Publications

Activated macrophages kill bacteria, a function known to depend on the expression of NF-IL-6. Here, it is demonstrated that the attenuated Brucella abortus vaccine strain 19 replicates much better in NF-IL-6-/- than in NF-IL-6(+/+) and NF-IL-6(+/+)-activated murine macrophages and at levels comparable to those observed in normal macrophages infected with the pathogenic strain 2308. The role of NF-IL-6 in the inhibition of intracellular bacterial replication is related to its control of endocytosis and membrane fusion between endosomes and Brucella-containing phagosomes. Addition of the granulocyte-CSF (G-CSF), whose induction is impaired in NF-IL-6(-/-) macrophages, restores both endocytosis and the morphology of endosomes, together with bactericidal activity. Regulation of membrane traffic in endocytosis by G-CSF whose expression is controlled by NF-IL-6 may explain how a host cell can control intracellular bacterial replication.

Summary Whooping cough, a contagious respiratory disease caused by Bordetella pertussis , is characterized by paroxysmal coughing; however, the mechanism has not been studied because of the lack of versatile animal models that reproduce the cough. Here, we present a mouse model that reproduces coughing after intranasal inoculation with the bacteria or its components and demonstrate that lipooligosaccharide (LOS), pertussis toxin (PTx), and Vag8 of the bacteria cooperatively function to cause coughing. LOS-induced bradykinin sensitized a transient receptor potential ion channel, TRPV1, which acts as a sensor to evoke the cough reflex. Vag8 further increased bradykinin levels by inhibiting the C1 esterase inhibitor, the major downregulator of the contact system, which generates bradykinin. PTx inhibits intrinsic negative regulation systems for TRPV1 through inactivation of G i GTPases. Our findings provide a basis for answering long-standing questions on the pathophysiology of the pertussis cough.

When host cells are infected by an RNA virus, pattern-recognition receptors (PRRs) recognize the viral RNA and induce the antiviral innate immunity. Toll-like receptor 7 (TLR7) detects the genomic RNA of incoming murine leukemia virus (MLV) in endosomes and mediates the antiviral response. However, the RNA-sensing PRR that recognizes the MLV in the cytosol is not fully understood. Here, we definitively demonstrate that zinc-finger antiviral protein (ZAP) acts as a cytosolic RNA sensor, inducing the degradation of the MLV transcripts by the exosome, an RNA degradation system, on RNA granules. Although the retinoic acid inducible gene I (RIG-I)–like receptors (RLRs) RIG-I and melanoma differentiation-associated protein 5 detect various RNA viruses in the cytosol and induce the type I IFN-dependent antiviral response, RLR loss does not alter the replication efficiency of MLV. In sharp contrast, the loss of ZAP greatly enhances the replication efficiency of MLV. ZAP localizes to RNA granules, where the processing-body and stress-granule proteins assemble. ZAP induces the recruitment of the MLV transcripts and exosome components to the RNA granules. The CCCH-type zinc-finger domains of ZAP, which are RNA-binding motifs, mediate its localization to RNA granules and MLV transcripts degradation by the exosome. Although ZAP was known as a regulator of RIG-I signaling in a human cell line, ZAP deficiency does not affect the RIG-I–dependent production of type I IFN in mouse cells. Thus, ZAP is a unique member of the cytosolic RNA-sensing PRR family that targets and eliminates intracellular RNA viruses independently of TLR and RLR family members.

Although both microsomal prostaglandin E synthase (mPGES)-1 and cyclooxygenase (COX)-2 are critical factors in stroke injury, but the interactions between these enzymes in the ischaemic brain is still obscure. This study examines the hypothesis that mPGES-1 activity is required for COX-2 to cause neuronal damage in ischaemic injury.We used a glutamate-induced excitotoxicity model in cultures of rat or mouse hippocampal slices and a mouse middle cerebral artery occlusion-reperfusion model in vivo. The effect of a COX-2 inhibitor on neuronal damage in mPGES-1 knockout (KO) mice was compared with that in wild-type (WT) mice.In rat hippocampal slices, glutamate-induced excitotoxicity, as well as prostaglandin (PG) E(2) production and PGES activation, was significantly attenuated by either MK-886 or NS-398, inhibitors of mPGES-1 and COX-2 respectively; however, co-application of these inhibitors had neither an additive nor a synergistic effect. The protective effect of NS-398 on the excitotoxicity observed in WT slices was completely abolished in mPGES-1 KO slices, which showed less excitotoxicity than WT slices. In the transient focal ischaemia model, mPGES-1 and COX-2 were co-localized in the infarct region of the cortex. Injection of NS-398 reduced not only ischaemic PGE(2) production, but also ischaemic injuries in WT mice, but not in mPGES-1 KO mice, which showed less dysfunction than WT mice.Microsomal prostaglandin E synthase-1 and COX-2 are co-induced by excess glutamate in ischaemic brain. These enzymes are co-localized and act together to exacerbate stroke injury, by excessive PGE(2) production.

NK cells play essential roles in eliminating virally infected cells and tumor cells. Polyinosinic-polycytidylic acid (poly I:C), a double-stranded RNA analog recognized by melanoma-differentiation associated gene 5 (MDA5) and TLR3, activates NK cells in vivo. MDA5 and TLR3 signal through distinct adaptor molecules, IFN-promoter stimulator-1 (IPS-1) and Toll/IL-1R domain-containing adaptor inducing IFN-β (TRIF), respectively. However, it remains unclear how NK cells are activated by poly I:C in vivo. In this study, we demonstrate that the IPS-1-dependent and the TRIF-dependent pathways are essential for NK cell activation to poly I:C stimulation in mice, whereas deficiency in either IPS-1 or TRIF only modestly impairs the poly I:C-induced NK cell activation. Furthermore, both IPS-1 and TRIF contributed to suppression of implanted B16 tumor growth in response to poly I:C administration via NK cell activation. Presence of IPS-1 and TRIF in dendritic cells (DCs), but not NK cells, was required for production of IFN-γ to poly I:C in NK cells in vitro. Moreover CD8α+ conventional dendritic cells (cDCs), but not CD8α− cDCs, expressed genes for type I IFNs, IL-6, and IL-12p40 in response to poly I:C stimulation, and were also responsible for inducing IFN-γ production in NK cells. Taken together, poly I:C activates the IPS-1- and TRIF-dependent pathways in CD8α+ cDCs, which in turn leads to NK cell activation.

No abstract is provided for this article.

No abstract is provided for this article.

The Mycobacterium bovis bacillus Calmette-Guérin cell-wall skeleton (BCG-CWS) activates Toll-like receptor (TLR) 2 and TLR4, but unlike the typical TLR4 agonist bacterial lipopolysaccharide barely induces type 1 IFN. BCG-CWS has been used for adjuvant immunotherapy for patients with cancer. We investigated the adjuvant potential of BCG-CWS for induction of CTLs subsequent to TLR-mediated dendritic cell (DC) maturation, using a syngeneic mouse tumor model (B16 melanoma in C57BL/6). We evaluated the retardation of tumor growth and cytotoxic response in wild-type and MyD88−/− mice immunized with tumor debris and/or BCG-CWS. Delays in tumor growth and cytotoxic response were induced by immunization with a mixture of BCG-CWS emulsion and the tumor. BCG-CWS was capable of activating DCs ex vivo by the criteria of CD80/CD86 up-regulation and cytokine (interleukin-12, tumor necrosis factor-α) induction. Efficient tumor suppression and ex vivo cytokine induction did not occur in MyD88-deficient mice and cells, suggesting that the MyD88 adapter is crucial for induction of tumor cytotoxicity. Because TLR4 is involved in both MyD88-dependent and -independent pathways and the latter affects DC maturation, our findings indicate that both pathways cooperate to induce CTL-based tumor immunity.

Optimal vaccine efficacy requires not only a protective antigen, but also a strong immune activator as an adjuvant. Most viral vaccines, such as influenza vaccines and nonviral genetic vaccines (e.g., DNA vaccines), contain nucleic acids, which appear to act as essential 'built-in' adjuvants. Specific receptors, including Toll-like receptors, retinoic acid-inducible protein-I-like receptors, and nucleotide-binding oligomerization domain-like receptors can detect specific nucleic acid patterns, depending on the immunized tissue, cell type and intracellular localization. The resulting immune activation is uniquely regulated by intra- and intercellular signaling pathways, which are indispensable for the ensuing vaccine immunogenicity, such as antigen-specific T- and B-cell responses. Thus, elucidation and manipulation of immune signaling and interactions by nucleic acid adjuvants are essential for maximizing the immunogenicity and safety of viral and DNA vaccines.

Permanent exposure to pathogens requires decisions toward tolerance or immunity as a prime task of dendritic cells. The molecular mechanisms preventing uncontrolled immune responses are not completely clear. We investigated the regulatory function of Ncf1, an organizing protein of NADPH oxidase, in the signaling cascade of Toll-like receptors. TLR9-stimulated spleen cells from both Ncf1-deficient and B10.Q mice with a point mutation in exon 8 of Ncf1 exhibited increased IL-12p70 secretion compared with controls. This finding was restricted to stimulatory CpG2216 and not induced by CpG2088. Because only CpG/TLR9-induced IL-12p70 was regulated by Ncf1, we used TRIF(-/-) and MyD88(-/-) cells to show that TLR9/MyD88 was primarily affected. Interestingly, additional experiments revealed that spleen cells from NOX2/gp91(phox)-deficient mice and the blocking of electron transfer by diphenylene iodonium had no influence on CpG-induced IL-12p70, confirming an NADPH oxidase-independent function of Ncf1. Finally, proving the in vivo relevance CpG adjuvant-guided OVA immunization resulted in a strong augmentation of IL-12p70-dependent Th1 IFN-gamma response only in Ncf1-deficient mice. These data suggest for the first time an important role for Ncf1 in the fine tuning of the TLR9/MyD88 pathway in vitro and in vivo that is independent of its role as an activator of NOX2.

Double-stranded ribonucleic acid (dsRNA) serves as a danger signal associated with viral infection and leads to stimulation of innate immune cells. In contrast, the immunostimulatory potential of single-stranded RNA (ssRNA) is poorly understood and innate immune receptors for ssRNA are unknown. We report that guanosine (G)- and uridine (U)-rich ssRNA oligonucleotides derived from human immunodeficiency virus–1 (HIV-1) stimulate dendritic cells (DC) and macrophages to secrete interferon-α and proinflammatory, as well as regulatory, cytokines. By using Toll-like receptor (TLR)–deficient mice and genetic complementation, we show that murine TLR7 and human TLR8 mediate species-specific recognition of GU-rich ssRNA. These data suggest that ssRNA represents a physiological ligand for TLR7 and TLR8.

OK-432 is a Streptococcus-derived immunotherapeutic agent for malignancies. Our group has tried to identify the effective components of OK-432 and has succeeded in isolating a lipoteichoic acid–related preparation designated as OK-PSA, which is a strong inducer of T helper 1 (TH1) cells, and elicits an anticancer effect via Toll-like receptor (TLR) 4. Conversely, bacterial DNA with unmethylated CpG motifs can stimulate a TH1-type host response via TLR9. The unmethylated CpG DNA contained in OK-432 may play a role in its anticancer effect. In the current study, we investigated the effect of OK-432–derived DNA (OK-DNA) in augmenting the anticancer immune response. Analysis of OK-DNA with the restriction enzymes Hpa II and MspI revealed that OK-DNA contained unmethylated CpG motifs. OK-DNA induced TH1-type cytokines such as interferon-γ, tumor necrosis factor-α, interleukin (IL)-12, and IL-18 and augmented killer cell activities in vitro on human peripheral blood mononuclear cells, whereas the methylated OK-DNA did not. Cytokines were also produced by OK-DNA–stimulated splenocytes derived from wild-type mice but not from TLR9-deficient mice. In the in vivo study, peritumoral administration of OK-DNA resulted in a significant inhibition of tumor growth in syngeneic tumor-bearing wild-type and TLR4-deficient mice but not in TLR9-deficient mice. The antitumor effect of OK-432 in TLR9-deficient mice was significantly but partially reduced compared with that in wild-type mice, whereas the effect of OK-432 was almost completely eliminated in TLR4-deficient mice. These findings suggest that unmethylated CpG DNA in OK-432 functions as an active component in OK-432–induced anticancer immunity via TLR9.