Cytoplasmic dsRNA induces the expression of OCT3/4 and NANOG mRNAs in differentiated human cells.

Cytoplasmic dsRNA is recognized by RNA helicase RIG-I (RIG-I) and melanoma differentiation-associated protein 5 (MDA5), triggering induction of the innate immune response via the mitochondrial antiviral signaling protein (MAVS). In contrast, extracellular dsRNA is internalized into endosomes and recognized by Toll-like receptor 3 (TLR3), which triggers signaling via the Toll-like receptor adaptor molecule 1 (TICAM-1). Poly(I:C) is a synthetic dsRNA analog and increases the expression of octamer-binding protein 3/4 (OCT3/4), NANOG, and SRY-box (SOX) mRNAs during pluripotency induction. However, the mechanism underlying this increase is unclear. Here, we focused on the mechanism of poly(I:C)-induced expression of stem cell-specific genes in human somatic cells. Addition of poly(I:C) to human fibroblast culture medium did not increase OCT3/4 mRNA expression, but poly(I:C) transfection markedly increased OCT3/4 expression and induced nuclear localization of the OCT3/4 protein, implying that not TLR3, but RIG-I and MDA5 are required for OCT3/4 expression. Moreover, although cytoplasmic dsRNA increased OCT3/4 mRNA, cytoplasmic dsDNAs, such as salmon sperm DNA and poly(dA:dT), did not. Interestingly, the expression of NANOG, SOX2, Krüppel-like factor 4 (KLF4), and proto-oncogene c-Myc was also increased by cytoplasmic dsRNA. Of note, siRNAs that silenced MAVS and interferon regulatory factor 1 (IRF1) expression reduced OCT3/4 levels after stimulation with poly(I:C); however, an NF-κB inhibitor and siRNA-mediated knockdown of proto-oncogene c-Jun did not significantly reduce the mRNA levels. We conclude that cytoplasmic dsRNA increases the expression of stem cell-specific genes in human somatic cells in a MAVS- and IRF1-dependent manner.

Extracellular RNA Sensing by Pattern Recognition Receptors.

RNA works as a genome and messenger in RNA viruses, and it sends messages in most of the creatures of the Earth, including viruses, bacteria, fungi, plants, and animals. The human innate immune system has evolved to detect single- and double-stranded RNA molecules from microbes by pattern recognition receptors and induce defense reactions against infections such as the production of type I interferons and inflammatory cytokines. To avoid cytokine toxicity causing chronic inflammation or autoimmunity by sensing self-RNA, the activation of RNA sensors is strictly regulated. All of the Toll-like receptors that recognize RNA are localized to endosomes/lysosomes, which require internalization of RNA for sensing through an endocytic pathway. RIG-I-like receptors sense RNA in cytosol. These receptors are expressed in a cell type-specific fashion, enabling sensing of RNA for a wide range of microbial invasions. At the same time, both endosomal and cytoplasmic receptors have strategies to respond only to RNA of pathogenic microorganisms or dying cells. RNA are potential vaccine adjuvants for immune enhancement against cancer and provide a benefit for vaccinations. Understanding the detailed molecular mechanisms of the RNA-sensing system will help us to broaden the clinical utility of RNA adjuvants for patients with incurable diseases.

Type I Interferon-Independent Dendritic Cell Priming and Antitumor T Cell Activation Induced by a Mycoplasma fermentans Lipopeptide.

Mycoplasma fermentans-derived diacylated lipoprotein M161Ag (MALP404) is recognized by human/mouse toll-like receptor (TLR) 2/TLR6. Short proteolytic products including macrophage-activating lipopeptide 2 (MALP2) have been utilized as antitumor immune-enhancing adjuvants. We have chemically synthesized a short form of MALP2 named MALP2s (S-[2,3-bis(palmitoyloxy)propyl]-CGNNDE). MALP2 and MALP2s provoke natural killer (NK) cell activation in vitro but only poorly induce tumor regression using in vivo mouse models loading NK-sensitive tumors. Here, we identified the functional mechanism of MALP2s on dendritic cell (DC)-priming and cytotoxic T lymphocyte (CTL)-dependent tumor eradication using CTL-sensitive tumor-implant models EG7 and B16-OVA. Programmed death ligand-1 (PD-L1) blockade therapy in combination with MALP2s + ovalbumin (OVA) showed a significant additive effect on tumor growth suppression. MALP2s increased co-stimulators CD80/86 and CD40, which were totally MyD88-dependent, with no participation of toll-IL-1R homology domain-containing adaptor molecule-1 or type I interferon signaling in DC priming. MALP2s + OVA consequently augmented proliferation of OVA-specific CTLs in the spleen and at tumor sites. Chemokines and cytolytic factors were upregulated in the tumor. Strikingly, longer duration and reinvigoration of CTLs in spleen and tumors were accomplished by the addition of MALP2s + OVA to α-PD-L1 antibody (Ab) therapy compared to α-PD-L1 Ab monotherapy. Then, tumors regressed better in the MALP2s/OVA combination than in the α-PD-L1 Ab monotherapy. Hence, MALP2s/tumor-associated antigens combined with α-PD-L1 Ab is a good therapeutic strategy in some mouse models. Unfortunately, numerous patients are still resistant to PD-1/PD-L1 blockade, and good DC-priming adjuvants are desired. Cytokine toxicity by MALP2s remains to be settled, which should be improved by chemical modification in future studies.

The TLR3/TICAM-1 signal constitutively controls spontaneous polyposis through suppression of c-Myc in Apc Min/+ mice.

BACKGROUND: Intestinal tumorigenesis is promoted by myeloid differentiation primary response gene 88 (MyD88) activation in response to the components of microbiota in Apc Min/+ mice. Microbiota also contains double-stranded RNA (dsRNA), a ligand for TLR3, which activates the toll-like receptor adaptor molecule 1 (TICAM-1, also known as TRIF) pathway. METHODS: We established Apc Min/+ Ticam1 -/- mice and their survival was compared to survival of Apc Min/+ Myd88 -/- and wild-type (WT) mice. The properties of polyps were investigated using immunofluorescence staining and RT-PCR analysis. RESULTS: We demonstrate that TICAM-1 is essential for suppression of polyp formation in Apc Min/+ mice. TICAM-1 knockout resulted in shorter survival of mice compared to WT mice or mice with knockout of MyD88 in the Apc Min/+ background. Polyps were more frequently formed in the distal intestine of Apc Min/+ Ticam1 -/- mice than in Apc Min/+ mice. Infiltration of immune cells such as CD11b+ and CD8α+ cells into the polyps was detected histologically. CD11b and CD8α mRNAs were increased in polyps of Apc Min/+ Ticam1 -/- mice compared to Apc Min/+ mice. Gene expression of inducible nitric oxide synthase (iNOS), interferon (IFN)-γ, CXCL9 and IL-12p40 was increased in polyps of Apc Min/+ Ticam1 -/- mice. mRNA and protein expression of c-Myc, a critical transcription factor for inflammation-associated polyposis, were increased in polyps of Apc Min/+ Ticam1 -/- mice. A Lactobacillus strain producing dsRNA was detected in feces of Apc Min/+ mice. CONCLUSION: These results imply that the TLR3/TICAM-1 pathway inhibits polyposis through suppression of c-Myc expression and supports long survival in Apc Min/+ mice.

Functional interfaces between TICAM-2/TRAM and TICAM-1/TRIF in TLR4 signaling.

Toll-like receptor 4 (TLR4) recognizes lipopolysaccharide (LPS), produces pro-inflammatory cytokines and type I interferons, and associates with a trigger of endotoxin shock. TLR4 is interacted with a TIR domain-containing adaptor molecule-2 (TICAM-2)/TRAM [TRIF (TIR domain-containing adaptor-inducing interferon-ß)-related adaptor molecule] via its Toll-interleukin-1 receptor homology (TIR) domain. TICAM-2 acts as a scaffold protein and activates TIR domain-containing adaptor molecule-1 (TICAM-1)/TRIF. According to the structural analysis by NMR, TICAM-2 interacts with TICAM-1 by the acidic amino acids motif, E87/D88/D89. The TIR domain of TICAM-2 couples with the dimer of TIR domain of TLR4 beneath the membrane, and TICAM-2 itself also forms dimer and constitutes a binding site with TICAM-1. Endosomal localization of TICAM-2 is essential for TLR4-mediated type I interferon-inducing signal from the endosome. N-terminal myristoylation allows TICAM-2 to anchor to the endosomal membrane. Additionally, we have identified two acidic amino acids, D91/E92, as a functional motif that cooperatively determines endosomal localization of TICAM-2. This structural information of TICAM-2 suggests that the specific structure is indispensable for the endosomal localization and type I interferon production of TICAM-2. Taken together with the knowledge on cytoplasmic sensors for LPS, TICAM-2/TICAM-1 may conform to a signal network on TLR4 to facilitate induction of cytokine disorders.

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.

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/ß 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.

The dataset of proteins specifically interacted with activated TICAM-1.

The presented data are related with our paper entitled "14-3-3-zeta participates in TLR3-mediated TICAM-1 signal-platform formation" (Funami et al., 2016) [1]. These data show the proteins which specifically bind to the activated (oligomerized) TICAM-1. Fifty-three proteins were identified as specifically interacted with oligomerized TICAM-1. Mutant TICAM-1 cannot form the active oligomer, so the proteins interacted with mutant TICAM-1 are dispensable for TICAM-1-signaling. Among 53 proteins, 14-3-3-zeta specifically interacts with oligomerized TICAM-1 to corroborate TICAM-1 signalosome.

Double-stranded RNA analog and type I interferon regulate expression of Trem paired receptors in murine myeloid cells.

BACKGROUND: Triggering receptors expressed on myeloid cells (Trem) proteins are a family of cell surface receptors used to control innate immune responses such as proinflammatory cytokine production in mice. Trem genes belong to a rapidly expanding family of receptors that include activating and inhibitory paired-isoforms. RESULTS: By comparative genomic analysis, we found that Trem4, Trem5 and Trem-like transcript-6 (Treml6) genes typically paired receptors. These paired Trem genes were murine-specific and originated from an immunoreceptor tyrosine-based inhibition motif (ITIM)-containing gene. Treml6 encoded ITIM, whereas Trem4 and Trem5 lacked the ITIM but possessed positively-charged residues to associate with DNAX activating protein of 12 kDa (DAP12). DAP12 was directly associated with Trem4 and Trem5, and DAP12 coupling was mandatory for their expression on the cell surface. In bone marrow-derived dendritic cells (BMDCs) and macrophages (BMDMs), and splenic DC subsets, polyinosinic-polycytidylic acid (polyI:C) followed by type I interferon (IFN) production induced Trem4 and Treml6 whereas polyI:C or other TLR agonists failed to induce the expression of Trem5. PolyI:C induced Treml6 and Trem4 more efficiently in BMDMs than BMDCs. Treml6 was more potentially up-regulated in conventional DC (cDCs) and plasmacytoid DC (pDCs) than Trem4 in mice upon in vivo stimulation with polyI:C. DISCUSSION: Treml6-dependent inhibitory signal would be dominant in viral infection compared to resting state. Though no direct ligands of these Trem receptors have been determined, the results infer that a set of Trem receptors are up-regulated in response to viral RNA to regulate myeloid cell activation through modulation of DAP12-associated Trem4 and ITIM-containing Treml6.

14-3-3-zeta participates in TLR3-mediated TICAM-1 signal-platform formation.

Recognition of pathogen-associated molecular patterns (PAMPs) by pattern-recognition receptors (PRRs) is important in innate immune signaling. Toll-like receptors (TLRs) are well-characterized PRRs and are pivotal in antiviral and antitumor host defense. TIR domain-containing adaptor molecule 1 (TICAM-1, also called TRIF) is an adapter molecule in TLR3- and TLR4-mediated IRF3 activation, late-phase NF-κB activation and MAPK-mediated AP-1 activation. When a TLR3 ligand is added to TLR3-positive cells, TICAM-1 transiently interacts with TLR3 and forms multimers in the cytosol. However, the precise mechanism of TICAM-1 multimer formation remains unknown. In this study, we identified 14-3-3-zeta as a molecule that functions in TLR3-mediated signaling. Knockdown of 14-3-3-zeta reduced production of type I interferon and inflammatory cytokines, nuclear translocation of IRF3 and phosphorylation of IκB via the TLR3-TICAM-1 pathway. Furthermore, TICAM-1 multimerization by ligand stimulation was prohibited by 14-3-3-zeta knockdown. These results suggest that 14-3-3-zeta is involved in the TLR3-TICAM-1 pathway in promoting multimerization of TICAM-1 for the formation of a TICAM-1 signalosome.

Raftlin Controls Lipopolysaccharide-Induced TLR4 Internalization and TICAM-1 Signaling in a Cell Type-Specific Manner.

The clathrin-dependent endocytic pathway is crucial for endosomal TLR3- and TLR4-mediated Toll-IL-1R domain-containing adaptor molecule-1 (TICAM-1) signaling. TLR4 uses a different signaling platform, plasma membrane and endosomes, for activation of TIRAP-MyD88 and TICAM-2-TICAM-1, respectively. LPS-induced endocytosis of TLR4 is mandatory for TICAM-1-mediated signaling including IFN-ß production. Several molecules/mechanisms such as CD14, clathrin, and phosphatidylinositol metabolism have been reported to act as inducers of TLR4 translocation. However, the molecular mechanism of spatiotemporal regulation of TLR4 signaling remains unresolved. We have previously shown that Raftlin is essential for clathrin-dependent endocytosis of TLR3 ligand in human epithelial cells and myeloid dendritic cells (DCs). In this article, we demonstrate that Raftlin also mediated LPS-induced TLR4 internalization and TICAM-1 signaling in human monocyte-derived DCs and macrophages (Mo-Mϕs). When Raftlin was knocked down, LPS-induced TLR4-mediated IFN-ß promoter activation, but not NF-κB activation, was decreased in HEK293 cells overexpressing TLR4/MD-2 or TLR4/MD-2/CD14. LPS-induced IFN-ß production by monocyte-derived DCs and Mo-Mϕs was significantly decreased by knockdown of Raftlin. Upon LPS stimulation, Raftlin moved from the cytoplasm to the plasma membrane in Mo-Mϕs, where it colocalized with TLR4. Raftlin associated with clathrin-associated adaptor protein-2 in resting cells and transiently bound to TLR4 and clathrin at the cell surface in response to LPS. Thus, Raftlin appears to modulate cargo selection as an accessary protein of clathrin-associated adaptor protein-2 in clathrin-mediated endocytosis of TLR3/4 ligands.

Interferon (IFN) and Cellular Immune Response Evoked in RNA-Pattern Sensing During Infection with Hepatitis C Virus (HCV).

Hepatitis C virus (HCV) infects hepatocytes but not dendritic cells (DCs), but DCs effectively mature in response to HCV-infected hepatocytes. Using gene-disrupted mice and hydrodynamic injection strategy, we found the MAVS pathway to be crucial for induction of type III interferons (IFNs) in response to HCV in mouse. Human hepatocytes barely express TLR3 under non-infectious states, but frequently express it in HCV infection. Type I and III IFNs are induced upon stimulation with polyI:C, an analog of double-stranded (ds)RNA. Activation of TLR3 and the TICAM-1 pathway, followed by DC-mediated activation of cellular immunity, is augmented during exposure to viral RNA. Although type III IFNs are released from replication-competent human hepatocytes, DC-mediated CTL proliferation and NK cell activation hardly occur in response to the released type III IFNs. Yet, type I IFNs and HCV-infected hepatocytes can induce maturation of DCs in either human or mouse origin. In addition, mouse CD8+ DCs mature in response to HCV-infected hepatocytes unless the TLR3/TICAM-1 pathway is blocked. We found the exosomes containing HCV RNA in the supernatant of the HCV-infected hepatocytes act as a source of TLR3-mediated DC maturation. Here we summarize our view on the mechanism by which DCs mature to induce NK and CTL in a status of HCV infection.

LRRC59 Regulates Trafficking of Nucleic Acid-Sensing TLRs from the Endoplasmic Reticulum via Association with UNC93B1.

Compartmentalization of nucleic acid (NA)-sensing TLR3, 7, 8, and 9 is strictly regulated to direct optimal response against microbial infection and evade recognition of host-derived NAs. Uncoordinated 93 homolog B1 (UNC93B1) is indispensable for trafficking of NA-sensing TLRs from the endoplasmic reticulum (ER) to endosomes/lysosomes. UNC93B1 controls loading of the TLRs into COPII vesicles to exit from the ER and traffics with the TLRs in the steady state. Ligand-induced translocation also happens on NA-sensing TLRs. However, the molecular mechanism for ligand-dependent trafficking of TLRs from the ER to endosomes/lysosomes remains unclear. In this study, we demonstrated that leucine-rich repeat containing protein (LRRC) 59, an ER membrane protein, participated in trafficking of NA-sensing TLRs from the ER. Knockdown of LRRC59 reduced TLR3-, 8-, and 9-mediated, but not TLR4-mediated, signaling. Upon ligand stimulation, LRRC59 associated with UNC93B1 in a TLR-independent manner, which required signals induced by ligand internalization. Endosomal localization of endogenous TLR3 was decreased by silencing of LRRC59, suggesting that LRRC59 promotes UNC93B1-mediated translocation of NA-sensing TLRs from the ER upon infection. These findings help us understand how NA-sensing TLRs control their proper distribution in the infection/inflammatory state.

Identification of a Regulatory Acidic Motif as the Determinant of Membrane Localization of TICAM-2.

TLR4 triggers LPS signaling through the adaptors Toll/IL-1R domain-containing adaptor molecule (TICAM)-2 (also called TRAM) and TICAM-1 (also called TRIF), together with Toll/IL-1R domain-containing adaptor protein (TIRAP) and MyD88. The MyD88 pathway mediates early phase responses to LPS on the plasma membrane, whereas the TICAM pathway mediates late-phase responses, which induce the production of type I IFN and activation of inflammasomes. TICAM-2 bridges TLR4 and TICAM-1 for LPS signaling in the endosome. Recently, we identified an acidic motif, E87/D88/D89 in TICAM-2, that provides the interaction surfaces between TICAM-2 and TICAM-1. In the present study, we found additional D91/E92 in TICAM-2, conserved across species, that is crucial for TICAM-1 activation. The D91A/E92A mutant protein was distributed largely to the cytosol, despite myristoylation, suggesting its importance for assistance of membrane localization of TICAM-2. An ectopically expressed D91A/E92A mutant per se failed to activate TICAM-1, unlike its wild-type counterpart that forms self-aggregation, but it still retained the ability to pass LPS-mediated IFN regulatory factor (IRF)3 activation. In a TICAM-2 knockout human cell line expressing TLR4/MD-2 with or without CD14, overexpression of the D91A/E92A mutant did not activate IRF3, but upon LPS stimulation, it induced sufficient TLR4-mediated IRF3 activation with high coefficient colocalization. Hence, the D91/E92 motif guides TICAM-2 membrane localization and self-activation for signaling. Our results suggest the presence of two distinct steps underlying endosomal LPS signaling on TICAM-2 for TICAM-1 activation: TICAM-2 assembling in TLR4 and/or TICAM-2 self-activation. D91A/E92A of TICAM-2 selectively associates the TLR4-dependent TICAM-2 assembling, but not cytosolic TICAM-2 self-aggregation, to activate TICAM-1.

Endosomal localization of TLR8 confers distinctive proteolytic processing on human myeloid cells.

Nucleic acid-sensing TLRs are involved in both antimicrobial immune responses and autoimmune inflammation. TLR8 is phylogenetically and structurally related to TLR7 and TLR9, which undergo proteolytic processing in the endolysosomes to generate functional receptors. Recent structural analyses of human TLR8 ectodomain and its liganded form demonstrated that TLR8 is also cleaved, and both the N- and C-terminal halves contribute to ligand binding. However, the structures and ssRNA recognition mode of endogenous TLR8 in human primary cells are largely unknown. In this study, we show that proteolytic processing of TLR8 occurs in human monocytes and macrophages in a different manner compared with TLR7/9 cleavage. The insertion loop between leucine-rich repeats 14 and 15 in TLR8 is indispensable for the cleavage and stepwise processing that occurs in the N-terminal fragment. Both furin-like proprotein convertase and cathepsins contribute to TLR8 cleavage in the early/late endosomes. TLR8 recognizes viral ssRNA and endogenous RNA, such as microRNAs, resulting in the production of proinflammatory cytokines. Hence, localization sites of the receptors are crucial for the nucleic acid-sensing mode and downstream signaling.

Assessment of the Toll-like receptor 3 pathway in endosomal signaling.

The innate immune system plays key roles in antimicrobial responses by developing the pattern-recognition receptors that recognize microbial components. The endosomal Toll-like receptors (TLRs) and cytosolic RIG-I-like receptors (RLRs) both recognize viral nucleic acids and are essential for antiviral immunity. Recent evidence suggests that compartmentalization of the receptors, and also their adaptor molecule, is important for discrimination between self and nonself and for distinct innate immune signals. TLR3 is a type I transmembrane protein that localizes in the endosomal membrane in myeloid dendritic cells (DCs) and fibroblasts/epithelial cells. TLR3 recognizes extracellular viral double-stranded RNA (dsRNA) and the synthetic dsRNA, poly(I:C). On recognition of dsRNA in the endosomes, TLR3 oligomerizes and induces type I interferon and proinflammatory cytokine production via an adaptor molecule, TICAM-1 (also known as TRIF). Additionally, the TLR3 signal in DCs triggers gene transcription required for DC maturation and the activation of natural killer cells and cytotoxic T lymphocytes. Remarkably, it has been reported that extracellular dsRNA is also recognized by cytosolic RLR. Making a distinction between TLR3-mediated endosomal signaling and RLR-mediated signaling is key to understanding the role of these receptors in innate immunity.

Structures and interface mapping of the TIR domain-containing adaptor molecules involved in interferon signaling.

Homotypic and heterotypic interactions between Toll/interleukin-1 receptor (TIR) domains in Toll-like receptors (TLRs) and downstream adaptors are essential to evoke innate immune responses. However, such oligomerization properties present intrinsic difficulties in structural studies of TIR domains. Here, using BB-loop mutations that disrupt homotypic interactions, we determined the structures of the monomeric TIR domain-containing adaptor molecule (TICAM)-1 and TICAM-2 TIR domains. Docking of the monomeric structures, together with yeast two hybrid-based mutagenesis assays, reveals that the homotypic interaction between TICAM-2 TIR is indispensable to present a scaffold for recruiting the monomeric moiety of the TICAM-1 TIR dimer. This result proposes a unique idea that oligomerization of upstream TIR domains is crucial for binding of downstream TIR domains. Furthermore, the bivalent nature of each TIR domain dimer can generate a large signaling complex under the activated TLRs, which would recruit downstream signaling molecules efficiently. This model is consistent with previous reports that BB-loop mutants completely abrogate downstream signaling.

Hepatitis C virus infection induces inflammatory cytokines and chemokines mediated by the cross talk between hepatocytes and stellate cells.

Inflammatory cytokines and chemokines play important roles in inflammation during viral infection. Hepatitis C virus (HCV) is a hepatotropic RNA virus that is closely associated with chronic liver inflammation, fibrosis, and hepatocellular carcinoma. During the progression of HCV-related diseases, hepatic stellate cells (HSCs) contribute to the inflammatory response triggered by HCV infection. However, the underlying molecular mechanisms that mediate HSC-induced chronic inflammation during HCV infection are not fully understood. By coculturing HSCs with HCV-infected hepatocytes in vitro, we found that HSCs stimulated HCV-infected hepatocytes, leading to the expression of proinflammatory cytokines and chemokines such as interleukin-6 (IL-6), IL-8, macrophage inflammatory protein 1α (MIP-1α), and MIP-1ß. Moreover, we found that this effect was mediated by IL-1α, which was secreted by HSCs. HCV infection enhanced production of CCAAT/enhancer binding protein (C/EBP) ß mRNA, and HSC-dependent IL-1α production contributed to the stimulation of C/EBPß target cytokines and chemokines in HCV-infected hepatocytes. Consistent with this result, knockdown of mRNA for C/EBPß in HCV-infected hepatocytes resulted in decreased production of cytokines and chemokines after the addition of HSC conditioned medium. Induction of cytokines and chemokines in hepatocytes by the HSC conditioned medium required a yet to be identified postentry event during productive HCV infection. The cross talk between HSCs and HCV-infected hepatocytes is a key feature of inflammation-mediated, HCV-related diseases.

Toll-IL-1-receptor-containing adaptor molecule-1: a signaling adaptor linking innate immunity to adaptive immunity.

The innate immune system senses microbial infections using pattern-recognition receptors and signals to activate adaptive immunity. Type I transmembrane protein Toll-like receptors (TLRs) play important roles in antimicrobial immune responses. Upon the recognition of pathogen-associated molecular patterns, TLRs homo- or heterodimerize and recruit distinct adaptor molecules to the intracellular TIR domains. Toll-IL-1-receptor-containing adaptor molecule-1 (TICAM-1) is a signaling adaptor downstream of TLRs 3 and 4 that recognizes virus-derived double-stranded RNA and lipopolysaccharide, respectively. TLR3 is expressed on the endosomal membrane in myeloid DCs, where TLR3-mediated signaling is initiated. Once TICAM-1 is activated, transcription factors, IRF-3, NF-κB, and AP-1, are activated, leading to production of IFN-ß and proinflammatory cytokines and maturation of dendritic cells, which are capable of activating NK cells and cytotoxic T cells. Hence, TICAM-1 signaling appears to link innate immunity to adaptive immunity. In this review, we summarize the current knowledge on TICAM-1 and discuss its role in virus infection and antitumor immunity.