Cytotoxic Tph-like cells are involved in persistent tissue damage in IgG4-related disease.

OBJECTIVES: The aim of this study was to determine pathological features of T peripheral helper (Tph)-like (PD-1+CXCR5-CD4+ T) cells in IgG4-related disease (IgG4-RD). METHODS: Tph-like cells in the blood and submandibular glands (SMGs) from IgG4-RD patients were analyzed by flow cytometry. Correlations between level of a Tph-like cell subset and clinical parameters of IgG4-RD were investigated. The cytotoxic capacity of Tph-like cells was also examined. Expression profiles of a molecule related to a Tph-like cell subset in IgG4-RD SMGs were assessed by immunohistochemistry. RESULTS: Tph-like cells from IgG4-RD patients highly expressed a fractalkine receptor, CX3CR1. Percentages of circulating CX3CR1+ Tph-like cells were significantly correlated with clinical parameters including IgG4-RD Responder Index, number of involved organs, and serum level of soluble IL-2 receptor. CX3CR1+ Tph-like cells abundantly possessed cytotoxic T lymphocyte-related molecules such as granzyme A, perforin, and G protein-coupled receptor 56. Functional assays revealed their cytotoxic potential against vascular endothelial cells and ductal epithelial cells. Immunohistochemistry showed that fractalkine was markedly expressed in vascular endothelial cells and ductal epithelial cells in IgG4-RD SMGs. CONCLUSION: CX3CR1+ Tph-like cells are thought to contribute to persistent tissue injury in IgG4-RD and are a potential clinical marker and/or therapeutic target for inhibiting progression of IgG4-RD.

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.

cGAMP Promotes Germinal Center Formation and Production of IgA in Nasal-Associated Lymphoid Tissue.

Induction of immunoglobulin (Ig) A in the mucosa of the upper respiratory tract and the nasal cavity protects against influenza virus infection. Cyclic dinucleotides (CDNs) are used as mucosal adjuvants to enhance the immunogenicity of intranasal influenza hemagglutinin (HA) vaccines. The adjuvant activity of 2'3' cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) on Ig production was investigated in nasal-associated lymphoid tissue (NALT), serum of wild-type C57BL/6J, and stimulator of interferon genes (STING)-deficient mice, which do not recognize cGAMP. Mice were vaccinated intranasally with a HA vaccine with or without the cGAMP adjuvant. IgA and IgG production, T-cell responses, germinal center formation, and cytokine expression in NALT were assayed. cGAMP enhanced IgA and IgG production, and promoted T-cell responses. Intranasal administration of cGAMP activated both NALT and systemic immune cells, induced a favorable cytokine environment for IgA induction, and promoted germinal center formation. The cGAMP effect was STING-dependent. Taken together, cGAMP as an HA vaccine adjuvant promoted a STING-dependent NALT environment suitable for the enhancement of IgA production.

Tumor cell death by pattern-sensing of exogenous RNA: Tumor cell TLR3 directly induces necroptosis by poly(I:C) in vivo, independent of immune effector-mediated tumor shrinkage.

Poly(I:C) acts on dendritic cells to induce potent antitumor effects through the production of cytokines/interferons, activation of natural killer cells and proliferation of cytotoxic T lymphocytes. In some tumor or myeloid lineages, poly(I:C) seemed to induce necroptosis in concert with a pan-caspase inhibitor by directly acting on toll-like receptor (TLR) 3 in both in vivo and in vitro models.

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.

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.

Evolution of the DEAD box helicase family in chicken: chickens have no DHX9 ortholog.

Viral RNA represents a pattern molecule that can be recognized by RNA sensors in innate immunity. Humans and mice possess cytoplasmic DNA/RNA sensors for detecting viral replication. There are a number of DEAD (Asp-Glu-Ala-Asp; DExD/H) box-type helicases in mammals, among which retinoic acid-inducible gene 1 (RIG-I) and melanoma differentiation-associated protein 5 (MDA50) are indispensable for RNA sensing; however, they are functionally supported by a number of sensors that directly bind viral RNA or replicative RNA intermediates to convey signals to RIG-I and MDA5. Some DEAD box helicase members recognize DNA irrespective of the origin. These sensors transmit IFN-inducing signals through adaptors, including mitochondrial antiviral signaling. Viral double-stranded RNAs are reportedly sensed by the helicases DDX1, DDX21, DHX36, DHX9, DDX3, DDX41, LGP2 and DDX60, in addition to RIG-I and MDA5, and induce type I IFNs, thereby blocking viral replication. Humans and mice have all nucleic acid sensors listed here. In the RNA sensing system in chicken, it was found in the present study that most DEAD box helicases are conserved; however, DHX9 is genetically deficient in addition to reported RIG-I. Based on the current genome databases, similar DHX9 deficiency was observed in ducks and several other bird species. Because chicken, but not duck, was found to be deficient in RIG-I, the RNA-sensing system of chicken lacks RIG-I and DHX9 and is thus more fragile than that of duck or mammal. DHX9 may generally compensate for the function of RIG-I and deficiency of DHX9 possibly participates in exacerbations of viral infection such as influenza in chickens.

RIOK3-mediated phosphorylation of MDA5 interferes with its assembly and attenuates the innate immune response.

MDA5 is a cytoplasmic viral double-stranded RNA (dsRNA) sensor and triggers type I interferon (IFN) production. MDA5 assembles along viral dsRNA, leading to the formation of an MDA5 filament required for activating the MAVS adaptor. A recent study has revealed that PP1α and PP1γ phosphatases are responsible for dephosphorylating MDA5 and are essential for its activation. Here, we identified RIO kinase 3 (RIOK3) as a protein kinase that phosphorylates the MDA5 C-terminal region. RIOK3 knockout strongly enhanced type I IFN and IFN-inducible gene expression following measles virus infection. Conversely, the ectopic expression of RIOK3 or a phosphomimetic MDA5-S828D mutation attenuated MDA5-mediated signaling. Moreover, RIOK3-mediated MDA5 phosphorylation impaired MDA5 multimer formation, indicating that MDA5 C-terminal phosphorylation interferes with MDA5 filament formation and suppresses its signaling. Our data revealed a regulatory mechanism underlying the activation of the cytoplasmic viral RNA sensor MDA5 in both uninfected and virus-infected cells.

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

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.

MAVS-dependent IRF3/7 bypass of interferon ß-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/ß were not induced in MV-infected CD150Tg/Mavs(-/-)BMDCs, while IFN-ß 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-ß 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.

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.

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-ß-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-ß 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-ß 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-ß 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-ß 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-ß-induction for their attenuation: V protein mutation and production of defective interference (DI) RNA.

Oligomerized TICAM-1 (TRIF) in the cytoplasm recruits nuclear BS69 to enhance NF-kappaB 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-kappaB and IFN-regulatory factor (IRF)-3. BS69 was involved in the TICAM-1 complex, and the activation of NF-kappaB/IRF-3 followed by cytokine production was augmented in the presence of BS69 overexpression. Knockdown of endogenous BS69 resulted in a decrease of IFN-beta 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-kappaB activation by EBV-derived latent membrane protein 1, suggest that BS69 harbors dual modes of cytoplasmic NF-kappaB regulation, positively in the TICAM-1 pathway and negatively in the latent membrane protein 1 pathway.

Deletion of the SOCS3 gene in liver parenchymal cells promotes hepatitis-induced hepatocarcinogenesis.

BACKGROUND & AIMS: A recent study has suggested that the methylation silencing of the suppressor of cytokine signaling-3 (SOCS3), a negative regulator of interleukin-6-related cytokines, could be involved in hepatocellular carcinoma (HCC). However, the roles of SOCS3 in hepatocellular carcinogenesis and hepatitis have not been established. We investigated the effect of deleting the SOCS3 gene on the development of hepatitis and HCC in hepatitis C virus-infected patients and mouse models. METHODS: The expression of SOCS genes in HCC and non-HCC regions of patient samples was determined by real-time reverse-transcription polymerase chain reaction and immunoblotting. The conditional knockout approach in mice was used to determine the hepatocyte-specific roles of SOCS3. To generate a liver-specific deletion, floxed SOCS3 (SOCS3(fl/fl)) mice were crossed with albumin-Cre transgenic mice. Hepatitis and HCC were induced by administering concanavalin A and diethylnitrosamine, respectively. RESULTS: SOCS3 expression was reduced in the HCC regions compared with the non-HCC regions. Carcinogen-induced hepatic tumor development was enhanced by deletion of the SOCS3 gene, which was associated with higher levels of the targets of signal transducers and activators of transcription (ie, B-cell lymphoma-XL, B-cell lymphoma-2, C-myelocytomatosis, cyclin D1, and vascular endothelial growth factor). In the concanavalin A-mediated hepatitis model, deletion of the SOCS3 gene in the hepatocytes protected against liver injury through suppression of interferon-gamma signaling and induction of the antiapoptotic protein Bcl-XL. CONCLUSIONS: Deletion of the SOCS3 gene in hepatocytes promotes the activation of STAT3, resistance to apoptosis, and an acceleration of proliferation, resulting in enhanced hepatitis-induced hepatocarcinogenesis.

TGF-beta1 suppresses IFN-gamma-induced NO production in macrophages by suppressing STAT1 activation and accelerating iNOS protein degradation.

TGF-beta1 is a well-known immunosuppressive cytokine; however, little is known of the effect of TGF-beta1 on antigen-presenting cells (APCs). In this report, we investigated the molecular mechanisms of the suppressive effects of TGF-beta1 on APCs including dendritic cells and macrophages. Although TGF-beta1 did not greatly affect the activation of APCs, as assessed by the induction of IL-12 or the upregulation of CD40 in response to LPS, it strongly inhibited IFN-gamma-induced nitric oxide (NO) production from macrophages and dendritic cells. Using murine macrophage-like cell line RAW 264.7, we demonstrated that TGF-beta1 not only reduced the inducible NO synthase (iNOS) protein stability but also suppressed the iNOS gene transcription. We also found that TGF-beta1 directly inhibited IFN-gamma-induced STAT1 activation by reducing STAT1 tyrosine phosphorylation. The IFN-gamma Type I receptor (IFNGR1) was found to be associated with the TGF-beta1 Type I receptor (TGF-betaRI) and was phosphorylated by the TGF-betaRI. Reduced activation of STAT1 by TGF-beta1 was abrogated by the mutation in the IFNGR1 in which the serine residues of potential sites of phosphorylation by TGF-betaRI were replaced by alanine residues. Thus, multiple mechanisms are present for the TGF-beta1-mediated reduction of iNOS production, and we propose a novel mechanism for regulating inflammatory cytokine by an anti-inflammatory cytokine, TGF-beta1; i.e. suppression of IFN-gamma-induced STAT1 activation by an association of the IFNGR1 with the TGF-betaRI.

IFNgamma-dependent, spontaneous development of colorectal carcinomas in SOCS1-deficient mice.

Approximately 20% of human cancers are estimated to develop from chronic inflammation. Recently, the NF-kappaB pathway was shown to play an essential role in promoting inflammation-associated cancer, but the role of the JAK/STAT pathway, another important signaling pathway of proinflammatory cytokines, remains to be investigated. Suppressor of cytokine signaling-1 (SOCS1) acts as an important physiological regulator of cytokine responses, and silencing of the SOCS1 gene by DNA methylation has been found in several human cancers. Here, we demonstrated that SOCS1-deficient mice (SOCS1-/- Tg mice), in which SOCS1 expression was restored in T and B cells on a SOCS1-/- background, spontaneously developed colorectal carcinomas carrying nuclear beta-catenin accumulation and p53 mutations at 6 months of age. However, interferon (IFN)gamma-/- SOCS1-/- mice and SOCS1-/- Tg mice treated with anti-IFNgamma antibody did not develop such tumors. STAT3 and NF-kappaB activation was evident in SOCS1-/- Tg mice, but these were not sufficient for tumor development because these are also activated in IFNgamma-/- SOCS1-/- mice. However, colons of SOCS1-/- Tg mice, but not IFNgamma-/- SOCS1-/- mice, showed hyperactivation of STAT1, which resulted in the induction of carcinogenesis-related enzymes, cyclooxygenase-2 and inducible nitric oxide synthase. These data strongly suggest that SOCS1 is a unique antioncogene which prevents chronic inflammation-mediated carcinogenesis by regulation of the IFNgamma/STAT1 pathways.

A novel Zinc finger protein, ZCCHC11, interacts with TIFA and modulates TLR signaling.

Toll-like receptors (TLRs) play an important role as a sensor of microbial pathogens in the innate immune response. TLRs transmit signals through the recruitment of adaptor proteins including tumor necrosis factor-associated factor 6 (TRAF6), which mediates the activation of IkappaB kinase (IKK). TIFA (TRAF-interacting protein with a forkhead-associated (FHA) domain) has been shown to bind to TRAF6 and activate IKK by promoting the oligomerization and ubiquitin-ligase activity of TRAF6. FHA domains preferentially bind to phospho-threonine residues in their targets. Here, we identified a novel zinc finger protein, ZCCHC11, that interacts with TIFA from phosphoproteins of a macrophage cell line, RAW 264.7, by using affinity purification with GST-TIFA and mass spectrometric analysis. By a search of the EST database, we found a 200kDa full-length form (ZCCHC11L). ZCCHC11L was mostly located to the nucleus, but translocated into the cytoplasm in response to LPS and bound to TIFA. Overexpression and knockdown by siRNA indicated that ZCCHC11 functions as a negative regulator of TLR-mediated NF-kappaB activation. The N-terminal region (ZCCHC11S) including C2H2-type [corrected] Zn-finger motif was sufficient for suppression of NF-kappaB. We propose that ZCCHC11 is a unique TLR signal regulator, which interacts with TIFA after LPS treatment and suppresses the TRAF6-dependent activation of NF-kappaB.

FLN29, a novel interferon- and LPS-inducible gene acting as a negative regulator of toll-like receptor signaling.

Lipopolysaccharide (LPS) activates macrophages through toll-like receptor (TLR) 4. Although the mechanism of the TLR signaling pathway has been well documented, the mechanism of the negative regulation in response to LPS, particularly LPS tolerance, is still poorly understood. In this study we identified and characterized a novel interferon- and LPS-inducible gene, FLN29, which contains a TRAF6-related zinc finger motif and TRAF family member-associated NF-kappaB activator-related sequences. The induction of FLN29 was dependent on STAT1. The forced expression of FLN29 in macrophage-like RAW cells resulted in the suppression of TLR-mediated NF-kappaB and mitogen-activated protein kinase activation, while a reduced expression of FLN29 by small interfering RNA partly cancelled the down-regulation of LPS signaling. Furthermore, we demonstrated that NF-kappaB activation induced by TRAF6 and TAB2 was impaired by co-expression of FLN29, suggesting FLN29 may regulate the downstream of TRAF6. Taken together, FLN29 is a new negative feedback regulator of TLR signaling.