Tyrosine in the hinge region of the pore-forming motif regulates oligomeric ß-barrel pore formation by Vibrio cholerae cytolysin.

ß-barrel pore-forming toxins perforate cell membranes by forming oligomeric ß-barrel pores. The most crucial step is the membrane-insertion of the pore-forming motifs that create the transmembrane ß-barrel scaffold. Molecular mechanism that regulates structural reorganization of these pore-forming motifs during ß-barrel pore-formation still remains elusive. Using Vibrio cholerae cytolysin as an archetypical example of the ß-barrel pore-forming toxin, we show that a key tyrosine residue (Y321) in the hinge region of the pore-forming motif plays crucial role in this process. Mutation of Y321 abrogates oligomerization of the membrane-bound toxin protomers, and blocks subsequent steps of pore-formation. Our study suggests that the presence of Y321 in the hinge region of the pore-forming motif is crucial for the toxin molecule to sense membrane-binding, and to trigger essential structural rearrangements required for the subsequent oligomerization and pore-formation process. Such a regulatory mechanism of pore-formation by V. cholerae cytolysin has not been documented earlier in the structurally related ß-barrel pore-forming toxins.

Interleukin-18 and cytotoxic impairment are independent and synergistic causes of murine virus-induced hyperinflammation.

Hemophagocytic lymphohistiocytosis (HLH) and macrophage activation syndrome (MAS) are life-threatening hyperinflammatory syndromes typically associated with underlying hematologic and rheumatic diseases, respectively. Familial HLH is associated with genetic cytotoxic impairment and thereby to excessive antigen presentation. Extreme elevation of serum interleukin-18 (IL-18) has been observed specifically in patients with MAS, making it a promising therapeutic target, but how IL-18 promotes hyperinflammation remains unknown. In an adjuvant-induced MAS model, excess IL-18 promoted immunopathology, whereas perforin deficiency had no effect. To determine the effects of excess IL-18 on virus-induced immunopathology, we infected Il18-transgenic (Il18tg) mice with lymphocytic choriomeningitis virus (LCMV; strain Armstrong). LCMV infection is self-limited in wild-type mice, but Prf1-/- mice develop prolonged viremia and fatal HLH. LCMV-infected Il18-transgenic (Il18tg) mice developed cachexia and hyperinflammation comparable to Prf1-/- mice, albeit with minimal mortality. Like Prf1-/- mice, immunopathology was largely rescued by CD8 depletion or interferon-γ (IFNg) blockade. Unlike Prf1-/- mice, they showed normal target cell killing and normal clearance of viral RNA and antigens. Rather than impairing cytotoxicity, excess IL-18 acted on T lymphocytes to amplify their inflammatory responses. Surprisingly, combined perforin deficiency and transgenic IL-18 production caused spontaneous hyperinflammation specifically characterized by CD8 T-cell expansion and improved by IFNg blockade. Even Il18tg;Prf1-haplosufficient mice demonstrated hyperinflammatory features. Thus, excess IL-18 promotes hyperinflammation via an autoinflammatory mechanism distinct from, and synergistic with, cytotoxic impairment. These data establish IL-18 as a potent, independent, and modifiable driver of life-threatening innate and adaptive hyperinflammation and support the rationale for an IL-18-driven subclass of hyperinflammation.

Impaired immune surveillance accelerates accumulation of senescent cells and aging.

Cellular senescence is a stress response that imposes stable cell-cycle arrest in damaged cells, preventing their propagation in tissues. However, senescent cells accumulate in tissues in advanced age, where they might promote tissue degeneration and malignant transformation. The extent of immune-system involvement in regulating age-related accumulation of senescent cells, and its consequences, are unknown. Here we show that Prf1-/- mice with impaired cell cytotoxicity exhibit both higher senescent-cell tissue burden and chronic inflammation. They suffer from multiple age-related disorders and lower survival. Strikingly, pharmacological elimination of senescent-cells by ABT-737 partially alleviates accelerated aging phenotype in these mice. In LMNA+/G609G progeroid mice, impaired cell cytotoxicity further promotes senescent-cell accumulation and shortens lifespan. ABT-737 administration during the second half of life of these progeroid mice abrogates senescence signature and increases median survival. Our findings shed new light on mechanisms governing senescent-cell presence in aging, and could motivate new strategies for regenerative medicine.

Polymicrobial sepsis influences NK-cell-mediated immunity by diminishing NK-cell-intrinsic receptor-mediated effector responses to viral ligands or infections.

The sepsis-induced cytokine storm leads to severe lymphopenia and reduced effector capacity of remaining/surviving cells. This results in a prolonged state of immunoparalysis, that contributes to enhanced morbidity/mortality of sepsis survivors upon secondary infection. The impact of sepsis on several lymphoid subsets has been characterized, yet its impact on NK-cells remains underappreciated-despite their critical role in controlling infection(s). Here, we observed numerical loss of NK-cells in multiple tissues after cecal-ligation-and-puncture (CLP)-induced sepsis. To elucidate the sepsis-induced lesions in surviving NK-cells, transcriptional profiles were evaluated and indicated changes consistent with impaired effector functionality. A corresponding deficit in NK-cell capacity to produce effector molecules following secondary infection and/or cytokine stimulation (IL-12,IL-18) further suggested a sepsis-induced NK-cell intrinsic impairment. To specifically probe NK-cell receptor-mediated function, the activating Ly49H receptor, that recognizes the murine cytomegalovirus (MCMV) m157 protein, served as a model receptor. Although relative expression of Ly49H receptor did not change, the number of Ly49H+ NK-cells in CLP hosts was reduced leading to impaired in vivo cytotoxicity and the capacity of NK-cells (on per-cell basis) to perform Ly49H-mediated degranulation, killing, and effector molecule production in vitro was also severely reduced. Mechanistically, Ly49H adaptor protein (DAP12) activation and clustering, assessed by TIRF microscopy, was compromised. This was further associated with diminished AKT phosphorylation and capacity to flux calcium following receptor stimulation. Importantly, DAP12 overexpression in NK-cells restored Ly49H/D receptors-mediated effector functions in CLP hosts. Finally, as a consequence of sepsis-dependent numerical and functional lesions in Ly49H+ NK-cells, host capacity to control MCMV infection was significantly impaired. Importantly, IL-2 complex (IL-2c) therapy after CLP improved numbers but not a function of NK-cells leading to enhanced immunity to MCMV challenge. Thus, the sepsis-induced immunoparalysis state includes numerical and NK-cell-intrinsic functional impairments, an instructive notion for future studies aimed in restoring NK-cell immunity in sepsis survivors.

Cholangiocarcinoma-derived exosomes inhibit the antitumor activity of cytokine-induced killer cells by down-regulating the secretion of tumor necrosis factor-α and perforin.

OBJECTIVE: The aim of our study is to observe the impact of cholangiocarcinoma-derived exosomes on the antitumor activities of cytokine-induced killer (CIK) cells and then demonstrate the appropriate mechanism. METHODS: Tumor-derived exosomes (TEXs), which are derived from RBE cells (human cholangiocarcinoma line), were collected by ultracentrifugation. CIK cells induced from peripheral blood were stimulated by TEXs. Fluorescence-activated cell sorting (FACS) was performed to determine the phenotypes of TEX-CIK and N-CIK (normal CIK) cells. The concentrations of tumor necrosis factor-α (TNF-α) and perforin in the culture medium supernatant were examined by using an enzyme-linked immunosorbent assay (ELISA) kit. A CCK-8 kit was used to evaluate the cytotoxic activity of the CIK cells to the RBE cell line. RESULTS: The concentrations of TNF-α and perforin of the group TEX-CIK were 138.61 pg/ml and 2.41 ng/ml, respectively, lower than those of the group N-CIK 194.08 pg/ml (P<0.01) and 3.39 ng/ml (P<0.05). The killing rate of the group TEX-CIK was 33.35%, lower than that of the group N-CIK (47.35% (P<0.01)). The population of CD3(+), CD8(+), NK (CD56(+)), and CD3(+)CD56(+) cells decreased in the TEX-CIK group ((63.2±6.8)%, (2.5±1.0)%, (0.53±0.49)%, (0.45±0.42)%) compared with the N-CIK group ((90.3±7.3)%, (65.7±3.3)%, (4.2±1.2)%, (15.2±2.7)%), P<0.01. CONCLUSIONS: Our results suggest that RBE cells-derived exosomes inhibit the antitumor activity of CIK cells by down-regulating the population of CD3(+), CD8(+), NK (CD56(+)), and CD3(+)CD56(+) cells and the secretion of TNF-α and perforin. TEX may play an important role in cholangiocarcinoma immune escape.

Introduction: brief historical overview.

Membranes are essential in defining the border and ensuring function of all living cells. As such they are vulnerable and have been a preferred target of attack throughout evolution. The most powerful way of damaging a membrane is through the insertion of pore-forming proteins. Research over the last decades shows that such proteins are produced by bacteria to attack bacterial or eukaryotic cells, vertebrates to kill invading organisms or infected cells, and by eukaryotic cells to "kill" mitochondria and trigger apoptosis. The breadth of effect of these proteins is bringing together, in a very exciting way, research communities that used to be unaware of each other.

Perforin: a key pore-forming protein for immune control of viruses and cancer.

Perforin (PFN) is the key pore-forming molecule in the cytotoxic granules of immune killer cells. Expressed only in killer cells, PFN is the rate-limiting molecule for cytotoxic function, delivering the death-inducing granule serine proteases (granzymes) into target cells marked for immune elimination. In this chapter we describe our current understanding of how PFN accomplishes this task. We discuss where PFN is expressed and how its expression is regulated, the biogenesis and storage of PFN in killer cells and how they are protected from potential damage, how it is released, how it delivers Granzymes into target cells and the consequences of PFN deficiency.

Perforin and human diseases.

Natural killer (NK) cells and cytotoxic T lymphocytes (CTL) use a highly toxic pore-forming protein perforin (PFN) to destroy cells infected with intracellular pathogens and cells with pre-cancerous transformations. However, mutations of PFN and defects in its expression can cause an abnormal function of the immune system and difficulties in elimination of altered cells. As discussed in this chapter, deficiency of PFN due to the mutations of its gene, PFN1, can be associated with malignancies and severe immune disorders such as familial hemophagocytic lymphohistiocytosis (FHL) and macrophage activation syndrome. On the other hand, overactivity of PFN can turn the immune system against autologous cells resulting in other diseases such as systemic lupus erythematosus, polymyositis, rheumatoid arthritis and cutaneous inflammation. PFN also has a crucial role in the cellular rejection of solid organ allografts and destruction of pancreatic ß-cells resulting in type 1 diabetes. These facts highlight the importance of understanding the biochemical characteristics of PFN.

The role of MACPF proteins in the biology of malaria and other apicomplexan parasites.

Apicomplexans are eukaryotic parasites of major medical and veterinary importance. They have complex life cycles through frequently more than one host, interact with many cell types in their hosts, and can breach host cell membranes during parasite traversal of, or egress from, host cells. Some of these parasites make a strikingly heavy use of the pore-forming MACPF domain, and encode up to 10 different MACPF domain-containing proteins. In this chapter, we focus on the two most studied and medically important apicomplexans, Plasmodium and Toxoplasma, and describe the known functions of their MACPF polypeptide arsenal. Apicomplexan MACPF proteins appear to be involved in a variety of membrane-damaging events, making them an attractive model to dissect the structure-function relationships of the MACPF domain.

Chlamydial MACPF protein CT153.

Chlamydiae are obligate intracellular bacterial parasites that infect a wide range of metazoan hosts. Some Chlamydia species are important causes of chronic inflammatory diseases of the ocular, genital and respiratory tracts in humans. Genes located in a variable region of chlamydial genomes termed the plasticity zone are known to be key determinants of pathogenic diversity. The plasticity zone protein CT153, present only in select species, contains a membrane attack complex/perforin (MACPF) domain, which may mediate chlamydial interactions with the host cell. CT153 is present throughout the C. trachomatis developmental cycle and is processed into polypeptides that interact with membranes differently than does the parent protein. Chlamydiae interact extensively with membranes from the time of invasion until they eventually exit host cells, so numerous roles for a MACPF protein in pathogenesis of these pathogens are conceivable. Here, we present an overview of what is known about CT153 and highlight potential roles of a MACPF family protein in a group of pathogens whose intracellular development is marked by a series of interactions with host cell membranes and organelles. Finally, we identify new strategies for identifying CT153 functions made feasible by the recent development of a basic toolset for genetic manipulation of chlamydiae.

Fungal MACPF-like proteins and aegerolysins: bi-component pore-forming proteins?

Proteins with membrane-attack complex/perforin (MACPF) domains are found in almost all kingdoms of life, and they have a variety of biological roles, including defence and attack, organism development, and cell adhesion and signalling. The distribution of these proteins in fungi appears to be restricted to some Pezizomycotina and Basidiomycota species only, in correlation with another group of proteins with unknown biological function, known as aegerolysins. These two protein groups coincide in only a few species, and they might operate in concert as cytolytic bi-component pore-forming agents. Representative proteins here include pleurotolysin B, which has a MACPF domain, and the aegerolysin-like protein pleurotolysin A, and the very similar ostreolysin A, which have been purified from oyster mushroom (Pleurotus ostreatus). These have been shown to act in concert to perforate natural and artificial lipid membranes with high cholesterol and sphingomyelin content. The aegerolysin-like proteins provide the membrane cholesterol/sphingomyelin selectivity and recruit oligomerised pleurotolysin B molecules, to create a membrane-inserted pore complex. The resulting protein structure has been imaged with electron microscopy, and it has a 13-meric rosette-like structure, with a central lumen that is ~4-5 nm in diameter. The opened transmembrane pore is non-selectively permeable for ions and smaller neutral solutes, and is a cause of cytolysis of a colloid-osmotic type. The biological significance of these proteins for the fungal life-style is discussed.

Perforin- and granulysin-mediated cytotoxicity and interleukin 15 play roles in neurocognitive impairment in patients with acute lymphoblastic leukaemia.

Acute lymphoblastic leukaemia (ALL) is an aggressive disease. The course of disease is regulated by pro-inflammatory agents, and malignant cell infiltration of tissues plays a deleterious role in disease progression, greatly impacting quality of life, especially in the cognitive domains. Our hypothesis is that significant serum concentrations of interleukin 15 (IL-15) are responsible for higher expression of adhesion molecules on endothelial cells of blood-brain barrier (BBB) which allow leukaemia cells and/or normal lymphocytes the infiltration into the brain. In brain tissue these cells could be stimulated to release perforin and granulysin causing induction of apoptosis in brain cells that are involved in complex neural signalling mediated by neurotransmitters, and consequent fine cognitive impairment. Such changes could be detected early, even before notable clinical psycho-neurological or radiological changes in patients with ALL. To evaluate this hypothesis we propose measuring cognitive function using Complex Reactiometer Drenovac (CRD) scores in patients with ALL. The expression of different adhesion molecules on BBB as well as presence and distribution of different lymphocytes in brain tissue will be analyzed. We will then correlate CRD scores with levels of IL-15 and the percentages of T cells, natural killer T cells, and natural killer cells expressing perforin and/or granulysin proteins. CRD is a scientifically recognised and highly sensitive psychometric laboratory test based on the complex chronometric mathematical measuring of speed of reaction to various stimuli. It provides an objective assessment of cognitive functions from the most complex mental activities to the simplest reaction reflexes. Early recognition of cognitive dysfunction might be important when selecting the most appropriate chemotherapy and/or radiotherapy regimens, and could allow for the implementation of preventive measures against further deterioration in cognitive function and quality of life in patients with ALL.

Perforin and granzyme B have separate and distinct roles during atherosclerotic plaque development in apolipoprotein E knockout mice.

The granzyme B/perforincytotoxic pathway is a well established mechanism of initiating target cell apoptosis. Previous studies have suggested a role for the granzyme B/perforin cytotoxic pathway in vulnerable atherosclerotic plaque formation. In the present study, granzyme B deficiency resulted in reduced atherosclerotic plaque development in the descending aortas of apolipoprotein E knockout mice fed a high fat diet for 30 weeks while perforindeficiency resulted in greater reduction in plaque development with significantly less plaque area than granzyme Bdeficient mice. In contrast to the descending aorta, no significant change in plaque size was observed in aortic roots from either granzyme Bdeficient or perforindeficient apolipoprotein E knockout mice. However, atherosclerotic plaques in the aortic roots did exhibit significantly more collagen in granzyme B, but not perforin deficient mice. Together these results suggest significant, yet separate roles for granzyme B and perforin in the pathogenesis of atherosclerosis that go beyond the traditional apoptotic pathway with additional implications in plaque development, stability and remodelling of extracellular matrix.

Perforin deficiency impairs a critical immunoregulatory loop involving murine CD8(+) T cells and dendritic cells.

Humans and mice with impaired perforin-dependent cytotoxic function may develop excessive T-cell activation and the fatal disorder hemophagocytic lymphohistiocytosis (HLH) after infection. Though cytotoxic lymphocytes can kill antigen-presenting cells, the physiological mechanism of perforin-mediated immune regulation has never been demonstrated in a disease-relevant context. We used a murine model of HLH to examine how perforin controls immune activation, and we have defined a feedback loop that is critical for immune homeostasis. This endogenous feedback loop involves perforin-dependent elimination of rare, antigen-presenting dendritic cells (DCs) by CD8(+) T cells and has a dominant influence on the magnitude of T-cell activation after viral infection. Antigen presentation by a minor fraction of DCs persisted in T-cell- or perforin-deficient animals and continued to drive T-cell activation well beyond initial priming in the latter animals. Depletion of DCs or transfer of perforin-sufficient T cells dampened endogenous DC antigen presentation and T-cell activation, demonstrating a reciprocal relationship between perforin in CD8(+) T cells and DC function. Thus, selective cytotoxic "pruning" of DC populations by CD8(+) T cells limits T-cell activation and protects against the development of HLH and potentially other immunopathological conditions.

Controversies in granzyme biology.

Granzymes (Grz) are a family of serine proteases found in the granules of cytotoxic lymphocytes and are emerging as an important group of proteins involved in immune function and surveillance. Grz have both cytotoxic and more recently reported non-cytotoxic roles, however these functions are still subject to thorough investigation. The significance of the cytotoxic and importantly the non-cytotoxic roles of Grz will be discussed in this review, detailing accepted and controversial functions.

Naturally occurring regulatory T cells: markers, mechanisms, and manipulation.

Naturally occurring CD4(+)CD25(high) forkhead box protein 3 (FOXP3)(+) regulatory T cells (nTregs) are key mediators of immunity, which orchestrate and maintain tolerance to self and foreign antigens. In the recent 1.5 decades, a multitude of studies have aimed to define the phenotype and function of nTregs and to assess their therapeutic potential for modulating immune mediated disorders such as autoimmunity, allergy, and episodes of transplant rejection. In this review, we summarize the current knowledge on the biology of nTregs. We address the exact definition of nTregs by specific markers and combinations thereof, which is a prerequisite for the state-of-the-art isolation of defined nTreg populations. Furthermore, we discuss the mechanism by which nTregs mediate immunosuppression and how this knowledge might translate into novel therapeutic modalities. With first clinical studies of nTreg-based therapies being finished, questions concerning the reliable sources of nTregs are becoming more and more eminent. Consequently, approaches allowing conversion of CD4(+) T cells into nTregs by coculture with antigen-presenting cells, cytokines, and/or pharmacological agents are discussed. In addition, genetic engineering approaches for the generation of antigen-specific nTregs are described.

A possible role for perforin and granzyme B in resveratrol-enhanced radiosensitivity of prostate cancer.

Perforin and granzyme B are expressed primarily by activated lymphocytes (cytotoxic T cells, natural killer cells, and natural killer T cells) and function together to induce apoptosis of target cells. Typically, these proteins are not expressed in tumor cells. In the present study, we established the constitutive expression of perforin and granzyme B by the PC-3 and DU145 prostate cancer (PCA) cell lines with reverse transcription polymerase chain reaction, immunohistochemistry, Western blot, or a combination of techniques. The combination of radiation and resveratrol (XRT/RSV) additively/synergistically decreased survival of PCA because, at least in part, of increased apoptosis. We further demonstrated that treatment with RSV up-regulated the expression of both perforin and granzyme B, whereas treatment with XRT up-regulated the expression of granzyme B, but not that of perforin. Combined XRT/RSV treatment of PCA cells further increased the expression of both perforin and granzyme B compared with RSV or XRT alone. Thus, increased radiosensitivity of prostate cancer cells induced by RSV correlated with up-regulation of perforin and granzyme B, demonstrating a possible mechanism for tumor apoptosis. These findings might be helpful in devising new strategies for treating PCA.

Critical role for perforin and Fas-dependent killing of dendritic cells in the control of inflammation.

After stimulation of antigen-specific T cells, dendritic cell (DCs) are susceptible to killing by these activated T cells that involve perforin and Fas-dependent mechanisms. Fas-dependent DC apoptosis has been shown to limit DC accumulation and prevent the development of autoimmunity. However, a role for perforin in the maintenance of DC homeostasis for immune regulation remains to be determined. Here we show that perforin deficiency in mice, together with the deletion of Fas in DCs (perforin(-/-)DC-Fas(-/-)), led to DC accumulation, uncontrolled T-cell activation, and IFN-γ production by CD8+ T cells, resulting in the development of lethal hemophagocytic lymphohistiocytosis. Consistently, adoptive transfer of Fas(-/-) DCs induced over-activation and IFN-γ production in perforin(-/-) CD8+ T cells. Neutralization of IFN-γ prevented the spreading of inflammatory responses to different cell types and protected the survival of perforin(-/-)DC-Fas(-/-) mice. Our data suggest that perforin and Fas synergize in the maintenance of DC homeostasis to limit T cell activation, and prevent the initiation of an inflammatory cascade.

Perforin activity at membranes leads to invaginations and vesicle formation.

The cytotoxic cell granule secretory pathway is essential for immune defence. How the pore-forming protein perforin (PFN) facilitates the cytosolic delivery of granule-associated proteases (granzymes) remains enigmatic. Here we show that PFN is able to induce invaginations and formation of complete internal vesicles in giant unilamellar vesicles. Formation of internal vesicles depends on native PFN and calcium and antibody labeling shows the localization of PFN at the invaginations. This vesiculation is recapitulated in large unilamellar vesicles and in this case PFN oligomers can be seen associated with the necks of the invaginations. Capacitance measurements show PFN is able to increase a planar lipid membrane surface area in the absence of pore formation, in agreement with the ability to induce invaginations. Finally, addition of PFN to Jurkat cells causes the formation of internal vesicles prior to pore formation. PFN is capable of triggering an endocytosis-like event in addition to pore formation, suggesting a new paradigm for its role in delivering apoptosis-inducing granzymes into target cells.