Interferon-gamma reverses the evasion of Birc1e/Naip5 gene mediated murine macrophage immunity by Legionella pneumophila mutant lacking flagellin

Legionella pneumophila is the etiologic agent of Legionnaires' disease. This bacterium contains a single monopolar flagellum, of which the FlaA subunit is a major protein constituent. The murine macrophage resistance against this bacterium is controlled by the Birc1e/Naip5 gene, which belongs to the NOD family. We evaluated the intracellular growth of the flaA mutant bacteria as well as another aflagellated fliA mutant, within bone marrow-derived macrophages from mice with an intact (C57BL/6, BALB/c) or mutated (A/J) Birc1e/Naip5 gene. The flaA mutant L. pneumophila multiplied within C57BL/6 and BALB/c macrophages while the wild-type strain did not. Cell viability was not impaired until 3 days after infection when the flaA mutant bacteria replicated 10(2-3)-fold in macrophages, implying that L. pneumophila inhibited host cell death during the early phase of intracellular replication. The addition of recombinant interferon-gamma (IFN-gamma) to the infected macrophages restricted replication of the flaA mutant within macrophages; these treated cells also showed enhanced nitric oxide production, although inhibition of nitric oxide production did not affect the IFN-gamma induced inhibition of Legionella replication. These findings suggested that IFN-gamma activated macrophages to restrict the intracellular growth of the L. pneumophila flaA mutant by a NO independent pathway.     

Naip5/Birc1e and susceptibility to Legionella pneumophila

Genetic analysis in mice is a powerful approach for the identification of genes and proteins that have a key role at the interface of the host-pathogen interaction. The Lgn1 locus has been found to control the intracellular replication of Legionella pneumophila in murine macrophages. Using functional complementation in transgenic mice, the Naip5/Birc1e gene has been identified as responsible for the Lgn1 effect. The classification of Naip5/Birc1e as a member of the NLR protein family suggests that Naip5/Birc1e acts as an intracellular sensor of L. pneumophila. The nature of the signal transduced by Naip5/Birc1e in response to Legionella products is of great interest but is currently unknown. Here, several possible scenarios are presented.     

The Nod-like receptor family member Naip5/Birc1e restricts Legionella pneumophila growth independently of caspase-1 activation

Similar to Ipaf and caspase-1, the Nod-like receptor protein Naip5 restricts intracellular proliferation of Legionella pneumophila, the causative agent of a severe form of pneumonia known as Legionnaires' disease. Thus, Naip5 has been suggested to regulate Legionella replication inside macrophages through the activation of caspase-1. In this study, we show that cytosolic delivery of recombinant flagellin activated caspase-1 in A/J macrophages carrying a mutant Naip5 allele, and in C57BL/6 (B6) macrophages congenic for the mutant Naip5 allele (B6-Naip5(A/J)), but not in Ipaf(-/-) cells. In line with these results, A/J and B6-Naip5(A/J) macrophages induced high levels of caspase-1 activation and IL-1beta secretion when infected with Legionella. In addition, transgenic expression of a functional Naip5 allele in A/J macrophages did not alter Legionella-induced caspase-1 activation and IL-1beta secretion. Notably, defective Naip5 signaling renders B6-Naip5(A/J) macrophages permissive for Legionella proliferation despite normal caspase-1 activation. These results indicate that the restriction of intracellular Legionella replication is more complex than previously appreciated and requires both Ipaf-dependent caspase-1 activation as well as functional Naip5 signaling.     

Birc1e/Naip5 rapidly antagonizes modulation of phagosome maturation by Legionella pneumophila

Legionella survives intracellularly by preventing fusion with lysosomes, due to phagosome escape from the endocytic pathway at an early stage of phagosome maturation, and by creating a replicative organelle that acquires endoplasmic reticulum (ER) characteristics through sustained interactions and fusion with the ER. Intracellular replication of Legionella pneumophila in mouse macrophages is controlled by the Lgn1 locus. Functional complementation in vivo has identified the Birc1e/Naip5 gene as being responsible for the Lgn1 effect. To understand the function and temporal site of action of Birc1e/Naip5 in susceptibility to L. pneumophila, we examined the biogenesis of Legionella-containing vacuoles (LCVs) formed in permissive A/J macrophages and in their Birc1e/Naip5 transgenic non-permissive counterpart. Birc1e/Naip5 effects on acquisition of lysosomal and ER markers were evident within 1-2 h following infection. A significantly higher proportion of LCVs formed in Birc1e/Naip5 transgenic macrophages had acquired the lysosomal markers cathepsin D and Lamp1 by 2 h post infection, whereas a significantly higher proportion of LCVs formed in permissive macrophages were positively stained for the ER markers BAP31 and calnexin, 6 h post infection. Likewise, studies by electron microscopy showed acquisition of lysosomal contents (horseradish peroxidase), within the first hour following phagocytic uptake, by LCVs formed in Birc1e/Naip5 transgenic macrophages and delivery of the ER marker glucose 6-phosphatase (G6Pase) only to the lumen of LCVs formed in A/J macrophages. Finally, a larger proportion of LCVs formed in A/J macrophages were studded with ribosomes 24 h post infection, compared with LCVs formed in Birc1e/Naip5 transgenic macrophages. These results suggest that sensing of L. pneumophila products by Birc1e/Naip5 in macrophages occurs rapidly following phagocytosis, a process that antagonizes the ability of L. pneumophila to remodel its phagosome into a specialized vacuole with ER characteristics.     

Naip5 affects host susceptibility to the intracellular pathogen Legionella pneumophila

BACKGROUND: Legionella pneumophila is a gram-negative bacterial pathogen that is the cause of Legionnaires' Disease. Legionella produces disease because it can replicate inside a specialized compartment of host macrophages. Macrophages isolated from various inbred mice exhibit large differences in permissiveness for intracellular replication of Legionella. A locus affecting this host-resistance phenotype, Lgn1, has been mapped to chromosome 13, but the responsible gene has not been identified. RESULTS: Here, we report that Naip5 (also known as Birc1e) influences susceptibility to Legionella. Naip5 encodes a protein that is homologous to plant innate immunity (so-called "resistance") proteins and has been implicated in signaling pathways related to apoptosis regulation. Detailed recombination mapping and analysis of expression implicates Naip5 in the Legionella permissiveness differences among mouse strains. A bacterial artificial chromosome (BAC) transgenic line expressing a nonpermissive allele of Naip5 exhibits a reduction in macrophage Legionella permissiveness. In addition, morpholino-based antisense inhibition of Naip5 causes an increase in the Legionella permissiveness of macrophages. CONCLUSIONS: We conclude that polymorphisms in Naip5 are involved in the permissiveness differences of mouse macrophages for intracellular Legionella replication. We speculate that Naip5 is a functional mammalian homolog of plant "resistance" proteins that monitor for, and initiate host response to, the presence of secreted bacterial virulence proteins.     

E1A oncogene enhancement of caspase-2-mediated mitochondrial injury sensitizes cells to macrophage nitric oxide-induced apoptosis

The adenovirus E1A oncogene induces innate immune rejection of tumors by sensitizing tumor cells to apoptosis in response to injuries, such as those inflicted by macrophage-produced TNF alpha and NO. E1A sensitizes cells to TNF by repressing its activation of NF-kappaB-dependent, antiapoptotic defenses. This suggested the hypothesis that E1A blockade of the NF-kappaB activation response might be the central mechanism of E1A induced cellular sensitivity to other proapoptotic injuries, such as macrophage-produced NO. However, creation of E1A-positive NIH-3T3 mouse cell variants with high-level, NF-kappaB-dependent resistance to TNF did not coselect for resistance to apoptosis induced by either macrophage-NO or chemical-NO, as the hypothesis would predict. E1A expression did block cellular recovery from NO-induced mitochondrial injury and converted the reversible, NO-induced cytostasis response of cells to an apoptotic response. This viral oncogene-induced phenotypic conversion of the cellular injury response of mouse and human cells was mediated by an E1A-related increase in NO-induced activation of caspase-2, an apical initiator of intrinsic apoptosis. Blocking caspase-2 activation or expression eliminated the NO-induced apoptotic response of E1A-positive cells. These results define an NF-kappaB-independent pathway through which the E1A gene of human adenovirus sensitizes mouse and human cells to apoptosis by enhancement of caspase-2-mediated mitochondrial injury.     

Restriction of Legionella pneumophila growth in macrophages requires the concerted action of cytokine and Naip5/Ipaf signalling pathways

Macrophages from the C57BL/6 (B6) mouse strain restrict intracellular growth of Legionella pneumophila, whereas A/J macrophages are highly permissive. The mechanism by which B6 macrophages restrict Legionella growth remains poorly understood, but is known to require the cytosolic microbe sensors Naip5 (Birc1e) and Ipaf. We hypothesized that Naip5 and Ipaf may act in partnership with other antimicrobial signalling pathways in macrophages. Indeed, we found that macrophages lacking either tumour necrosis factor (TNF)-alpha or type I interferon (IFN) signalling are permissive for growth of L. pneumophila, even in the presence of functional Naip5 and Ipaf alleles. Similarly, macrophages lacking Naip5 and/or Ipaf signalling were permissive even though we found that Naip5 or Ipaf were not required for induction of TNF-alpha and type I IFN. Therefore, our data suggest that the mechanism by which B6 macrophages restrict intracellular replication of L. pneumophila is more complex than previously appreciated, and involves the concerted action of cytokine and intracellular microbe sensor signalling pathways.     

Salmonella induces macrophage death by caspase-1-dependent necrosis

We provide evidence that Salmonella typhimurium kills phagocytes by an unusual proinflammatory mechanism of necrosis that is distinguishable from apoptosis. Infection stimulated a distinctly diffuse pattern of DNA fragmentation in macrophages, which contrasted with the marked nuclear condensation displayed by control cells undergoing chemically induced apoptosis. In apoptotic cells, DNA fragmentation and nuclear condensation result from caspase-3-mediated proteolysis; caspases also subvert necrotic cell death by cleaving and inactivating poly ADP-ribose polymerase (PARP). Caspase-3 was not activated during Salmonella infection, and PARP remained in its active, uncleaved state. Another hallmark of apoptosis is sustained membrane integrity during cell death; yet, infected macrophages rapidly lost membrane integrity, as indicated by simultaneous exposure of phosphatidylserine with the uptake of vital dye and the release of the cytoplasmic enzyme lactate dehydrogenase. During experimentally induced necrosis, lethal ion fluxes through the plasma membrane can be prevented by exogenous glycine; similarly, glycine completely blocked Salmonella-induced cytotoxicity. Finally, inhibition of the interleukin (IL)-1-converting enzyme caspase-1 blocked the death of infected macrophages, but not control cells induced to undergo apoptosis or necrosis. Thus, Salmonella-infected macrophages are killed by an unusual caspase-1-dependent mechanism of necrosis.     

Revisiting IRF1-mediated antiviral innate immunity

Many studies have been conducted over the last few decades to understand better the functions of IRF3 and IRF7 in antiviral immune responses. However, the precise underlying molecular mechanism of IRF1-mediated immune response remains largely unknown. Recent studies indicate that IRF1 exerts strong antiviral activities against several viral infections through diverse mechanisms, both in IFN-dependent and IFN-independent manners. Nevertheless, the efficacy and kinetics of inducing IFNs and ISGs remain unknown. Here we summarize the recent advances in IRF1 research and highlight its potential roles in initiating IFN immune responses and subsequent IRF1-triggering antiviral responses. Challenges regarding the IFN positive feedback mediated by IRF7 during infection will be discussed; this classical loop may also be mediated in part by IRF1. Therefore, we propose a revised model that may help decipher the functional roles of IRF1 in antiviral immunity.     

Active caspase-1-mediated secretion of retinoic acid inducible gene-I

Caspase-1 is an inflammatory caspase that controls the activation and secretion of the inflammatory cytokines, IL-1beta and IL-18. We observed that cellular levels of retinoic acid-inducible gene-I (RIG-I) were enhanced when the pan-caspase inhibitor Z-VAD-fmk or caspase-1-specific inhibitor Z-WEHD-fmk blocked caspase activity. Overexpression of caspase-1 reduced cellular levels of RIG-I and inhibited RIG-I-mediated signaling activity. Enzymatic activity of caspase-1 was necessary to control RIG-I, although it was not a substrate of proteolytic cleavage by caspase-1. Caspase-1 physically interacted with full length RIG-I, but not with mutant forms lacking either the amino- or carboxyl-terminal domains. RIG-I was present in the supernatant of cells transfected with active caspase-1 but not with caspase-4. Stimulating cells with LPS and ATP also induced secretion of endogenous RIG-I in macrophages. Our data suggest a novel mechanism that negatively regulates RIG-I-mediated signaling activity via caspase-1-dependent secretion of RIG-I protein.     

Effects of ALS-related SOD1 mutants on dynein- and KIF5-mediated retrograde and anterograde axonal transport

Transport of material and signals between extensive neuronal processes and the cell body is essential to neuronal physiology and survival. Slowing of axonal transport has been shown to occur before the onset of symptoms in amyotrophic lateral sclerosis (ALS). We have previously shown that several familial ALS-linked copper–zinc superoxide dismutase (SOD1) mutants (A4V, G85R, and G93A) interacted and colocalized with the retrograde dynein–dynactin motor complex in cultured cells and affected tissues of ALS mice. We also found that the interaction between mutant SOD1 and the dynein motor played a critical role in the formation of large inclusions containing mutant SOD1. In this study, we showed that, in contrast to the dynein situation, mutant SOD1 did not interact with anterograde transport motors of the kinesin-1 family (KIF5A, B and C). Using dynein and kinesin accumulation at the sciatic nerve ligation sites as a surrogate measurement of axonal transport, we also showed that dynein mediated retrograde transport was slower in G93A than in WT mice at an early presymptomatic stage. While no decrease in KIF5A-mediated anterograde transport was detected, the slowing of anterograde transport of dynein heavy chain as a cargo was observed in the presymptomatic G93A mice. The results from this study along with other recently published work support that mutant SOD1 might only interact with and interfere with some kinesin members, which, in turn, could result in the impairment of a selective subset of cargos. Although it remains to be further investigated how mutant SOD1 affects different axonal transport motor proteins and various cargos, it is evident that mutant SOD1 can induce defects in axonal transport, which, subsequently, contribute to the propagation of toxic effects and ultimately motor neuron death in ALS.     

Protective role of Cop in Rip2/caspase-1/caspase-4-mediated HeLa cell death

CARD only protein (Cop) was recently identified as a protein with significant homology with the CARD of caspase-1. We have conducted functional studies on Cop and report on its role as an inhibitor of cell death in a broad range of cell death paradigms. A notable exception in the ability of Cop to inhibit cell death pertains to its inability to inhibit ER stress-mediated cell death. Furthermore, in addition to the known interaction of Cop and caspase-1, we demonstrated a novel interaction of Cop with caspase-4. We propose that Cop's action to prevent TNF-alpha-induced cell death may operate independently of the mitochondrial death pathway. Furthermore, Cop overexpression inhibits Bid cleavage. In summary, Cop inhibition of cell death, at least to a certain extent, results from its interference with the activation of caspase-1 and caspase-4. Understanding the mechanistic details modulating caspase cell death pathways should provide important information for the development of therapies for diseases featuring aberrant caspase activation. Cop, as an inhibitor of an important apical caspase cell death axis, may provide a tool for modulating pathological cell death.     

Induction of interleukin 1 by Legionella pneumophila in murine peritoneal macrophage cultures

Legionella pneumophila-induced production of both membrane-associated and secreted interleukin 1 (mIL-1 and sIL-1, respectively) was examined utilizing peritoneal macrophages from BALB/c mice. The Legionella preparations for these studies included viable bacteria and formalin-killed whole cell preparations. Both of the preparations induced mIL-1 and sIL-1 in a dose-dependent fashion. However, the viable bacteria required about 1 log lower concentrations than the formalin-killed bacteria to induce the same level of IL-1 activity measured in the thymocyte proliferation assay. Kinetic studies showed that mIL-1 and sIL-1 were detectable within 4 hr after addition of either of the L. pneumophila preparations to the peritoneal macrophage cultures, with peak levels achieved within 24 hr. These results indicate that L. pneumophila is a potent inducer of both mIL-1 and sIL-1 in normal mouse peritoneal macrophage cultures.     

PKC and MEK pathways inhibit caspase-9/-3-mediated cytotoxicity in differentiated cells

Many studies have indicated that differentiated cells inhibit drug-induced cytotoxicity but undifferentiated cells do not, though the mechanisms are unclear. Currently, HL-60 cells are induced to differentiate into macrophage-like cells with Phorbol-12-myristate-13-acetate (TPA) treatment (TPA-differentiated cells). Our study shows that caspase-9/-3-mediated cytotoxicity can be induced in undifferentiated HL-60 cells but not in TPA-differentiated HL-60 cells. However, caspase-9/-3-mediated cytotoxicity can be induced in TPA-differentiated cells if they are pretreated with a protein kinase C (PKC) or a mitogen activated protein kinase (MEK) inhibitor. Taken together, this study demonstrates that TPA-differentiated HL-60 cells inhibit caspases-9/-3-mediated cytotoxicity through the PKC and MEK signaling pathways.     

Salmonella-induced macrophage death: the role of caspase-1 in death and inflammation.

Salmonella typhimurium invades host macrophages and can induce either an almost immediate cell death or establish an intracellular niche within the phagocytic vacuole. Rapid cell death depends on the Salmonella pathogenicity island SPI1 and the host protein caspase-1, a member of the pro-apoptotic caspase family of proteases. Caspase-1-dependent cell death leads to the activation of the potent pro-inflammatory cytokines interleukin (IL)-1beta and IL-18 to produce bioactive cytokines. Animal studies indicate that the activation of these cytokines is necessary for efficient colonization of the mouse gastrointestinal tract. Salmonella that reside in the phagocytic vacuole do not cause this early cell death and can trigger a macrophage death at a much later time point. This late-phase cell death is dependent on SPI2-encoded genes and ompR.     

A small molecule binding HMGB1 inhibits caspase-11-mediated lethality in sepsis

Caspase-11, a cytosolic lipopolysaccharide (LPS) receptor, mediates lethal immune responses and coagulopathy in sepsis, a leading cause of death worldwide with limited therapeutic options. We previously showed that over-activation of caspase-11 is driven by hepatocyte-released high mobility group box 1 (HMGB1), which delivers extracellular LPS into the cytosol of host cells during sepsis. Using a phenotypic screening strategy with recombinant HMGB1 and peritoneal macrophages, we discovered that FeTPPS, a small molecule selectively inhibits HMGB1-mediated caspase-11 activation. The physical interaction between FeTPPS and HMGB1 disrupts the HMGB1-LPS binding and decreases the capacity of HMGB1 to induce lysosomal rupture, leading to the diminished cytosolic delivery of LPS. Treatment of FeTPPS significantly attenuates HMGB1- and caspase-11-mediated immune responses, organ damage, and lethality in endotoxemia and bacterial sepsis. These findings shed light on the development of HMGB1-targeting therapeutics for lethal immune disorders and might open a new avenue to treat sepsis.Subject terms: Cell death and immune response, Sepsis     

NOS1-mediated macrophage and endothelial cell interaction in the progression of atherosclerosis

Atherosclerosis is a chronic inflammatory disease arising due to an imbalance in lipid metabolism and maladaptive immune response driven by the accumulation of cholesterol-laden macrophages in the artery wall. Interactions between monocytes/macrophages and endothelial cells play an essential role in the pathogenesis of atherosclerosis. In our current study, nitric oxide synthase 1 (NOS1)-derived nitric oxide (NO) has been identified as a regulator of macrophage and endothelial cell interaction. Oxidized LDL (OxLDL) activates NOS1, which results in the expression of CD40 ligand in macrophages. OxLDL-stimulated macrophages produce some soluble factors which increase the CD40 receptor expression in endothelial cells. This increases the interaction between the macrophages and endothelial cells, which leads to an increase in the inflammatory response. Inhibition of NOS1-derived NO might serve as an effective strategy to reduce foam cell formation and limit the extent of atherosclerotic plaque expansion.     

Involvement of cannabinoid receptor CB2 in dectin-1-mediated macrophage phagocytosis

Cannabinoid receptors are expressed in macrophages, but little is known of their roles. We here examined their involvement in phagocytosis. The presence of 2-arachidonylglycerol, an endocannabinoid, augmented the phagocytosis of zymosan by mouse macrophages, while the phagocytosis of Escherichia coli, Staphylococcus aureus, apoptotic cells or latex beads remained unaffected. An agonist of the cannabinoid receptors CB1 and CB2 also stimulated the phagocytosis of zymosan. The stimulatory effect of 2-arachidonylglycerol was abolished when phagocytosis reactions were carried out in the presence of an antagonist of CB2 but not of CB1. Furthermore, the phagocytosis of zymosan in the presence of 2-arachidonylglycerol was severely inhibited by the addition of a beta-glucan-containing carbohydrate or antibody neutralizing dectin-1, a beta-glucan-recognizing phagocytosis receptor. These results suggested that the activation of CB2 in macrophages leads to the stimulation of dectin-1-mediated phagocytosis.