RESUMO
My path to becoming a scientist has taken many twists and turns. This is perhaps not unusual to hear. Indeed, in discussions with my colleagues it seems that for many of us the path was never a straight one. Certainly, for me there have been moments when my whole world was encompassed by science and at other times, I have felt strongly that my time in science was up. I like to think that as scientists we ask a lot of questions and, for many of us, those questions extend to our very purpose as a scientist. My intention with this article is not to document my career path in detail or to provide very specific advice. Rather, I hope to describe how questions have defined my journey and to inspire others to occasionally pause and ask themselves what a career in science means to them. Today, I am an Assistant Professor at a major Canadian university, and here are the questions I asked along the way.
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Cross-presentation of dead cell-associated antigens by conventional dendritic cells type 1 (cDC1s) is critical for CD8+ T cells response against many tumors and viral infections. It is facilitated by DNGR-1 (CLEC9A), an SYK-coupled cDC1 receptor that detects dead cell debris. Here, we report that DNGR-1 engagement leads to rapid activation of CBL and CBL-B E3 ligases to cause K63-linked ubiquitination of SYK and terminate signaling. Genetic deletion of CBL E3 ligases or charge-conserved mutation of target lysines within SYK abolishes SYK ubiquitination and results in enhanced DNGR-1-dependent antigen cross-presentation. We also find that cDC1 deficient in CBL E3 ligases are more efficient at cross-priming CD8+ T cells to dead cell-associated antigens and promoting host resistance to tumors. Our findings reveal a role for CBL-dependent ubiquitination in limiting cross-presentation of dead cell-associated antigens and highlight an axis of negative regulation of cDC1 activity that could be exploited to increase anti-tumor immunity.
Assuntos
Apresentação Cruzada , Ubiquitina-Proteína Ligases , Linfócitos T CD8-Positivos , Proteínas Proto-Oncogênicas c-cbl , Ubiquitinação , Células Dendríticas , Quinase SykRESUMO
Dual-wavelength and dual-fluorophore ratiometric imaging has become a powerful tool for the study of pH in intracellular compartments. It allows for the dynamic imaging of live cells while accounting for changes in the focal plane, differential loading of the fluorescent probe, and photobleaching caused by repeated image acquisitions. Ratiometric microscopic imaging has the added advantage over whole-population methods of being able to resolve individual cells and even individual organelles. In this chapter, we provide a detailed discussion of the basic principles of ratiometric imaging and its application to the measurement of phagosomal pH, including probe selection, the necessary instrumentation, and calibration methods.
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Corantes Fluorescentes , Fagossomos , Microscopia de Fluorescência/métodos , Ionóforos , Concentração de Íons de Hidrogênio , Espectrometria de FluorescênciaRESUMO
The phagosome is a redox-active organelle. Numerous reductive and oxidative systems play both direct and indirect roles in phagosomal function. With the advent of newer methodologies to study these redox events in live cells, the details of how redox conditions change within the maturing phagosome, how they are regulated, and how they influence other phagosomal functions can be investigated. In this chapter, we detail phagosome-specific, fluorescence-based assays that measure disulfide reduction and the production of reactive oxygen species in live phagocytes such as macrophages and dendritic cells, in real time.
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Macrófagos , Fagossomos , Fagossomos/metabolismo , Oxirredução , Espécies Reativas de Oxigênio/metabolismo , Estresse OxidativoRESUMO
Recruited neutrophils are among the first phagocytic cells to interact with the phagosomal pathogen Leishmania following inoculation into the mammalian dermis. Analysis of Leishmania-infected neutrophils has revealed alterations in neutrophil viability, suggesting that the parasite can both induce or inhibit apoptosis. In this study, we demonstrate that entry of Leishmania major into murine neutrophils is dependent on the neutrophil surface receptor CD11b (CR3/Mac-1) and is enhanced by parasite opsonization with C3. Infected neutrophils underwent robust NADPH oxidase isoform 2 (NOX2)-dependent respiratory burst based on detection of reactive oxygen species within the phagolysosome but largely failed to eliminate the metacyclic promastigote life cycle stage of the parasite. Infected neutrophils displayed an "apoptotic" phosphatidylserine (PS)-positive phenotype, which was induced by both live and fixed parasites but not latex beads, suggesting that PS expression was parasite specific but does not require active infection. In addition, neutrophils from parasite/neutrophil coculture had increased viability, decreased caspase 3, 8, and 9 gene expression, and reduced protein levels of both the pro and cleaved forms of the classical apoptosis-inducing executioner caspase, Caspase 3. Our data suggest that CD11b-mediated Leishmania internalization initiates respiratory burst and PS externalization, followed by a reduction in both the production and cleavage of caspase 3, resulting in a phenotypic state of "stalled apoptosis."
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Leishmania major , Parasitos , Animais , Camundongos , Apoptose , Caspase 3/metabolismo , Leishmania major/metabolismo , Antígeno de Macrófago 1/metabolismo , Mamíferos/metabolismo , Neutrófilos/metabolismo , Parasitos/metabolismo , Explosão RespiratóriaRESUMO
Phagocytes, such as macrophages and dendritic cells, possess the ability to ingest large quantities of exogenous material into membrane-bound endocytic organelles such as macropinosomes and phagosomes. Typically, the ingested material, which consists of diverse macromolecules such as proteins and nucleic acids, is delivered to lysosomes where it is digested into smaller molecules like amino acids and nucleosides. These smaller molecules can then be exported out of the lysosomes by transmembrane transporters for incorporation into the cell's metabolic pathways or for export from the cell. There are, however, exceptional instances when undigested macromolecules escape degradation and are instead delivered across the membrane of endocytic organelles into the cytosol of the phagocyte. For example, double stranded DNA, a damage associated molecular pattern shed by necrotic tumor cells, is endocytosed by phagocytes in the tumor microenvironment and delivered to the cytosol for detection by the cytosolic "danger" sensor cGAS. Other macromolecular "danger" signals including lipopolysaccharide, intact proteins, and peptidoglycans can also be actively transferred from within endocytic organelles to the cytosol. Despite the obvious biological importance of these processes, we know relatively little of how macromolecular "danger" signals are transferred across endocytic organelle membranes for detection by cytosolic sensors. Here we review the emerging evidence for the active cytosolic transfer of diverse macromolecular "danger" signals across endocytic organelle membranes. We will highlight developing trends and discuss the potential molecular mechanisms driving this emerging phenomenon.
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Endossomos , Fagossomos , Citosol/metabolismo , Endocitose , Endossomos/metabolismo , Substâncias Macromoleculares/metabolismo , Macrófagos/metabolismo , Fagossomos/metabolismoRESUMO
Phagocytes play critical roles in the maintenance of organismal homeostasis and immunity. Central to their role is their ability to take up and process exogenous material via the related processes of phagocytosis and macropinocytosis. The mechanisms and functions underlying macropinocytosis have remained severely understudied relative to phagocytosis. In recent years, however, there has been a renaissance in macropinocytosis research. Phagocytes can engage in various forms of macropinocytosis including an "induced" form and a "constitutive" form. This chapter, however, will focus on constitutive macropinocytosis and its role in the maintenance of immunity. Functions previously attributed to macropinocytosis, including antigen presentation and immune surveillance, will be revisited in light of recent revelations and emerging concepts will be highlighted.
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Fagócitos , Pinocitose , Apresentação de Antígeno , Homeostase , FagocitoseRESUMO
The membranes of endosomes, phagosomes and macropinosomes can become damaged by the physical properties of internalized cargo, by active pathogenic invasion or by cellular processes, including endocytic maturation. Loss of membrane integrity is often deleterious and is, therefore, prevented by mitigation and repair mechanisms. However, it can occasionally be beneficial and actively induced by cells. Here, we summarize the mechanisms by which cells, in particular phagocytes, try to prevent membrane damage and how, when this fails, they repair or destroy damaged endocytic organelles. We also detail how one type of phagocyte, the dendritic cell, can deliberately trigger localized damage to endocytic organelles to allow for major histocompatibility complex class I presentation of exogenous antigens and initiation of CD8+ T-cell responses to viruses and tumours. Our review highlights mechanisms for the regulation of endocytic organelle membrane integrity at the intersection of cell biology and immunology that could be co-opted for improving vaccination and intracellular drug delivery.
Assuntos
Endossomos/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Fagócitos/metabolismo , Animais , Apresentação de Antígeno , Linfócitos T CD8-Positivos/metabolismo , Apresentação Cruzada , HumanosRESUMO
In recent years, the field of macropinocytosis has grown rapidly. Macropinocytosis has emerged as a central mechanism by which innate immune cells maintain organismal homeostasis and immunity. Simultaneously, and in contrast to its homeostatic role, it can also drive various pathologies, including cancer and viral infections. Unlike other modes of endocytosis, the tools developed for studying the maturation of macropinosomes remain underdeveloped. Here the protocol describes newly developed tools for studying the redox environment within the lumen of early and maturing macropinosomes. Methodologies for using ratiometric fluorescence microscopy in assessing the pH, production of reactive oxygen species, and the degradative capacity within the lumen of individual macropinosomes in live cells are described. Single organelle measurements offer the advantage of revealing spatiotemporal heterogeneity, which is often lost with population-based approaches. Emphasis is placed on the basic principles of dual fluorophore ratiometric microscopy, including probe selection, instrumentation, calibration, and single-cell versus population-based methods.
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Endossomos , Pinocitose , Endossomos/metabolismo , Concentração de Íons de Hidrogênio , Microscopia de Fluorescência , OxirreduçãoRESUMO
The ongoing pandemic of SARS-CoV-2 calls for rapid and cost-effective methods to accurately identify infected individuals. The vast majority of patient samples is assessed for viral RNA presence by RT-qPCR. Our biomedical research institute, in collaboration between partner hospitals and an accredited clinical diagnostic laboratory, established a diagnostic testing pipeline that has reported on more than 252,000 RT-qPCR results since its commencement at the beginning of April 2020. However, due to ongoing demand and competition for critical resources, alternative testing strategies were sought. In this work, we present a clinically-validated procedure for high-throughput SARS-CoV-2 detection by RT-LAMP in 25 minutes that is robust, reliable, repeatable, sensitive, specific, and inexpensive.
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Cross-presentation of antigens from dead tumor cells by type 1 conventional dendritic cells (cDC1s) is thought to underlie priming of anti-cancer CD8+ T cells. cDC1 express high levels of DNGR-1 (a.k.a. CLEC9A), a receptor that binds to F-actin exposed by dead cell debris and promotes cross-presentation of associated antigens. Here, we show that secreted gelsolin (sGSN), an extracellular protein, decreases DNGR-1 binding to F-actin and cross-presentation of dead cell-associated antigens by cDC1s. Mice deficient in sGsn display increased DNGR-1-dependent resistance to transplantable tumors, especially ones expressing neoantigens associated with the actin cytoskeleton, and exhibit greater responsiveness to cancer immunotherapy. In human cancers, lower levels of intratumoral sGSN transcripts, as well as presence of mutations in proteins associated with the actin cytoskeleton, are associated with signatures of anti-cancer immunity and increased patient survival. Our results reveal a natural barrier to cross-presentation of cancer antigens that dampens anti-tumor CD8+ T cell responses.
Assuntos
Apresentação Cruzada/imunologia , Gelsolina/metabolismo , Imunidade , Lectinas Tipo C/metabolismo , Neoplasias/imunologia , Receptores Imunológicos/metabolismo , Receptores Mitogênicos/metabolismo , Actinas/metabolismo , Sequência de Aminoácidos , Animais , Antígenos de Neoplasias/metabolismo , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Apresentação Cruzada/efeitos dos fármacos , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Gelsolina/química , Gelsolina/deficiência , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inibidores de Checkpoint Imunológico/farmacologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Imunidade/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Mutação/genética , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/patologia , Ligação Proteica/efeitos dos fármacos , Análise de SobrevidaRESUMO
Type 1 conventional dendritic (cDC1) cells are necessary for cross-presentation of many viral and tumor antigens to CD8+ T cells. cDC1 cells can be identified in mice and humans by high expression of DNGR-1 (also known as CLEC9A), a receptor that binds dead-cell debris and facilitates XP of corpse-associated antigens. Here, we show that DNGR-1 is a dedicated XP receptor that signals upon ligand engagement to promote phagosomal rupture. This allows escape of phagosomal contents into the cytosol, where they access the endogenous major histocompatibility complex class I antigen processing pathway. The activity of DNGR-1 maps to its signaling domain, which activates SYK and NADPH oxidase to cause phagosomal damage even when spliced into a heterologous receptor and expressed in heterologous cells. Our data reveal the existence of innate immune receptors that couple ligand binding to endocytic vesicle damage to permit MHC class I antigen presentation of exogenous antigens and to regulate adaptive immunity.
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Apresentação de Antígeno , Apresentação Cruzada , Células Dendríticas/metabolismo , Lectinas Tipo C/metabolismo , Fagossomos/metabolismo , Receptores Imunológicos/metabolismo , Receptores Mitogênicos/metabolismo , Linfócitos T/metabolismo , Animais , Morte Celular , Técnicas de Cocultura , Células Dendríticas/imunologia , Células HEK293 , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Lectinas Tipo C/genética , Ligantes , Camundongos , NADPH Oxidases/metabolismo , Fagossomos/genética , Fagossomos/imunologia , Fosforilação , Células RAW 264.7 , Espécies Reativas de Oxigênio/metabolismo , Receptores Imunológicos/genética , Receptores Mitogênicos/genética , Transdução de Sinais , Quinase Syk/metabolismo , Linfócitos T/imunologiaRESUMO
Macropinocytosis has received increasing attention in recent years for its various roles in nutrient acquisition, immune surveillance, and virus and cancer pathologies. In most cases macropinocytosis is initiated by the sudden increase in an external stimulus such as a growth factor. This "induced" form of macropinocytosis has been the subject of much of the work addressing its mechanism and function over the years. An alternative, "constitutive" form of macropinocytosis restricted to primary innate immune cells also exists, although its mechanism has remained severely understudied. This mini-review focuses on the very recent advances that have shed new light on the initiation, formation and functional relevance of constitutive macropinocytosis in primary innate immune cells. An emphasis is placed on how this new understanding of constitutive macropinocytosis is helping to define the sentinel function of innate immune cells including polarized macrophages and dendritic cells.
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Imunidade Inata , Vigilância Imunológica , Pinocitose/fisiologia , Animais , Apresentação de Antígeno/imunologia , Biomarcadores , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Humanos , Macrófagos/imunologia , Macrófagos/metabolismo , Receptores de Reconhecimento de Padrão/metabolismo , Transdução de SinaisRESUMO
Human mesenchymal stem/stromal cells (MSCs) have been reported to produce an M2-like, alternatively activated phenotype in macrophages. In addition, MSCs mediate effective bacterial clearance in pre-clinical sepsis models. Thus, MSCs have a paradoxical antimicrobial and anti-inflammatory response that is not understood.Here, we studied the phenotypic and functional response of monocyte-derived human macrophages to MSC exposure in vitroMSCs induced two distinct, coexistent phenotypes: M2-like macrophages (generally elongated morphology, CD163+, acute phagosomal acidification, low NOX2 expression and limited phagosomal superoxide production) and M1-like macrophages characterised by high levels of phagosomal superoxide production. Enhanced phagosomal reactive oxygen species production was also observed in alveolar macrophages from a rodent model of pneumonia-induced sepsis. The production of M1-like macrophages was dependent on prostaglandin E2 and phosphatidylinositol 3-kinase. MSCs enhanced human macrophage phagocytosis of unopsonised bacteria and enhanced bacterial killing compared with untreated macrophages. Bacterial killing was significantly reduced by blockade of NOX2 using diphenyleneiodonium, suggesting that M1-like cells are primarily responsible for this effect. MSCs also enhanced phagocytosis and polarisation of M1-like macrophages derived from patients with severe sepsis.The enhanced antimicrobial capacity (M1-like) and inflammation resolving phenotype (M2-like) may account for the paradoxical effect of these cells in sepsis in vivo.
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Infecções por Escherichia coli/imunologia , Macrófagos Alveolares/citologia , Células-Tronco Mesenquimais/citologia , NADPH Oxidase 2/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Sepse/imunologia , Animais , Diferenciação Celular , Técnicas de Cocultura , Humanos , Ativação de Macrófagos , Macrófagos Alveolares/microbiologia , Células-Tronco Mesenquimais/microbiologia , Fagocitose , Ratos Sprague-DawleyRESUMO
Macropinocytosis mediates the uptake of antigens and of nutrients that dictate the regulation of cell growth by mechanistic target of rapamycin complex 1 (mTORC1). Because these functions differ in proinflammatory and anti-inflammatory macrophages, we compared the macropinocytic ability of two extreme polarization states. We found that anti-inflammatory macrophages perform vigorous macropinocytosis constitutively, while proinflammatory cells are virtually inactive. The total cellular content of Rho-family GTPases was higher in anti-inflammatory cells, but this disparity failed to account for the differential macropinocytic activity. Instead, reduced activity of Rac/RhoG was responsible for the deficient macropinocytosis of proinflammatory macrophages, as suggested by the stimulatory effects of heterologously expressed guanine nucleotide-exchange factors or of constitutively active (but not wild-type) forms of these GTPases. Similarly, differences in the activation state of phosphatidylinositol 3-kinase (PtdIns3K) correlated with the macropinocytic activity of pro- and anti-inflammatory macrophages. Differences in PtdIns3K and Rho-GTPase activity were attributable to the activity of calcium-sensing receptors (CaSRs), which appear to be functional only in anti-inflammatory cells. However, agonists of PtdIns3K, including cytokines, chemokines, and LPS, induced macropinocytosis in proinflammatory cells. Our findings revealed a striking difference in the macropinocytic ability of pro- and anti-inflammatory macrophages that correlates with their antigen-presenting and metabolic activity.
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Inflamação/patologia , Macrófagos/patologia , Pinocitose , Biomarcadores/metabolismo , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Polaridade Celular/efeitos dos fármacos , Células Cultivadas , Citocinas/farmacologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Humanos , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Fenótipo , Fosfatidilinositol 3-Quinases/metabolismo , Pinocitose/efeitos dos fármacos , Receptores de Detecção de Cálcio/metabolismo , Proteínas rho de Ligação ao GTP/metabolismoRESUMO
Professional phagocytes continuously extend dynamic, actin-driven membrane protrusions. These protrusions, often referred to as membrane ruffles, serve a critical role in the essential phagocyte processes of macropinocytosis and phagocytosis. Small GTPases, such as RAC1/2, spatially and temporally regulate membrane ruffle formation. We have recently shown that extracellular calcium regulates the elaboration of membrane ruffles primarily through the synthesis of phosphatidic acid (PtdOH) at the plasma membrane. RAC1/2 guanine nucleotide exchange factors harbouring polybasic stretches are recruited by PtdOH to sites of ruffle formation. Here we discuss our findings and offer perspectives on how the regulation of dynamic actin structures at the plasma membrane by small GTPases is a critical component of phagocyte function.
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Actinas/metabolismo , Cálcio/metabolismo , Membrana Celular/metabolismo , Fagócitos/citologia , Espaço Extracelular/metabolismo , Humanos , Proteínas Monoméricas de Ligação ao GTP/metabolismoRESUMO
Dual wavelength ratiometric imaging has become a powerful tool for the study of pH in intracellular compartments. It allows for the dynamic imaging of live cells while accounting for changes in the focal plane, differential loading of the fluorescent probe, and photobleaching caused by repeated image acquisitions. Ratiometric microscopic imaging has the added advantage over whole population methods of being able to resolve individual cells and even individual organelles. In this chapter we provide a detailed discussion of the basic principles of ratiometric imaging and its application to the measurement of phagosomal pH, including probe selection, the necessary instrumentation, and calibration methods.
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Microscopia de Fluorescência/métodos , Fagossomos/metabolismo , Animais , Técnicas Biossensoriais , Calibragem , Humanos , Concentração de Íons de Hidrogênio , Imageamento Tridimensional , Camundongos , Proteínas Opsonizantes/metabolismo , Fagocitose , Células RAW 264.7 , Coloração e Rotulagem , Zimosan/metabolismoRESUMO
Phagocytosis, the regulated uptake of large particles (>0.5 µm in diameter), is essential for tissue homeostasis and is also an early, critical component of the innate immune response. Phagocytosis can be conceptually divided into three stages: phagosome, formation, maturation, and resolution. Each of these involves multiple reactions that require exquisite spatial and temporal orchestration. The molecular events underlying these stages are being unraveled and the current state of knowledge is briefly summarized in this article.