Ablation of IL-17A leads to severe colitis in IL-10-deficient mice: implications of myeloid-derived suppressor cells and NO production.

IL-10 is an immune regulatory cytokine and its genetic defect leads to gastrointestinal inflammation in humans and mice. Moreover, the IL-23/Th17 axis is known to be involved in these inflammatory disorders. IL-17A, a representative cytokine produced by Th17 cells, has an important role for the pathological process of inflammatory diseases. However, the precise function of IL-17A in inflammatory bowel disease (IBD) remains controversial. In this study, we evaluated the effect of IL-17A on colitis in IL-10-deficient (Il10-/-) mice. Mice lacking both IL-10 and IL-17A (Il10-/-Il17a-/-) suffered from fatal wasting and manifested more severe colitis compared with Il10-/-Il17a+/- mice. Moreover, we found that CD11b+Gr-1+ myeloid-derived suppressor cells (MDSCs) accumulated in the bone marrow, spleen and peripheral blood of Il10-/-Il17a-/- mice. These MDSCs highly expressed inducible nitric oxide synthase (iNOS) (Nos2) and suppressed the T-cell response in vitro in a NOS-dependent manner. In correlation with these effects, the concentration of nitric oxide was elevated in the serum of Il10-/-Il17a-/- mice. Surprisingly, the severe colitis observed in Il10-/-Il17a-/- mice was ameliorated in Il10-/-Il17a-/-Nos2-/- mice. Our findings suggest that IL-17A plays suppressive roles against spontaneous colitis in Il10-/- mice in an iNOS-dependent manner and inhibits MDSC differentiation and/or proliferation.

Protective mucosal immunity mediated by epithelial CD1d and IL-10.

The mechanisms by which mucosal homeostasis is maintained are of central importance to inflammatory bowel disease. Critical to these processes is the intestinal epithelial cell (IEC), which regulates immune responses at the interface between the commensal microbiota and the host. CD1d presents self and microbial lipid antigens to natural killer T (NKT) cells, which are involved in the pathogenesis of colitis in animal models and human inflammatory bowel disease. As CD1d crosslinking on model IECs results in the production of the important regulatory cytokine interleukin (IL)-10 (ref. 9), decreased epithelial CD1d expression--as observed in inflammatory bowel disease--may contribute substantially to intestinal inflammation. Here we show in mice that whereas bone-marrow-derived CD1d signals contribute to NKT-cell-mediated intestinal inflammation, engagement of epithelial CD1d elicits protective effects through the activation of STAT3 and STAT3-dependent transcription of IL-10, heat shock protein 110 (HSP110; also known as HSP105), and CD1d itself. All of these epithelial elements are critically involved in controlling CD1d-mediated intestinal inflammation. This is demonstrated by severe NKT-cell-mediated colitis upon IEC-specific deletion of IL-10, CD1d, and its critical regulator microsomal triglyceride transfer protein (MTP), as well as deletion of HSP110 in the radioresistant compartment. Our studies thus uncover a novel pathway of IEC-dependent regulation of mucosal homeostasis and highlight a critical role of IL-10 in the intestinal epithelium, with broad implications for diseases such as inflammatory bowel disease.

Hyper-reactive cloned mice generated by direct nuclear transfer of antigen-specific CD4+ T cells.

T-cell receptor (TCR)-transgenic mice have been employed for evaluating antigen-response mechanisms, but their non-endogenous TCR might induce immune response differently than the physiologically expressed TCR Nuclear transfer cloning produces animals that retain the donor genotype in all tissues including germline and immune systems. Taking advantage of this feature, we generated cloned mice that carry endogenously rearranged TCR genes from antigen-specific CD4+ T cells. We show that T cells of the cloned mice display distinct developmental pattern and antigen reactivity because of their endogenously pre-rearranged TCRα (rTα) and TCRß (rTß) alleles. These alleles were transmitted to the offspring, allowing us to establish a set of mouse lines that show chronic-type allergic phenotypes, that is, bronchial and nasal inflammation, upon local administrations of the corresponding antigens. Intriguingly, the existence of either rTα or rTß is sufficient to induce in vivo hypersensitivity. These cloned mice expressing intrinsic promoter-regulated antigen-specific TCR are a unique animal model with allergic predisposition for investigating CD4+ T-cell-mediated pathogenesis and cellular commitment in immune diseases.

Identification of biomarker sets for predicting the efficacy of sublingual immunotherapy against pollen-induced allergic rhinitis.

Sublingual immunotherapy (SLIT) is effective against allergic rhinitis, although a substantial proportion of individuals is refractory. Herein, we describe a predictive modality to reliably identify SLIT non-responders (NRs). We conducted a 2-year clinical study in 193 adult patients with Japanese cedar pollinosis, with biweekly administration of 2000 Japanese allergy units of cedar pollen extract as the maintenance dose. After identifying high-responder (HR) patients with improved severity scores and NR patients with unchanged or exacerbated symptoms, differences in 33 HR and 34 NR patients were evaluated in terms of peripheral blood cellular profiles by flow cytometry and serum factors by ELISA and cytokine bead array, both pre- and post-SLIT. Improved clinical responses were seen in 72% of the treated patients. Pre-therapy IL-12p70 and post-therapy IgG1 serum levels were significantly different between HR and NR patients, although these parameters alone failed to distinguish NR from HR patients. However, the analysis of serum parameters in the pre-therapy samples with the Adaptive Boosting (AdaBoost) algorithm distinguished NR patients with high probability within the training data set. Cluster analysis revealed a positive correlation between serum Th1/Th2 cytokines and other cytokines/chemokines in HR patients after SLIT. Thus, processing of pre-therapy serum parameters with AdaBoost and cluster analysis can be reliably used to develop a prediction method for HR/NR patients.

Efficient generation of functional hepatocytes from human embryonic stem cells and induced pluripotent stem cells by HNF4α transduction.

Hepatocyte-like cells from human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) are expected to be a useful source of cells drug discovery. Although we recently reported that hepatic commitment is promoted by transduction of SOX17 and HEX into human ESC- and iPSC-derived cells, these hepatocyte-like cells were not sufficiently mature for drug screening. To promote hepatic maturation, we utilized transduction of the hepatocyte nuclear factor 4α (HNF4α) gene, which is known as a master regulator of liver-specific gene expression. Adenovirus vector-mediated overexpression of HNF4α in hepatoblasts induced by SOX17 and HEX transduction led to upregulation of epithelial and mature hepatic markers such as cytochrome P450 (CYP) enzymes, and promoted hepatic maturation by activating the mesenchymal-to-epithelial transition (MET). Thus HNF4α might play an important role in the hepatic differentiation from human ESC-derived hepatoblasts by activating the MET. Furthermore, the hepatocyte like-cells could catalyze the toxication of several compounds. Our method would be a valuable tool for the efficient generation of functional hepatocytes derived from human ESCs and iPSCs, and the hepatocyte-like cells could be used for predicting drug toxicity.

Induction of type I interferon by adenovirus-encoded small RNAs.

Transduction with replication-incompetent recombinant adenovirus (Ad) vectors results in a rapid activation of innate immune responses, such as inflammatory cytokine production and subsequent tissue damage. The precise mechanisms of the innate immune responses induced by Ad vectors remain to be clarified. Possible components of Ad vectors that activate innate immune responses are the capsid protein, the viral genome (DNA), and viral transcripts. In the present study, we demonstrate that virus-associated RNAs (VA-RNAs), which are small RNAs transcribed by RNA polymerase III, induce the production of type I IFN (IFN-α and IFN-ß), but they do not induce the production of inflammatory cytokines (IL-6 and IL-12), in mouse embryonic fibroblasts (MEFs) and granulocyte-macrophage colony-stimulating factor-generated bone marrow-derived dendritic cells (GM-DCs). We also show that IFN-ß promoter stimulator-1 is involved in VA-RNA-dependent IFN-ß production in MEFs and is partially involved in type I IFN production in GM-DCs. This study provides important insight into the mechanisms of Ad vector-triggered innate immune responses, which may lead to more advanced and rational Ad vector designs for gene therapies and vaccine applications.

Novel Adjuvant S-540956 Targets Lymph Nodes and Reduces Genital Recurrences and Vaginal Shedding of HSV-2 DNA When Administered with HSV-2 Glycoprotein D as a Therapeutic Vaccine in Guinea Pigs.

Herpes simplex virus type 2 (HSV-2) is a leading cause of genital ulcer disease and a major risk factor for acquisition and transmission of HIV. Frequent recurrent genital lesions and concerns about transmitting infection to intimate partners affect the quality of life of infected individuals. Therapeutic vaccines are urgently needed to reduce the frequency of genital lesions and transmission. S-540956 is a novel vaccine adjuvant that contains CpG oligonucleotide ODN2006 annealed to its complementary sequence and conjugated to a lipid that targets the adjuvant to lymph nodes. Our primary goal was to compare S-540956 administered with HSV-2 glycoprotein D (gD2) with no treatment in a guinea pig model of recurrent genital herpes (studies 1 and 2). Our secondary goals were to compare S-540956 with oligonucleotide ODN2006 (study1) or glucopyranosyl lipid A in a stable oil-in-water nano-emulsion (GLA-SE) (study 2). gD2/S-540956 reduced the number of days with recurrent genital lesions by 56%, vaginal shedding of HSV-2 DNA by 49%, and both combined by 54% compared to PBS, and was more efficacious than the two other adjuvants. Our results indicate that S-540956 has great potential as an adjuvant for a therapeutic vaccine for genital herpes, and merits further evaluation with the addition of potent T cell immunogens.

Critical roles of the cAMP-responsive element-binding protein-mediated pathway in disorganized epithelial phenotypes caused by mitochondrial dysfunction.

In most human cancers, somatic mutations have been identified in the mtDNA; however, their significance remains unclear. We recently discovered that NMuMG mouse mammary epithelial cells, when deprived of mitochondria or following inhibition of respiratory activity, undergo epithelial morphological disruption accompanied with irregular edging of E-cadherin, the appearance of actin stress fibers, and an altered gene expression profile. In this study, using the mtDNA-less pseudo ρ0 cells obtained from NMuMG mouse mammary epithelial cells, we examined the roles of two mitochondrial stress-associated transcription factors, cAMP-responsive element-binding protein (CREB) and C/EBP homologous protein-10 (CHOP), in the disorganization of epithelial phenotypes. We found that the expression of matrix metalloproteinase-13 and that of GADD45A, SNAIL and integrin α1 in the ρ0 cells were regulated by CHOP and CREB, respectively. Of note, knockdown and pharmacological inhibition of CREB ameliorated the disrupted epithelial morphology. It is interesting to note that the expression of high mobility group AT-hook 2 (HMGA2), a non-histone chromatin protein implicated in malignant neoplasms, was increased at the protein level through the CREB pathway. Here, we reveal how the activation of the CREB/HMGA2 pathway is implicated in the repression of integrin α1 expression in HepG2 human cancer cells, highlighting the importance of the CREB/HMGA2 pathway in malignant transformation associated with mitochondrial dysfunction, thereby raising the possibility that the pathway indirectly interferes with the cell-cell adhesion structure by influencing the cell-extracellular matrix adhesion status. Overall, the data suggest that mitochondrial dysfunction potentially contributes to neoplastic transformation of epithelial cells through the activation of these transcriptional pathways.

Role of leukotriene B4 receptor signaling in human preadipocyte differentiation.

We investigated the role of leukotriene B(4) (LTB(4))-leukotriene receptor (BLT) signaling in preadipocyte differentiation into mature adipocytes. Blockade of BLT signaling by treatment with lipoxygenase inhibitors, a BLT antagonist, and small interfering RNAs for BLTs in human and mouse preadipocytes isolated from adipose tissues showed acceleration of differentiation into mature adipocytes. DNA microarray analysis revealed regulation of transforming growth factor, beta-induced 68 kDa (TGFBI) expression through the BLT signaling pathway during adipocyte differentiation. Knockdown of TGFBI also showed acceleration of preadipocyte differentiation. The LTB(4)-BLT signaling pathway may negatively regulate preadipocyte differentiation via induction of TGFBI expression as a rate-limiting system to control adipocyte differentiation.

Type-I IFN signaling is required for the induction of antigen-specific CD8(+) T cell responses by adenovirus vector vaccine in the gut-mucosa.

Adenovirus vector (Adv) vaccination at a systemic site, such as intramuscular (i.m.) immunization, can induce antigen-specific CD8(+) T cell responses in both systemic and mucosal compartments. It remains unclear, however, how antigen-specific CD8(+) T cell response is induced in the mucosa. In this study, we found that type-I IFN signaling is required for the induction of mRNA expression of retinal dehydrogenase in the draining lymph nodes following the i.m. Adv vaccination. We show that type-I IFN signaling is required for the induction of antigen-specific CD8(+) T cell response in the gut-mucosal compartment following the i.m. Adv vaccination.

Further reduction in adenovirus vector-mediated liver transduction without largely affecting transgene expression in target organ by exploiting microrna-mediated regulation and the Cre-loxP recombination system.

In order to detarget undesirable transduction in the liver by an adenovirus (Ad) vector, we previously demonstrated that insertion of sequences perfectly complementary to liver-specific miR-122a into the 3'-untranslated region (UTR) of transgene specifically reduced the transgene expression in the liver by approximately 100-fold; however, a certain level of residual transgene expression was still found in the liver. In order to further suppress the hepatic transduction, we developed a two-Ad vector system that uses the microRNA (miRNA)-regulated transgene expression system and the Cre-loxP recombination system, i.e., insertion of miR-122a target sequences and loxP sites into the transgene expression cassette and coadministration of a Cre recombinase-expressing Ad vector. In addition, to maintain as much as possible the transgene expression in the spleen, which is the target organ of this study, spleen-specific miR-142-3p target sequences were inserted into the 3'-UTR of the Cre recombinase gene to suppress Cre recombinase expression in the spleen. The spleen is an attractive target for immunotherapy because the spleen plays important roles in the immune system. Coadministration of Ad vector possessing CMV promoter-driven Cre recombinase expression cassette with miR-142-3p target sequences resulted in a further 24-fold reduction in the hepatic transgene expression by the Ad vector containing miR-122a target sequences and loxP sites, compared with coadministration of control Ad vector. On the other hand, there was no significant reduction of transgene expression in the spleen.

Efficient generation of hepatoblasts from human ES cells and iPS cells by transient overexpression of homeobox gene HEX.

Human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have the potential to differentiate into all cell lineages, including hepatocytes, in vitro. Induced hepatocytes have a wide range of potential application in biomedical research, drug discovery, and the treatment of liver disease. However, the existing protocols for hepatic differentiation of PSCs are not very efficient. In this study, we developed an efficient method to induce hepatoblasts, which are progenitors of hepatocytes, from human ESCs and iPSCs by overexpression of the HEX gene, which is a homeotic gene and also essential for hepatic differentiation, using a HEX-expressing adenovirus (Ad) vector under serum/feeder cell-free chemically defined conditions. Ad-HEX-transduced cells expressed α-fetoprotein (AFP) at day 9 and then expressed albumin (ALB) at day 12. Furthermore, the Ad-HEX-transduced cells derived from human iPSCs also produced several cytochrome P450 (CYP) isozymes, and these P450 isozymes were capable of converting the substrates to metabolites and responding to the chemical stimulation. Our differentiation protocol using Ad vector-mediated transient HEX transduction under chemically defined conditions efficiently generates hepatoblasts from human ESCs and iPSCs. Thus, our methods would be useful for not only drug screening but also therapeutic applications.

Interleukin-1 family cytokines as mucosal vaccine adjuvants for induction of protective immunity against influenza virus.

A safe and potent adjuvant is needed for development of mucosal vaccines against etiological agents, such as influenza virus, that enter the host at mucosal surfaces. Cytokines are potential adjuvants for mucosal vaccines because they can enhance primary and memory immune responses enough to protect against some infectious agents. For this study, we tested 26 interleukin (IL) cytokines as mucosal vaccine adjuvants and compared their abilities to induce antigen (Ag)-specific immune responses against influenza virus. In mice intranasally immunized with recombinant influenza virus hemagglutinin (rHA) plus one of the IL cytokines, IL-1 family cytokines (i.e., IL-1α, IL-1ß, IL-18, and IL-33) were found to increase Ag-specific immunoglobulin G (IgG) in plasma and IgA in mucosal secretions compared to those after immunization with rHA alone. In addition, high levels of both Th1- and Th2-type cytokines were observed in mice immunized with rHA plus an IL-1 family cytokine. Furthermore, mice intranasally immunized with rHA plus an IL-1 family cytokine had significant protection against a lethal influenza virus infection. Interestingly, the adjuvant effects of IL-18 and IL-33 were significantly decreased in mast cell-deficient W/W(v) mice, indicating that mast cells have an important role in induction of Ag-specific mucosal immune responses induced by IL-1 family cytokines. In summary, our results demonstrate that IL-1 family cytokines are potential mucosal vaccine adjuvants and can induce Ag-specific immune responses for protection against pathogens like influenza virus.

Quantitative analysis of the leaky expression of adenovirus genes in cells transduced with a replication-incompetent adenovirus vector.

Theoretically, adenovirus (Ad) genes should not be expressed following transduction with a replication-incompetent Ad vector because the E1A gene, which is essential for the expression of other viral gene, is deleted in a replication-incompetent Ad vector. However, leaky expression of viral genes is known to occur following transduction with an E1-deleted Ad vector, leading to an induction of cellular immunity against Ad proteins. To date, no detailed analysis of the leaky expression profiles of Ad genes has been performed. In this study, we systematically examined the expression profiles of Ad genes in cells following transduction with a replication-incompetent Ad vector (Ad-L2) at multiplicities of infection (MOIs) of 10 and 100 using real-time RT-PCR. Significant expression was found for the E4 and pIX genes following transduction with Ad-L2 in cultured cells. The expression levels of the E4 and pIX genes were approximately 30- to 600-fold lower than those of the transgene (firefly luciferase), and 50- to 5000-fold lower than those of the E4 and pIX genes following transduction at the same MOI with the wild-type Ad. Unexpectedly, expression levels of the major capsid proteins were approximately the same as, or even slightly above, the background levels (Ad gene expression levels in mock-transduced cells). This study provides valuable information for the design of a safe and efficient replication-incompetent Ad vector.

S-540956, a CpG Oligonucleotide Annealed to a Complementary Strand With an Amphiphilic Chain Unit, Acts as a Potent Cancer Vaccine Adjuvant by Targeting Draining Lymph Nodes.

Robust induction of cancer-antigen-specific CD8+ T cells is essential for the success of cancer peptide vaccines, which are composed of a peptide derived from a cancer-specific antigen and an immune-potentiating adjuvant, such as a Toll-like receptor (TLR) agonist. Efficient delivery of a vaccine antigen and an adjuvant to antigen-presenting cells in the draining lymph nodes (LNs) holds key to maximize vaccine efficacy. Here, we developed S-540956, a novel TLR9-agonistic adjuvant consisting of B-type CpG ODN2006 (also known as CpG7909), annealed to its complementary sequence oligodeoxynucleotide (ODN) conjugated to a lipid; it could target both a cancer peptide antigen and a CpG-adjuvant in the draining LNs. S-540956 accumulation in the draining LNs and activation of plasmacytoid dendritic cells (pDCs) were significantly higher than that of ODN2006. Mechanistic analysis revealed that S-540956 enhanced the induction of MHC class I peptide-specific CD8+ T cell responses via TLR9 in a CD4+ T cell-independent manner. In mice, the therapeutic effect of S-540956-adjuvanted with a human papillomavirus (HPV)-E7 peptide vaccine against HPV-E7-expressing TC-1 tumors was significantly better than that of an ODN2006-adjuvanted vaccine. Our findings demonstrate a novel adjuvant discovery with the complementary strand conjugated to a lipid, which enabled draining LN targeting and increased ODN2006 accumulation in draining LNs, thereby enhancing the adjuvant effect. Our findings imply that S-540956 is a promising adjuvant for cancer peptide vaccines and has a high potential for applications in various vaccines, including recombinant protein vaccines.

[PPARgamma-gene therapy].

In recent years, there has been increasing evidence that peroxisome proliferators-activated receptor gamma (PPARgamma) plays crucial roles in various pathophysiological processes, some of which are associated with a decrease in the expression of PPARgamma. Our previous study demonstrated that gene delivery of PPARgamma could be used to restore and/or enhance endogenous anti-inflammatory processes that are normally operative in mammalian tissues such as colon. In this review, we describe the outline of recent progress in the development of PPARgamma-gene therapy and PPARgamma molecular pathways, along with the results obtained in our previous experiments and those reported by others.

TNF superfamily member, TL1A, is a potential mucosal vaccine adjuvant.

The identification of cytokine adjuvants capable of inducing an efficient mucosal immune response against viral pathogens has been long anticipated. Here, we attempted to identify the potential of tumor necrosis factor superfamily (TNFS) cytokines to function as mucosal vaccine adjuvants. Sixteen different TNFS cytokines were used to screen mucosal vaccine adjuvants, after which their immune responses were compared. Among the TNFS cytokines, intranasal immunization with OVA plus APRIL, TL1A, and TNF-alpha exhibited stronger immune response than those immunized with OVA alone. TL1A induced the strongest immune response and augmented OVA-specific IgG and IgA responses in serum and mucosal compartments, respectively. The OVA-specific immune response of TL1A was characterized by high levels of serum IgG1 and increased production of IL-4 and IL-5 from splenocytes of immunized mice, suggesting that TL1A might induce Th2-type responses. These findings indicate that TL1A has the most potential as a mucosal adjuvant among the TNFS cytokines.

Proteomic Approach to FcepsilonRI aggregation-initiated signal transduction cascade in human mast cells.

BACKGROUND: Mast cells (MCs) play a central role in allergic reactions through high-affinity IgE receptor (FcepsilonRI)-mediated responses. Many attempts have been performed to investigate MC functions, though molecular bases of the intracellular signaling cascade through FcepsilonRI, especially in human MCs, remain scant and unexplored. METHODS: Human MCs were differentiated from CD34+ cells by culture with stem cell factor, IL-6 and IL-3. The differential phosphorylation profiles of protein tyrosine residues in the resulting MCs with or without FcepsilonRI aggregation were examined by two-dimensional gel electrophoresis. The candidate phosphoproteins of interest were picked, in-gel digested and mass spectrometry fingerprinted. RESULTS: Approximately 40 proteins in MCs were phosphorylated on their tyrosine residues in response to activation and some of them were identified. Particularly IL-31 receptor alpha, solute carrier family 39, syntaxin 5 and heterogeneous nuclear ribonucleoprotein are newly identified as phosphoproteins that are potentially involved in the MC signaling cascade through FcepsilonRI. CONCLUSION: Our present phosphoproteome data may provide the clue to understand the molecular mechanisms for the activation of human MCs.