[Show abstract][Hide abstract] ABSTRACT: Toxoplasma gondii possesses sets of dense granule proteins (GRAs) that either assemble at, or cross the parasitophorous vacuole membrane (PVM) and exhibit motifs resembling the HT/PEXEL previously identified in a repertoire of exported Plasmodium proteins. Within Plasmodium spp., cleavage of the HT/PEXEL motif by the endoplasmic reticulum-resident protease Plasmepsin V precedes trafficking to and export across the PVM of proteins involved in pathogenicity and host cell remodelling. Here, we have functionally characterized the T. gondii aspartyl protease 5 (ASP5), a Golgi-resident protease that is phylogenetically related to Plasmepsin V. We show that deletion of ASP5 causes a significant loss in parasite fitness in vitro and an altered virulence in vivo. Furthermore, we reveal that ASP5 is necessary for the cleavage of GRA16, GRA19 and GRA20 at the PEXEL-like motif. In the absence of ASP5, the intravacuolar nanotubular network disappears and several GRAs fail to localize to the PVM, while GRA16 and GRA24, both known to be targeted to the host cell nucleus, are retained within the vacuolar space. Additionally, hypermigration of dendritic cells and bradyzoite cyst wall formation are impaired, critically impacting on parasite dissemination and persistence. Overall, the absence of ASP5 dramatically compromises the parasite's ability to modulate host signalling pathways and immune responses.
[Show abstract][Hide abstract] ABSTRACT: Cancer cachexia, which is characterized by decreased food intake, weight loss and systemic inflammation, increases patient's morbidity and mortality. We previously showed that rikkunshito (RKT), a Japanese traditional herbal medicine (Kampo), ameliorated the symptoms of cancer cachexia through ghrelin signaling-dependent and independent pathways. To investigate other mechanisms of RKT action in cancer cachexia, we performed metabolome analysis of plasma in a rat model bearing the Yoshida AH-130 hepatoma. A total of 110 metabolites were detected in plasma and RKT treatment significantly altered levels of 23 of those metabolites in cachexia model rats. Among them, glucarate, which is known to have anticarcinogenic activity through detoxification of carcinogens via inhibition of β-glucuronidase, was increased in plasma following administration of RKT. In our AH-130 ascites-induced cachexia rat model, administration of glucarate delayed onset of weight loss, improved muscle atrophy, and reduced ascites content. Additionally, glucarate reduced levels of plasma interferon-γ (IFN-γ) in tumor-bearing rats and was also found to suppress LPS-induced IFN-γ expression in splenocytes in vitro. These results suggest that glucarate has anti-inflammatory activity via a direct effect on immune host cells and suggest that RKT may also ameliorate inflammation partly through the elevation of glucarate in plasma.
Evidence-based Complementary and Alternative Medicine 10/2015; 2015(5):871832. DOI:10.1155/2015/871832 · 1.88 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Also known as Sqstm1, p62 is a selective autophagy adaptor with a ubiquitin-binding domain. However, the role of p62 in the host defense against Toxoplasma gondii infection is unclear. Here, we show that interferon γ (IFN-γ) stimulates ubiquitin and p62 recruitment to T. gondii parasitophorous vacuoles (PVs). Some essential autophagy-related proteins, but not all, are required for this recruitment. Regardless of normal IFN-γ-induced T. gondii clearance activity and ubiquitination, p62 deficiency in antigen-presenting cells (APCs) and mice diminishes the robust IFN-γ-primed activation of CD8(+) T cells that recognize the T. gondii-derived antigen secreted into PVs. Because the expression of Atg3 and Irgm1/m3 in APCs is essential for PV disruption, ubiquitin and p62 recruitment, and vacuolar-antigen-specific CD8(+) T cell activation, IFN-γ-mediated ubiquitination and the subsequent recruitment of p62 to T. gondii are specifically required for the acquired immune response after PV disruption by IFN-γ-inducible GTPases.
[Show abstract][Hide abstract] ABSTRACT: The data is related to the research article entitled “Arf6 mediates Schwann cell differentiation and myelination” . To further investigate the role of Arf6 in promoting myelination by Schwann cells in vivo, we have characterized an another line (#2) of small-hairpin (sh)RNA transgenic mice targeting Arf6. The number of transgenes per one allele in this line was very low (2 transgenes), comparing with high copies in the previous line (#1, 20 transgenes) . In 4 days of neonatal age, transgenic mice exhibited decreased myelin thickness; however, decreased levels were not as much as those in the line #1, likely depending on transgene copy number. In 60-day-old mice, the difference became smaller. On the other hand, transgene׳s effect was not related to cell proliferation and apoptosis. These data support the key role of Arf6 in Schwann cell myelination, especially in the initiation.
[Show abstract][Hide abstract] ABSTRACT: A pharmaceutical grade Japanese traditional medicine, daikenchuto (TU-100), consisting of Japanese pepper, processed ginger, and ginseng, has been widely used for various intestinal disorders in Japan and now under development as a new therapeutic drug in the US. It is suggested that TU-100 ingredients exert pharmacological effects on intestines via two routes, from the luminal side before absorption and the peripheral blood stream after absorption. Therefore, in order to fully understand the pharmacological actions of TU-100, it is critically important to know the intraluminal amounts and forms of ingested TU-100 ingredients. In the present study, after administrating TU-100 to rats, the concentrations of TU-100 ingredients and their conjugates in the peripheral and portal blood and ileal contents were determined by LC-MS/MS. Next, TU-100 was administered to patients with ileostomy bags, but whose small intestines are diagnosed as healthy, and the ingredients/conjugates in the ileal effluent were analyzed. The results suggest that: (1) Pepper ingredients hydroxysanshools are rapidly absorbed and enter systemic circulation, (2) Ginseng ingredients ginsenosides are transported to the colon with the least absorption, (3) Ginger ingredients gingerols are absorbed and some conjugated in the small intestine and transported via the portal vein. While only a small amount of gingerols/gingerol conjugates enter systemic circulation, considerable amounts reappear in the small intestine. Thus, the effect of TU-100 on the intestines is believed to be a composite of multiple actions by multiple compounds supplied via multiple routes.
[Show abstract][Hide abstract] ABSTRACT: Interferon (IFN)-inducible Guanylate Binding Proteins (GBPs) mediate cell-autonomous host resistance to bacterial pathogens and promote inflammasome activation. The prevailing model postulates that these two GBP-controlled activities are directly linked through GBP-dependent vacuolar lysis. It was proposed that rupture of pathogen-containing vacuoles (PVs) by GBPs destroyed the microbial refuge and simultaneously contaminated the host cell cytosol with microbial activators of inflammasomes. Here, we demonstrate that GBP-mediated host resistance and GBP-mediated inflammatory responses can be uncoupled. We show that PVs formed by the rodent pathogen Chlamydia muridarum, so-called inclusions, remain free of GBPs and that C. muridarum is impervious to GBP-mediated restrictions on bacterial growth. Although GBPs neither bind to C. muridarum inclusions nor restrict C. muridarum growth, we find that GBPs promote inflammasome activation in C. muridarum-infected macrophages. We demonstrate that C. muridarum infections induce GBP-dependent pyroptosis through both caspase-11-dependent noncanonical and caspase-1-dependent canonical inflammasomes. Amongst canonical inflammasomes we find that C. muridarum and the human pathogen Chlamydia trachomatis not only activate NLRP3, as previously reported, but also AIM2. Our data show that GBPs support fast-kinetics processing and secretion of IL-1β and IL-18 by the NLRP3 inflammasome but are dispensable for the secretion of the same cytokines at later times post-infection. Because IFNγ fails to induce IL-1β transcription, GBP-dependent fast-kinetics inflammasome activation can drive the preferential processing of constitutively expressed IL-18 in IFNγ-primed macrophages in the absence of prior TLR stimulation. Together, our results reveal that GBPs control the kinetics of inflammasome activation and thereby shape macrophage responses to Chlamydia infections.
Infection and Immunity 09/2015; DOI:10.1128/IAI.00856-15 · 3.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Herbal medicines have been used in Japan for more than 1500 years and traditional Japanese medicines (Kampo medicines) are now fully integrated into the modern healthcare system. In total, 148 Kampo formulae are officially approved as prescription drugs and covered by the national health insurance system in Japan. However, despite their long track record of clinical use, the multi-targeted, multi-component properties of Kampo medicines, which are fundamentally different from Western medicines, have made it difficult to create a suitable framework for conducting well-designed, large-scale clinical trials. In turn, this has led to misconceptions among western trained physicians concerning the paucity of scientific evidence for the beneficial effects of Kampo medicines. Fortunately, there has been a recent surge in scientifically robust data from basic and clinical studies for some of the Kampo medicines, e.g., daikenchuto (TU-100). Numerous basic and clinical studies on TU-100, including placebo-controlled double-blind studies for various gastrointestinal disorders, and absorption, distribution, metabolism and excretion (ADME) studies, have been conducted or are in the process of being conducted in both Japan and the USA. Clinical studies suggest that TU-100 is beneficial for postoperative complications, especially ileus and abdominal bloating. ADME and basic studies indicate that the effect of TU-100 is a composite of numerous actions mediated by multiple compounds supplied via multiple routes. In addition to known mechanisms of action via enteric/sensory nerve stimulation, novel mechanisms via the TRPA1 channel and two pore domain potassium channels have recently been elucidated. TU-100 compounds target these channels with and without absorption, both before and after metabolic activation by enteric flora, with different timings and possibly with synergism.
Frontiers in Pharmacology 08/2015; 6:159. DOI:10.3389/fphar.2015.00159 · 3.80 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: IFN-γ orchestrates cell-autonomous host defense against various intracellular vacuolar pathogens. IFN-γ-inducible GTPases, such as p47 immunity-related GTPases (IRGs) and p65 guanylate-binding proteins (GBPs), are recruited to pathogen-containing vacuoles, which is important for disruption of the vacuoles, culminating in the cell-autonomous clearance. Although the positive regulation for the proper recruitment of IRGs and GBPs to the vacuoles has been elucidated, the suppressive mechanism is unclear. Here, we show that Rab GDP dissociation inhibitor α (RabGDIα), originally identified as a Rab small GTPase inhibitor, is a negative regulator of IFN-γ-inducible GTPases in cell-autonomous immunity to the intracellular pathogen Toxoplasma gondii. Overexpression of RabGDIα, but not of RabGDIβ, impaired IFN-γ-dependent reduction of T. gondii numbers. Conversely, RabGDIα deletion in macrophages and fibroblasts enhanced the IFN-γ-induced clearance of T. gondii. Furthermore, upon a high dose of infection by T. gondii, RabGDIα-deficient mice exhibited a decreased parasite burden in the brain and increased resistance in the chronic phase than did control mice. Among members of IRGs and GBPs important for the parasite clearance, Irga6 and Gbp2 alone were more frequently recruited to T. gondii-forming parasitophorous vacuoles in RabGDIα-deficient cells. Notably, Gbp2 positively controlled Irga6 recruitment that was inhibited by direct and specific interactions of RabGDIα with Gbp2 through the lipid-binding pocket. Taken together, our results suggest that RabGDIα inhibits host defense against T. gondii by negatively regulating the Gbp2-Irga6 axis of IFN-γ-dependent cell-autonomous immunity.
Proceedings of the National Academy of Sciences 08/2015; 112(33). DOI:10.1073/pnas.1510031112 · 9.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Hepatocellular carcinoma develops in either chronically injured or seemingly intact livers. To explore the tumorigenic mechanisms underlying these different conditions, we compared the mRNA expression profiles of mouse hepatocellular tumors induced by the repeated injection of CCl4 or a single diethylnitrosamine (DEN) injection using a cDNA microarray. We identified tumor-associated genes that were expressed differentially in the cirrhotic CCl4 model (H19, Igf2, Cbr3, and Krt20) and the noncirrhotic DEN model (Tff3, Akr1c18, Gpc3, Afp, and Abcd2) as well as genes that were expressed comparably in both models (Ly6d, Slpi, Spink3, Scd2, and Cpe). The levels and patterns of mRNA expression of these genes were validated by RT-qPCR analyses. Most of these genes were highly expressed in mouse livers during the fetal/neonatal periods. We also examined the mRNA expression of these genes in mouse tumors induced by thioacetamide, another cirrhotic inducer, and those that developed spontaneously in noncirrhotic livers and found that they shared a similar expression profile as that observed in CCl4 -induced and DEN-induced tumors, respectively. There was a close relationship between the expression levels of Igf2 and H19 mRNA, which were activated in the cirrhotic models. Our results show that mouse liver tumors reactivate fetal/neonatal genes, some of which are specific to cirrhotic or noncirrhotic modes of pathogenesis. This article is protected by copyright. All rights reserved.
This article is protected by copyright. All rights reserved.
Cancer Science 05/2015; 106(8). DOI:10.1111/cas.12700 · 3.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Inflammasomes are critical for mounting host defense against pathogens. The molecular mechanisms that control activation of the AIM2 inflammasome in response to different cytosolic pathogens remain unclear. Here we found that the transcription factor IRF1 was required for activation of the AIM2 inflammasome during infection with the Francisella tularensis subspecies novicida (F. novicida), whereas engagement of the AIM2 inflammasome by mouse cytomegalovirus (MCMV) or transfected double-stranded DNA did not require IRF1. Infection of F. novicida detected by the DNA sensor cGAS and its adaptor STING induced type I interferon-dependent expression of IRF1, which drove the expression of guanylate-binding proteins (GBPs); this led to intracellular killing of bacteria and DNA release. Our results reveal a specific requirement for IRF1 and GBPs in the liberation of DNA for sensing by AIM2 depending on the pathogen encountered by the cell.
[Show abstract][Hide abstract] ABSTRACT: The AIM2 inflammasome detects double-stranded DNA in the cytosol and induces caspase-1-dependent pyroptosis as well as release of the inflammatory cytokines interleukin 1β (IL-1β) and IL-18. AIM2 is critical for host defense against DNA viruses and bacteria that replicate in the cytosol, such as Francisella tularensis subspecies novicida (F. novicida). The activation of AIM2 by F. novicida requires bacteriolysis, yet whether this process is accidental or is a host-driven immunological mechanism has remained unclear. By screening nearly 500 interferon-stimulated genes (ISGs) through the use of small interfering RNA (siRNA), we identified guanylate-binding proteins GBP2 and GBP5 as key activators of AIM2 during infection with F. novicida. We confirmed their prominent role in vitro and in a mouse model of tularemia. Mechanistically, these two GBPs targeted cytosolic F. novicida and promoted bacteriolysis. Thus, in addition to their role in host defense against vacuolar pathogens, GBPs also facilitate the presentation of ligands by directly attacking cytosolic bacteria.