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ABSTRACT: A recent study published in Nature by Keestra and colleagues addresses how the immune system detects the pathogenic potential of microbes and provides evidence that one strategy involves NOD1, which monitors the activation state of the RhoGTPases that are targeted by virulence effectors produced by pathogenic microbes. Interestingly, their findings reveal striking similarities with previous observations made in flies and plants, establishing the evolutionary conservation of this detection system in the innate immune arsenal in many taxa.
Cell Research 05/2013; · 8.19 Impact Factor
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Anna Sokolovska,
Christine E Becker,
W K Eddie Ip,
Vijay A K Rathinam,
Matthew Brudner,
Nicholas Paquette,
Antoine Tanne,
Sivapriya K Vanaja,
Kathryn J Moore,
Katherine A Fitzgerald,
Adam Lacy-Hulbert, Lynda M Stuart
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ABSTRACT: Phagocytosis is a fundamental cellular process that is pivotal for immunity as it coordinates microbial killing, innate immune activation and antigen presentation. An essential step in this process is phagosome acidification, which regulates many functions of these organelles that allow phagosomes to participate in processes that are essential to both innate and adaptive immunity. Here we report that acidification of phagosomes containing Gram-positive bacteria is regulated by the NLRP3 inflammasome and caspase-1. Active caspase-1 accumulates on phagosomes and acts locally to control the pH by modulating buffering by the NADPH oxidase NOX2. These data provide insight into a mechanism by which innate immune signals can modify cellular defenses and establish a new function for the NLRP3 inflammasome and caspase-1 in host defense.
Nature Immunology 05/2013; · 26.01 Impact Factor
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Michael K Mansour,
Jenny M Tam,
Nida S Khan,
Michael Seward,
Peter J Davids,
Sravanthi Puranam,
Anna Sokolovska,
David B Sykes,
Zeina Dagher,
Christine Becker,
Antoine Tanne,
Jennifer L Reedy, Lynda M Stuart,
Jatin M Vyas
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ABSTRACT: Elimination of fungal pathogens by phagocytes requires phagosome maturation, a process that involves the recruitment and fusion of intracellular proteins. The role of Dectin-1, a β-1,3-glucan receptor, critical for fungal recognition and triggering of Th17 responses, to phagosomal maturation has not been defined. We show that GFP-Dectin-1 translocates to the fungal phagosome, but its signal decays after two hours. Inhibition of acidification results in retention of GFP-Dectin-1 to phagosome membranes highlighting the requirement for an acidic pH. Following β-1,3-glucan recognition, GFP-Dectin-1 undergoes tyrosine phosphorylation by Src kinases with subsequent Syk activation. Our results demonstrate that Syk is activated independently of intraphagosomal pH. Inhibition of Src or Syk results in prolonged retention of GFP-Dectin-1 to the phagosome signifying a link between Syk and intraphagosomal pH. β-1,3-glucan phagosomes expressing a signaling incompetent Dectin-1 failed to mature as demonstrated by prolonged Dectin-1 retention, presence of Rab5B, failure to acquire LAMP-1 and inability to acidify. Phagosomes containing C. albicans also require Dectin-1-dependent Syk activation for phagosomal maturation. Taken together, these results support a model where Dectin-1 not only controls internalization of β-1,3-glucan containing cargo and triggers proinflammatory cytokines, but also acts as a master regulator for subsequent phagolysosomal maturation through Syk activation.
Journal of Biological Chemistry 04/2013; · 4.77 Impact Factor
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ABSTRACT: A fundamental question regarding any immune system is how it can discriminate between pathogens and non-pathogens. Here, we discuss how this discrimination can be mediated by a surveillance system distinct from pattern-recognition receptors that recognize conserved microbial patterns. It can be based instead on the ability of the host to sense perturbations in host cells induced by bacterial toxins or 'effectors' that are encoded by pathogenic microorganisms. Such 'effector-triggered immunity' was previously demonstrated mainly in plants, but recent data confirm that animals can also use this strategy.
Nature Reviews Immunology 03/2013; 13(3):199-206. · 32.25 Impact Factor
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ABSTRACT: Phagocytes are an important part of host defense, playing a critical role in innate immune responses against pathogens and in the initiation of adaptive immunity. One of the main characteristics of these cells is their ability to recognize and internalize invading microorganisms into a phagosome. The internalized microbe is rapidly delivered into a mature phagolysosome where it is killed and degraded. However, numerous pathogens have evolved complex mechanisms to manipulate these intracellular organelles to establish a survival niche. Here, we describe several methods to assess important properties of phagosomes in macrophages, such as phagocytosis, acidification of the phagosome contents during the maturation process, and the ability of phagosomes to inactivate and kill pathogens. Phagocytosis and phagosome acidification assays are FACS-based assays where labeled bacteria are used as probes to monitor internalization into a phagosome and to detect the pH of the phagosome environment. The killing assay is based on the counting of bacterial colonies after recovery of internalized bacteria from macrophages. Curr. Protoc. Immunol. 99:14.30.1-14.30.12. © 2012 by John Wiley & Sons, Inc.
Current protocols in immunology / edited by John E. Coligan ... [et al.] 11/2012; Chapter 14:Unit14.30.
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ABSTRACT: In this issue of Immunity, Irving et al. (2012) show that protein kinase R (PKR) regulates the cytoskeleton via an interaction with gelsolin. This alternative role for PKR prevents penetration of virions into the cell.
Immunity 05/2012; 36(5):695-6. · 21.64 Impact Factor
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ABSTRACT: Immunosuppression via cell-cell contact with apoptotic cells is a well studied immunological phenomenon. Although the original studies of immune repression used primary cells, which undergo spontaneous cell death or apoptosis in response to irradiation, more recent studies have relied on chemotherapeutic agents to induce apoptosis in cell lines. In this work, we demonstrate that Jurkat cells induced to die with actinomycin D suppressed inflammatory cytokine production by macrophages, whereas cells treated with etoposide did not. This immune repression mediated by actinomycin D-treated cells did not require phagocytosis or cell-cell contact and thus occurs through a different mechanism from that seen with primary apoptotic neutrophils. Moreover, cells induced to die with etoposide and then treated for a short time with actinomycin D also suppressed macrophage responses, indicating that suppression was mediated by actinomycin D independent of the mechanism of cell death. Finally, phagocytosis of actinomycin D-treated cells caused apoptosis in macrophages, and suppression could be blocked by inhibition of caspase activity in the target macrophage. Together, these data indicate that apoptotic cells act as "Trojan horses," delivering actinomycin D to engulfing macrophages. Suppression of cytokine production by macrophages is therefore due to exposure to actinomycin D from apoptotic cells and is not the result of cell-receptor interactions. These data suggest that drug-induced death may not be an appropriate surrogate for the immunosuppressive activity of apoptotic cells. Furthermore, these effects of cytotoxic drugs on infiltrating immune phagocytes may have clinical ramifications for their use as antitumor therapies.
Journal of Biological Chemistry 03/2012; 287(19):16029-36. · 4.77 Impact Factor
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Janine M van Gils,
Merran C Derby,
Luciana R Fernandes,
Bhama Ramkhelawon,
Tathagat D Ray,
Katey J Rayner,
Sajesh Parathath,
Emilie Distel,
Jessica L Feig,
Jacqueline I Alvarez-Leite,
Alistair J Rayner,
Thomas O McDonald,
Kevin D O'Brien, Lynda M Stuart,
Edward A Fisher,
Adam Lacy-Hulbert,
Kathryn J Moore
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ABSTRACT: Atherosclerotic plaque formation is fueled by the persistence of lipid-laden macrophages in the artery wall. The mechanisms by which these cells become trapped, thereby establishing chronic inflammation, remain unknown. Here we found that netrin-1, a neuroimmune guidance cue, was secreted by macrophages in human and mouse atheroma, where it inactivated the migration of macrophages toward chemokines linked to their egress from plaques. Acting via its receptor, UNC5b, netrin-1 inhibited the migration of macrophages directed by the chemokines CCL2 and CCL19, activation of the actin-remodeling GTPase Rac1 and actin polymerization. Targeted deletion of netrin-1 in macrophages resulted in much less atherosclerosis in mice deficient in the receptor for low-density lipoprotein and promoted the emigration of macrophages from plaques. Thus, netrin-1 promoted atherosclerosis by retaining macrophages in the artery wall. Our results establish a causative role for negative regulators of leukocyte migration in chronic inflammation.
Nature Immunology 02/2012; 13(2):136-43. · 26.01 Impact Factor
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ABSTRACT: A common defining characteristic of pathogenic bacteria is the expression of a repertoire of effector molecules that have been named virulence factors. These bacterial factors include a -variety of proteins, such as toxins that are internalized by receptors and translocate across endosomal membranes to reach the cytosol, as well as others that are introduced directly into the cell by means of bacterial secretory apparatuses. Given the importance of these effectors for understanding bacterial pathogenicity, significant effort has been made to dissect their molecular mechanisms of action and their respective roles during infection. Herein we will discuss how Drosophila have been used as a model system to study these important microbial effectors, and to understand their contribution to pathogenicity.
Advances in experimental medicine and biology 01/2012; 710:29-36. · 1.09 Impact Factor
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Laurent Boyer,
Lorin Magoc,
Stephanie Dejardin,
Michael Cappillino,
Nicholas Paquette,
Charlotte Hinault,
Guillaume M Charriere,
W K Eddie Ip,
Shannon Fracchia,
Elizabeth Hennessy,
Deniz Erturk-Hasdemir,
Jean-Marc Reichhart,
Neal Silverman,
Adam Lacy-Hulbert, Lynda M Stuart
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ABSTRACT: Although infections with virulent pathogens often induce a strong inflammatory reaction, what drives the increased immune response to pathogens compared to nonpathogenic microbes is poorly understood. One possibility is that the immune system senses the level of threat from a microorganism and augments the response accordingly. Here, focusing on cytotoxic necrotizing factor 1 (CNF1), an Escherichia coli-derived effector molecule, we showed the host indirectly sensed the pathogen by monitoring for the effector that modified RhoGTPases. CNF1 modified Rac2, which then interacted with the innate immune adaptors IMD and Rip1-Rip2 in flies and mammalian cells, respectively, to drive an immune response. This response was protective and increased the ability of the host to restrict pathogen growth, thus defining a mechanism of effector-triggered immunity that contributes to how metazoans defend against microbes with pathogenic potential.
Immunity 10/2011; 35(4):536-49. · 21.64 Impact Factor
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ABSTRACT: Immune responses in the intestine are controlled by regulatory T cells (Tregs), which prevent inflammation in response to commensal bacteria. A specific population of intestinal dendritic cells (DCs), marked by expression of CD103, generate Tregs more efficiently than other DC populations through mechanisms that involve retinoic acid and transforming growth factor (TGF)-β. However, it is not clear how CD103(+) DCs are specialized for this function. We investigated the ability of CD103(+) DCs to promote Treg generation through activation of TGF-β and the role of integrins with the αv subunit in this process.
Naïve T cells were cultured with purified DCs from mesenteric lymph nodes (MLNs) or intestines of wild-type and αv conditional knockout mice to assess generation of Tregs. Antigens were administered orally to mice, and antigen-specific generation of Tregs was measured in intestinal tissues. Expression of the integrin αv subunit was measured in purified subpopulations of DCs by quantitative polymerase chain reaction and immunoblot analyses.
In vitro, CD103(+) DCs generated more Tregs in the presence of latent TGF-β than other MLN DCs. Efficient generation of Tregs required expression of the integrin αv subunit by DCs; mice that lacked αv in immune cells did not convert naïve T cells to intestinal Tregs in response to oral antigen. CD103(+) DCs derived from the MLNs selectively expressed high levels of integrin αvβ8 compared with other populations of DCs.
Expression of αvβ8 is required for CD103(+) DCs to become specialized and activate latent TGF-β and generate Tregs during the induction of tolerance to intestinal antigens in mice.
Gastroenterology 07/2011; 141(5):1813-20. · 11.68 Impact Factor
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ABSTRACT: Th17 cells are a distinct lineage of T helper cells that protect the body from bacterial and fungal infection. However, Th17 cells also contribute to inflammatory and autoimmune disorders such as multiple sclerosis. Th17 cell generation requires exposure of naive T cells to the cytokine TGF-β in combination with proinflammatory cytokines. Here we show that differentiation of Th17 cells is also critically dependent on αv integrins. In mice, lack of integrin αv in the immune system resulted in loss of Th17 cells in the intestine and lymphoid tissues. It also led to protection from experimental autoimmune encephalomyelitis (EAE). Further analysis indicated that αv integrins on DCs activated latent TGF-β during T cell stimulation and thereby promoted differentiation of Th17 cells. Furthermore, pharmacologic inhibition of αv integrins using cyclic RGD peptides blocked TGF-β activation and Th17 cell generation in vitro and protected mice from EAE. These data demonstrate that activation of TGF-β by αv-expressing myeloid cells may be a critical step in the generation of Th17 cells and suggest that αv integrins could be therapeutic targets in autoimmune disease.
The Journal of clinical investigation 12/2010; 120(12):4445-52. · 15.39 Impact Factor
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Guillaume M Charrière,
Wk Eddie Ip,
Stéphanie Dejardin,
Laurent Boyer,
Anna Sokolovska,
Michael P Cappillino,
Bobby J Cherayil,
Daniel K Podolsky,
Koichi S Kobayashi,
Neal Silverman,
Adam Lacy-Hulbert, Lynda M Stuart
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ABSTRACT: NOD2 (nucleotide-binding oligomerization domain containing 2) is an important cytosolic pattern recognition receptor that activates NF-kappaB and other immune effector pathways such as autophagy and antigen presentation. Despite its intracellular localization, NOD2 participates in sensing of extracellular microbes such as Staphylococcus aureus. NOD2 ligands similar to the minimal synthetic ligand muramyl dipeptide (MDP) are generated by internalization and processing of bacteria in hydrolytic phagolysosomes. However, how these derived ligands exit this organelle and access the cytosol to activate NOD2 is poorly understood. Here, we address how phagosome-derived NOD2 ligands access the cytosol in human phagocytes. Drawing on data from Drosophila phagosomes, we identify an evolutionarily conserved role of SLC15A transporters, Drosophila Yin and PEPT2, as MDP transporters in fly and human phagocytes, respectively. We show that PEPT2 is highly expressed by human myeloid cells. Ectopic expression of both Yin and PEPT2 increases the sensitivity of NOD2-dependent NF-kappaB activation. Additionally, we show that PEPT2 associates with phagosome membranes. Together, these data identify Drosophila Yin and PEPT2 as evolutionarily conserved phagosome-associated transporters that are likely to be of particular importance in delivery of bacteria-derived ligands generated in phagosomes to cytosolic sensors recruited to the vicinity of these organelles.
Journal of Biological Chemistry 06/2010; 285(26):20147-54. · 4.77 Impact Factor
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Guillaume M. Charrière,
WK Eddie Ip,
Stéphanie Dejardin,
Laurent Boyer,
Anna Sokolovska,
Michael P. Cappillino,
Bobby J. Cherayil,
Daniel K. Podolsky,
Koichi S. Kobayashi,
Neal Silverman,
Adam Lacy-Hulbert, Lynda M. Stuart
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ABSTRACT: NOD2 (nucleotide-binding oligomerization domain containing 2) is an important cytosolic pattern recognition receptor that activates NF-κB and other immune effector pathways such as autophagy
and antigen presentation. Despite its intracellular localization, NOD2 participates in sensing of extracellular microbes such
as Staphylococcus aureus. NOD2 ligands similar to the minimal synthetic ligand muramyl dipeptide (MDP) are generated by internalization and processing
of bacteria in hydrolytic phagolysosomes. However, how these derived ligands exit this organelle and access the cytosol to
activate NOD2 is poorly understood. Here, we address how phagosome-derived NOD2 ligands access the cytosol in human phagocytes.
Drawing on data from Drosophila phagosomes, we identify an evolutionarily conserved role of SLC15A transporters, Drosophila Yin and PEPT2, as MDP transporters in fly and human phagocytes, respectively. We show that PEPT2 is highly expressed by human
myeloid cells. Ectopic expression of both Yin and PEPT2 increases the sensitivity of NOD2-dependent NF-κB activation. Additionally,
we show that PEPT2 associates with phagosome membranes. Together, these data identify Drosophila Yin and PEPT2 as evolutionarily conserved phagosome-associated transporters that are likely to be of particular importance
in delivery of bacteria-derived ligands generated in phagosomes to cytosolic sensors recruited to the vicinity of these organelles.
Journal of Biological Chemistry 06/2010; 285(26):20147-20154. · 4.77 Impact Factor
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W K Eddie Ip,
Anna Sokolovska,
Guillaume M Charriere,
Laurent Boyer,
Stephanie Dejardin,
Michael P Cappillino,
L Michael Yantosca,
Kazue Takahashi,
Kathryn J Moore,
Adam Lacy-Hulbert, Lynda M Stuart
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ABSTRACT: Innate immunity is vital for protection from microbes and is mediated by humoral effectors, such as cytokines, and cellular immune defenses, including phagocytic cells (e.g., macrophages). After internalization by phagocytes, microbes are delivered into a phagosome, a complex intracellular organelle with a well-established and important role in microbial killing. However, the role of this organelle in cytokine responses and microbial sensing is less well defined. In this study, we assess the role of the phagosome in innate immune sensing and demonstrate the critical interdependence of phagocytosis and pattern recognition receptor signaling during response to the Gram-positive bacteria Staphylococcus aureus. We show that phagocytosis is essential to initiate an optimal MyD88-dependent response to Staphylococcus aureus. Prior to TLR-dependent cytokine production, bacteria must be engulfed and delivered into acidic phagosomes where acid-activated host enzymes digest the internalized bacteria to liberate otherwise cryptic bacterial-derived ligands that initiate responses from the vacuole. Importantly, in macrophages in which phagosome acidification is perturbed, the impaired response to S. aureus can be rescued by the addition of lysostaphin, a bacterial endopeptidase active at neutral pH that can substitute for the acid-activated host enzymes. Together, these observations delineate the interdependence of phagocytosis with pattern recognition receptor signaling and suggest that therapeutics to augment functions and signaling from the vacuole may be useful strategies to increase host responses to S. aureus.
The Journal of Immunology 06/2010; 184(12):7071-81. · 5.79 Impact Factor
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Cameron R Stewart, Lynda M Stuart,
Kim Wilkinson,
Janine M van Gils,
Jiusheng Deng,
Annett Halle,
Katey J Rayner,
Laurent Boyer,
Ruiqin Zhong,
William A Frazier,
Adam Lacy-Hulbert,
Joseph El Khoury,
Douglas T Golenbock,
Kathryn J Moore
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ABSTRACT: In atherosclerosis and Alzheimer's disease, deposition of the altered self components oxidized low-density lipoprotein (LDL) and amyloid-beta triggers a protracted sterile inflammatory response. Although chronic stimulation of the innate immune system is believed to underlie the pathology of these diseases, the molecular mechanisms of activation remain unclear. Here we show that oxidized LDL and amyloid-beta trigger inflammatory signaling through a heterodimer of Toll-like receptors 4 and 6. Assembly of this newly identified heterodimer is regulated by signals from the scavenger receptor CD36, a common receptor for these disparate ligands. Our results identify CD36-TLR4-TLR6 activation as a common molecular mechanism by which atherogenic lipids and amyloid-beta stimulate sterile inflammation and suggest a new model of TLR heterodimerization triggered by coreceptor signaling events.
Nature Immunology 02/2010; 11(2):155-61. · 26.01 Impact Factor
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ABSTRACT: Innate immunity is the earliest response to invading microbes and acts to contain infection in the first minutes to hours of challenge. Unlike adaptive immunity that relies upon clonal expansion of cells that emerge days after antigenic challenge, the innate immune response is immediate. Soluble mediators, including complement components and the mannose binding lectin (MBL) make an important contribution to innate immune protection and work along with epithelial barriers, cellular defenses such as phagocytosis, and pattern-recognition receptors that trigger pro-inflammatory signaling cascades. These four aspects of the innate immune system act in concert to protect from pathogen invasion. Our work has focused on understanding the protection provided by this complex defense system and, as discussed in this review, the particular contribution of soluble mediators such as MBL and phagocytic cells. Over the past two decades both human epidemiological data and mouse models have indicated that MBL plays a critical role in innate immune protection against a number of pathogens. As demonstrated by our recent in vitro work, we show that MBL and the innate immune signaling triggered by the canonical pattern-recognition receptors (PRRs), the Toll-like receptors (TLRs), are linked by their spatial localization to the phagosome. These observations demonstrated a novel role for MBL as a TLR co-receptor and establishes a new paradigm for the role of opsonins, which we propose to function not only to increase microbial uptake but also to spatially coordinate, amplify, and synchronize innate immune defenses mechanism. In this review we discuss both the attributes of MBL that make it a unique soluble pattern recognition molecule and also highlight its broader role in coordinating innate immune activation.
Immunological Reviews 08/2009; 230(1):9-21. · 11.15 Impact Factor
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ABSTRACT: Phagocytosis, the engulfment of material by cells, is a highly conserved process that arose before the development of multicellularity. Phagocytes have a key role in embryogenesis and also guard the portals of potential pathogen entry. They discriminate between diverse particles through the array of receptors expressed on their surface. In higher species, arguably the most sophisticated function of phagocytes is the processing and presentation of antigens derived from internalized material to stimulate lymphocytes and long-lived specific immunity. Central to these processes is the generation of a phagosome, the organelle that forms around internalized material. As we discuss in this Review, over the past two decades important insights into phagocytosis have been gleaned from studies in the model organism Drosophila melanogaster.
Nature Reviews Immunology 03/2008; 8(2):131-41. · 32.25 Impact Factor
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ABSTRACT: Innate immunity is the first-line defense against pathogens and relies on phagocytes, soluble components, and cell-surface and cytosolic pattern recognition receptors. Despite using hard-wired receptors and signaling pathways, the innate immune response demonstrates surprising specificity to different pathogens. We determined how combinatorial use of innate immune defense mechanisms defines the response. We describe a novel cooperation between a soluble component of the innate immune system, the mannose-binding lectin, and Toll-like receptor 2 that both specifies and amplifies the host response to Staphylococcus aureus. Furthermore, we demonstrate that this cooperation occurs within the phagosome, emphasizing the importance of engulfment in providing the appropriate cellular environment to facilitate the synergy between these defense pathways.
Journal of Experimental Medicine 02/2008; 205(1):169-81. · 13.85 Impact Factor
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ABSTRACT: The pattern recognition receptor CD36 initiates a signaling cascade that promotes microglial activation and recruitment to beta-amyloid deposits in the brain. In the present study we identify the focal adhesion-associated proteins p130Cas, Pyk2, and paxillin as novel members of the tyrosine kinase signaling pathway downstream of CD36 and show that assembly of this complex is essential for microglial migration. In primary microglia and macrophages exposed to beta-amyloid, the scaffolding protein p130Cas is rapidly tyrosine-phosphorylated and co-localizes with CD36 to membrane ruffles contemporaneous with F-actin polymerization. These beta-amyloid-stimulated events are not detected in CD36 null cells and are dependent on CD36 activation of Src family tyrosine kinases. Fyn, a Src kinase known to interact with CD36, co-precipitates with p130Cas and is an essential upstream intermediate in the signaling pathways leading to phosphorylation of the p130Cas substrate domain. Furthermore, the p130Cas-interacting kinase Pyk2 and the cytoskeletal adapter protein paxillin also demonstrate CD36-dependent phosphorylation, identifying these focal adhesion molecules as additional members of this beta-amyloid signaling cascade. Disruption of this p130Cas complex by small interfering RNA silencing inhibits p44/42 mitogen-activated protein kinase phosphorylation and microglial migration, illustrating the importance of this pathway in microglial activation and recruitment. Together, these data are the first to identify the signaling cascade that directly links CD36 to the actin cytoskeleton and, thus, implicates it in diverse processes such as cellular migration, adhesion, and phagocytosis.
Journal of Biological Chemistry 10/2007; 282(37):27392-401. · 4.77 Impact Factor
