Miwa Sasai

Absence of autophagy results in reactive oxygen species-dependent amplification of RLR signaling

Autophagy is a highly conserved process that maintains homeostasis by clearing damaged organelles and long-lived proteins. The consequences of deficiency in autophagy manifest in a variety of pathological states including neurodegenerative diseases, inflammatory disorders, and cancer. Here, we studied the role of autophagy in the homeostatic regulation of innate antiviral defense. Single-stranded RNA viruses are recognized by the members of the RIG-I-like receptors (RLRs) in the cytosol. RLRs signal through IPS-1, resulting in the production of the key antiviral cytokines, type I IFNs. Autophagy-defective Atg5−/− cells exhibited enhanced RLR signaling, increased IFN secretion, and resistance to infection by vesicular stomatitis virus. In the absence of autophagy, cells accumulated dysfunctional mitochondria, as well as mitochondria-associated IPS-1. Reactive oxygen species (ROS) associated with the dysfunctional mitochondria were largely responsible for the enhanced RLR signaling in Atg5−/− cells, as antioxidant treatment blocked the excess RLR signaling. In addition, autophagy-independent increase in mitochondrial ROS by treatment of cells with rotenone was sufficient to amplify RLR signaling in WT cells. These data indicate that autophagy contributes to homeostatic regulation of innate antiviral defense through the clearance of dysfunctional mitochondria, and revealed that ROS associated with mitochondria play a key role in potentiating RLR signaling.

Pathogen Recognition Receptors: Ligands and Signaling Pathways by Toll-Like Receptors

Toll-like receptors (TLRs) play critical roles in host defense against microbes. In the past decade, growing numbers of in vitro, in vivo and in silico studies have been performed and revealed the physiological significance and structural basis of their ligands and signal transduction, which involves various extracellular, membrane-bound, cytoplasmic and nuclear signaling molecules for the activation of TLR signaling. However, negative regulation of TLR-mediated responses is also essential for the prevention of autoimmunity and is mediated by a number of molecules. In this review, we will introduce recent advances in the understanding of TLR biology in terms of their ligands and signaling pathways.

Spatiotemporal Mobilization of Toll/IL-1 Receptor Domain-Containing Adaptor Molecule-1 in Response to dsRNA

TLR3 recognizes viral dsRNA and induces antiviral immune responses. TLR3-mediated cell activation relies on Toll/IL-1R (TIR) domain-containing adaptor molecule-1 (TICAM-1, also named TIR domain-containing adaptor inducing IFN-β or TRIF), which recruits downstream signaling molecules to activate the transcription factors IFN regulatory factor 3 (IRF-3) and NF-κB. The mechanisms by which TICAM-1 is activated and transmits signals remain largely unknown. In this study we show that TICAM-1 alters its distribution profile from a diffuse cytoplasmic form to a speckle-like structure in response to dsRNA. The receptor-interacting protein 1 (RIP1), a crucial signaling molecule for TICAM-1-mediated NF-κB activation, accumulated in the TICAM-1 speckles. In addition, NF-κB-activating kinase-associated protein 1 (NAP1), a downstream molecule linking TICAM-1 and the IRF-3-activating kinase TBK1 (TANK-binding kinase 1), was also recruited to the TICAM-1 speckles. Notably, a transient colocalization of TICAM-1 and TLR3 was observed before the extensive formation of the TICAM-1 speckles. Thus, the spatiotemporal mobilization of TICAM-1 in response to dsRNA and the formation of the TICAM-1 speckles containing RIP1 and NAP1 are important for the activation of the TLR3-TICAM-1 pathway.

Role of Mouse and Human Autophagy Proteins in IFN-γ–Induced Cell-Autonomous Responses against Toxoplasma gondii

IFN-γ mediates cellular innate immunity against an intracellular parasite, Toxoplasma gondii, by inducing immunity-related GTPases such as p47 IFN-γ–regulated GTPases (IRGs) and p65 guanylate-binding proteins (GBPs), which also participate in antibacterial responses via autophagy. An essential autophagy protein, Atg5, was previously shown to play a critical role in anti–T. gondii cell-autonomous immunity. However, the involvement of other autophagy proteins remains unknown. In this study, we show that essential autophagy proteins differentially participate in anti–T. gondii cellular immunity by recruiting IFN-γ–inducible GTPases. IFN-γ–induced suppression of T. gondii proliferation and recruitment of an IRG Irgb6 and GBPs are profoundly impaired in Atg7- or Atg16L1-deficient cells. In contrast, cells lacking other essential autophagy proteins, Atg9a and Atg14, are capable of mediating the anti–T. gondii response and recruiting Irgb6 and GBPs to the parasites. Although IFN-γ also stimulates anti–T. gondii cellular immunity in humans, whether this response requires GBPs and human autophagy proteins remains to be seen. To analyze the role of human ATG16L1 and GBPs in IFN-γ–mediated anti–T. gondii responses, human cells lacking ATG16L1 or GBPs were generated by the Cas9/CRISPR genome-editing technique. Although both ATG16L1 and GBPs are dispensable for IFN-γ–induced inhibition of T. gondii proliferation in the human cells, human ATG16L1 is also required for the recruitment of GBPs. Taken together, human ATG16L1 and mouse autophagy components Atg7 and Atg16L1, but not Atg9a and Atg14, participate in the IFN-γ–induced recruitment of the immunity-related GTPases to the intracellular pathogen.

Homo-oligomerization Is Essential for Toll/Interleukin-1 Receptor Domain-containing Adaptor Molecule-1-mediated NF-κB and Interferon Regulatory Factor-3 Activation

Toll-IL-1 receptor (TIR) domain-containing adaptor molecule-1 (TICAM-1, also named TIR domain-containing adaptor-inducing interferon (IFN)-β or TRIF)) is a signaling adaptor of Toll-like receptor (TLR) 3/4 that activates the transcription factors, interferon regulatory factor-3 (IRF-3) and NF-κB leading to inducing IFN-β production. The mechanisms by which TICAM-1 is activated by TLR3/4 to serve as a signaling platform are unknown. In this study, we show that homo-oligomerization of TICAM-1 is critical for TICAM-1-mediated activation of NF-κB and IRF-3. Both TIR and C-terminal domain of TICAM-1 mediated TICAM-1 oligomerization. Pro434 located in the TIR domain and the C-terminal region, with the exception of the RIP homotypic-interacting motif, were determinants of TICAM-1 oligomerization. Mutation of TIR domain (P434H) or deletion of C-terminal domain greatly reduced TICAM-1-mediated NF-κB and IFN-β promoter activation. TICAM-1 oligomerization at either the TIR domain or the C-terminal region resulted in recruitment of tumor necrosis factor receptor-associated factor 3, a downstream signaling molecule essential for TICAM-1-mediated IRF-3 activation, but not recruitment of the IRF-3 kinase complex, NF-κB-activating kinase-associated protein 1 and TANK-binding kinase 1. In addition, RIP homotypic-interacting motif mutant, which possesses two oligomerization motifs but not the RIP1 binding motif, also failed to recruit NF-κB-activating kinase-associated protein 1 and TANK-binding kinase 1. Thus, full activation and formation of TICAM-1 signalosomes requires oligomerization induced at two different sites and RIP1 binding.

Selective and strain-specific NFAT4 activation by the Toxoplasma gondii polymorphic dense granule protein GRA6

Ma et al. show that the Toxoplasma gondii polymorphic dense granule protein GRA6 triggers the activation of the host transcription factor NFAT4, thus affecting the host immune response and maximizing parasite virulence.

p62 Plays a Specific Role in Interferon-γ-Induced Presentation of a Toxoplasma Vacuolar Antigen

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.

Essential role for GABARAP autophagy proteins in interferon-inducible GTPase-mediated host defense

Mammalian autophagy-related 8 (Atg8) homologs consist of LC3 proteins and GABARAPs, all of which are known to be involved in canonical autophagy. In contrast, the roles of Atg8 homologs in noncanonical autophagic processes are not fully understood. Here we show a unique role of GABARAPs, in particular gamma-aminobutyric acid (GABA)-A-receptor-associated protein-like 2 (Gabarapl2; also known as Gate-16), in interferon-γ (IFN-γ)-mediated antimicrobial responses. Cells that lacked GABARAPs but not LC3 proteins and mice that lacked Gate-16 alone were defective in the IFN-γ-induced clearance of vacuolar pathogens such as Toxoplasma. Gate-16 but not LC3b specifically associated with the small GTPase ADP-ribosylation factor 1 (Arf1) to mediate uniform distribution of interferon-inducible GTPases. The lack of GABARAPs reduced Arf1 activation, which led to formation of interferon-inducible GTPase-containing aggregates and hampered recruitment of interferon-inducible GTPases to vacuolar pathogens. Thus, GABARAPs are uniquely required for antimicrobial host defense through cytosolic distribution of interferon-inducible GTPases.

Innate immune response induced by baculovirus attenuates transgene expression in mammalian cells

ABSTRACT The baculovirus Autographa californica nucleopolyhedrovirus (AcNPV) has been widely used to achieve a high level of foreign gene expression in insect cells, as well as for efficient gene transduction into mammalian cells without any replication. In addition to permitting efficient gene delivery, baculovirus has been shown to induce host innate immune responses in various mammalian cells and in mice. In this study, we examined the effects of the innate immune responses on gene expression by recombinant baculoviruses in cultured cells. The reporter gene expression in IRF3-deficient mouse embryonic fibroblasts (MEFs) infected with the recombinant baculovirus was shown to be enhanced in accordance with the suppression of beta interferon (IFN-β) production. Furthermore, efficient gene transduction by the recombinant baculovirus was achieved in MEFs deficient for stimulator of interferon genes (STING), TANK binding kinase 1 (TBK1), IFN regulatory factor 3 (IRF3), or IFN-β promoter stimulator 1 (IPS-1), but not in those deficient for IRF7, MyD88, or Z-DNA binding protein 1 (ZBP1)/DAI. Enhancement of gene expression by the recombinant baculovirus was also observed in human hepatoma cell lines replicating hepatitis C virus (HCV), in which innate immunity was impaired by the cleavage of IPS-1 by the viral protease. In addition, infection with the recombinant baculovirus expressing the BH3-only protein, BIMS, a potent inducer of apoptosis, resulted in a selective cell death in the HCV replicon cells. These results indicate that innate immune responses induced by infection with baculovirus attenuate transgene expression, and this characteristic might be useful for a selective gene transduction into cells with impaired innate immunity arising from infection with various viruses.

RabGDIα is a negative regulator of interferon-γ–inducible GTPase-dependent cell-autonomous immunity to Toxoplasma gondii

Significance IFN-γ is a proinflammatory cytokine and stimulates induction of ∼2,000 genes, including IFN-γ–inducible GTPases, such as immunity-related GTPases (IRGs) and guanylate-binding proteins (GBPs), that are critically required for cell-autonomous host defense against the vacuolar pathogen Toxoplasma gondii. Mechanisms of how recruitment of these GTPases to the vacuoles is positively regulated have been gradually elucidated. However, the negative regulation remains unknown. Here, we show that Rab GDP dissociation inhibitor α (RabGDIα) acts as a suppressor of IFN-γ–inducible GTPases, such as Gbp2 and Irga6. RabGDIα deficiency resulted in enhanced IFN-γ–mediated T. gondii clearance in vitro and in vivo. Furthermore, RabGDIα inhibited the act of Gbp2 and Irga6 through the lipid-binding pocket. Thus, our current study demonstrates a negative regulatory mechanism for IFN-γ–inducible GTPase-dependent cell-autonomous immunity. 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.