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Complex interactions between keratinocytes and various cell types, such as inflammatory cells and stromal cells, contribute to the pathogenesis of chronic inflammatory skin lesions. In proinflammatory cytokine‒mediated disease settings, IL-9 plays a pathological role in inflammatory dermatitis. However, IL-9‒related mechanisms remain incompletely understood. In this study, we established tamoxifen-induced keratinocyte-specific IL-9RA-deficient mice (K14

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, Kruppel-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.

POU domain class 2-associating factor 1 (also called Bob1), which is mainly expressed in B cells, regulates B cell homeostasis and controls humoral immune responses. Although Bob1 is known to function reliably in T cell subsets including follicular helper T cells, Th1 cells and Th2 cells, it is unknown whether Bob1 functions in other T cell subsets. In this study, we found that Bob1 knock out (KO) mice are resistant to experimental autoimmune encephalomyelitis (EAE) induced by MOG35-55 peptide and that Bob1 KO T cells are defective in Th17 differentiation. Importantly, Bob1 interacts with retinoid acid receptor-related orphan receptor (ROR) gamma t (RORγt), a signature transcription factor for Th17 cells, through the ligand-binding domain of RORγt, thereby enhancing IL-17A transcription activity. IL-17A induction by Bob1 requires the ability for its formation of a DNA-Oct1-Bobl ternary complex. Thus, our findings demonstrate that Bob1 enhances IL-17A expression in vivo and in vitro by interacting with RORγt in Th17 cells, suggesting that Bob1 plays a pivotal role in Th17-mediated autoimmune disease.

Adjuvant stimulates pattern-recognition receptors (PRRs) expressed by dendritic cells, which causes immune-enhancing of T lymphocytes. Adjuvant also induces innate immune response in whole-body cells via PRRs to evoke cytokinemia. A cytokine-mediated immune response is important for the systemic protection of a host from microbial infections. Using an influenza subcomponent vaccine in a mouse model, we intranasally administered a TLR3-specific adjuvant ARNAX + HA split vaccine to mice. ARNAX efficiently induced mucosal IgA and systemic IgG production by nasal drop. Moreover, ARNAX + HA simultaneously induced CD8 and CD4 T cell activation. We have previously shown that ARNAX does not induce harmful systemic cytokine production. Thus, our findings indicate that the ARNAX + HA vaccine is a harmless prophylactic vaccine for flu that induces HA-specific T cell activation and IgA/IgG production. These results suggested that ARNAX + antigen enhanced the immune response without inducing inflammatory toxicity for vaccination against infectious diseases.

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. 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. 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. 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.

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.

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.

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.

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.

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.

Suppressor of cytokine signaling (SOCS)3 is a major negative feedback regulator of signal transducer and activator of transcription (STAT)3-activating cytokines. Transgenic mouse studies indicate that high levels of SOCS3 in T cells result in type 2 T helper cell (Th2) skewing and lead to hypersensitivity to allergic diseases. To define the physiological roles of SOCS3 in T cells, we generated T cell–specific SOCS3 conditional knockout mice. We found that the mice lacking SOCS3 in T cells showed reduced immune responses not only to ovalbumin-induced airway hyperresponsiveness but also to Leishmania major infection. In vitro, SOCS3-deficient CD4+ T cells produced more transforming growth factor (TGF)-β1 and interleukin (IL)-10, but less IL-4 than control T cells, suggesting preferential Th3-like differentiation. We found that STAT3 positively regulates TGF-β1 promoter activity depending on the potential STAT3 binding sites. Furthermore, chromatin immunoprecipitation assay revealed that more STAT3 was recruited to the TGF-β1 promoter in SOCS3-deficient T cells than in control T cells. The activated STAT3 enhanced TGF-β1 and IL-10 expression in T cells, whereas the dominant-negative form of STAT3 suppressed these. From these findings, we propose that SOCS3 regulates the production of the immunoregulatory cytokines TGF-β1 and IL-10 through modulating STAT3 activation.

Caveolin-1 (Cav-1) has been suggested to function as a negative regulator of mitogen-stimulated proliferation and the Ras-p42/44 ERK (MAP kinase) pathway in a variety of cell types. However, the molecular basis of this suppression has not been clarified. Spred/Sprouty family proteins are also negative regulators of the ERK pathway by interacting with Raf-1. The Spred/Sprouty family proteins contain a cysteine-rich (CR) domain at the C-terminus, which is thought to be palmitoylated like Cav-1 and necessary for membrane anchoring. In this study, we demonstrated that Spred-1 localized in cholesterol-rich membrane raft/caveola fractions and interacted with Cav-1. To clarify the biological effect of Cav-1/Spred-1 interaction, we used hematopoietic cells that lacked expression of caveolins but expressed Spred-1. Forced expression of Cav-1 suppressed SCF- and IL-3-induced proliferation and ERK activation. Furthermore, forced expression of exogenous Spred-1 in Cav-1-expressing cells further suppressed proliferation and ERK activation. These data suggest that Spred-1 inhibits ERK activation in collaboration with Cav-1.

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 DC's differential susceptibility to MV. (C) 2014 Elsevier Ltd. All rights reserved.

It has been shown that transforming growth factor beta1 (TGF-beta1) is critical in the generation of CD4(+)CD25(+)Foxp3(+)-inducible regulatory T cells (iTregs) from naïve CD4(+)T cells. However, in contrast to natural Tregs, TGF-beta1-induced iTregs rapidly lose both Foxp3 expression and suppression activity. We found that TGF-beta1-induced Foxp3 levels were maintained by the addition of the anti-interleukin 4 (IL-4) antibody or by STAT6 gene deletion. Thus, IL-4 is an important suppressor of Foxp3 induction, and T helper 2 development is a major cause for the disappearance of iTreg during long culture. Using promoter analysis in EL4 cells and primary T cells, we identified a silencer region containing a STAT6 binding site. STAT6 binding to this site reduced TGF-beta1-mediated Foxp3 promoter activation and chromatin modification. Retinoic acid has also been shown to suppress loss of Foxp3 induced by TGF-beta1. Retinoic acid in the presence of TGF-beta1 reduced STAT6 binding to the Foxp3 promoter and enhanced histone acetylation, thereby reverting the effect of IL-4. We propose that antagonistic agents for neutralizing IL-4 could be a novel strategy to facilitate inducible Treg cell generation and the promotion of tolerance in Th2-dominated diseases such as allergy.

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.

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.

Syndecan-1, -2, -3, and -4 are heparan sulfate proteoglycans that are differentially expressed during development and wound repair. To determine whether syndecans are also involved in brain injury, we examined the expression of syndecan core proteins genes in cryo-injured mouse brain, using in situ hybridization. All syndecan mRNA transcripts were similarly expressed in the region surrounding the necrotic tissue, exhibiting peak levels at day 7 after injury. Comparison with cellular markers showed that reactive astrocytes were the primary source of syndecans. Syndecans serve as co-receptors for fibroblast growth factor (FGF) and as a reservoir for another heparin-binding growth factor, pleiotrophin (PTN, or heparin-binding growth-associated molecule. In our model, FGF receptor1 (FGFR1) and PTN mRNA levels were upregulated in reactive astrocytes. The distribution patterns of FGFR1 and PTN overlapped considerably with those of syndecan-1 and -3 mRNAs, respectively. These results suggest that syndecans are expressed primarily in reactive astrocytes, and may provide a supportive environment for regenerating axons in concert with heparin-binding growth factors (e.g., FGF and PTN) in the injured brain.