Mitochondrial reactive zones in antiviral innate immunity.

Mitochondria are multi-functioning organelles that participate in a wide range of biologic processes from energy metabolism to cellular suicide. Mitochondria are also involved in the cellular innate immune response against microorganisms or environmental irritants, particularly in mammals. Mitochondrial-mediated innate immunity is achieved by the activation of two discrete signaling pathways, the NLR family pyrin domain-containing 3 inflammasomes and the retinoic acid-inducible gene I-like receptor pathway. In both pathways, a mitochondrial outer membrane adaptor protein, called mitochondrial antiviral signaling MAVS, and mitochondria-derived components play a key role in signal transduction. In this review, we discuss current insights regarding the fundamental phenomena of mitochondrial-related innate immune responses, and review the specific roles of various mitochondrial subcompartments in fine-tuning innate immune signaling events. We propose that specific targeting of mitochondrial functions is a potential therapeutic approach for the management of infectious diseases and autoinflammatory disorders with an excessive immune response.

The microRNAs miR-302b and miR-372 regulate mitochondrial metabolism via the SLC25A12 transporter, which controls MAVS-mediated antiviral innate immunity.

MicroRNAs (miRNAs) are small noncoding RNAs that suppress the expression of multiple genes and are involved in numerous biologic functions and disorders, including human diseases. Here, we report that two miRNAs, miR-302b and miR-372, target mitochondrial-mediated antiviral innate immunity by regulating mitochondrial dynamics and metabolic demand. Using human cell lines transfected with the synthetic analog of viral dsRNA, poly(I-C), or challenged with Sendai virus, we found that both miRNAs are up-regulated in the cells late after viral infection and ultimately terminate the production of type I interferons and inflammatory cytokines. We found that miR-302b and miR-372 are involved in dynamin-related protein 1 (DRP1)-dependent mitochondrial fragmentation and disrupt mitochondrial metabolism by attenuating solute carrier family 25 member 12 (SLC25A12), a member of the SLC25 family. Neutralizing the effects of the two miRNAs through specific inhibitors re-established the mitochondrial dynamics and the antiviral responses. We found that SLC25A12 contributes to regulating the antiviral response by inducing mitochondrial-related metabolite changes in the organelle. Structure-function analysis indicated that SLC25A12, as part of a prohibitin complex, associates with the mitochondrial antiviral-signaling protein in mitochondria, providing structural insight into the regulation of the mitochondrial-mediated antiviral response. Our results contribute to the understanding of how miRNAs modulate the innate immune response by altering mitochondrial dynamics and metabolic demand. Manipulating the activities of miR-302b and miR-372 may be a potential therapeutic approach to target RNA viruses.

Immunogenic cell death in cancer therapy: Present and emerging inducers.

In the tumour microenvironment (TME), immunogenic cell death (ICD) plays a major role in stimulating the dysfunctional antitumour immune system. Chronic exposure of damage-associated molecular patterns (DAMPs) attracts receptors and ligands on dendritic cells (DCs) and activates immature DCs to transition to a mature phenotype, which promotes the processing of phagocytic cargo in DCs and accelerates the engulfment of antigenic components by DCs. Consequently, via antigen presentation, DCs stimulate specific T cell responses that kill more cancer cells. The induction of ICD eventually results in long-lasting protective antitumour immunity. Through the exploration of ICD inducers, recent studies have shown that there are many novel modalities with the ability to induce immunogenic cancer cell death. In this review, we mainly discussed and summarized the emerging methods for inducing immunogenic cancer cell death. Concepts and molecular mechanisms relevant to antitumour effects of ICD are also briefly discussed.

BinCARD2 as a positive regulator of interferon response in innate immunity.

Innate immunity is a system that recognizes primarily and excludes pathogenic microorganism. MAVS/IPS-1/Cardif/Visa functions as an adapter protein for RIG-I like receptors (RLRs) and plays a key role in the production of antiviral proteins, interferons (IFNs), for RNA viruses. However, the activation mechanism is not fully understood. Here, we show that BinCARD isoform2 (BinCARD2), carrying CARD domain structure like MAVS, functions in innate immune response. Knockdown of BinCARD2 reduced the RLR ligand-induced expression of IFN-ß mRNA and activation of the IFNB promoter. The activation of the IFNB promoter by overexpression of MAVS or TBK1 was suppressed by silencing of BinCARD2, but no effect on IFNB promoter activation by overexpression of TRIF or constitutive activated IRF-3. Furthermore, we confirmed that BinCARD2 protein associated with MAVS but not TBK1 by immunoprecipitation and colocalized with MAVS. Accordingly, we investigated whether BinCARD2 was involved in MAVS activation and showed that siBinCARD2 did not affect RIG-I/MAVS binding but impaired the MAVS oligomerization. Moreover, we infected A549 cells with vesicular stomatitis virus (VSV) and found that induction of IFN-ß and IL-6 mRNA after VSV infection was decreased by BinCARD2 knockdown. Thus, these data may suggest that BinCARD2 associates with MAVS to positively modulate the oligomerization in the RIG-I like receptors pathway and activates innate immune response.

Preventive effect of apigenin against isoproterenol-induced apoptosis in cardiomyoblasts.

We investigated the effect of apigenin, a dietary flavonoid, on isoproterenol hydrochloride (ISO)-induced apoptotic signaling in cardiomyoblast H9C2 cells. The results showed that apigenin treatment (10 µM) prevented ISO (31.25 µM)-induced lipid peroxidative levels and antioxidants status in H9C2 cells. Furthermore, apigenin inhibited expression of inflammatory markers in ISO-treated cells. In addition, apigenin prevented ISO-induced DNA damage and apoptotic signaling through modulating the expression of Bax, caspase-3, -8 and -9, cytochrome c, and Fas proteins in H9C2 cells. It is concluded that apigenin prevents ISO-induced antioxidants depletion, oxidative DNA damage, inflammatory, and apoptotic signaling in H9C2 cells. Thus, the present results demonstrated that apigenin has a cardioprotective effect on cardiomyoblasts cells.

Mitochondria maintain controlled activation state of epithelial-resident T lymphocytes.

Epithelial-resident T lymphocytes, such as intraepithelial lymphocytes (IELs) located at the intestinal barrier, can offer swift protection against invading pathogens. Lymphocyte activation is strictly regulated because of its potential harmful nature and metabolic cost, and most lymphocytes are maintained in a quiescent state. However, IELs are kept in a heightened state of activation resembling effector T cells but without cytokine production or clonal proliferation. We show that this controlled activation state correlates with alterations in the IEL mitochondrial membrane, especially the cardiolipin composition. Upon inflammation, the cardiolipin composition is altered to support IEL proliferation and effector function. Furthermore, we show that cardiolipin makeup can particularly restrict swift IEL proliferation and effector functions, reducing microbial containment capability. These findings uncover an alternative mechanism to control cellular activity, special to epithelial-resident T cells, and a novel role for mitochondria, maintaining cells in a metabolically poised state while enabling rapid progression to full functionality.

The role of the mitochondria and the endoplasmic reticulum contact sites in the development of the immune responses.

Mitochondria and endoplasmic reticulum (ER) contact sites (MERCs) are dynamic modules enriched in subset of lipids and specialized proteins that determine their structure and functions. The MERCs regulate lipid transfer, autophagosome formation, mitochondrial fission, Ca2+ homeostasis and apoptosis. Since these functions are essential for cell biology, it is therefore not surprising that MERCs also play a critical role in organ physiology among which the immune system stands by its critical host defense function. This defense system must discriminate and tolerate host cells and beneficial commensal microorganisms while eliminating pathogenic ones in order to preserve normal homeostasis. To meet this goal, the immune system has two lines of defense. First, the fast acting but unspecific innate immune system relies on anatomical physical barriers and subsets of hematopoietically derived cells expressing germline-encoded receptors called pattern recognition receptors (PRR) recognizing conserved motifs on the pathogens. Second, the slower but very specific adaptive immune response is added to complement innate immunity. Adaptive immunity relies on another set of specialized cells, the lymphocytes, harboring receptors requiring somatic recombination to be expressed. Both innate and adaptive immune cells must be activated to phagocytose and process pathogens, migrate, proliferate, release soluble factors and destroy infected cells. Some of these functions are strongly dependent on lipid transfer, autophagosome formation, mitochondrial fission, and Ca2+ flux; this indicates that MERCs could regulate immunity.

Mitochondria-associated membranes (MAMs): An emerging platform connecting energy and immune sensing to metabolic flexibility.

Living organisms have the capacity to sense both nutrients and immune signals in order to adapt their metabolism to the needs, and both metabolic inflexibility and exacerbated immune responses are associated with metabolic diseases. Over the past decade, mitochondria emerged as key nutrient and immune sensors regulating numerous signalling pathways, and mitochondria dysfunction has been extensively implicated in metabolic diseases. Interestingly, mitochondria interact physically and functionally with the endoplasmic reticulum (ER, in contact sites named mitochondria-associated membranes (MAMs), in order to exchange metabolites and calcium and regulate cellular homeostasis. Emerging evidences suggest that MAMs provide a platform for hormone and nutrient signalling pathways and for innate immune responses, then regulating mitochondrial bioenergetics and apoptosis. Here, I thus propose the concept that MAMs could be attractive nutrient and immune sensors that regulate mitochondria physiology in order to adapt metabolism and cell fate, and that organelle miscommunication could be involved in the metabolic inflexibility and the pro-inflammatory status associated with metabolic diseases.

Mitochondria-Endoplasmic Reticulum Contact Sites Mediate Innate Immune Responses.

Mitochondria and the endoplasmic reticulum (ER) are fundamental organelles that coordinate high-order cell functions. Mitochondria are centers of energy production, whereas the ER is responsible for folding, transport, and degradation of proteins. In addition to their specific functions, mitochondria and ER actively communicate with each other to promote a variety of cellular events, such as material transfer and signal transduction. Recent studies have shown the critical involvement of these organelles in regulation of the innate immune system, which functions in host defense. The innate immune system utilizes a wide range of germ-line-encoded pattern recognition receptors (PRRs) to recognize pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) and induces inflammatory and antiviral responses. Contact sites between mitochondria and the ER function in assembly of the NLR family pyrin domain containing 3 (NLRP3)-inflammasome to promote the inflammatory response. The NLRP3-inflammasome is a protein complex composed of the receptor NLRP3 on the ER side and the adaptor apoptosis-associated speck-like protein containing a CARD on the mitochondrial side; it induces caspase-1-dependent maturation of proinflammatory cytokines such as interleukin (IL)-1ß and IL-18. Furthermore, ER-mitochondria contact sites function in initiation and mediation of signal transduction pathways downstream of intracellular PRRs, such as retinoic acid-inducible gene I-like receptor and cyclic GMP-AMP synthase, to promote the antiviral response. Therefore, ER-mitochondria contact sites, also known as mitochondria-associated membranes, play key roles in regulation of innate immune responses.

Mitochondrial permeabilization engages NF-κB-dependent anti-tumour activity under caspase deficiency.

Apoptosis represents a key anti-cancer therapeutic effector mechanism. During apoptosis, mitochondrial outer membrane permeabilization (MOMP) typically kills cells even in the absence of caspase activity. Caspase activity can also have a variety of unwanted consequences that include DNA damage. We therefore investigated whether MOMP-induced caspase-independent cell death (CICD) might be a better way to kill cancer cells. We find that cells undergoing CICD display potent pro-inflammatory effects relative to apoptosis. Underlying this, MOMP was found to stimulate NF-κB activity through the downregulation of inhibitor of apoptosis proteins. Strikingly, engagement of CICD displays potent anti-tumorigenic effects, often promoting complete tumour regression in a manner dependent on intact immunity. Our data demonstrate that by activating NF-κB, MOMP can exert additional signalling functions besides triggering cell death. Moreover, they support a rationale for engaging caspase-independent cell death in cell-killing anti-cancer therapies.

Mitochondria are the powerhouses of immunity.

Recent evidence indicates that mitochondria lie at the heart of immunity. Mitochondrial DNA acts as a danger-associated molecular pattern (DAMP), and the mitochondrial outer membrane is a platform for signaling molecules such as MAVS in RIG-I signaling, and for the NLRP3 inflammasome. Mitochondrial biogenesis, fusion and fission have roles in aspects of immune-cell activation. Most important, Krebs cycle intermediates such as succinate, fumarate and citrate engage in processes related to immunity and inflammation, in both innate and adaptive immune cells. These discoveries are revealing mitochondrial targets that could potentially be exploited for therapeutic gain in inflammation and cancer.

Group A Streptococcus Modulates Host Inflammation by Manipulating Polymorphonuclear Leukocyte Cell Death Responses.

Polymorphonuclear leukocyte (PMN) cell death strongly influences the resolution of inflammatory episodes, and may exacerbate adverse pathologies in response to infection. We investigated PMN cell death mechanisms following infection by virulent group A Streptococcus (GAS). Human PMNs were infected in vitro with a clinical, virulent GAS isolate and an avirulent derivative strain, and compared for phagocytosis, the production of reactive oxygen species (ROS), mitochondrial membrane depolarization and apoptotic markers. C57BL/6J mice were then infected, in order to observe the effects on murine PMNs in vivo. Human PMNs phagocytosed virulent GAS less efficiently, produced less ROS and underwent reduced mitochondrial membrane depolarization compared with phagocytosis of avirulent GAS. Morphological and biochemical analyses revealed that PMNs infected with avirulent GAS exhibited nuclear fragmentation and caspase-3 activation consistent with an anti-inflammatory apoptotic phenotype. Conversely, virulent GAS induced PMN vacuolization and plasma membrane permeabilization, leading to a necrotic form of cell death. Infection of the mice with virulent GAS engendered significantly higher systemic pro-inflammatory cytokine release and localized infiltration of murine PMNs, with cells associated with virulent GAS infection exhibiting reduced apoptotic potential. Avirulent GAS infection was associated with lower levels of proinflammatory cytokines and tissue PMN apoptosis. We propose that the differences in PMN cell death mechanisms influence the inflammatory responses to infection by GAS.

CD24-triggered caspase-dependent apoptosis via mitochondrial membrane depolarization and reactive oxygen species production of human neutrophils is impaired in sepsis.

Apoptosis is the most common pathway of neutrophil death under both physiological and inflammatory conditions. In this study, we describe an apoptotic pathway in human neutrophils that is triggered via the surface molecule CD24. In normal neutrophils, CD24 ligation induces death through depolarization of the mitochondrial membrane in a manner dependent on caspase-3 and caspase-9 and reactive oxygen species. Proinflammatory cytokines such as TNF-α, IFN-γ, and GM-CSF upregulated the expression of CD24 in vitro, favoring the emergence of a new CD16(high)/CD24(high) subset of cultured neutrophils. We observed that CD24 expression (at both mRNA and protein levels) was significantly downregulated in neutrophils from sepsis patients but not from patients with systemic inflammatory response syndrome. This downregulation was reproduced by incubation of neutrophils from healthy controls with corticosteroids or with plasma collected from sepsis patients, but not with IL-10 or TGF-ß. Decreased CD24 expression observed on sepsis neutrophils was associated with lack of functionality of the molecule, because cross-ligation of CD24 failed to trigger apoptosis in neutrophils from sepsis patients. Our results suggest a novel aspect of CD24-mediated immunoregulation and represent, to our knowledge, the first report showing the role of CD24 in the delayed/defective cell death in sepsis.

Mechanisms of MAVS regulation at the mitochondrial membrane.

Mitochondria have emerged as critical platforms for antiviral innate immune signaling. This is due in large part to the mitochondrial localization of the innate immune signaling adaptor MAVS (mitochondrial antiviral signaling protein), which coordinates signals received from two independent cytosolic pathogen recognition receptors (PRRs) to induce antiviral genes. The existence of a shared adaptor for two central PRRs presents an ideal target by which the host cell can prevent cellular damage induced by uncontrolled inflammation through alteration of MAVS expression and/or signaling. In this review, we focus on the MAVS regulome and review the cellular factors that regulate MAVS by (1) protein-protein interactions, (2) alterations in mitochondrial dynamics, and/or (3) post-translational modifications.

Antiphospholipid antibodies internalised by human syncytiotrophoblast cause aberrant cell death and the release of necrotic trophoblast debris.

Antiphospholipid antibodies (aPL) are the strongest maternal risk factor for pre-eclampsia, a hypertensive disease of human pregnancy. Pre-eclampsia is triggered by a toxic factor released from the placenta that activates the maternal endothelium. Antiphospholipid antibodies cause the release of necrotic trophoblast debris from the placental syncytiotrophoblast and this debris can activate endothelial cells. In this study, we investigated how aPL affects syncytiotrophoblast death and production of necrotic trophoblast debris by examining the interaction between aPL and human first trimester placental explants. Human polyclonal and murine monoclonal aPL, but not control antibodies, were rapidly internalised by the syncytiotrophoblast. Inhibitors of endocytosis or the low-density lipoprotein receptor (LDLR) family, but not toll-like receptors, decreased the internalisation of aPL and prevented the release of necrotic trophoblast debris from the syncytiotrophoblast. Once internalised, aPL increased inner mitochondrial membrane leak and Cytochrome c release while depressing oxidative flux through Complex IV of the electron transport system in syncytiotrophoblast mitochondria. These data suggest that the human syncytiotrophoblast internalises aPL by antigen-dependent endocytosis involving LDLR family members. Once internalised by the syncytiotrophoblast, aPL affects the death-regulating mitochondria, causing extrusion of necrotic trophoblast debris which can activate maternal endothelial cells thereby contributing to the pathogenesis of pre-eclampsia.

Stomatin-like protein 2 deficiency in T cells is associated with altered mitochondrial respiration and defective CD4+ T cell responses.

Stomatin-like protein 2 (SLP-2) is a mostly mitochondrial protein that regulates mitochondrial biogenesis and function and modulates T cell activation. To determine the mechanism of action of SLP-2, we generated T cell-specific SLP-2-deficient mice. These mice had normal numbers of thymocytes and T cells in the periphery. However, conventional SLP-2-deficient T cells had a posttranscriptional defect in IL-2 production in response to TCR ligation, and this translated into reduced CD4(+) T cell responses. SLP-2 deficiency was associated with impaired cardiolipin compartmentalization in mitochondrial membranes, decreased levels of the NADH dehydrogenase (ubiquinone) iron-sulfur protein 3, NADH dehydrogenase (ubiquinone) 1ß subcomplex subunit 8, and NADH dehydrogenase (ubiquinone) 1α subcomplex subunit 9 of respiratory complex I, and decreased activity of this complex as well as of complex II plus III of the respiratory chain. In addition, SLP-2-deficient T cells showed a significant increase in uncoupled mitochondrial respiration and a greater reliance on glycolysis. Based on these results, we propose that SLP-2 organizes the mitochondrial membrane compartmentalization of cardiolipin, which is required for optimal assembly and function of respiratory chain complexes. This function, in T cells, helps to ensure proper metabolic response during activation.

Inflammasome formation and IL-1ß release by human blood monocytes in response to silver nanoparticles.

In this study, the immunological effect of silver nanoparticles on innate immunity was investigated using primary human monocytes. After exposure to silver nanoparticles, production of IL-1ß, a critical cytokine involved in induction of innate immunity, significantly increased as particle size decreased. These results suggest that silver nanoparticles may evoke an immunologically active state. The size effect of silver nanoparticles on IL-1ß production was also further investigated. 5 nm and 28 nm silver nanoparticles induced inflammasome formation and subsequent caspase-1 activation. Using inhibitors, we found exposure to silver nanoparticles caused leakage of cathepsins from lysosomes and efflux of intracellular K(+). These two events induced superoxide within mitochondrial membranes, leading to inflammasome formation. 5 nm silver nanoparticles produced more hydrogen peroxide and were more cytotoxic than 28 nm silver nanoparticles, suggesting the balance between superoxide and hydrogen peroxide governs cell fate, death or activation. Moreover, these findings also suggest that the immunological significance of silver nanoparticles should be considered with respect to their capacity to synergistically activate immune responses.

Arenavirus nucleoprotein targets interferon regulatory factor-activating kinase IKKε.

Arenaviruses perturb innate antiviral defense by blocking induction of type I interferon (IFN) production. Accordingly, the arenavirus nucleoprotein (NP) was shown to block activation and nuclear translocation of interferon regulatory factor 3 (IRF3) in response to virus infection. Here, we sought to identify cellular factors involved in innate antiviral signaling targeted by arenavirus NP. Consistent with previous studies, infection with the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) prevented phosphorylation of IRF3 in response to infection with Sendai virus, a strong inducer of the retinoic acid-inducible gene I (RIG-I)/mitochondrial antiviral signaling (MAVS) pathway of innate antiviral signaling. Using a combination of coimmunoprecipitation and confocal microscopy, we found that LCMV NP associates with the IκB kinase (IKK)-related kinase IKKε but that, rather unexpectedly, LCMV NP did not bind to the closely related TANK-binding kinase 1 (TBK-1). The NP-IKKε interaction was highly conserved among arenaviruses from different clades. In LCMV-infected cells, IKKε colocalized with NP but not with MAVS located on the outer membrane of mitochondria. LCMV NP bound the kinase domain (KD) of IKKε (IKBKE) and blocked its autocatalytic activity and its ability to phosphorylate IRF3, without undergoing phosphorylation. Together, our data identify IKKε as a novel target of arenavirus NP. Engagement of NP seems to sequester IKKε in an inactive complex. Considering the important functions of IKKε in innate antiviral immunity and other cellular processes, the NP-IKKε interaction likely plays a crucial role in arenavirus-host interaction.

Translocase of outer mitochondrial membrane 70 induces interferon response and is impaired by hepatitis C virus NS3.

Hepatitis C virus (HCV) elevated expression of the translocase of outer mitochondrial membrane 70 (Tom70). Interestingly, overexpression of Tom70 induces interferon (IFN) synthesis in hepatocytes, and it was impaired by HCV. Here, we addressed the mechanism of this impairment. The HCV NS3/4A protein induced Tom70 expression. The HCV NS3 protein interacted in cells, and cleaved the adapter protein mitochondrial anti-viral signaling (MAVS). Ectopic overexpression of Tom70 could not inhibit this cleavage. As a result, IRF-3 phosphorylation was impaired and IFN-ß induction was suppressed. These results indicate that MAVS works upstream of Tom70 and the cleavage of MAVS by HCV NS3 protease suppresses signaling of IFN induction.