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.

γδ T Cells Kill Plasmodium falciparum in a Granzyme- and Granulysin-Dependent Mechanism during the Late Blood Stage.

Plasmodium spp., the causative agent of malaria, have a complex life cycle. The exponential growth of the parasites during the blood stage is responsible for almost all malaria-associated morbidity and mortality. Therefore, tight immune control of the intraerythrocytic replication of the parasite is essential to prevent clinical malaria. Despite evidence that the particular lymphocyte subset of γδ T cells contributes to protective immunity during the blood stage in naive hosts, their precise inhibitory mechanisms remain unclear. Using human PBMCs, we confirmed in this study that γδ T cells specifically and massively expanded upon activation with Plasmodium falciparum culture supernatant. We also demonstrate that these activated cells gain cytolytic potential by upregulating cytotoxic effector proteins and IFN-γ. The killer cells bound to infected RBCs and killed intracellular P. falciparum via the transfer of the granzymes, which was mediated by granulysin in a stage-specific manner. Several vital plasmodial proteins were efficiently destroyed by granzyme B, suggesting proteolytic degradation of these proteins as essential in the lymphocyte-mediated death pathway. Overall, these data establish a granzyme- and granulysin-mediated innate immune mechanism exerted by γδ T cells to kill late-stage blood-residing P. falciparum.

Silver-nanoparticles increase bactericidal activity and radical oxygen responses against bacterial pathogens in human osteoclasts.

Bone infections are difficult to treat and can lead to severe tissue destruction. Acute bone infections are usually caused by Staphylococcus aureus. Osteoclasts, which belong to the monocyte/macrophage lineage, are the key cells in bone infections. They are not well equipped for killing bacteria and may serve as a reservoir for bacterial pathogens. Silver has been known for centuries for its bactericidal activity. Here, we investigated the bactericidal effects of nano-silver particles in bacteria infected human osteoclasts. We found that nano-silver in per se non-toxic concentration enhanced the bactericidal activity in osteoclasts against intracellular Methicillin-resistant, virulent Staphylococcus aureus. The reduced bacterial survival in nano-silver pretreated cells correlated with increased reactive oxygen responses towards the invading pathogens. Overall, these results indicate that nano-silver compounds should be considered as an effective treatment and prevention option for bacterial bone and orthopedic implant infections.

Interactions of human microglia cells with Japanese encephalitis virus.

BACKGROUND: Japanese encephalitis virus (JEV) is a neurotropic flavivirus causing mortality and morbidity in humans. Severe Japanese encephalitis cases display strong inflammatory responses in the central nervous system and an accumulation of viral particles in specific brain regions. Microglia cells are the unique brain-resident immune cell population with potent migratory functions and have been proposed to act as a viral reservoir for JEV. Animal models suggest that the targeting of microglia by JEV is partially responsible for inflammatory reactions in the brain. Nevertheless, the interactions between human microglia and JEV are poorly documented. METHODS: Using human primary microglia and a new model of human blood monocyte-derived microglia, the present study explores the interaction between human microglia and JEV as well as the role of these cells in viral transmission to susceptible cells. To achieve this work, vaccine-containing inactivated JEV and two live JEV strains were applied on human microglia. RESULTS: Live JEV was non-cytopathogenic to human microglia but increased levels of CCL2, CXCL9 and CXCL10 in such cultures. Furthermore, human microglia up-regulated the expression of the fraktalkine receptor CX3CR1 upon exposure to both JEV vaccine and live JEV. Although JEV vaccine enhanced MHC class II on all microglia, live JEV enhanced MHC class II mainly on CX3CR1+ microglia cells. Importantly, human microglia supported JEV replication, but infectivity was only transmitted to neighbouring cells in a contact-dependent manner. CONCLUSION: Our findings suggest that human microglia may be a source of neuronal infection and sustain JEV brain pathogenesis.

Leukocyte protease binding to nucleic acids promotes nuclear localization and cleavage of nucleic acid binding proteins.

Killer lymphocyte granzyme (Gzm) serine proteases induce apoptosis of pathogen-infected cells and tumor cells. Many known Gzm substrates are nucleic acid binding proteins, and the Gzms accumulate in the target cell nucleus by an unknown mechanism. In this study, we show that human Gzms bind to DNA and RNA with nanomolar affinity. Gzms cleave their substrates most efficiently when both are bound to nucleic acids. RNase treatment of cell lysates reduces Gzm cleavage of RNA binding protein targets, whereas adding RNA to recombinant RNA binding protein substrates increases in vitro cleavage. Binding to nucleic acids also influences Gzm trafficking within target cells. Preincubation with competitor DNA and DNase treatment both reduce Gzm nuclear localization. The Gzms are closely related to neutrophil proteases, including neutrophil elastase (NE) and cathepsin G. During neutrophil activation, NE translocates to the nucleus to initiate DNA extrusion into neutrophil extracellular traps, which bind NE and cathepsin G. These myeloid cell proteases, but not digestive serine proteases, also bind DNA strongly and localize to nuclei and neutrophil extracellular traps in a DNA-dependent manner. Thus, high-affinity nucleic acid binding is a conserved and functionally important property specific to leukocyte serine proteases. Furthermore, nucleic acid binding provides an elegant and simple mechanism to confer specificity of these proteases for cleavage of nucleic acid binding protein substrates that play essential roles in cellular gene expression and cell proliferation.

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.

Biocompatible Rhamnolipid Self-Assemblies with pH-Responsive Antimicrobial Activity.

There is an urgent need for alternative antimicrobial materials due to the growing challenge of bacteria becoming resistant to conventional antibiotics. This study demonstrates the creation of a biocompatible pH-switchable antimicrobial material by combining bacteria-derived rhamnolipids (RL) and food-grade glycerol monooleate (GMO). The integration of RL into dispersed GMO particles, with an inverse-type liquid crystalline cubic structure in the core, leads to colloidally stable supramolecular materials. The composition and pH-triggered structural transformations are studied with small-angle X-ray scattering, cryogenic transmission electron microscopy, and dynamic light scattering. The composition-structure-activity relationship is analyzed and optimized to target bacteria at acidic pH values of acute wounds. The new RL/GMO dispersions reduce Staphylococcus aureus (S. aureus) populations by 7-log after 24 h of treatment with 64 µg mL-1 of RL and prevent biofilm formation at pH = 5.0, but have no activity at pH = 7.0. Additionally, the system is active against methicillin-resistant S. aureus (MRSA) with minimum inhibitory concentration of 128 µg mL-1 at pH 5.0. No activity is found against several Gram-negative bacteria at pH 5.0 and 7.0. The results provide a fundamental understanding of lipid self-assembly and the design of lipid-based biomaterials, which can further guide the development of alternative bio-based solutions to combat bacteria.

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.

Validation of Effective Extracellular Vesicles Isolation Methods Adapted to Field Studies in Malaria Endemic Regions.

Malaria affects the poorer regions of the world and is of tremendous health and economic burden for developing countries. Extracellular vesicles (EVs) are small vesicles released by almost any cells in the human body, including malaria infected red blood cells. Recent evidence shows that EVs might contribute to the pathogenesis of malaria. In addition, EVs hold considerable value in biomarker discovery. However, there are still significant gaps in our understanding of EV biology. So far most of our knowledge about EVs in malaria comes from in vitro work. More field studies are required to gain insight into their contribution to the disease and pathogenesis under physiological conditions. However, to perform research on EVs in low-income regions might be challenging due to the lack of appropriate equipment to isolate EVs. Therefore, there is a need to develop and validate EV extraction protocols applicable to poorly equipped laboratories. We established and validated two protocols for EV isolation from cell culture supernatants, rodent and human plasma. We compared polyethylene glycol (PEG) and salting out (SA) with sodium acetate for precipitation of EVs. We then characterized the EVs by Transmission Electron Microscopy (TEM), Western Blot, Size-exclusion chromatography (SEC), bead-based flow cytometry and protein quantification. Both protocols resulted in efficient purification of EVs without the need of expensive material or ultracentrifugation. Furthermore, the procedure is easily scalable to work with large and small sample volumes. Here, we propose that both of our approaches can be used in resource limited countries, therefore further helping to close the gap in knowledge of EVs during malaria.

Improved Purification of Human Granzyme A/B and Granulysin Using a Mammalian Expression System.

Cytotoxic lymphocytes release proteins contained within the cytoplasmic cytolytic granules after recognition of infected or tumor target cells. These cytotoxic granular proteins (namely granzymes, granulysin, and perforin) are key immunological mediators within human cellular immunity. The availability of highly purified cytotoxic proteins has been fundamental for understanding their function in immunity and mechanistic involvement in sepsis and autoimmunity. Methods for recovery of native cytotoxic proteins can be problematic leading to: 1) the co-purification of additional proteins, confounding interpretation of function, and 2) low yields of highly purified proteins. Recombinant protein expression of individual cytolytic components can overcome these challenges. The use of mammalian expression systems is preferred for optimal post-translational modifications and avoidance of endotoxin contamination. Some of these proteins have been proposed for host directed human therapies (e.g. - granzyme A), or treatment of systemic infections or tumors as in granulysin. We report here a novel expression system using HEK293T cells for cost-effective purification of high yields of human granzymes (granzyme A and granzyme B) and granulysin with enhanced biological activity than previous reports. The resulting proteins are free of native contaminants, fold correctly, and remain enzymatically active. Importantly, these improvements have also led to the first purification of biologically active recombinant human granulysin in high yields from a mammalian system. This method can be used as a template for purification of many other secreted cellular proteins and may lead to advances for human medicine.

Involvement of CD252 (CD134L) and IL-2 in the expression of cytotoxic proteins in bacterial- or viral-activated human T cells.

Regulation of cytotoxic effector molecule expression in human CTLs after viral or bacterial activation is poorly understood. By using human autologous dendritic cells (DCs) to prime T lymphocytes, we found perforin only highly up-regulated in virus- (HSV-1, vaccinia virus) but not in intracellular bacteria- (Listeria innocua, Listeria monocytogenes, Mycobacterium tuberculosis, Chlamydophila pneumoniae) activated CTLs. In contrast, larger quantities of IFN-gamma and TNF-alpha were produced in Listeria-stimulated cultures. Granzyme B and granulysin were similarly up-regulated by all tested viruses and intracellular bacteria. DCs infected with HSV-1 showed enhanced surface expression of the costimulatory molecule CD252 (CD134L) compared with Listeria-infected DC and induced enhanced secretion of IL-2. Adding blocking CD134 or neutralizing IL-2 Abs during T cell activation reduced the HSV-dependent up-regulation of perforin. These data indicate a distinct CTL effector function in response to intracellular pathogens triggered via differing endogenous IL-2 production upon costimulation through CD252.

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.

Granzyme A Produced by γ9δ2 T Cells Activates ER Stress Responses and ATP Production, and Protects Against Intracellular Mycobacterial Replication Independent of Enzymatic Activity.

Mycobacterium tuberculosis (Mtb), the pathological agent that causes tuberculosis (TB) is the number one infectious killer worldwide with one fourth of the world's population currently infected. Data indicate that γ9δ2 T cells secrete Granzyme A (GzmA) in the extracellular space triggering the infected monocyte to inhibit growth of intracellular mycobacteria. Accordingly, deletion of GZMA from γ9δ2 T cells reverses their inhibitory capacity. Through mechanistic studies, GzmA's action was investigated in monocytes from human PBMCs. The use of recombinant human GzmA expressed in a mammalian system induced inhibition of intracellular mycobacteria to the same degree as previous human native protein findings. Our data indicate that: 1) GzmA is internalized within mycobacteria-infected cells, suggesting that GzmA uptake could prevent infection and 2) that the active site is not required to inhibit intracellular replication. Global proteomic analysis demonstrated that the ER stress response and ATP producing proteins were upregulated after GzmA treatment, and these proteins abundancies were confirmed by examining their expression in an independent set of patient samples. Our data suggest that immunotherapeutic host interventions of these pathways may contribute to better control of the current TB epidemic.

A Profound Membrane Reorganization Defines Susceptibility of Plasmodium falciparum Infected Red Blood Cells to Lysis by Granulysin and Perforin.

Malaria remains one of the most serious health problems in developing countries. The causative agent of malaria, Plasmodium spp., have a complex life cycle involving multiple developmental stages as well as different morphological, biochemical and metabolic requirements. We recently found that γδ T cells control parasite growth using pore-forming proteins to deliver their cytotoxic proteases, the granzymes, into blood residing parasites. Here, we follow up on the molecular mechanisms of parasite growth inhibition by human pore-forming proteins. We confirm that Plasmodium falciparum infection efficiently depletes the red blood cells of cholesterol, which renders the parasite surrounding membranes susceptible to lysis by prokaryotic membrane disrupting proteins, such as lymphocytic granulysin or the human cathelicidin LL-37. Interestingly, not the cholesterol depletion but rather the simultaneous exposure of phosphatidylserine, a negatively charged phospholipid, triggers resistance of late stage parasitized red blood cells towards the eukaryotic pore forming protein perforin. Overall, by revealing the molecular events we establish here a pathogen-host interaction that involves host cell membrane remodeling that defines the susceptibility towards cytolytic molecules.

Role of Extracellular Vesicles in Cellular Cross Talk in Malaria.

Malaria infection caused by the Plasmodium species is a complex disease in which a fine balance between host and parasite factors determine the disease severity. While in some individuals, the infection will trigger only a mild and uncomplicated disease, other individuals will develop severe complications which lead to death. Extracellular vesicles (EVs) secreted by infected red blood cells (iRBCs), as well as other host cells, are important regulators of the balance that determines the disease outcome. In addition, EVs constitute a robust mode of cell-to-cell communication by transferring signaling cargoes between parasites, and between parasites and host, without requiring cellular contact. The transfer of membrane and cytosolic proteins, lipids, DNA, and RNA through EVs not only modulate the immune response, it also mediates cellular communication between parasites to synchronize the transmission stage. Here, we review the recent progress in understanding EV roles during malaria.

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.

Connexin45 expression in the human obstructed detrusor muscle.

PURPOSE: Patients with benign prostatic hyperplasia (BPH) and bladder outlet obstruction (BOO) frequently develop lower urinary tract symptoms (LUTS). To elucidate the underlying pathomechanisms we focused on altered cellular communication between detrusor cells. METHODS: Bladder biopsies were collected from eight BPH patients with compensated BOO and from eight non-obstructed patients. Detrusor areas were separated by laser capture microdissection microscopy, and extracted RNA was subjected to quantitative RT-PCR for connexin43 and connexin45. Furthermore, localization of connexin45 and lysosome membrane associated protein 1 was studied by immunohistochemistry. RESULTS: We found the human detrusor to express connexin45 rather than connexin43. Compared to controls, connexin45 expression was not significantly changed in detrusors of obstructed patients. However, connexin45 protein patterns were focally altered in obstruction. CONCLUSIONS: Our study is the first to provide evidence that connexin45-coupling of detrusor cells may be regionally impaired in patients with BOO due to BPH. The altered connexin45 coupling may contribute to LUTS.