Nam-Hyuk Cho

A multifunctional core–shell nanoparticle for dendritic cell-based cancer immunotherapy

Dendritic cell-based cancer immunotherapy requires tumour antigens to be delivered efficiently into dendritic cells and their migration to be monitored in vivo. Nanoparticles have been explored as carriers for antigen delivery, but applications have been limited by the toxicity of the solvents used to make nanoparticles, and by the need to use transfection agents to deliver nanoparticles into cells. Here we show that an iron oxide-zinc oxide core-shell nanoparticle can deliver carcinoembryonic antigen into dendritic cells while simultaneously acting as an imaging agent. The nanoparticle-antigen complex is efficiently taken up by dendritic cells within one hour and can be detected in vitro by confocal microscopy and in vivo by magnetic resonance imaging. Mice immunized with dendritic cells containing the nanoparticle-antigen complex showed enhanced tumour antigen specific T-cell responses, delayed tumour growth and better survival than controls.

The Orientia tsutsugamushi genome reveals massive proliferation of conjugative type IV secretion system and host–cell interaction genes

Scrub typhus is caused by the obligate intracellular rickettsia Orientia tsutsugamushi (previously called Rickettsia tsutsugamushi). The bacterium is maternally inherited in trombicuid mites and transmitted to humans by feeding larvae. We report here the 2,127,051-bp genome of the Boryong strain, which represents the most highly repeated bacterial genome sequenced to date. The repeat density of the scrub typhus pathogen is 200-fold higher than that of its close relative Rickettsia prowazekii, the agent of epidemic typhus. A total of 359 tra genes for components of conjugative type IV secretion systems were identified at 79 sites in the genome. Associated with these are >200 genes for signaling and host–cell interaction proteins, such as histidine kinases, ankyrin-repeat proteins, and tetratrico peptide-repeat proteins. Additionally, the O. tsutsugamushi genome contains >400 transposases, 60 phage integrases, and 70 reverse transcriptases. Deletions and rearrangements have yielded unique gene combinations as well as frequent pseudogenization in the tra clusters. A comparative analysis of the tra clusters within the genome and across strains indicates sequence homogenization by gene conversion, whereas complexity, diversity, and pseudogenization are acquired by duplications, deletions, and transposon integrations into the amplified segments. The results suggest intragenomic duplications or multiple integrations of a massively proliferating conjugative transfer system. Diversifying selection on host–cell interaction genes along with repeated population bottlenecks may drive rare genome variants to fixation, thereby short-circuiting selection for low complexity in bacterial genomes.

Expression of chemokine genes in murine macrophages infected with Orientia tsutsugamushi.

ABSTRACT Scrub typhus, caused by Orientia tsutsugamushiinfection, is characterized by local as well as systemic inflammatory manifestations. Inflammation is initiated by O. tsutsugamushi-infected macrophages and endothelial cells in the dermis. We investigated the regulation of chemokine induction in macrophage cell line J774A.1 in response to O. tsutsugamushi infection. The mRNAs for macrophage inflammatory proteins 1α/β (MIP-1α/β), MIP-2, and macrophage chemoattractant protein 1 were induced within 30 min, and their levels showed a transitory peak for 3 to 12 h. However, the lymphotactin, eotaxin, gamma interferon-inducible protein 10, and T-cell activation gene 3 mRNAs were not detected by RNase protection assays. Heat-killedO. tsutsugamushi induced a similar extent of chemokine responses. Induction of the chemokine genes was not blocked by the eukaryotic protein synthesis inhibitor cycloheximide, suggesting that de novo synthesis of host cell protein is not required for these transcriptional responses. The induction of chemokine mRNAs by O. tsutsugamushi was blocked by the inhibitors of NF-κB activation. Furthermore, O. tsutsugamushi induced the nuclear translocation and activation of NF-κB. These results demonstrate that heat-stable molecules of O. tsutsugamushiinduce a subset of chemokine genes and that induction involves activation of the transcription factor NF-κB.

Characterization of the Kaposi's Sarcoma-Associated Herpesvirus K1 Signalosome

ABSTRACT Kaposis sarcoma (KS) is a multifocal angiogenic tumor and appears to be a hyperplastic disorder caused, in part, by local production of inflammatory cytokines. The K1 lymphocyte receptor-like protein of KS-associated herpesvirus (KSHV) efficiently transduces extracellular signals to elicit cellular activation events through its cytoplasmic immunoreceptor tyrosine-based activation motif (ITAM). To further delineate K1-mediated signal transduction, we purified K1 signaling complexes and identified its cellular components. Upon stimulation, the K1 ITAM was efficiently tyrosine phosphorylated and subsequently interacted with cellular Src homology 2 (SH2)-containing signaling proteins Lyn, Syk, p85, PLCγ2, RasGAP, Vav, SH2 domain-containing protein tyrosine phosphatase 1/2, and Grab2 through its phosphorylated tyrosine residues. Mutational analysis demonstrated that each tyrosine residue of K1 ITAM contributed to the interactions with cellular signaling proteins in distinctive ways. Consequently, these interactions led to the marked augmentation of cellular signal transduction activity, evidenced by the increase of cellular tyrosine phosphorylation and intracellular calcium mobilization, the activation of NF-AT and AP-1 transcription factor activities, and the production of inflammatory cytokines. These results demonstrate that KSHV K1 effectively recruits a set of cellular SH2-containing signaling molecules to form the K1 signalosome, which elicits downstream signal transduction and induces inflammatory cytokine production.

Kaposi's Sarcoma-Associated Herpesvirus K7 Protein Targets a Ubiquitin-Like/Ubiquitin-Associated Domain-Containing Protein To Promote Protein Degradation

ABSTRACT Pathogens exploit host machinery to establish an environment that favors their propagation. Because of their pivotal roles in cellular physiology, protein degradation pathways are common targets for viral proteins. Protein-linking integrin-associated protein and cytoskeleton 1 (PLIC1), also called ubiquilin, contains an amino-terminal ubiquitin-like (UBL) domain and a carboxy-terminal ubiquitin-associated (UBA) domain. PLIC1 is proposed to function as a regulator of the ubiquitination complex and proteasome machinery. Kaposis sarcoma-associated herpesvirus (KSHV) contains a small membrane protein, K7, that protects cells from apoptosis induced by various stimuli. We report here that cellular PLIC1 is a K7-interacting protein and that the central hydrophobic region of K7 and the carboxy-terminal UBA domain of PLIC1 are responsible for their interaction. Cellular PLIC1 formed a dimer and bound efficiently to polyubiquitinated proteins through its carboxy-terminal UBA domain, and this activity correlated with its ability to stabilize cellular IκB protein. In contrast, K7 interaction prevented PLIC1 from forming a dimer and binding to polyubiquitinated proteins, leading to the rapid degradation of IκB. Furthermore, K7 expression promoted efficient degradation of the p53 tumor suppressor, resulting in inhibition of p53-mediated apoptosis. These results indicate that KSHV K7 targets a regulator of the ubiquitin- and proteasome-mediated degradation machinery to deregulate cellular protein turnover, which potentially provides a favorable environment for viral reproduction.

Fibronectin Facilitates the Invasion of Orientia tsutsugamushi into Host Cells through Interaction with a 56-kDa Type-Specific Antigen

BACKGROUND Orientia tsutsugamushi, the causative agent of scrub typhus, is an obligate intracellular bacterium. The pathogens mechanism of cellular invasion is poorly characterized. METHODS Through ligand immunoblots, glutathione S-transferase (GST) pull-down assays, and in vitro inhibition assays of intracellular invasion, a bacterial ligand was identified and was shown to interact with fibronectin (Fn) to enhance the intracellular invasion of O. tsutsugamushi. RESULTS O. tsutsugamushi can bind to immobilized Fn in vitro, and exogenous Fn stimulates bacterial invasion of mammalian host cells. Bacterial invasion in the presence of Fn was abrogated by the addition of Arg-Gly-Asp peptides or by an anti-alpha5beta1 integrin antibody. Through a ligand immunoblot and GST pull-down assay, a 56-kDa type-specific antigen (TSA56) was identified as the bacterial ligand responsible for the interaction with Fn. Antigenic domain III and the adjacent C-terminal region (aa 243-349) of TSA56 interacted with Fn. Furthermore, we found that the enhanced invasion of the pathogen was abrogated by the addition of purified recombinant peptides derived from TSA56. CONCLUSION Fn facilitates the invasion of O. tsutsugamushi through its interaction with TSA56.

Expression of Chemokine Genes in Human Dermal Microvascular Endothelial Cell Lines Infected with Orientia tsutsugamushi

ABSTRACT Scrub typhus, caused by Orientia tsutsugamushi, is characterized by local as well as systemic inflammatory manifestations. The main pathologic change is focal or disseminated multiorgan vasculitis, which is caused by the destruction of endothelial cells and perivascular infiltration of leukocytes. We investigated the regulation of chemokine induction in transformed human dermal microvascular endothelial cells (HMEC-1) in response to O. tsutsugamushiinfection. The monocyte chemoattractant protein-1 (MCP-1) and interleukin 8 (IL-8) mRNAs were induced, and their levels showed a transitory peak at 3 and 6 h, respectively. The RANTES transcript was detected at 6 h after infection, with increased levels evident by 48 h. The induction of the MCP-1 and IL-8 genes was not blocked by cycloheximide, suggesting that de novo protein synthesis of host cell proteins is not required for their transcriptional activation. Heat- or UV-inactivated O. tsutsugamushi induced a similar extent of MCP-1 and IL-8 responses. The induction of MCP-1 and IL-8 transcripts in the endothelial cells by O. tsutsugamushiwas not blocked by the inhibitors of NF-κB. Furthermore, the activation of NF-κB was not detected in HMEC-1 stimulated withO. tsutsugamushi. These results demonstrate that heat-stable molecules of O. tsutsugamushi induce the MCP-1 and IL-8 genes and the induction of the chemokine genes may be mediated by an NF-κB independent mechanism. We also showed that another major transcription factor, activator protein-1 (AP-1), was up-regulated in HMEC-1 after O. tsutsugamushi infection. This suggests the possible involvement of AP-1 in the chemokine gene expression.

Selective Gαi Subunits as Novel Direct Activators of Transient Receptor Potential Canonical (TRPC)4 and TRPC5 Channels

Background: Activation of TRPC4/5 channels is mediated by GPCR activation. Results: TRPC4/5 was activated by the Gαi/o-coupled receptor and the Gαi protein, which interacted directly with each other. Conclusion: Gαi proteins play an essential role as novel activators of TRPC4/5. Significance: Our findings provide new insights into the activation mechanism of inhibitory Gα proteins. The ubiquitous transient receptor potential canonical (TRPC) channels function as non-selective, Ca2+-permeable channels and mediate numerous cellular functions. It is commonly assumed that TRPC channels are activated by stimulation of Gαq-PLC-coupled receptors. However, whether the Gαq-PLC pathway is the main regulator of TRPC4/5 channels and how other Gα proteins may regulate these channels are poorly understood. We previously reported that TRPC4/TRPC5 can be activated by Gαi. In the current work, we found that Gαi subunits, rather than Gαq, are the primary and direct activators of TRPC4 and TRPC5. We report a novel molecular mechanism in which TRPC4 is activated by several Gαi subunits, most prominently by Gαi2, and TRPC5 is activated primarily by Gαi3. Activation of Gαi by the muscarinic M2 receptors or expression of the constitutively active Gαi mutants equally and fully activates the channels. Moreover, both TRPC4 and TRPC5 are activated by direct interaction of their conserved C-terminal SESTD (SEC14-like and spectrin-type domains) with the Gαi subunits. Two amino acids (lysine 715 and arginine 716) of the TRPC4 C terminus were identified by structural modeling as mediating the interaction with Gαi2. These findings indicate an essential role of Gαi proteins as novel activators for TRPC4/5 and reveal the molecular mechanism by which G-proteins activate the channels.

Deregulation of DNA Damage Signal Transduction by Herpesvirus Latency-Associated M2

ABSTRACT Infected cells recognize viral replication as a DNA damage stress and elicit a DNA damage response that ultimately induces apoptosis as part of host immune surveillance. Here, we demonstrate a novel mechanism where the murine gamma herpesvirus 68 (γHV68) latency-associated, anti-interferon M2 protein inhibits DNA damage-induced apoptosis by interacting with the DDB1/COP9/cullin repair complex and the ATM DNA damage signal transducer. M2 expression constitutively induced DDB1 nuclear localization and ATM kinase activation in the absence of DNA damage. Activated ATM subsequently induced Chk activation and p53 phosphorylation and stabilization without eliciting H2AX phosphorylation and MRN recruitment to foci upon DNA damage. Consequently, M2 expression inhibited DNA repair, rendered cells resistant to DNA damage-induced apoptosis, and induced a G1 cell cycle arrest. Our results suggest that γHV68 M2 blocks apoptosis-mediated intracellular innate immunity, which might ultimately contribute to its role in latent infection.

Induction of the Gene Encoding Macrophage Chemoattractant Protein 1 by Orientia tsutsugamushi in Human Endothelial Cells Involves Activation of Transcription Factor Activator Protein 1

ABSTRACT Human macrophage chemoattractant protein 1 (MCP-1) is a potent mediator of macrophage migration and therefore plays an essential role in early events of inflammation. In endothelial cells, at least three independent pathways regulate MCP-1 expression by NF-κB and AP-1. Orientia tsutsugamushi causes vasculitis in humans by replicating inside macrophages and endothelial cells. In the present study, we investigated the cis-acting and trans-acting elements involved in O. tsutsugamushi-induced MCP-1 gene expression in human umbilical vein endothelial cells (HUVEC). Although NF-κB activation was observed in HUVEC infected with O. tsutsugamushi, inhibition of NF-κB activation did not affect the MCP-1 expression. However, treatment of HUVEC with extracellular signal-regulated kinase (ERK) kinase inhibitor or p38 mitogen-activated protein kinase (MAPK) inhibitor suppressed expression of MCP-1 mRNA concomitant with downregulation of activator protein 1 (AP-1) activation. Deletion of triphorbol acetate response elements (TRE) at position −69 to −63 of MCP-1 gene abolished inducible promoter activity. Deletion of TRE at position −69 to −63−96 to −90 or deletion of NF-κB-binding site at position −69 to −63−88 to −79 did not affect the inducibility of promoter. Site-directed mutagenesis of the NF-κB binding sites at positions −2640 to −2632, −2612 to −2603 in the enhancer region, or the AP-1 biding site at position −2276 to −2270 decreased the inducible activity of the promoter. Taken together, AP-1 activation by both the ERK pathway and the p38 MAPK pathway as well as their binding to TRE at position −69 to −63 in proximal promoter and TRE at position −2276 to −2270 in enhancer region is altogether essential in induction of MCP-1 mRNA in HUVEC infected with O. tsutsugamushi. Although NF-κB activation is not essential per se, the κB site in the enhancer region is important in MCP-1 induction of HUVEC. This discrepancy in the involvement of the NF-κB may be due to the function of chromatin structures and other transcription cofactors in the regulation of MCP-1 gene expression in response to O. tsutsugamushi infectioin.