Publications

Toll-like receptors (TLRs) play an important role in the control of infection with Borrelia burgdorferi . Deficiencies in TLR-2 or the shared TLR adapter molecule MyD88 have been shown to result in greatly increased bacterial burdens in mice. However, although in vitro studies have shown that the activation of TLR pathways by B. burgdorferi results in the release of inflammatory cytokines, studies in deficient mice have shown either no change or increased rather than decreased inflammation in infected animals. In this study, we looked at mechanisms to explain the increase in inflammation in the absence of MyD88. We found that MyD88-deficient mice infected with B. burgdorferi did not show increased inflammation at sites typically associated with Lyme disease (joints and heart). However, there was markedly increased inflammation in the muscles, kidneys, pancreas, and lungs of deficient animals. Muscle inflammation was typically seen perivascularly and perineuronally similar to that seen in infected humans. Chemotactic chemokines and cytokines were greatly increased in the muscle and kidneys of MyD88-deficient animals but not in the joints or heart tissue, suggesting that MyD88-independent pathways for recognizing B. burgdorferi and inducing these chemokines are present in the muscle and kidneys. Interleukin-18 signaling through MyD88 does not appear to play a role in either control of infection or inflammation.

Sphingobacterium spiritivorum has five unusual sphingophospholipids (SPLs). Our previous study determined the complete chemical structures of these SPLs. The compositions of the long-chain bases/fatty acids in the ceramide portion, isoheptadecasphingosine/isopentadecanoate or isoheptadecasphingosine/2-hydroxy isopentadecanoate, are characteristic. The immune response against bacterial lipid components is considered to play important roles in microbial infections. It is reported that several bacterial sphingolipids composed of ceramide are recognized by CD1-restricted T and NKT cells and that a non-peptide antigen is recognized by γδ T cells. In this study, we demonstrated that these bacterial SPLs activated murine bone marrow macrophages (BMMs) via Toll-like receptor (TLR) 4 but not TLR2, although they slightly activated CD1d-restricted NKT and γδT cells. Interestingly, this TLR 4-recognition pathway of bacterial SPLs involves the fatty acid composition of ceramide in addition to the sugar moiety. A non-hydroxy fatty acid composed of ceramide was necessary to activate murine BMMs. The bacterial survival was significantly higher in TLR4-KO mice than in TLR2-KO and wild-type mice. The results indicate that activation of the TLR4-dependent pathway of BMMs by SPLs induced an innate immune response and contributed to bacterial clearance.

No abstract is provided for this article.

Rationale: To identify the mechanisms by which a novel antedrug Toll like receptor 7 (TLR7) agonist, AZ12441970, mediates the long-lasting suppression of Th2 immune responses. Methods: Long-lasting efficacy of AZ12441970 against bronchoalveolar lavage (BAL) eosinophilia was investigated utilizing Ovalbumin (OVA)/Alum-sensitised mice or OVA-specific Th2 cells adoptively transferred into mice. The mechanism by which AZ12441970 mediated long-lasting efficacy was investigated utilizing T cell-deficient nude mice and several knock-out (KO) mice including type I IFN receptor KO mice, B cell KO mice and rag2 KO mice lacking B and T cells. Results: Weekly lung doses of AZ12441970 for 6 weeks significantly inhibited BAL eosinophilia for at least 6 weeks following cessation of dosing. The long-lasting efficacy observed with the compound was largely abolished in type I IFN receptor KO mice, rag2 KO mice or nude mice, but maintained in B cell KO mice. T cells were shown to be involved in the mechanism of efficacy in a T cell reconstituted nude mouse model of allergic airways inflammation. Nose only exposure of AZ12441970 also resulted in long-term efficacy in the lung. Conclusions: We show that intranasal dosing of AZ12441970 to achieve lung exposure in mice induces a long-lasting efficacy against Th2 immune responses in the lung. The efficacy in the lung is also achieved by nose only exposure of the TLR7 agonist. Furthermore, our mechanism studies demonstrate that the long-lasting inhibition of the Th2 immune responses with the compound is IFN-α- and T-cell-dependent. These data suggest that novel antedrug TLR7 agonists may have potential in the treatment of allergic disease.

Pyelonephritis, in which renal tubular epithelial cells are directly exposed to bacterial component, is a major predisposing cause of renal insufficiency. Although previous studies have suggested C-C chemokines are involved in the pathogenesis, the exact source and mechanisms of the chemokine secretion remain ambiguous. In this study, we evaluated the involvement of Toll-like receptors (TLRs) in C-C chemokine production by mouse primary renal tubular epithelial cells (MTECs). MTECs constitutively expressed mRNA for TLR1, 2, 3, 4, and 6, but not for TLR5 or 9. MTECs also expressed MD-2, CD14, myeloid differentiation factor 88, and Toll receptor-IL-1R domain-containing adapter protein/myeloid differentiation factor 88-adapter-like. Synthetic lipid A and lipoprotein induced monocyte chemoattractant protein 1 (MCP-1) and RANTES production in MTECs, which strictly depend on TLR4 and TLR2, respectively. In contrast, MTECs were refractory to CpG-oligodeoxynucleotide in chemokine production, consistently with the absence of TLR9. LPS-mediated MCP-1 and RANTES production in MTECs was abolished by NF-κB inhibition, but unaffected by extracellular signal-regulated kinase inhibition. In LPS-stimulated MTECs, inhibition of c-Jun N-terminal kinase and p38 mitogen-activated protein kinase significantly decreased RANTES, but did not affect MCP-1 mRNA induction. Thus, MTECs have a distinct expression pattern of TLR and secrete C-C chemokines in response to direct stimulation with a set of bacterial components.

Hepatitis C virus (HCV) utilizes cellular factors for efficient propagation. Ubiquitin is covalently conjugated to the substrate to alter its stability or to modulate signal transduction. In this study, we examined the importance of ubiquitination for HCV propagation. We found that inhibition of deubiquitinating enzymes (DUBs) or overexpression of nonspecific DUBs impaired HCV replication, suggesting that ubiquitination regulates HCV replication. To identify specific DUBs involved in HCV propagation, we set up RNA interference (RNAi) screening against DUBs and successfully identified ubiquitin-specific protease 15 (USP15) as a novel host factor for HCV propagation. Our studies showed that USP15 is involved in translation of HCV RNA and production of infectious HCV particles. In addition, deficiency of USP15 in human hepatic cell lines (Huh7 and Hep3B/miR-122 cells) but not in a nonhepatic cell line (293T cells) impaired HCV propagation, suggesting that USP15 participates in HCV propagation through the regulation of hepatocyte-specific functions. Moreover, we showed that loss of USP15 had no effect on innate immune responses in vitro and in vivo We also found that USP15-deficient Huh7 cells showed reductions in the amounts of lipid droplets (LDs), and the addition of palmitic acids restored the production of infectious HCV particles. Taken together, these data suggest that USP15 participates in HCV propagation by regulating the translation of HCV RNA and the formation of LDs.IMPORTANCE Although ubiquitination has been shown to play important roles in the HCV life cycle, the roles of deubiquitinating enzymes (DUBs), which cleave ubiquitin chains from their substrates, in HCV propagation have not been investigated. Here, we identified USP15 as a DUB regulating HCV propagation. USP15 showed no interaction with viral proteins and no participation in innate immune responses. Deficiency of USP15 in Huh7 cells resulted in suppression of the translation of HCV RNA and reduction in the amounts of lipid droplets, and the addition of fatty acids partially restored the production of infectious HCV particles. These data suggest that USP15 participates in HCV propagation in hepatic cells through the regulation of viral RNA translation and lipid metabolism.

Toll-like receptor 4 (TLR4) of macrophages recognizes LPS of Gram-negative bacteria in cooperation with CD14, which is also involved in the recognition of apoptotic cells. In this study we asked whether TLR4 plays a role in the phagocytic clearance of apoptotic cells by macrophages. Macrophages were prepared from peritoneal fluid of thioglycolate-treated mice carrying either a wild-type or a disrupted TLR4-encoding gene and were examined for their ability to phagocytose apoptotic mouse thymocytes, apoptotic Jurkat T cells, Ig-opsonized mouse thymocytes, Ig-opsonized zymosan particles, and latex beads. Both populations of macrophages equally expressed CD14 on their surfaces and showed almost equal activities of binding to and engulfing all these targets. However, apoptotic thymocytes, apoptotic Jurkat cells, and opsonized thymocytes disappeared more rapidly in TLR4-deficient macrophages than in wild-type macrophages, and the fusion between endosomes/lysosomes and phagosomes containing any target cells or particles was accelerated in mutant macrophages. Activation of the transcription factor NF-κB appeared not to occur in wild-type macrophages after engulfment, and the rate of apoptotic cell degradation in wild-type macrophages remained the same regardless of the activation of NF-κB. Finally, immunohistochemical analyses showed that ectopically expressed TLR4 was associated with phagosomes in a macrophage-derived cell line. All these results collectively indicate that TLR4 negatively regulates the degradation of engulfed cells in macrophages via a pathway independent of NF-κB.

A single miRNA exhibits both an inhibitory effect on the antiviral response and an antiviral effect on viral replication.

To gain a clue to whether the transcription factor CCAAT/enhancer-binding protein β (C/EBPβ) is implicated in mouse behavior, the water maze performance of C/EBPβ(-/-) mice was compared with the performance of their wild type (WT) controls. The escape latencies of C/EBPβ(-/-) mice increased along with the number of trials within all training sessions in the hidden-platform tasks. Even in the visible-platform tasks, their escape latencies did not decrease along with the number of trials. The unusual performance in C/EBPβ(-/-) mice during later trials may allow us to presume that they suffer certain emotional vulnerability to stress from the water-maze training, although the involvement of other factors such as motor or immune system should be also considered.

No abstract is provided for this article.

No abstract is provided for this article.

To gain a clue to whether the transcription factor CCAAT/enhancer-binding protein β (C/EBPβ) is implicated in mouse behavior, the water maze performance of C/EBPβ(-/-) mice was compared with the performance of their wild type (WT) controls. The escape latencies of C/EBPβ(-/-) mice increased along with the number of trials within all training sessions in the hidden-platform tasks. Even in the visible-platform tasks, their escape latencies did not decrease along with the number of trials. The unusual performance in C/EBPβ(-/-) mice during later trials may allow us to presume that they suffer certain emotional vulnerability to stress from the water-maze training, although the involvement of other factors such as motor or immune system should be also considered.

The netrin family of proteins are involved in axon guidance during central nervous system development. In vertebrates, two membrane bound forms and five secreted forms of netrin have been reported. In addition to their critical role in neural morphogenesis, a growing number of reports suggest that netrin family proteins also play a role in inflammatory conditions, angiogenesis, and tumorigenesis. In these processes, Unc5 and DCC family proteins serve as receptors of netrin proteins. Recently, it was reported that some netrin family proteins may be involved in the pathogenesis of skeletal diseases including osteoporosis and arthritis. For example, administration of secreted netrin family proteins such as netrin 1 and netrin 4 has prophylactic potential in pathogenic bone degradation in mice. However, netrin 1 blocking antibody also protects mice from inflammatory bone destruction. Therefore, netrin family proteins are involved in the regulation of bone homeostasis, but their bona fide roles in the skeletal system remain controversial. In this review, we discuss the osteo-innate-immune functions of the netrin family of proteins, and summarize their therapeutic potential.

Herpes simplex virus type 1 ocular infection elicits a potent inflammatory response including the production of the chemokines, CXCL9 and CXCL10, in mice. Since HSV-1 nucleic acid is recognized by pattern receptors including Toll-like receptor (TLR) 9, we tested the hypothesis that TLR9 is necessary for the early augmentation of CXCL10 following HSV-1 infection. Similar to wild type controls, TLR9 deficient mice constitutively expressed CXCL10 in the cornea. Following infection or stimulation with the deoxycytidylate-phosphate-deoxyguanylate (CpG) motif, CXCL10 levels were significantly elevated in the cornea of wild type but not TLR9 or type I interferon receptor deficient mice. The reduced CXCL10 response in the cornea of TLR deficient mice was correlative with an increase in virus shedding and a reduction in neutrophil infiltration. This is the first report that shows enhanced CXCL10 expression following neurotropic viral replication requires both intact TLR 9 and type I interferon signaling pathways.

<div>Abstract<p>The immunoreceptor NKG2D originally identified in natural killer (NK) cells recognizes ligands that are upregulated on tumor cells. Expression of NKG2D ligands (NKG2DL) is induced by the DNA damage response (DDR), which is often activated constitutively in cancer cells, revealing them to NK cells as a mechanism of immunosurveillance. Here, we report that the induction of retinoic acid early transcript 1 (RAE1) ligands for NKG2D by the DDR relies on a STING-dependent DNA sensor pathway involving the effector molecules TBK1 and IRF3. Cytosolic DNA was detected in lymphoma cell lines that express RAE1 and its occurrence required activation of the DDR. Transfection of DNA into ligand-negative cells was sufficient to induce RAE1 expression. <i>Irf3<sup>+/−</sup></i>;Eμ-<i>Myc</i> mice expressed lower levels of RAE1 on tumor cells and showed a reduced survival rate compared with <i>Irf3<sup>+/+</sup></i>;Eμ-<i>Myc</i> mice. Taken together, our results suggest that genomic damage in tumor cells leads to activation of STING-dependent DNA sensor pathways, thereby activating RAE1 and enabling tumor immunosurveillance. <i>Cancer Res; 74(8); 2193–203. ©2014 AACR</i>.</p></div>