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.

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.

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.

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.

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.

STIM1 promotes migration, phagosomal maturation and antigen cross-presentation in dendritic cells.

Antigen cross-presentation by dendritic cells (DC) stimulates cytotoxic T cell activation to promote immunity to intracellular pathogens, viruses and cancer. Phagocytosed antigens generate potent T cell responses, but the signalling and trafficking pathways regulating their cross-presentation are unclear. Here, we show that ablation of the store-operated-Ca2+-entry regulator STIM1 in mouse myeloid cells impairs cross-presentation and DC migration in vivo and in vitro. Stim1 ablation reduces Ca2+ signals, cross-presentation, and chemotaxis in mouse bone-marrow-derived DCs without altering cell differentiation, maturation or phagocytic capacity. Phagosomal pH homoeostasis and ROS production are unaffected by STIM1 deficiency, but phagosomal proteolysis and leucyl aminopeptidase activity, IRAP recruitment, as well as fusion of phagosomes with endosomes and lysosomes are all impaired. These data suggest that STIM1-dependent Ca2+ signalling promotes the delivery of endolysosomal enzymes to phagosomes to enable efficient cross-presentation.

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.

Granzyme B enters the mitochondria in a Sam50-, Tim22- and mtHsp70-dependent manner to induce apoptosis.

We have found that granzyme B (GB)-induced apoptosis also requires reactive oxygen species resulting from the alteration of mitochondrial complex I. How GB, which does not possess a mitochondrial targeting sequence, enter this organelle is unknown. We show that GB enters the mitochondria independently of the translocase of the outer mitochondrial membrane complex, but requires instead Sam50, the central subunit of the sorting and assembly machinery that integrates outer membrane ß-barrel proteins. Moreover, GB breaches the inner membrane through Tim22, the metabolite carrier translocase pore, in a mitochondrial heat-shock protein 70 (mtHsp70)-dependent manner. Granzyme A (GA) and caspase-3 use a similar route to the mitochondria. Finally, preventing GB from entering the mitochondria either by mutating lysine 243 and arginine 244 or depleting Sam50 renders cells more resistant to GB-mediated reactive oxygen species and cell death. Similarly, Sam50 depletion protects cells from GA-, GM- and caspase-3-mediated cell death. Therefore, cytotoxic molecules enter the mitochondria to induce efficiently cell death through a noncanonical Sam50-, Tim22- and mtHsp70-dependent import pathway.

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.

SIRT6 Suppresses Cancer Stem-like Capacity in Tumors with PI3K Activation Independently of Its Deacetylase Activity.

Cancer stem cells (CSCs) have high tumorigenic capacity. Here, we show that stem-like traits of specific human cancer cells are reduced by overexpression of the histone deacetylase sirtuin 6 (SIRT6). SIRT6-sensitive cancer cells bear mutations that activate phosphatidylinositol-3-kinase (PI3K) signaling, and overexpression of SIRT6 reduces growth, progression, and grade of breast cancer in a mouse model with PI3K activation. Tumor metabolomic and transcriptomic analyses reveal that SIRT6 overexpression dampens PI3K signaling and stem-like characteristics and causes metabolic rearrangements in this cancer model. Ablation of a PI3K activating mutation in otherwise isogenic cancer cells is sufficient to convert SIRT6-sensitive into SIRT6-insensitive cells. SIRT6 overexpression suppresses PI3K signaling at the transcriptional level and antagonizes tumor sphere formation independent of its histone deacetylase activity. Our data identify SIRT6 as a putative molecular target that hinders stemness of tumors with PI3K activation.

(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.

Low Dose BCG Infection as a Model for Macrophage Activation Maintaining Cell Viability.

Mycobacterium bovis BCG, the current vaccine against tuberculosis, is ingested by macrophages promoting the development of effector functions including cell death and microbicidal mechanisms. Despite accumulating reports on M. tuberculosis, mechanisms of BCG/macrophage interaction remain relatively undefined. In vivo, few bacilli are sufficient to establish a mycobacterial infection; however, in vitro studies systematically use high mycobacterium doses. In this study, we analyze macrophage/BCG interactions and microenvironment upon infection with low BCG doses and propose an in vitro model to study cell activation without affecting viability. We show that RAW macrophages infected with BCG at MOI 1 activated higher and sustained levels of proinflammatory cytokines and transcription factors while MOI 0.1 was more efficient for early stimulation of IL-1ß, MCP-1, and KC. Both BCG infection doses induced iNOS and NO in a dose-dependent manner and maintained nuclear and mitochondrial structures. Microenvironment generated by MOI 1 induced macrophage proliferation but not MOI 0.1 infection. In conclusion, BCG infection at low dose is an efficient in vitro model to study macrophage/BCG interactions that maintains macrophage viability and mitochondrial structures. This represents a novel model that can be applied to BCG research fields including mycobacterial infections, cancer immunotherapy, and prevention of autoimmunity and allergies.

Decitabine Treatment of Glioma-Initiating Cells Enhances Immune Recognition and Killing.

Malignant gliomas are aggressive brain tumours with very poor prognosis. The majority of glioma cells are differentiated (glioma-differentiated cells: GDCs), whereas the smaller population (glioma-initiating cells, GICs) is undifferentiated and resistant to conventional therapies. Therefore, to better target this pool of heterogeneous cells, a combination of diverse therapeutic approaches is envisaged. Here we investigated whether the immunosensitising properties of the hypomethylating agent decitabine can be extended to GICs. Using the murine GL261 cell line, we demonstrate that decitabine augments the expression of the death receptor FAS both on GDCs and GICs. Interestingly, it had a higher impact on GICs and correlated with an enhanced sensitivity to FASL-mediated cell death. Moreover, the expression of other critical molecules involved in cognate recognition by cytotoxic T lymphocytes, MHCI and ICAM-1, was upregulated by decitabine treatment. Consequently, T-cell mediated killing of both GDCs and GICs was enhanced, as was T cell proliferation after reactivation. Overall, although GICs are described to resist classical therapies, our study shows that hypomethylating agents have the potential to enhance glioma cell recognition and subsequent destruction by immune cells, regardless of their differentiation status. These results support the development of combinatorial treatment modalities including epigenetic modulation together with immunotherapy in order to treat heterogenous malignancies such as glioblastoma.

Glioma Stemlike Cells Enhance the Killing of Glioma Differentiated Cells by Cytotoxic Lymphocytes.

Glioblastoma multiforme, the most aggressive primary brain tumor, is maintained by a subpopulation of glioma cells with self-renewal properties that are able to recapitulate the entire tumor even after surgical resection or chemo-radiotherapy. This typifies the vast heterogeneity of this tumor with the two extremes represented on one end by the glioma stemlike cells (GSC) and on the other by the glioma differentiated cells (GDC). Interestingly, GSC are more sensitive to immune effector cells than the GDC counterpart. However, how GSC impact on the killing on the GDC and vice versa is not clear. Using a newly developed cytotoxicity assay allowing to simultaneously monitor cytotoxic lymphocytes-mediated killing of GSC and GDC, we found that although GSC were always better killed and that their presence enhanced the killing of GDC. In contrast, an excess of GDC had a mild protective effect on the killing of GSC, depending on the CTL type. Overall, our results suggest that during combination therapy, immunotherapy would be the most effective after prior treatment with conventional therapies.

A High Yield and Cost-efficient Expression System of Human Granzymes in Mammalian Cells.

When cytotoxic T lymphocytes (CTL) or natural killer (NK) cells recognize tumor cells or cells infected with intracellular pathogens, they release their cytotoxic granule content to eliminate the target cells and the intracellular pathogen. Death of the host cells and intracellular pathogens is triggered by the granule serine proteases, granzymes (Gzms), delivered into the host cell cytosol by the pore forming protein perforin (PFN) and into bacterial pathogens by the prokaryotic membrane disrupting protein granulysin (GNLY). To investigate the molecular mechanisms of target cell death mediated by the Gzms in experimental in-vitro settings, protein expression and purification systems that produce high amounts of active enzymes are necessary. Mammalian secreted protein expression systems imply the potential to produce correctly folded, fully functional protein that bears posttranslational modification, such as glycosylation. Therefore, we used a cost-efficient calcium precipitation method for transient transfection of HEK293T cells with human Gzms cloned into the expression plasmid pHLsec. Gzm purification from the culture supernatant was achieved by immobilized nickel affinity chromatography using the C-terminal polyhistidine tag provided by the vector. The insertion of an enterokinase site at the N-terminus of the protein allowed the generation of active protease that was finally purified by cation exchange chromatography. The system was tested by producing high levels of cytotoxic human Gzm A, B and M and should be capable to produce virtually every enzyme in the human body in high yields.

Novel Cell-Penetrating Peptide-Based Vaccine Induces Robust CD4+ and CD8+ T Cell-Mediated Antitumor Immunity.

Vaccines that can coordinately induce multi-epitope T cell-mediated immunity, T helper functions, and immunologic memory may offer effective tools for cancer immunotherapy. Here, we report the development of a new class of recombinant protein cancer vaccines that deliver different CD8(+) and CD4(+) T-cell epitopes presented by MHC class I and class II alleles, respectively. In these vaccines, the recombinant protein is fused with Z12, a novel cell-penetrating peptide that promotes efficient protein loading into the antigen-processing machinery of dendritic cells. Z12 elicited an integrated and multi-epitopic immune response with persistent effector T cells. Therapy with Z12-formulated vaccines prolonged survival in three robust tumor models, with the longest survival in an orthotopic model of aggressive brain cancer. Analysis of the tumor sites showed antigen-specific T-cell accumulation with favorable modulation of the balance of the immune infiltrate. Taken together, the results offered a preclinical proof of concept for the use of Z12-formulated vaccines as a versatile platform for the development of effective cancer vaccines.