Cytotoxic cells kill intracellular bacteria through granulysin-mediated delivery of granzymes.

When killer lymphocytes recognize infected cells, perforin delivers cytotoxic proteases (granzymes) into the target cell to trigger apoptosis. What happens to intracellular bacteria during this process is unclear. Human, but not rodent, cytotoxic granules also contain granulysin, an antimicrobial peptide. Here, we show that granulysin delivers granzymes into bacteria to kill diverse bacterial strains. In Escherichia coli, granzymes cleave electron transport chain complex I and oxidative stress defense proteins, generating reactive oxygen species (ROS) that rapidly kill bacteria. ROS scavengers and bacterial antioxidant protein overexpression inhibit bacterial death. Bacteria overexpressing a GzmB-uncleavable mutant of the complex I subunit nuoF or strains that lack complex I still die, but more slowly, suggesting that granzymes disrupt multiple vital bacterial pathways. Mice expressing transgenic granulysin are better able to clear Listeria monocytogenes. Thus killer cells play an unexpected role in bacterial defense.

Perforin pores in the endosomal membrane trigger the release of endocytosed granzyme B into the cytosol of target cells.

How the pore-forming protein perforin delivers apoptosis-inducing granzymes to the cytosol of target cells is uncertain. Perforin induces a transient Ca2+ flux in the target cell, which triggers a process to repair the damaged cell membrane. As a consequence, both perforin and granzymes are endocytosed into enlarged endosomes called 'gigantosomes'. Here we show that perforin formed pores in the gigantosome membrane, allowing endosomal cargo, including granzymes, to be gradually released. After about 15 min, gigantosomes ruptured, releasing their remaining content. Thus, perforin delivers granzymes by a two-step process that involves first transient pores in the cell membrane that trigger the endocytosis of granzyme and perforin and then pore formation in endosomes to trigger cytosolic release.

Granzyme A cleaves a mitochondrial complex I protein to initiate caspase-independent cell death.

The killer lymphocyte protease granzyme A (GzmA) triggers caspase-independent target cell death with morphological features of apoptosis. We previously showed that GzmA acts directly on mitochondria to generate reactive oxygen species (ROS) and disrupt the transmembrane potential (DeltaPsi(m)) but does not permeabilize the mitochondrial outer membrane. Mitochondrial damage is critical to GzmA-induced cell death since cells treated with superoxide scavengers are resistant to GzmA. Here we find that GzmA accesses the mitochondrial matrix to cleave the complex I protein NDUFS3, an iron-sulfur subunit of the NADH:ubiquinone oxidoreductase complex I, after Lys56 to interfere with NADH oxidation and generate superoxide anions. Target cells expressing a cleavage site mutant of NDUFS3 are resistant to GzmA-mediated cell death but remain sensitive to GzmB.

ER-mitochondria contacts control surface glycan expression and sensitivity to killer lymphocytes in glioma stem-like cells.

Glioblastoma is a highly heterogeneous aggressive primary brain tumor, with the glioma stem-like cells (GSC) being more sensitive to cytotoxic lymphocyte-mediated killing than glioma differentiated cells (GDC). However, the mechanism behind this higher sensitivity is unclear. Here, we found that the mitochondrial morphology of GSCs modulates the ER-mitochondria contacts that regulate the surface expression of sialylated glycans and their recognition by cytotoxic T lymphocytes and natural killer cells. GSCs displayed diminished ER-mitochondria contacts compared to GDCs. Forced ER-mitochondria contacts in GSCs increased their cell surface expression of sialylated glycans and reduced their susceptibility to cytotoxic lymphocytes. Therefore, mitochondrial morphology and dynamism dictate the ER-mitochondria contacts in order to regulate the surface expression of certain glycans and thus play a role in GSC recognition and elimination by immune effector cells. Targeting the mitochondrial morphology, dynamism, and contacts with the ER could be an innovative strategy to deplete the cancer stem cell compartment to successfully treat glioblastoma.

SAM50, a side door to the mitochondria: The case of cytotoxic proteases.

SAM50, a 7-8 nm diameter ß-barrel channel of the mitochondrial outer membrane, is the central channel of the sorting and assembly machinery (SAM) complex involved in the biogenesis of ß-barrel proteins. Interestingly, SAM50 is not known to have channel translocase activity; however, we have recently found that this channel is necessary and sufficient for mitochondrial entry of cytotoxic proteases. Cytotoxic lymphocytes eliminate cells that pose potential hazards, such as virus- and bacteria-infected cells as well as cancer cells. They induce cell death following the delivery of granzyme cytotoxic proteases into the cytosol of the target cell. Although granzyme A and granzyme B (GA and GB), the best characterized of the five human granzymes, trigger very distinct apoptotic cascades, they share the ability to directly target the mitochondria. GA and GB do not have a mitochondrial targeting signal, yet they enter the target cell mitochondria to disrupt respiratory chain complex I and induce mitochondrial reactive oxygen species (ROS)-dependent cell death. We found that granzyme mitochondrial entry requires SAM50 and the translocase of the inner membrane 22 (TIM22). Preventing granzymes' mitochondrial entry compromises their cytotoxicity, indicating that this event is unexpectedly an important step for cell death. Although mitochondria are best known for their roles in cell metabolism and energy conversion, these double-membrane organelles are also involved in Ca2+ homeostasis, metabolite transport, cell cycle regulation, cell signaling, differentiation, stress response, redox homeostasis, aging, and cell death. This multiplicity of functions is matched with the complexity and plasticity of the mitochondrial proteome as well as the organelle's morphological and structural versatility. Indeed, mitochondria are extremely dynamic and undergo fusion and fission events in response to diverse cellular cues. In humans, there are 1500 different mitochondrial proteins, the vast majority of which are encoded in the nuclear genome and translated by cytosolic ribosomes, after which they must be imported and properly addressed to the right mitochondrial compartment. To this end, mitochondria are equipped with a very sophisticated and highly specific protein import machinery. The latter is centered on translocase complexes embedded in the outer and inner mitochondrial membranes working along five different import pathways. We will briefly describe these import pathways to put into perspective our finding regarding the ability of granzymes to enter the mitochondria.

Identification of a novel population of highly cytotoxic c-Met-expressing CD8+ T lymphocytes.

CD8+ cytotoxic T lymphocytes (CTLs) are critical mediators of anti-tumor immunity, and controlling the mechanisms that govern CTL functions could be crucial for enhancing patient outcome. Previously, we reported that hepatocyte growth factor (HGF) limits effective murine CTL responses via antigen-presenting cells. Here, we show that a fraction of murine effector CTLs expresses the HGF receptor c-Met (c-Met+ CTLs). Phenotypic and functional analysis of c-Met+ CTLs reveals that they display enhanced cytolytic capacities compared to their c-Met- CTL counterparts. Furthermore, HGF directly restrains the cytolytic function of c-Met+ CTLs in cell-mediated cytotoxicity reactions in vitro and in vivo and abrogates T-cell responses against metastatic melanoma in vivo Finally, we establish in three murine tumor settings and in human melanoma tissues that c-Met+ CTLs are a naturally occurring CD8+ T-cell population. Together, our findings suggest that the HGF/c-Met pathway could be exploited to control CD8+ T-cell-mediated anti-tumor immunity.

(Neuro)degenerated Mitochondria-ER contacts.

In the last years, a considerable amount of experimental evidence has highlighted the association between neurodegenerative disorders (NDD) and the biology of mitochondria-Endoplasmic Reticulum contacts (MERCs). In this review, we summarize the most recent findings on this topic. We underline that dysregulation of MERCs can contribute to the neurodegenerative process either by altering directly the functionality of neurons and their response to stress stimuli and metabolic shifts or by indirectly influencing the neuroinflammatory response that accompanies NDD. Our overview of the current literature suggest that defective MERCs could be a common determinant to the "hypergeneration" and "neurodegeneration" programs, leading respectively to tumours and NDD.

Granzyme B-activated p53 interacts with Bcl-2 to promote cytotoxic lymphocyte-mediated apoptosis.

Granzyme B (GzmB) plays a major role in CTLs and NK cell-mediated elimination of virus-infected cells and tumors. Human GzmB preferentially induces target cell apoptosis by cleaving the proapoptotic Bcl-2 family member Bid, which, together with Bax, induces mitochondrial outer membrane permeabilization. We previously showed that GzmB also induces a rapid accumulation of the tumor-suppressor protein p53 within target cells, which seems to be involved in GzmB-induced apoptosis. In this article, we show that GzmB-activated p53 accumulates on target cell mitochondria and interacts with Bcl-2. This interaction prevents Bcl-2 inhibitory effect on both Bax and GzmB-truncated Bid, and promotes GzmB-induced mitochondrial outer membrane permeabilization. Consequently, blocking p53-Bcl-2 interaction decreases GzmB-induced Bax activation, cytochrome c release from mitochondria, and subsequent effector caspases activation leading to a decreased sensitivity of target cells to both GzmB and CTL/NK-mediated cell death. Together, our results define p53 as a new important player in the GzmB apoptotic signaling pathway and in CTL/NK-induced apoptosis.

Alterations in RNA processing during immune-mediated programmed cell death.

During immune-mediated death, death-inducing granzyme (Gzm) proteases concentrate in the nucleus of cells targeted for immune elimination, suggesting that nuclear processes are important targets. Here we used differential 2D proteomics of GzmA-treated nuclei to identify potential GzmA substrates. Of 44 candidates, 33 were RNA-binding proteins important in posttranscriptional RNA processing, including 14 heterogeneous nuclear ribonucleoproteins (hnRNP). Multiple hnRNPs were degraded in cells undergoing GzmA-, GzmB-, or caspase-mediated death. GzmA and caspase activation impaired nuclear export of newly synthesized RNA and disrupted pre-mRNA splicing. Expressing GzmA-resistant hnRNP A1 inhibited GzmA-mediated cell death and rescued pre-mRNA splicing, suggesting that hnRNP A1 is an important GzmA substrate. Cellular stresses are known to inhibit initiation of cap-dependent translation. Disrupting pre-mRNA processing should block further new protein synthesis and promote death by interfering with pathways induced to protect cells from death.

Natural autoantibodies to Fcγ receptors in intravenous immunoglobulins.

A considerable progress has been achieved in the comprehension of the cellular and molecular mechanisms that account for the therapeutic benefit of intravenous immunoglobulin (IVIg) in several autoimmune and inflammatory conditions. However, the precise mechanisms responsible for such a wide range of biological activities have not been proven unambiguously. A wide range of specificities have been identified within IVIg including idiotypes of immunoglobulins, T cell receptor, HLA molecules, several cell surface molecules of immunological importance such as CD4, CD5, Fas, BAFF, cytokines and cytokine receptors, chemokine receptors, CD40 among others. Here we identify and characterize the natural autoantibodies of IgG isotype directed against the human Fc receptors. We show that the F(ab')2 of IVIg recognize the FcγRIII (CD16) and FcγRII (CD32). Interestingly, the immunopurified anti-FcγIII and anti-FcγII antibodies isolated from IVIg bind soluble and membrane-bound FcR and inhibit rosette formation. Altogether, these results along with previous reports provide pointers on the existence of functionally relevant natural autoantibodies towards a wide range of self-motifs that may participate in regulation of the immune response. Their presence in the therapeutic immunoglobulin preparations may explain at least in part, the beneficial effect of IVIg in autoimmune diseases.

Granzymes in health and diseases: the good, the bad and the ugly.

Granzymes are a family of serine proteases, composed of five human members: GA, B, H, M and K. They were first discovered in the 1980s within cytotoxic granules released during NK cell- and T cell-mediated killing. Through their various proteolytic activities, granzymes can trigger different pathways within cells, all of which ultimately lead to the same result, cell death. Over the years, the initial consideration of granzymes as mere cytotoxic mediators has changed due to surprising findings demonstrating their expression in cells other than immune effectors as well as new intracellular and extracellular activities. Additional roles have been identified in the extracellular milieu, following granzyme escape from the immunological synapse or their release by specific cell types. Outside the cell, granzyme activities mediate extracellular matrix alteration via the degradation of matrix proteins or surface receptors. In certain contexts, these processes are essential for tissue homeostasis; in others, excessive matrix degradation and extensive cell death contribute to the onset of chronic diseases, inflammation, and autoimmunity. Here, we provide an overview of both the physiological and pathological roles of granzymes, highlighting their utility while also recognizing how their unregulated presence can trigger the development and/or worsening of diseases.

Perforin activates clathrin- and dynamin-dependent endocytosis, which is required for plasma membrane repair and delivery of granzyme B for granzyme-mediated apoptosis.

Cytotoxic T lymphocytes and natural killer cells destroy target cells via the polarized exocytosis of lytic effector proteins, perforin and granzymes, into the immunologic synapse. How these molecules enter target cells is not fully understood. It is debated whether granzymes enter via perforin pores formed at the plasma membrane or whether perforin and granzymes are first endocytosed and granzymes are then released from endosomes into the cytoplasm. We previously showed that perforin disruption of the plasma membrane induces a transient Ca(2+) flux into the target cell that triggers a wounded membrane repair response in which lysosomes and endosomes donate their membranes to reseal the damaged membrane. Here we show that perforin activates clathrin- and dynamin-dependent endocytosis, which removes perforin and granzymes from the plasma membrane to early endosomes, preserving outer membrane integrity. Inhibiting clathrin- or dynamin-dependent endocytosis shifts death by perforin and granzyme B from apoptosis to necrosis. Thus by activating endocytosis to preserve membrane integrity, perforin facilitates granzyme uptake and avoids the proinflammatory necrotic death of a membrane-damaged cell.

Cleavage of transaldolase by granzyme B causes the loss of enzymatic activity with retention of antigenicity for multiple sclerosis patients.

Multiple sclerosis (MS) is an autoimmune demyelinating disease of the CNS resulting from a progressive loss of oligodendrocytes. Transaldolase (TAL) is expressed at selectively high levels in oligodendrocytes of the brain, and postmortem sections show concurrent loss of myelin basic protein and TAL from sites of demyelination. Infiltrating CD8(+) CTLs are thought to play a key role in oligodendrocyte cell death. Cleavage by granzyme B (GrB) is predictive for autoantigenicity of self-proteins, thereby further implicating CTL-induced death in the initiation and propagation of autoimmunity. The precursor frequency and CTL activity of HLA-A2-restricted TAL 168-176-specific CD8(+) T cells is increased in MS patients. In this paper, we show that TAL, but not myelin basic protein, is specifically cleaved by human GrB. The recognition site of GrB that resulted in the cleavage of a dominant TAL fragment was mapped to a VVAD motif at aa residue 27 by N-terminal sequencing and confirmed by site-directed mutagenesis. The major C-terminal GrB cleavage product, residues 28-337, had no enzymatic activity but retained the antigenicity of full-length TAL, effectively stimulating the proliferation and CTL activity of PBMCs and of CD8(+) T cell lines from patients with MS. Sera of MS patients exhibited similar binding affinity to wild-type and GrB-cleaved TAL. Because GrB mediates the killing of target cells and cleavage by GrB is predictive of autoantigen status of self proteins, GrB-cleaved TAL-specific T cell-mediated cytotoxicity may contribute to the progressive destruction of oligodendrocytes in patients with MS.

The cytotoxic T lymphocyte protease granzyme A cleaves and inactivates poly(adenosine 5'-diphosphate-ribose) polymerase-1.

Granzyme A (GzmA) in killer cells induces caspase-independent programmed cell death. In this study, we show that GzmA cleaves the DNA damage sensor poly(adenosine 5'-diphosphate-ribose) polymerase-1 (PARP-1) after Lys(498) in its automodification domain, separating the DNA binding domain from the catalytic domain, which interferes with repair of GzmA-induced DNA damage and enhances susceptibility to GzmA-mediated death. Overexpressing K498A PARP-1 reduces GzmA-mediated death and drives dying cells to necrosis rather than apoptosis. Conversely, inhibiting or genetically disrupting PARP-1 enhances cell vulnerability. The N-terminal GzmA cleavage fragment of PARP-1 acts as a PARP-1 dominant negative, binding to DNA and blocking DNA repair. Disrupting PARP-1, which is also a caspase target, is therefore required for efficient apoptosis by both caspase-independent and caspase-dependent pathways.

Reactive Oxygen Species: Do They Play a Role in Adaptive Immunity?

The immune system protects the host from a plethora of microorganisms and toxins through its unique ability to distinguish self from non-self. To perform this delicate but essential task, the immune system relies on two lines of defense. The innate immune system, which is by nature fast acting, represents the first line of defense. It involves anatomical barriers, physiological factors as well as a subset of haematopoietically-derived cells generically call leukocytes. Activation of the innate immune response leads to a state of inflammation that serves to both warn about and combat the ongoing infection and delivers the antigenic information of the invading pathogens to initiate the slower but highly potent and specific second line of defense, the adaptive immune system. The adaptive immune response calls on T lymphocytes as well as the B lymphocytes essential for the elimination of pathogens and the establishment of the immunological memory. Reactive oxygen species (ROS) have been implicated in many aspects of the immune responses to pathogens, mostly in innate immune functions, such as the respiratory burst and inflammasome activation. Here in this mini review, we focus on the role of ROS in adaptive immunity. We examine how ROS contribute to T-cell biology and discuss whether this activity can be extrapolated to B cells.

Oxidative and Non-Oxidative Antimicrobial Activities of the Granzymes.

Cell-mediated cytotoxicity is an essential immune defense mechanism to fight against viral, bacterial or parasitic infections. Upon recognition of an infected target cell, killer lymphocytes form an immunological synapse to release the content of their cytotoxic granules. Cytotoxic granules of humans contain two membrane-disrupting proteins, perforin and granulysin, as well as a homologous family of five death-inducing serine proteases, the granzymes. The granzymes, after delivery into infected host cells by the membrane disrupting proteins, may contribute to the clearance of microbial pathogens through different mechanisms. The granzymes can induce host cell apoptosis, which deprives intracellular pathogens of their protective niche, therefore limiting their replication. However, many obligate intracellular pathogens have evolved mechanisms to inhibit programed cells death. To overcome these limitations, the granzymes can exert non-cytolytic antimicrobial activities by directly degrading microbial substrates or hijacked host proteins crucial for the replication or survival of the pathogens. The granzymes may also attack factors that mediate microbial virulence, therefore directly affecting their pathogenicity. Many mechanisms applied by the granzymes to eliminate infected cells and microbial pathogens rely on the induction of reactive oxygen species. These reactive oxygen species may be directly cytotoxic or enhance death programs triggered by the granzymes. Here, in the light of the latest advances, we review the antimicrobial activities of the granzymes in regards to their cytolytic and non-cytolytic activities to inhibit pathogen replication and invasion. We also discuss how reactive oxygen species contribute to the various antimicrobial mechanisms exerted by the granzymes.

Cdk5 and GSK3ß inhibit fast endophilin-mediated endocytosis.

Endocytosis mediates the cellular uptake of micronutrients and cell surface proteins. Fast Endophilin-mediated endocytosis, FEME, is not constitutively active but triggered upon receptor activation. High levels of growth factors induce spontaneous FEME, which can be suppressed upon serum starvation. This suggested a role for protein kinases in this growth factor receptor-mediated regulation. Using chemical and genetic inhibition, we find that Cdk5 and GSK3ß are negative regulators of FEME. They antagonize the binding of Endophilin to Dynamin-1 and to CRMP4, a Plexin A1 adaptor. This control is required for proper axon elongation, branching and growth cone formation in hippocampal neurons. The kinases also block the recruitment of Dynein onto FEME carriers by Bin1. As GSK3ß binds to Endophilin, it imposes a local regulation of FEME. Thus, Cdk5 and GSK3ß are key regulators of FEME, licensing cells for rapid uptake by the pathway only when their activity is low.

Granzyme B Attenuates Bacterial Virulence by Targeting Secreted Factors.

Pathogenic bacteria secrete virulence factors that interact with the human host to establish infections. The human immune system evolved multiple mechanisms to fight bacterial invaders, including immune proteases that were demonstrated to contribute crucially to antibacterial defense. Here we show that granzyme B degrades multiple secreted virulence mediators from Listeria monocytogenes, Salmonella typhimurium, and Mycobacteria tuberculosis. Pathogenic bacteria, when infected in the presence of granzyme B or granzyme-secreting killer cells, fail to grow in human macrophages and epithelial cells owing to their crippled virulence. A granzyme B-uncleavable mutant form of the major Listeria virulence factor, listeriolysin O, rescued the virulence defect in response to granzyme treatment. Hence, we link the degradation of a single factor with the observed decrease in virulent bacteria growth. Overall, we reveal here an innate immune barrier function of granzyme B by disrupting bacterial virulence to facilitate bacteria clearance by bystander immune and non-immune cells.

Mitochondrial Entry of Cytotoxic Proteases: A New Insight into the Granzyme B Cell Death Pathway.

The mitochondria represent an integration and amplification hub for various death pathways including that mediated by granzyme B (GB), a granule enzyme expressed by cytotoxic lymphocytes. GB activates the proapoptotic B cell CLL/lymphoma 2 (Bcl-2) family member BH3-interacting domain death agonist (BID) to switch on the intrinsic mitochondrial death pathway, leading to Bcl-2-associated X protein (Bax)/Bcl-2 homologous antagonist/killer- (Bak-) dependent mitochondrial outer membrane permeabilization (MOMP), the dissipation of mitochondrial transmembrane potential (ΔΨm), and the production of reactive oxygen species (ROS). GB can also induce mitochondrial damage in the absence of BID, Bax, and Bak, critical for MOMP, indicating that GB targets the mitochondria in other ways. Interestingly, granzyme A (GA), GB, and caspase 3 can all directly target the mitochondrial respiratory chain complex I for ROS-dependent cell death. Studies of ROS biogenesis have revealed that GB must enter the mitochondria for ROS production, making the mitochondrial entry of cytotoxic proteases (MECP) an unexpected critical step in the granzyme death pathway. MECP requires an intact ΔΨm and is mediated though Sam50 and Tim22 channels in a mtHSP70-dependent manner. Preventing MECP severely compromises GB cytotoxicity. In this review, we provide a brief overview of the canonical mitochondrial death pathway in order to put into perspective this new insight into the GB action on the mitochondria to trigger ROS-dependent cell death.