Publications

Although the mucosal and the systemic immune compartments are structurally and functionally independent, they engage in cross-talk under specific conditions. To investigate this cross-talk, we vaccinated mice with tetanus toxoid together with cholera toxin with s.c. priming followed by intrarectal (IR) boosting. Interestingly, higher numbers of Ag-specific IgA and IgG Ab-secreting cells (ASCs) were detected in the lamina propria of the large intestine of mice vaccinated s.c.-IR. Ag-specific ASCs from the colon migrated to SDF-1α/CXCL12 and mucosae-associated epithelial chemokine/CCL28, suggesting that CXCR4+ and/or CCR10+ IgA ASCs found in the large intestine after s.c.-IR are of systemic origin. In the colonic patches-null mice, IgA ASCs in the large intestine were completely depleted. Furthermore, the accumulation of IgA ASCs in the colonic patches by inhibition of their migration with FTY720 revealed that colonic patches are the IgA class-switching site after s.c.-IR. Most interestingly, s.c.-IR induced numbers of Ag-specific IgA ASCs in the large intestine of TLR2−/−, TLR4−/−, MyD88−/−, and TRIF−/− mice that were comparable with those of wild-type mice. Taken together, our results suggest the possibility that cross-talk could occur between the large intestine and the systemic immune compartments via the colonic patches without the assistance of innate immunity.

Epstein–Barr virus latent membrane protein 1 (LMP1) activation of NF-κB is critical for Epstein–Barr virus-infected B lymphocyte survival. LMP1 activates the IκB kinase complex and NF-κB through two cytoplasmic signaling domains that engage tumor necrosis factor receptor-associated factor (TRAF)1/2/3/5 or TRADD and RIP. We now use cells lacking expression of TRAF2, TRAF5, TRAF6, IKKα, IKKβ, IKKγ, TAB2, IL-1 receptor-associated kinase (IRAK)1, or IRAK4 to assess their roles in LMP1-mediated NF-κB activation. LMP1-induced RelA nuclear translocation was similar in IKKα knockout (KO) and WT murine embryo fibroblasts (MEFs) but substantially deficient in IKKβ KO MEFs. NF-κB-dependent promoter responses were also substantially deficient in IKKβ KO MEFs but were hyperactive in IKKα KO MEFs. More surprisingly, NF-κB responses were near normal in TRAF2 and TRAF5 double-KO MEFs, IKKγ KO MEFs, TAB2 KO MEFs, and IRAK4 KO MEFs but were highly deficient in TRAF6 KO MEFs and IRAK1 KO HEK293 cells. Consistent with the importance of TRAF6, LMP1-induced NF-κB activation in HEK293 cells was inhibited by expression of dominant-negative TAB2 and Ubc13 alleles. These data extend a role for IKKα in IKKβ regulation, identify an unusual IKKβ-dependent and IKKγ-independent NF-κB activation, and indicate that IRAK1 and TRAF6 are essential for LMP1-induced NF-κB activation.

No abstract is provided for this article.

Summary The ability of mesenchymal stromal cells to modulate inflammation is at the basis of the ongoing interest in their therapeutic potential. Yet, reliable success in clinical trials is limited, possibly due to a limited understanding of their impact on the inflammatory milieu in physiological conditions. Here we show that, at steady state, mesenchymal progenitors regulate the balance between type 1 and type 2 inflammatory milieus by acting on innate immune cells through the TAK1-NFkB pathway. Suppressing the constitutive activity of this pathway in MPs leads to skewing of the immune system toward systemic Type 2 inflammation (Th2). These changes have significant effects on diseases with an important inflammatory component, leading to a worsening of disease in a preclinical model of Th2-dependent Asthma, and a reduction of symptoms associated with Th1/Th17-dependent experimental autoimmune encephalitis.

Taxol can mimic bacterial lipopolysaccharide (LPS) by activating mouse macrophages in a cell cycle-independent, LPS antagonist-inhibitable manner. Macrophages from C3H/HeJ mice, which have a spontaneous mutation in Toll-like receptor 4 (TLR4), are hyporesponsive to both LPS and Taxol, suggesting that LPS and Taxol may share a signaling pathway involving TLR4. To determine whether TLR4 and its interacting adaptor molecule MyD88 are necessary for Taxol's LPS mimetic actions, we examined Taxol responses of primary macrophages from genetically defective mice lacking either TLR4 (C57BL/10ScNCr) or MyD88 (MyD88 knockout). When stimulated with Taxol, macrophages from wild-type mice responded robustly by secreting both TNF and NO, while macrophages from either TLR4-deficient C57BL/10ScNCr mice or MyD88 knockout mice produced only minimal amounts of TNF and NO. Taxol-induced NF-kappa B-driven luciferase activity was reduced after transfection of RAW 264.7 macrophages with a dominant negative version of mouse MyD88. Taxol-induced microtubule-associated protein kinase (MAPK) activation and NF-kappa B nuclear translocation were absent from TLR4-null macrophages, but were preserved in MyD88 knockout macrophages with a slight delay in kinetics. Neither Taxol-induced NF-kappa B activation, nor I kappa B degradation was affected by the presence of phosphatidylinositol 3-kinase inhibitors. These results suggest that Taxol and LPS not only share a TLR4/MyD88-dependent pathway in generating inflammatory mediators, but also share a TLR4-dependent/MyD88-independent pathway leading to activation of MAPK and NF-kappa B.

Beclin 1 is a key regulator of autophagy and endocytosis. However, its autophagy-independent functions remain poorly understood. Here, we report that Beclin 1 regulates recycling endosome and is required for skin development in vivo. We first established keratinocyte-specific Beclin 1-knockout mice and found that these mutant mice died owing to severe impairment of epidermal barrier. Beclin 1 plays a role in autophagy and the endocytic pathway in cooperation with Atg14 and UVRAG, respectively, and keratinocyte-specific Atg14-knockout mice do not show any abnormal phenotypes, suggesting that Beclin 1 has a role in skin development via the endocytic pathway. Furthermore, we found that Beclin 1 deficiency causes mislocalization of integrins via a defect of recycling endosome, abnormal cell detachment of basal cells and their immature differentiation, and abnormal skin development. These results provide the first genetic evidence showing the roles of Beclin 1 in recycling endosome and skin development.

Macrophages recognize lipopolysaccharide (LPS) by Toll-like receptor 4 and activate inflammatory responses by inducing expression of various genes. TLR4 activates intracellular signaling pathways via TIR domain containing adaptor molecules, MyD88, and Toll/IL-1 domain containing adaptor inducing IFN-β (TRIF). Although macrophages lacking MyD88 or TRIF showed impaired cytokine production, activation of intracellular signaling molecules still occurred in response to LPS in these cells. In the present study, we implemented cDNA microarrays to investigate the contribution of MyD88 and TRIF in gene expression induced by LPS stimulation. Whereas wild-type macrophages induced 148 genes in response to LPS, macrophages lacking both MyD88 and TRIF did not upregulate any genes in response to LPS. Surprisingly, 80 LPS-inducible genes were redundantly regulated by either MyD88 or TRIF. In contrast, proinflammatory cytokines and chemokines were critically regulated by MyD88 or TRIF alone. Genes critically regulated by MyD88 alone tend to be induced quickly after LPS stimulation and regulated by mRNA stability as well as transcription. Genes known to be induced by type I interferons were simply dependent on TRIF for their expression. Taken together, MyD88 and TRIF play both redundant and distinct roles in LPS-induced gene expression.

Signal transducer and activator of transcription (Stat)-3 signals mediate many of the metabolic effects of the fat cell-derived hormone, leptin. In mice, brain-specific depletion of either the long form of the leptin receptor (Lepr) or Stat3 results in comparable obese phenotypes as does replacement of Lepr with an altered leptin receptor locus that codes for a Lepr unable to interact with Stat3. Among the multiple brain regions containing leptin-sensitive Stat3 sites, cells expressing feeding-related neuropeptides in the arcuate nucleus of the hypothalamus have received much of the focus. To determine the contribution to energy homeostasis of Stat3 expressed in agouti-related protein (Agrp)/neuropeptide Y (Npy) arcuate neurons, Stat3 was deleted specifically from these cells, and several metabolic indices were measured. It was found that deletion of Stat3 from Agrp/Npy neurons resulted in modest weight gain that was accounted for by increased adiposity. Agrp/Stat3-deficient mice also showed hyperleptinemia, and high-fat diet-induced hyperinsulinemia. Stat3 deletion in Agrp/Npy neurons also resulted in altered hypothalamic gene expression indicated by increased Npy mRNA and decreased induction of suppressor of cytokine signaling-3 in response to leptin. Agrp mRNA levels in the fed or fasted state were unaffected. Behaviorally, mice without Stat3 in Agrp/Npy neurons were mildly hyperphagic and hyporesponsive to leptin. We conclude that Stat3 in Agrp/Npy neurons is required for normal energy homeostasis, but Stat3 signaling in other brain areas also contributes to the regulation of energy homeostasis.

Tribbles pseudokinase 1 (Trib1) is a negative regulator of CCAAT/enhancer binding protein α (C/EBPα) and is known to induce granulopoiesis while suppressing monocyte differentiation. Loss of Trib1 was previously shown to increase the neutrophil population in the spleen but lead to M2‐like macrophage reduction. Because M2 macrophages are anti‐inflammatory and promote tissue repair by producing fibrogenic factors, we investigated liver fibrosis in Trib1‐deficient mice. Interestingly, loss of Trib1 suppressed fibrosis in the CCl4‐induced chronic liver injury model. Trib1 knockout increased neutrophils but had a minimal effect on the macrophage population in the liver. Hepatic expressions of neutrophil matrix metalloproteinases (Mmp)8 and Mmp9 were increased, but the production of fibrogenic factors, including transforming growth factor β1, was not affected by loss of Trib1. These results suggest that neutrophils are responsible for the suppression of fibrosis in Trib1‐deficient liver. Consistently, transplantation of Trib1‐deficient bone marrow cells into wild‐type mice alleviated CCl4‐induced fibrosis. Furthermore, expression of chemokine (C‐X‐C motif) ligand 1 (Cxcl1) by adeno‐associated viral vector in the normal liver recruited neutrophils and suppressed CCl4‐induced fibrosis; infusion of wild‐type neutrophils in CCl4‐treated mice also ameliorated fibrosis. Using recombinant adeno‐associated virus‐mediated expression of Mmp8 and Mmp9 alleviated liver fibrosis. Finally, neutrophil depletion by infusion of Ly6G antibody significantly enhanced CCl4‐induced fibrosis. Conclusion: While neutrophils are well known to exacerbate acute liver injury, our results demonstrate a beneficial role of neutrophils in chronic liver injury by promoting fibrolysis.

Based on its role as an energy storage compartment and endocrine organ, white adipose tissue (WAT) fulfills a critical function in the maintenance of whole-body energy homeostasis. Indeed, WAT dysfunction is connected to obesity-related type 2 diabetes triggered at least partly by an inflammatory response in adipocytes. The pseudokinase tribbles (TRB) 3 has been identified by us and others as a critical regulator of hepatic glucose homeostasis in type 2 diabetes and WAT lipid homeostasis. Therefore, this study aimed to test the hypothesis that the TRB gene family fulfills broader functions in the integration of metabolic and inflammatory pathways in various tissues.To determine the role of TRB family members for WAT function, we profiled the expression patterns of TRB13 under healthy and metabolic stress conditions. The differentially expressed TRB1 was functionally characterized in loss-of-function animal and primary adipocyte models.Here, we show that the expression of TRB1 was specifically upregulated during acute and chronic inflammation in WAT of mice. Deficiency of TRB1 was found to impair cytokine gene expression in white adipocytes and to protect against high-fat diet-induced obesity. In adipocytes, TRB1 served as a nuclear transcriptional coactivator for the nuclear factor kappaB subunit RelA, thereby promoting the induction of proinflammatory cytokines in these cells.As inflammation is typically seen in sepsis, insulin resistance, and obesity-related type 2 diabetes, the dual role of TRB1 as both a target and a (co) activator of inflammatory signaling might provide a molecular rationale for the amplification of proinflammatory responses in WAT in these subjects.

Event Abstract Back to Event Zinc-finger antiviral protein mediates RIG-I-like-receptor-independent antiviral response to murine leukemia virus Hanna Lee1, 2, Jun Komano3, Tatsuya Saitoh1, 2 and Shizuo Akira1, 2* 1 Osaka Unviresity, WPI Immunology frontier research center (IFReC), Japan 2 Osaka University, Research Institute for Microbial Disease, Japan 3 Osaka Prefectural Institue of Public Health, Department of Infection Disease, Japan Innate immunity is induced after the recognition of viral RNAs by pattern-recognition receptors (PRRs) and is the first line of the host defenses against a variety of RNA viruses (1, 2). Although Toll-like receptor 7 (TLR7) recognizes the genomic RNA of extracellular murine leukemia virus (MLV) to mediate the antiviral response (3-5), the RNA-sensing PRR that recognizes the MLV in the cytosol is not fully understood. Here, we show that zinc-finger antiviral protein (ZAP) is a cytosolic viral RNA sensor, inducing the degradation of the MLV transcripts by the exosome, an RNA degradation system, on RNA granules. Although loss of the RIG-I-like receptors (RLRs), which recognize various RNA viruses in the cytosol and induce the type I interferon (IFN)-dependent antiviral response, does not alter the replication efficiency of MLV, loss of ZAP greatly enhances the replication efficiency of MLV. ZAP localizes to RNA granules, where the processing-body and stress-granule proteins assemble. The CCCH-type zinc-finger domains of ZAP, which are RNA-binding motifs, mediate its localization to the RNA granules. ZAP recruits the MLV transcripts and an exosome component to the RNA granules leading to degradation of MLV transcripts. Although ZAP was known as a regulator of RLR signaling in human cells (6), ZAP does not regulate the RLR-dependent production of type I IFN in mouse cells. Thus, ZAP is a novel member of the cytosolic viral RNA-sensing PRR family that induces elimination of RNA viruses independently of TLR and RLR family members. Acknowledgements We thank Drs. H. Fan, D. Trono, H. Miyoshi, and T. Taniguchi for providing invaluable materials. This work was supported by a Japan Society for the Promotion of Science Grant-in-Aid for Challenging Exploratory Research (to T.S.); the Cabinet Office, Government of Japan, and the Japan Society for the Promotion of Science Funding Program for World-Leading Innovative R&D on Science and Technology ‘FIRST Program’ (to S.A.); and National Institutes of Health Grant PO1-AI070167 (to S.A.). References 1. Kawai T, Akira S (2009) The roles of TLRs, RLRs and NLRs in pathogen recognition. Int Immunol 21(4):317-337. 2. Iwasaki A (2012) A virological view of innate immune recognition. Annu Rev Microbiol 66:177-196. 3. Cella M, et al. (1999) Plasmacytoid monocytes migrate to inflamed lymph nodes and produce large amounts of type I interferon. Nat Med 5(8):919-923. 4. Diebold SS, et al. (2004) Innate antiviral responses by means of TLR7-mediated recognition of single-stranded RNA. Science 303(5663):1529-1531. 5. Heil F, et al. (2004) Species-specific recognition of single-stranded RNA via toll-like receptor 7 and 8. Science 303(5663):1526-1529. 6. Hayakawa S, et al. (2011) ZAPS is a potent stimulator of signaling mediated by the RNA helicase RIG-I during antiviral responses. Nat Immunol 12(1):37-44. Keywords: Antiviral protein, innate immunity, Retrovirus, RNA granule, CCCH-type zinc-finger-domain-containing protein Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Innate immunity Citation: Lee H, Komano J, Saitoh T and Akira S (2013). Zinc-finger antiviral protein mediates RIG-I-like-receptor-independent antiviral response to murine leukemia virus. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.00750 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 18 Jun 2013; Published Online: 22 Aug 2013. * Correspondence: Prof. Shizuo Akira, Osaka Unviresity, WPI Immunology frontier research center (IFReC), Osaka, Japan, sakira@biken.osaka-u.ac.jp Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Hanna Lee Jun Komano Tatsuya Saitoh Shizuo Akira Google Hanna Lee Jun Komano Tatsuya Saitoh Shizuo Akira Google Scholar Hanna Lee Jun Komano Tatsuya Saitoh Shizuo Akira PubMed Hanna Lee Jun Komano Tatsuya Saitoh Shizuo Akira Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

In myocardial ischemia/reperfusion injury, the innate immune and subsequent inflammatory responses play a crucial role in the extension of myocardial damage. Toll-like receptor 9 (TLR9) is a critical receptor for recognizing unmethylated CpG motifs that mitochondria contain in their DNA, and induces inflammatory responses. The aim of this study was to elucidate the role of TLR9 in myocardial ischemia/reperfusion injury. Isolated hearts from TLR9-deficient and control wild-type mice were subjected to 35 min of global ischemia, followed by 60 min of reperfusion with Langendorff apparatus. Furthermore, wild-type mouse hearts were infused with DNase I and subjected to ischemia/reperfusion. Ablation of TLR9-mediated signaling pathway attenuates myocardial ischemia/reperfusion injury and inflammatory responses, and digestion of extracellular mitochondrial DNA released from the infarct heart partially improved myocardial ischemia/reperfusion injury with no effect on inflammatory responses. TLR9 could be a therapeutic target to reduce myocardial ischemia/reperfusion injury.

Clinical and epidemiological studies have shown the contribution of viral infection to the development of allergic asthma. Many RNA viruses, pathogenic for the respiratory tract, generate double-stranded (ds)RNA during their replication. Typical innate immune responses triggered by dsRNA involve the endosomal and cytoplasmic pathways. The former is mediated by Toll/IL-1R domain-containing adaptor inducing IFN-β (TRIF), and the latter by IFN-β promoter stimulator 1 (IPS-1). We explored the effect of polyinocinic polycytidilic acid, a synthetic dsRNA, on the development of an asthma phenotype in mice. Administration of dsRNA during ovalbumin sensitization augmented airway eosinophilia and airway hyperresponsiveness after an antigen challenge, which was associated with enhanced induction of IL-13-producing CD8(+) T cells. The augmentation was induced in IPS-1-deficient mice but not in TRIF-deficient mice. The interactions between dendritic cells (DCs) and T cells are regulated by B7-family costimulatory molecules, including B7-H1 (also known as PD-L1), a putative ligand for programmed death-1 (PD-1). Treatment of bone marrow-derived DCs with dsRNA enhanced B7-H1 expression in a TRIF-dependent manner. Additionally, dsRNA increased B7-H1 expression on DCs in the draining lymph nodes of ovalbumin-sensitized mice. The augmentation of the asthma phenotype was prevented by the treatment of mice with anti-B7-H1 mAb but not with anti-PD-1 mAb. The augmentation was not induced in B7-H1-deficient mice. These results suggest that dsRNA-triggered activation of the innate immune system in sensitization leads to augmentation of the asthma phenotype via IL-13 mainly from CD8(+) T cells. B7-H1 plays a crucial role in the process without requiring interaction with PD-1.