The role of apoptosis-associated speck-Like protein (ASC) in the assembly of the inflammasome complex within macrophages has been elucidated in several studies. In this particular role, ASC functions as an adaptor protein by linking nod-like receptors (NLRs) and procaspase-1, thereby leading to the activation of caspase-1 to cleave inflammatory cytokines IL-1β and IL-18 and inducing pyroptosis. It has been noted that ASC maintains inflammasome-independent roles, including but not limited to controlling the expression of Dock2 and mitogen-activated protein kinases (MAPK/ERK2) and regulating the NF-κB pathway. This paper will emphasize the major roles of ASC during pathogen infection, the mechanisms by which it mediates inflammation, and discuss its more recently discovered functions.
apoptosis-associated speck-like protein (ASC); caspase-1; inflammasome; nod-like receptors (NLRs); pyroptosis
Cystic fibrosis (CF) is the most common inherited lethal disease in Caucasians which results in multiorgan dysfunction. However, 85% of the deaths are due to pulmonary infections. Infection by Burkholderia cenocepacia (B. cepacia) is a particularly lethal threat to CF patients because it causes severe and persistent lung inflammation and is resistant to nearly all available antibiotics. In CFTR ΔF508 (ΔF508) mouse macrophages, B. cepacia persists in vacuoles that do not fuse with the lysosomes and mediates increased production of IL-1β. It is believed that intracellular bacterial survival contributes to the persistence of the bacterium. Here we show for the first time that in wild-type but not in ΔF508 macrophages, many B. cepacia reside in autophagosomes that fuse with lysosomes at later stages of infection. Accordingly, association and intracellular survival of B. cepacia are higher in CFTR-ΔF508 macrophages than in WT macrophages. An autophagosome is a compartment that engulfs nonfunctional organelles and parts of the cytoplasm then delivers them to the lysosome for degradation to produce nutrients during periods of starvation or stress. Furthermore, we show that B. cepacia downregulates autophagy genes in WT and ΔF508 macrophages. However, autophagy dysfunction is more pronounced in ΔF508 macrophages since they already have compromised autophagy activity. We demonstrate that the autophagystimulating agent, rapamycin markedly decreases B. cepacia infection in vitro by enhancing the clearance of B. cepacia via induced autophagy. In vivo, rapamycin decreases bacterial burden in the lungs of CF mice and drastically reduces signs of lung inflammation. Together, our studies reveal that if efficiently activated, autophagy can control B. cepacia infection and ameliorate the associated inflammation. Therefore, autophagy is a novel target for new drug development for CF patients to control B. cepacia infection and accompanying inflammation.
autophagy; rapamycin; cystic fibrosis; host-pathogen interaction; Burkholderia cenocepacia; inflammation; macrophages
Burkholderia cenocepacia is an opportunistic pathogen that causes chronic infection and induces progressive respiratory inflammation in cystic fibrosis patients. Recognition of bacteria by mononuclear cells generally results in the activation of caspase-1 and processing of IL-1β, a major proinflammatory cytokine. In this study, we report that human pyrin is required to detect intracellular B. cenocepacia leading to IL-1β processing and release. This inflammatory response involves the host adapter molecule ASC and the bacterial type VI secretion system (T6SS). Human monocytes and THP-1 cells stably expressing either small interfering RNA against pyrin or YFP–pyrin and ASC (YFP–ASC) were infected with B. cenocepacia and analyzed for inflammasome activation. B. cenocepacia efficiently activates the inflammasome and IL-1β release in monocytes and THP-1. Suppression of pyrin levels in monocytes and THP-1 cells reduced caspase-1 activation and IL-1β release in response to B. cenocepacia challenge. In contrast, overexpression of pyrin or ASC induced a robust IL-1β response to B. cenocepacia, which correlated with enhanced host cell death. Inflammasome activation was significantly reduced in cells infected with T6SS-defective mutants of B. cenocepacia, suggesting that the inflammatory reaction is likely induced by an as yet uncharacterized effector(s) of the T6SS. Together, we show for the first time, to our knowledge, that in human mononuclear cells infected with B. cenocepacia, pyrin associates with caspase-1 and ASC forming an inflammasome that upregulates mononuclear cell IL-1β processing and release.
Endotoxin administration recapitulates many of the host responses to sepsis. Inhibitors of the cysteine protease caspase 1 have long been sought as a therapeutic because mice lacking caspase 1 are resistant to LPS-induced endotoxic shock. According to current thinking, caspase 1-mediated shock requires the proinflammatory caspase 1 substrates IL-1β and IL-18. We show, however, that mice lacking both IL-1β and IL-18 are normally susceptible to LPS-induced splenocyte apoptosis and endotoxic shock. This finding indicates the existence of another caspase 1-dependent mediator of endotoxemia. Reduced serum high mobility group box 1 (HMGB1) levels in caspase 1-deficient mice correlated with their resistance to LPS. A critical role for HMGB1 in endotoxemia was confirmed when mice deficient for IL-1β and IL-18 were protected from a lethal dose of LPS by pretreatment with HMGB1-neutralizing Abs. We found that HMGB1 secretion from LPS-primed macrophages required the inflammasome components apoptotic speck protein containing a caspase activation and recruitment domain (ASC), caspase 1 and Nalp3, whereas HMGB1 secretion from macrophages infected in vitro with Salmonella typhimurium was dependent on caspase 1 and Ipaf. Thus, HMGB1 secretion, which is critical for endotoxemia, occurs downstream of inflammasome assembly and caspase 1 activation.
Mycobacterium tuberculosis (M.tb), which causes tuberculosis, is a host-adapted intracellular pathogen of macrophages. Intracellular pattern recognition receptors in macrophages such as nucleotide-binding oligomerization domain (NOD) proteins regulate pro-inflammatory cytokine production. NOD2-mediated signalling pathways in response to M.tb have been studied primarily in mouse models and cell lines but not in primary human macrophages. Thus we sought to determine the role of NOD2 in regulating cytokine production and growth of virulent M.tb and attenuated Mycobacterium bovis BCG (BCG) in human macrophages. We examined NOD2 expression during monocyte differentiation and observed a marked increase in NOD2 transcript and protein following 2–3 days in culture. Pre-treatment of human monocyte-derived and alveolar macrophages with the NOD2 ligand muramyl dipeptide enhanced production of TNF-α and IL-1β in response to M.tb and BCG in a RIP2-dependent fashion. The NOD2-mediated cytokine response was significantly reduced following knock-down of NOD2 expression by using small interfering RNA (siRNA) in human macrophages. Finally, NOD2 controlled the growth of both M.tb and BCG in human macrophages, whereas controlling only BCG growth in murine macrophages. Together, our results provide evidence that NOD2 is an important intracellular receptor in regulating the host response to M.tb and BCG infection in human macrophages.
Nlrc4 is a member of the Nod-like receptors (NLRs), a family of cytosolic receptors involved in sensing bacterial molecules. NLRs are a group of proteins containing spans of leucine-rich repeats that senses bacterial factors within the eukaryotic cytosol. The recognition of bacterial factors provokes the formation of the inflammasome complex which includes specific NLRs. The inflammasome is responsible for caspase-1 activation which leads to the cleavage and maturation of inflammatory cytokines such as IL-1β and IL-18. Nlrc4 was considered to be a devoted flagellin sensor in eukaryotic cells. However, studies using a variety of pathogens such as Salmonella, Legionella, Shigella and Pseudomonas at high bacterial burdens revealed that Nlrc4 can mediate caspase-1 activation independent of bacterial flagellin. On the other hand, new reports showed that Nlrc4 can restrict bacterial infection independently of caspase-1. Therefore, Nlrc4 maybe involved in sensing more than one bacterial molecule and may participate in several immune complexes.
The ability of Legionella pneumophila to cause pneumonia is determined by its capability to evade the immune system and grow within human monocytes and their derived macrophages. Human monocytes efficiently activate caspase-1 in response to Salmonella but not to L. pneumophila. The molecular mechanism for the lack of inflammasome activation during L. pneumophila infection is unknown. Evaluation of the expression of several inflammasome components in human monocytes during L. pneumophila infection revealed that the expression of the apoptosis-associated speck-like protein (ASC) and the NOD-like receptor NLRC4 are significantly down-regulated in human monocytes. Exogenous expression of ASC maintained the protein level constant during L. pneumophila infection and conveyed caspase-1 activation and restricted the growth of the pathogen. Further depletion of ASC with siRNA was accompanied with improved NF-κB activation and enhanced L. pneumophila growth. Therefore, our data demonstrate that L. pneumophila manipulates ASC levels to evade inflammasome activation and grow in human monocytes. By targeting ASC, L. pneumophila modulates the inflammasome, the apoptosome, and NF-κB pathway simultaneously.
Caspase; Cellular Immune Response; Immunology; Inflammation; Innate Immunity; ASC; Legionella; NOD-like Receptors; Human Monocytes; Inflammasome
Macrophages activate autophagy as an immediate response to Legionella pneumophila infection, but what marks the pathogen phagosome as a target for the autophagy machinery is not known. Because a variety of bacteria, parasites, viruses, and toxins that associate with the endoplasmic reticulum enter host cells by a cholesterol-dependent route, we tested the hypothesis that autophagy is triggered when microbes engage components of lipid raft domains. As the intracellular respiratory pathogen L. pneumophila or the extracellular uropathogen FimH+ Escherichia coli entered macrophages by a cholesterol-sensitive mechanism, they immediatezly resided in vacuoles rich in glycosylphosphatidylinositol moieties and the autophagy enzyme Atg7. As expected for autophagosomes, the vacuoles sequentially acquired the endoplasmic reticulum protein BiP, the autophagy markers Atg8 and monodansyl-cadaverine, and the lysosomal protein LAMP-1. A robust macrophage response to the pathogens was cholesterol-dependent, since fewer Atg7-rich vacuoles were observed when macrophages were pretreated with methyl-β-cyclodextrin or filipin. A model in which macrophages exploit autophagy to capture pathogens within the lipid raft pathway for antigen presentation prior to disposal in lysosomes is discussed.
Legionella pneumophila; uropathogenic E. coli; cholesterol; endoplasmic reticulum; lysosomes
After ingestion by macrophages, Legionella pneumophila enter spacious vacuoles that are quickly enveloped by endoplasmic reticulum (ER), then slowly transferred to lysosomes. Here we demonstrate that the macrophage autophagy machinery recognizes the pathogen phagosome as cargo for lysosome delivery. The autophagy conjugation enzyme Atg7 immediately translocated to phagosomes harbouring virulent Legionella. Subsequently, Atg8, a second autophagy enzyme, and monodansyl-cadaverine (MDC), a dye that accumulates in acidic autophagosomes, decorated the pathogen vacuoles. The autophagy machinery responded to 10–30 kDa species released into culture supernatants by Type IV secretion-competent Legionella, as judged by the macrophages’ processing of Atg8 and formation of vacuoles that sequentially acquired Atg7, Atg8 and MDC. When compared with autophagosomes stimulated by rapamycin, Legionella vacuoles acquired Atg7, Atg8 and MDC more slowly, and Atg8 processing was also delayed. Moreover, compared with autophagosomes of Legionella-permissive naip5 mutant A/J macrophages, those of resistant C57BL/6 J macrophages matured quickly, preventing efficient Legionella replication. Accordingly, we discuss a model in which macrophages elevate autophagy as a barrier to infection, a decision influenced by regulatory interactions between Naip proteins and caspases.
The correct site for translation initiation for Escherichia coli WecA (Rfe), presumably involved in catalyzing the transfer of N-acetylglucosamine 1-phosphate to undecaprenylphosphate, was determined by using its FLAG-tagged derivatives. The N-terminal region containing three predicted transmembrane helices was found to be necessary for function but not for membrane localization of this protein.
Burkholderia cenocepacia is an opportunistic pathogen that survives intracellularly in macrophages and causes serious respiratory infections in patients with cystic fibrosis. We have previously shown that bacterial survival occurs in bacteria-containing membrane vacuoles (BcCVs) resembling arrested autophagosomes. Intracellular bacteria stimulate IL-1β secretion in a caspase-1-dependent manner and induce dramatic changes to the actin cytoskeleton and the assembly of the NADPH oxidase complex onto the BcCV membrane. A Type 6 secretion system (T6SS) is required for these phenotypes but surprisingly it is not required for the maturation arrest of the BcCV. Here, we show that macrophages infected with B. cenocepacia employ the NLRP3 inflammasome to induce IL-1β secretion and pyroptosis. Moreover, IL-1β secretion by B. cenocepacia-infected macrophages is suppressed in deletion mutants unable to produce functional Type VI, Type IV, and Type 2 secretion systems (SS). We provide evidence that the T6SS mediates the disruption of the BcCV membrane, which allows the escape of proteins secreted by the T2SS into the macrophage cytoplasm. This was demonstrated by the activity of fusion derivatives of the T2SS-secreted metalloproteases ZmpA and ZmpB with adenylcyclase. Supporting this notion, ZmpA and ZmpB are required for efficient IL-1β secretion in a T6SS dependent manner. ZmpA and ZmpB are also required for the maturation arrest of the BcCVs and bacterial intra-macrophage survival in a T6SS-independent fashion. Our results uncover a novel mechanism for inflammasome activation that involves cooperation between two bacterial secretory pathways, and an unanticipated role for T2SS-secreted proteins in intracellular bacterial survival.
The apoptosis-associated speck-like protein containing a caspase recruitment domain (Asc) is an adaptor molecule that mediates inflammatory and apoptotic signals. Legionella pneumophila is an intracellular bacterium and the causative agent of Legionnaire's pneumonia. L. pneumophila is able to cause pneumonia in immuno-compromised humans but not in most inbred mice. Murine macrophages that lack the ability to activate caspase-1, such as caspase-1−/− and Nlrc4−/− allow L. pneumophila infection. This permissiveness is attributed mainly to the lack of active caspase-1 and the absence of its down stream substrates such as caspase-7. However, the role of Asc in control of L. pneumophila infection in mice is unclear. Here we show that caspase-1 is moderately activated in Asc−/− macrophages and that this limited activation is required and sufficient to restrict L. pneumophila growth. Moreover, Asc-independent activation of caspase-1 requires bacterial flagellin and is mainly detected in cellular extracts but not in culture supernatants. We also demonstrate that the depletion of Asc from permissive macrophages enhances bacterial growth by promoting L. pneumophila-mediated activation of the NF-κB pathway and decreasing caspase-3 activation. Taken together, our data demonstrate that L. pneumophila infection in murine macrophages is controlled by several mechanisms: Asc-independent activation of caspase-1 and Asc-dependent regulation of NF-κB and caspase-3 activation.
inflammasome; caspase-1; Legionella pneumophila; Asc
The ability of Legionella pneumophila to proliferate within various protozoa in the aquatic environment and in macrophages indicates a remarkable evolution and microbial exploitation of evolutionarily conserved eukaryotic processes. Ankyrin B (AnkB) of L. pneumophila is a non-canonical F-box-containing protein, and is the only known Dot/Icm-translocated effector of L. pneumophila essential for intra-vacuolar proliferation within both macrophages and protozoan hosts. We show that the F-box domain of AnkB and the 9L10P conserved residues are essential for intracellular bacterial proliferation and for rapid acquisition of polyubiquitinated proteins by the Legionella-containing vacuole (LCV) within macrophages, Dictyostelium discoideum, and Acanthamoeba. Interestingly, translocation of AnkB and recruitment of polyubiquitinated proteins in macrophages and Acanthamoeba is rapidly triggered by extracellular bacteria within 5 min of bacterial attachment. Ectopically expressed AnkB within mammalian cells is localized to the periphery of the cell where it co-localizes with host SKP1 and recruits polyubiquitinated proteins, which results in restoration of intracellular growth to the ankB mutant similar to the parental strain. While an ectopically expressed AnkB-9L10P/AA variant is localized to the cell periphery, it does not recruit polyubiquitinated proteins and fails to trans-rescue the ankB mutant intracellular growth defect. Direct in vivo interaction of AnkB but not the AnkB-9L10P/AA variant with the host SKP1 is demonstrated. Importantly, RNAi-mediated silencing of expression of SKP1 renders the cells non-permissive for intracellular proliferation of L. pneumophila. The role of AnkB in exploitation of the polyubiquitination machinery is essential for intrapulmonary bacterial proliferation in the mouse model of Legionnaires' disease. Therefore, AnkB exhibits a novel molecular and functional mimicry of eukaryotic F-box proteins that exploits conserved polyubiquitination machinery for intracellular proliferation within evolutionarily distant hosts.
Legionella pneumophila is abundantly found in the aquatic environment within various protozoa and can cause a severe pneumonia called Legionnaires' disease when it invades human macrophages in the lung. The ability of L. pneumophila to invade and proliferate within macrophages and protozoa is dependent on the translocation of specific proteins into the invaded cell via a specialized secretory device, and these proteins modulate various host cell processes. Of these translocated proteins, AnkB is indispensable for intracellular growth of L. pneumophila within macrophages and protozoa. Here we show that AnkB is essential for establishing a favorable intracellular replicative niche by promoting the decoration of the Legionella containing vacuole (LCV) with polyubiquitinated proteins. The AnkB effector achieves this by mimicking the action of host cell F-box proteins, a highly conserved component of the SCF ubiquitin ligase complex that is found in both unicellular organisms and mammalian cells. Our study provides new insights into the ability of intracellular pathogens to hijack evolutionarily conserved host cell processes through molecular mimicry to establish a favorable replicative niche within various hosts and to cause disease in mammals.
Legionella pneumophila (L. pneumophila), the causative agent of a severe form of pneumonia called Legionnaires' disease, replicates in human monocytes and macrophages. Most inbred mouse strains are restrictive to L. pneumophila infection except for the A/J, Nlrc4−/− (Ipaf−/−), and caspase-1−/− derived macrophages. Particularly, caspase-1 activation is detected during L. pneumophila infection of murine macrophages while absent in human cells. Recent in vitro experiments demonstrate that caspase-7 is cleaved by caspase-1. However, the biological role for caspase-7 activation downstream of caspase-1 is not known. Furthermore, whether this reaction is pertinent to the apoptosis or to the inflammation pathway or whether it mediates a yet unidentified effect is unclear. Using the intracellular pathogen L. pneumophila, we show that, upon infection of murine macrophages, caspase-7 was activated downstream of the Nlrc4 inflammasome and required caspase-1 activation. Such activation of caspase-7 was mediated by flagellin and required a functional Naip5. Remarkably, mice lacking caspase-7 and its macrophages allowed substantial L. pneumophila replication. Permissiveness of caspase-7−/− macrophages to the intracellular pathogen was due to defective delivery of the organism to the lysosome and to delayed cell death during early stages of infection. These results reveal a new mechanism for caspase-7 activation downstream of the Nlrc4 inflammasome and present a novel biological role for caspase-7 in host defense against an intracellular bacterium.
Legionella pneumophila causes a severe form of pneumonia called Legionnaires' disease. In human macrophages, L. pneumophila establishes special vacuoles that do not fuse with the lysosome and grows intracellularly. However, in mouse macrophages, the bacteria are efficiently delivered to the lysosome for degradation. Importantly, caspase-1 is activated when L. pneumophila infects mouse macrophages, but not when it infects human cells. Caspase-1 activation promotes the fusion of the L. pneumophila vacuole with the lysosome and macrophage death. However, the caspase-1 substrate mediating such effects is unknown. Experiments performed in vitro demonstrate that caspase-7 is a substrate of caspase-1. Yet, it is not known if the reaction takes place within the macrophage, and it is unclear if it has any biological effect. In this study we show that, in mouse macrophages, caspase-7 is activated by L. pneumophila downstream of caspase-1 and requires the host receptors Nlrc4 and Naip5. Remarkably, caspase-7 activation during L. pneumophila infection restricts growth by promoting early macrophage death and efficient delivery of the organism to the lysosome. Consequently, L. pneumophila grows in the macrophages and the lungs of caspase-7−/− mice. Therefore, we demonstrate a novel caspase-7 activation pathway that contributes to the restriction of L. pneumophila infection.