo

Takemura and colleagues show that polyI:C+zVAD induced CT26 tumor necroptosis and regression in the absence of NK cells and CTLs, and caspase inhibitors directed to the tumor enhanced the efficacy of RNA adjuvant immunotherapy by modulating the tumor microenvironment and dendritic cell–mediated antitumor response. Double-stranded RNA directly acts on fibroblast and myeloid lineages to induce necroptosis as in TNFα. Here, we investigated whether this type of cell death occurred in cancer cells in response to polyinosinic–polycytidylic acid (polyI:C) and the pan-caspase inhibitor z-Val-Ala-Asp fluromethyl ketone (zVAD). We found that the colon cancer cell line CT26 is highly susceptible to necroptosis, as revealed by staining with annexin V/propidium iodide. CT26 cells possess RNA sensors, TLR3 and MDA5, which are upregulated by interferon (IFN)-inducing pathways and linked to receptor-interacting protein kinase (RIP) 1/3 activation via TICAM-1 or MAVS adaptor, respectively. Although exogenously added polyI:C alone marginally induced necroptosis in CT26 cells, a combined regimen of polyI:C and zVAD induced approximately 50% CT26 necroptosis in vitro without secondary effects of TNFα or type I IFNs. CT26 necroptosis depended on the TLR3–TICAM-1–RIP3 axis in the tumor cells to produce reactive oxygen species, but not on MDA5, MAVS, or the caspases/inflammasome activation. However, the RNA-derived necroptosis was barely reproduced in vivo in a CT26 tumor–implanted Balb/c mouse model with administration of polyI:C + zVAD. Significant shrinkage of CT26 tumors was revealed only when polyI:C (100 μg) was injected intraperitoneally and zVAD (1 mg) subcutaneously into tumor-bearing mice that were depleted of cytotoxic T lymphocytes and natural killer cells. The results were confirmed with immune-compromised mice with no lymphocytes. Although necroptosis-induced tumor growth retardation appears mechanistically complicated and dependent on the injection routes of polyI:C and zVAD, anti-caspase reagent directed to tumor cells will make RNA adjuvant immunotherapy more effective by modulating the formation of the tumoricidal microenvironment and dendritic cell–inducing antitumor immune system. Cancer Immunol Res; 3(8); 902–14. ©2015 AACR.

The MyD88 pathway in plasmacytoid and CD4+ dendritic cells primarily triggers type I IFN production against measles virus in a mouse infection model.

Infection by measles virus (MV) induces type I IFN via the retinoic acid–inducible gene I/melanoma differentiation–associated gene 5/mitochondrial antiviral signaling protein (MAVS) pathway in human cells. However, the in vivo role of the MAVS pathway in host defense against MV infection remains undetermined. CD150 transgenic (Tg) mice, which express human CD150, an entry receptor for MV, with the disrupting IFNR gene (Ifnar−/−), are susceptible to MV and serve as a model for MV infection. In this study, we generated CD150Tg/Mavs−/− mice and examined MV permissiveness compared with that in CD150Tg/Ifnar−/− mice. MV replicated mostly in the spleen of i.p.-infected CD150Tg/Ifnar−/− mice. Strikingly, CD150Tg/Mavs−/− mice were not permissive to MV in vivo because of substantial type I IFN induction. MV barely replicated in any other organs tested. When T cells, B cells, and dendritic cells (DCs) isolated from CD150Tg/Mavs−/− splenocytes were cultured with MV in vitro, only the DCs produced type I IFN. In vitro infection analysis using CD150Tg/Mavs−/− DC subsets revealed that CD4+ and plasmacytoid DCs, but not CD8α+ and CD8α−CD4− double negative DCs, were exclusively involved in type I IFN production in response to MV infection. Because CD150Tg/Mavs−/− mice turned permissive to MV by anti-IFNAR Ab, type I IFN produced by CD4+ DCs and plasmacytoid DCs plays a critical role in antiviral protection for neighboring cells expressing IFNAR. Induction of type I IFN in these DC subsets was abolished by the MyD88 inhibitory peptide. Thus, production of type I IFN occurs via the MyD88-dependent and MAVS-independent signaling pathway during MV infection.

Dendritic cell subsets involved in type I IFN induction in mouse measles virus infection models

Measles caused by measles virus (MV) infection remains important in child mortality. Although the natural host of MV is human, mouse models expressing MV entry receptors (human CD46, CD150) and disrupting the interferon (IFN) pathways work for investigating immune responses during early MV infection in vivo. Dendritic cells (DCs) are primary targets for MV in the mouse models and are efficiently infected with several MV strains in the respiratory tract in vivo. However, questions remain about what kind of DC in a variety of DC subsets is involved in initial MV infection and how the RNA sensors evoke circumventing signals against MV in infected DCs. Since type I IFN-inducing pathways are a pivotal defense system that leads to the restriction of systemic viral infection, we have generated CD150-transgenic mice with disrupting each of the IFN-inducing pathway, and clarified that DC subsets had subset-specific IFN-inducing systems, which critically determined the DCs differential susceptibility to MV.

Interferon-stimulated gene of 20 kDa protein (ISG20) degrades RNA of hepatitis B virus to impede the replication of HBV in vitro and in vivo

Hepatitis B virus (HBV) barely induces host interferon (IFN)-stimulated genes (ISGs), which allows efficient HBV replication in the immortalized mouse hepatocytes as per human hepatocytes. Here we found that transfection of Isg20 plasmid robustly inhibits the HBV replication in HBV-infected hepatocytes irrespective of IRF3 or IFN promoter activation. Transfection of Isg20 is thus effective to eradicate HBV in the infected hepatocytes. Transfection of HBV genome or ε-stem of HBV pgRNA (active pgRNA moiety) failed to induce Isg20 in the hepatocytes, while control polyI:C (a viral dsRNA analogue mimic) activated MAVS pathway leading to production of type I IFN and then ISGsg20 via the IFN-α/β receptor (IFNAR). Consistently, addition of IFN-α induced Isg20 and partially suppressed HBV replication in hepatocytes. Chasing HBV RNA, DNA and proteins by blotting indicated that ISG20 expression decreased HBV RNA and replicative DNA in HBV-transfected cells, which resulted in low HBs antigen production and virus titer. The exonuclease domains of ISG20 mainly participated in HBV-RNA decay. In vivo hydrodynamic injection, ISG20 was crucial for suppressing HBV replication without degrading host RNA in the liver. Taken together, ISG20 acts as an innate anti-HBV effector that selectively degrades HBV RNA and blocks replication of infectious HBV particles. ISG20 would be a critical effector for ameliorating chronic HBV infection in the IFN therapy.

A MAVS/TICAM-1-Independent Interferon-Inducing Pathway Contributes to Regulation of Hepatitis B Virus Replication in the Mouse Hydrodynamic Injection Model

Toll-like receptors (TLRs) and cytoplasmic RNA sensors have been reported to be involved in the regulation of hepatitis B virus (HBV) replication, but remain controversial due to the lack of a natural infectious model. Our current study sets out to characterize aspects of the role of the innate immune system in eliminating HBV using hydrodynamic-based injection of HBV replicative plasmid and knockout mice deficient in specific pathways of the innate system. The evidence indicated that viral replication was not affected by MAVS or TICAM-1 knockout, but absence of interferon regulatory factor 3 (IRF-3) and IRF-7 transcription factors, as well as the interferon (IFN) receptor, had an adverse effect on the inhibition of HBV replication, demonstrating the dispensability of MAVS and TICAM-1 pathways in the early innate response against HBV. Myd88-/- mice did not have a significant increase in the initial viremia, but substantial viral antigen persisted in the mice sera, a response similar to Rag2-/- mice, suggesting that the MyD88-dependent pathway participated in evoking an adaptive immune response against the clearance of intrahepatic HBV. Taken together, we show that the RNA-sensing pathways do not participate in the regulation of HBV replication in a mouse model; meanwhile MyD88 is implicated in the HBV clearance.

MAVS-dependent IRF3/7 bypass of interferon β-induction restricts the response to measles infection in CD150Tg mouse bone marrow-derived dendritic cells

Measles virus (MV) infects CD150Tg/Ifnar (IFN alpha receptor)(-/-) mice but not CD150 (a human MV receptor)-transgenic (Tg) mice. We have shown that bone marrow-derived dendritic cells (BMDCs) from CD150Tg/Ifnar(-/-) mice are permissive to MV in contrast to those from simple CD150Tg mice, which reveals a crucial role of type I interferon (IFN) in natural tropism against MV. Yet, the mechanism whereby BMDCs produce initial type I IFN has not been elucidated in MV infection. RNA virus infection usually allows cells to generate double-stranded RNA and induce activation of IFN regulatory factor (IRF) 3/7 transcription factors, leading to the production of type I IFN through the retinoic acid-inducible gene I (RIG-I)/melanoma differentiation-associated gene 5 (MDA5)-mitochondrial antiviral signaling protein (MAVS) pathway. In mouse experimental BMDCs models, we found CD150Tg/Mavs(-/-)BMDCs, but not CD150Tg/Irf3(-/-)/Irf7(-/-)BMDCs, permissive to MV. IFN-α/β were not induced in MV-infected CD150Tg/Mavs(-/-)BMDCs, while IFN-β was subtly induced in CD150Tg/Irf3(-/-)/Irf7(-/-)BMDCs. In vivo systemic infection was therefore established by transfer of MV-infected CD150Tg/Mavs(-/-) BMDCs to CD150Tg/Ifnar(-/-) mice. These data indicate that MAVS-dependent, IRF3/7-independent IFN-β induction triggers the activation of the IFNAR pathway so as to restrict the spread of MV by infected BMDCs. Hence, MAVS participates in the initial induction of type I IFN in BMDCs and IFNAR protects against MV spreading. We also showed the importance of IL-10-producing CD4(+) T cells induced by MV-infected BMDCs in vitro, which may account for immune modulation due to the functional aberration of DCs.

Oligomerized TICAM-1 (TRIF) in the cytoplasm recruits nuclear BS69 to enhance NF-κB activation and type I IFN induction

Although adenovirus 5 E1A‐binding protein (BS69) is a nuclear protein acting as a transcriptional repressor, we found by an yeast two‐hybrid and human cell immunoprecipitation another cytoplasmic function for this protein. BS69 bound Toll‐interleukin 1 receptor domain (TIR)‐containing adaptor molecule‐1 (TICAM‐1) (also named TRIF), an adaptor protein that couples with TLR3 around the endosome. BS69 translocated from the nucleus to the cytoplasm when cells were stimulated with dsRNA or transfected with TICAM‐1. Confocal analysis of cells with over‐expressed TICAM‐1 or those stimulated with dsRNA revealed the characteristic “TICAM‐1 speckle”, which reflects signalosome formation necessary for the activation of NF‐κB and IFN‐regulatory factor (IRF)‐3. BS69 was involved in the TICAM‐1 complex, and the activation of NF‐κB/IRF‐3 followed by cytokine production was augmented in the presence of BS69 overexpression. Knockdown of endogenous BS69 resulted in a decrease of IFN‐β induction, suggesting that BS69 is a positive regulator for the TLR3‐TICAM‐1 pathway. These results, together with a recent report showing the negative regulatory properties of BS69 in NF‐κB activation by EBV‐derived latent membrane protein 1, suggest that BS69 harbors dual modes of cytoplasmic NF‐κB regulation, positively in the TICAM‐1 pathway and negatively in the latent membrane protein 1 pathway.

The Anti-Oxidant Ergothioneine Augments the Immunomodulatory Function of TLR Agonists by Direct Action on Macrophages

L-Ergothioneine (EGT) is a naturally-occurring amino acid which is characterized by its antioxidant property; yet, the physiological role of EGT has yet to be established. We investigated the immune-enhancing properties of EGT, and found that it acts as a potentiator of toll-like receptor (TLR) signaling. When mouse bone marrow-derived macrophages (BMDMs) were pretreated with EGT, TLR signal-mediated cytokine production was augmented in BMDMs. The results were reproducible with TLR2, 3, 4 and 7 agonists. In particular, IL-6 and IL-12p40 were elevated further by pretreatment with EGT in BMDMs, suggesting the induction of M1 polarization. In co-culture assay with OT-II CD4+ T cells and splenic F4/80+ macrophages, EGT significantly induced Th17 skewing in CD4+ T cells. Thus, EGT is an immune modifier as well as a redox controller under TLR stimulation that induces M1 macrophages and a Th17 shift in inflammation.