Transient P2X7 receptor activation triggers macrophage death independent of Toll-like receptors 2 and 4, caspase-1, and pannexin-1 proteins

The function of P2X(7) receptors (ATP-gated ion channels) in innate immune cells is unclear. In the setting of Toll-like receptor (TLR) stimulation, secondary activation of P2X(7) ion channels has been linked to pro-caspase-1 cleavage and cell death. Here we show that cell death is a surprisingly early triggered event. We show using live-cell imaging that transient (1-4 min) stimulation of mouse macrophages with high extracellular ATP ([ATP]e) triggers delayed (hours) cell death, indexed as DEVDase (caspase-3 and caspase-7) activity. Continuous or transient high [ATP]e did not induce cell death in P2X(7)-deficient (P2X(7)(-/-)) macrophages or neutrophils (in which P2X(7) could not be detected). Blocking sustained Ca(2+) influx, a signature of P2X(7) ligation, was highly protective, whereas no protection was conferred in macrophages lacking caspase-1 or TLR2 and TLR4. Furthermore, pannexin-1 (Panx1) deficiency had no effect on transient ATP-induced delayed cell death or ATP-induced Yo-Pro-1 uptake (an index of large pore pathway formation). Thus, "transient" P2X(7) receptor activation and Ca(2+) overload act as a death trigger for native mouse macrophages independent of Panx1 and pro-inflammatory caspase-1 and TLR signaling.     

Activation of pannexin-1 mediates triglyceride-induced macrophage cell death

The accumulation of triglycerides (TGs) in macrophages induces cell death, a risk factor in the pathogenesis of atherosclerosis. We had previously reported that TG-induced macrophage death is triggered by caspase-1 and -2, therefore we investigated the mechanism underlying this phenomenon. We found that potassium efflux is increased in TG-treated THP-1 macrophages and that the inhibition of potassium efflux blocks TG-induced cell death as well as caspase-1 and -2 activation. Furthermore, reducing ATP concentration (known to induce potassium efflux), restored cell viability and caspase-1 and -2 activity. The activation of pannexin-1 (a channel that releases ATP), was increased after TG treatment in THP-1 macrophages. Inhibition of pannexin-1 activity using its inhibitor, probenecid, recovered cell viability and blocked the activation of caspase-1 and -2 in TG-treated macrophages. These results suggest that TG-induced THP-1 macrophage cell death is induced via pannexin-1 activation, which increases extracellular ATP, leading to an increase in potassium efflux.     

Pannexin-1-mediated intracellular delivery of muramyl dipeptide induces caspase-1 activation via cryopyrin/NLRP3 independently of Nod2

Muramyl dipeptide (MDP), the microbial activator of nucleotide-binding oligomerization domain 2 (Nod2), induces NF-kappaB and MAPK activation, leading to the production of multiple anti-bacterial and proinflammatory molecules. In addition, MDP has been implicated in IL-1beta secretion through the regulation of caspase-1. However, the mechanisms that mediate caspase-1 activation and IL-1beta secretion in response to MDP stimulation remain poorly understood. We show here that fluorescent MDP molecules are internalized in primary macrophages and accumulate in granular structures that colocalize with markers of acidified endosomal compartments. The uptake of MDP was Nod2-independent. Upon ATP stimulation, labeled MDP was rapidly released from acidified vesicles into the cytosol, a process that required functional pannexin-1. Caspase-1 activation induced by MDP and ATP required pannexin-1 and Cryopyrin but was independent of Nod2. Conversely, induction of pro-IL-1beta mRNA by MDP stimulation was abolished in Nod2-deficient macrophages but unimpaired in macrophages lacking Cryopyrin. These studies demonstrate a Nod2-independent mechanism mediated through pore-forming pannexin-1 that is required for intracellular delivery of MDP to the cytosol and caspase-1 activation. Furthermore, the work provides evidence for distinct roles of Nod2 and Cryopyrin in the regulation of MDP-induced caspase-1 activation and IL-1beta secretion.     

Priming of macrophages with lipopolysaccharide potentiates P2X7-mediated cell death via a caspase-1-dependent mechanism, independently of cytokine production.

ATP stimulation of cell surface P2X7 receptors results in cytolysis and cell death of macrophages. Activation of this receptor in bacterial lipopolysaccharide (LPS)-activated macrophages or monocytes also stimulates processing and release of the cytokine interleukin-1beta(IL-1beta) through activation of caspase-1. The cytokine interleukin 18 (IL-18) is also cleaved by caspase-1 and shares pro-inflammatory characteristics with IL-1beta. The objective of the present study was to test the hypothesis that IL-1beta, IL-18, and/or caspase-1 activation contribute directly to macrophage cell death induced by LPS and ATP. Macrophages were cultured from normal mice or those in which genes for the P2X7 receptor, IL-1beta, IL-1alpha, IL-18, or caspase-1 had been deleted. Our data confirm the importance of the P2X7 receptor in ATP-stimulated cell death and IL-1beta release from LPS-primed macrophages. We demonstrate that prolonged stimulation with ATP leads to cell death, which is partly dependent on LPS priming and caspase-1, but independent of cytokine processing and release. We also provide evidence that LPS priming of macrophages makes them highly susceptible to the toxic effects of brief exposure to ATP, which leads to rapid cell death by a mechanism that is dependent on caspase-1 but, again, independent of cytokine processing and release.     

P2X7 receptor differentially couples to distinct release pathways for IL-1beta in mouse macrophage

The proinflammatory IL-1 cytokines IL-1alpha, IL-1beta, and IL-18 are key mediators of the acute immune response to injury and infection. Mechanisms underlying their cellular release remain unclear. Activation of purinergic P2X(7) receptors (P2X(7)R) by extracellular ATP is a key physiological inducer of rapid IL-1beta release from LPS-primed macrophage. We investigated patterns of ATP-mediated release of IL-1 cytokines from three macrophage types in attempts to provide direct evidence for or against distinct release mechanisms. We used peritoneal macrophage from P2X(7)R(-/-) mice and found that release of IL-1alpha, IL-18, as well as IL-1beta, by ATP resulted exclusively from activation of P2X(7)R, release of all these IL-1 cytokines involved pannexin-1 (panx1), and that there was both a panx1-dependent and -independent component to IL-1beta release. We compared IL-1-release patterns from LPS-primed peritoneal macrophage, RAW264.7 macrophage, and J774A.1 macrophage. We found RAW264.7 macrophage readily release pro-IL-1beta independently of panx1 but do not release mature IL-1beta because they do not express apoptotic speck-like protein with a caspase-activating recruiting domain and so have no caspase-1 inflammasome activity. We delineated two distinct release pathways: the well-known caspase-1 cascade mediating release of processed IL-1beta that was selectively blocked by inhibition of caspase-1 or panx1, and a calcium-independent, caspase-1/panx1-independent release of pro-IL-1beta that was selectively blocked by glycine. None of these release responses were associated with cell damage or cytolytic effects. This provides the first direct demonstration of a distinct signaling mechanism responsible for ATP-induced release of pro-IL-1beta.     

ATP release from dying autophagic cells and their phagocytosis are crucial for inflammasome activation in macrophages

Pathogen-activated and damage-associated molecular patterns activate the inflammasome in macrophages. We report that mouse macrophages release IL-1β while co-incubated with pro-B (Ba/F3) cells dying, as a result of IL-3 withdrawal, by apoptosis with autophagy, but not when they are co-incubated with living, apoptotic, necrotic or necrostatin-1 treated cells. NALP3-deficient macrophages display reduced IL-1β secretion, which is also inhibited in macrophages deficient in caspase-1 or pre-treated with its inhibitor. This finding demonstrates that the inflammasome is activated during phagocytosis of dying autophagic cells. We show that activation of NALP3 depends on phagocytosis of dying cells, ATP release through pannexin-1 channels of dying autophagic cells, P(2)X(7) purinergic receptor activation, and on consequent potassium efflux. Dying autophagic Ba/F3 cells injected intraperitoneally in mice recruit neutrophils and thereby induce acute inflammation. These findings demonstrate that NALP3 performs key upstream functions in inflammasome activation in mouse macrophages engulfing dying autophagic cells, and that these functions lead to pro-inflammatory responses.     

Lipopolysaccharide-induced IL-18 secretion from murine Kupffer cells independently of myeloid differentiation factor 88 that is critically involved in induction of production of IL-12 and IL-1beta

IL-18, produced as biologically inactive precursor, is secreted from LPS-stimulated macrophages after cleavage by caspase-1. In this study, we investigated the mechanism underlying caspase-1-mediated IL-18 secretion. Kupffer cells constantly stored IL-18 and constitutively expressed caspase-1. Inhibition of new protein synthesis only slightly reduced IL-18 secretion, while it decreased and abrogated their IL-1beta and IL-12 secretion, respectively. Kupffer cells deficient in Toll-like receptor (TLR) 4, an LPS-signaling receptor, did not secrete IL-18, IL-1beta, and IL-12 upon LPS stimulation. In contrast, Kupffer cells lacking myeloid differentiation factor 88 (MyD88), an adaptor molecule for TLR-mediated-signaling, secreted IL-18 without IL-1beta and IL-12 production in a caspase-1-dependent and de novo synthesis-independent manner. These results indicate that MyD88 is essential for IL-12 and IL-1beta production from Kupffer cells while their IL-18 secretion is mediated via activation of endogenous caspase-1 without de novo protein synthesis in a MyD88-independent fashion after stimulation with LPS. In addition, infection with Listeria monocytogenes, products of which have the capacity to activate TLR, increased serum levels of IL-18 in wild-type and MyD88-deficient mice but not in caspase-1-deficient mice, whereas it induced elevation of serum levels of IL-12 in both wild-type and caspase-1-deficient mice but not in MyD88-deficient mice. Taken together, these results suggested caspase-1-dependent, MyD88-independent IL-18 release in bacterial infection.     

Activation of P2X(7) receptor by ATP plays an important role in regulating inflammatory responses during acute viral infection

Acute viral infection causes damages to the host due to uncontrolled viral replication but even replication deficient viral vectors can induce systemic inflammatory responses. Indeed, overactive host innate immune responses to viral vectors have led to devastating consequences. Macrophages are important innate immune cells that recognize viruses and induce inflammatory responses at the early stage of infection. However, tissue resident macrophages are not easily activated by the mere presence of virus suggesting that their activation requires additional signals from other cells in the tissue in order to trigger inflammatory responses. Previously, we have shown that the cross-talk between epithelial cells and macrophages generates synergistic inflammatory responses during adenoviral vector infection. Here, we investigated whether ATP is involved in the activation of macrophages to induce inflammatory responses during an acute adenoviral infection. Using a macrophage-epithelial cell co-culture system we demonstrated that ATP signaling through P2X(7) receptor (P2X(7)R) is required for induction of inflammatory mediators. We also showed that ATP-P2X(7)R signaling regulates inflammasome activation as inhibition or deficiency of P2X(7)R as well as caspase-1 significantly reduced IL-1β secretion. Furthermore, we found that intranasal administration of replication deficient adenoviral vectors in mice caused a high mortality in wild-type mice with symptoms of acute respiratory distress syndrome but the mice deficient in P2X(7)R or caspase-1 showed increased survival. In addition, wild-type mice treated with apyrase or inhibitors of P2X(7)R or caspase-1 showed higher rates of survival. The improved survival in the P2X(7)R deficient mice correlated with diminished levels of IL-1β and IL-6 and reduced neutrophil infiltration in the early phase of infection. These results indicate that ATP, released during viral infection, is an important inflammatory regulator that activates the inflammasome pathway and regulates inflammatory responses.     

Caspase-1-Dependent and -Independent Cell Death Pathways in Burkholderia pseudomallei Infection of Macrophages

The cytosolic pathogen Burkholderia pseudomallei and causative agent of melioidosis has been shown to regulate IL-1β and IL-18 production through NOD-like receptor NLRP3 and pyroptosis via NLRC4. Downstream signalling pathways of those receptors and other cell death mechanisms induced during B. pseudomallei infection have not been addressed so far in detail. Furthermore, the role of B. pseudomallei factors in inflammasome activation is still ill defined. In the present study we show that caspase-1 processing and pyroptosis is exclusively dependent on NLRC4, but not on NLRP3 in the early phase of macrophage infection, whereas at later time points caspase-1 activation and cell death is NLRC4- independent. In the early phase we identified an activation pathway involving caspases-9, -7 and PARP downstream of NLRC4 and caspase-1. Analyses of caspase-1/11-deficient infected macrophages revealed a strong induction of apoptosis, which is dependent on activation of apoptotic initiator and effector caspases. The early activation pathway of caspase-1 in macrophages was markedly reduced or completely abolished after infection with a B. pseudomallei flagellin FliC or a T3SS3 BsaU mutant. Studies using cells transfected with the wild-type and mutated T3SS3 effector protein BopE indicated also a role of this protein in caspase-1 processing. A T3SS3 inner rod protein BsaK mutant failed to activate caspase-1, revealed higher intracellular counts, reduced cell death and IL-1β secretion during early but not during late macrophage infection compared to the wild-type. Intranasal infection of BALB/c mice with the BsaK mutant displayed a strongly decreased mortality, lower bacterial loads in organs, and reduced levels of IL-1β, myeloperoxidase and neutrophils in bronchoalveolar lavage fluid. In conclusion, our results indicate a major role for a functional T3SS3 in early NLRC4-mediated caspase-1 activation and pyroptosis and a contribution of late caspase-1-dependent and -independent cell death mechanisms in the pathogenesis of B. pseudomallei infection.     

Pannexin-1 couples to maitotoxin- and nigericin-induced interleukin-1beta release through a dye uptake-independent pathway

Pannexin-1 is a recently identified membrane protein that can act as a nonselective pore permeable to dyes such as ethidium when ectopically expressed. Blockade of pannexin-1 in macrophage endogenously expressing the ATP-gated P2X7 receptor (P2X7R) blocks the initial dye uptake, but not the ionic current, and also blocks processing and release of interleukin-1beta (IL-1beta) in response to P2X7R activation. These results suggest that pannexin-1 may be a hemichannel activated by the P2X7R to provide the conduit for dye uptake and downstream signaling to processing and release of IL-1beta. We have pursued this hypothesis by measuring dye uptake and IL-1beta processing and release in mouse J774 macrophage in response to P2X7R activation and to maitotoxin and nigericin, two agents considered to evoke IL-1beta release via the same mechanism. The experiments were carried out over time periods during which no lactate dehydrogenase was released from cells to examine only noncytolytic pathways. P2X7R activation evoked dye uptake that could be separated into two components by pannexin-1 inhibition: an initial rapid phase and a slower pannexin-1-independent phase. Maitotoxin-evoked dye uptake was unaltered by pannexin-1 inhibition. Nigericin did not induce dye uptake. Inhibition of pannexin-1 blocked caspase-1 and IL-1beta processing and release in response to all three stimuli. Thus, although pannexin-1 is required for IL-1beta release in response to maitotoxin, nigericin, and ATP, a mechanism distinct from pannexin-1 hemichannel activation must underlie the former two processes.     

Altered cytokine production in mice lacking P2X(7) receptors

The P2X(7) receptor (P2X(7)R) is an ATP-gated ion channel expressed by monocytes and macrophages. To directly address the role of this receptor in interleukin (IL)-1 beta post-translational processing, we have generated a P2X(7)R-deficient mouse line. P2X(7)R(-/-) macrophages respond to lipopolysaccharide and produce levels of cyclooxygenase-2 and pro-IL-1 beta comparable with those generated by wild-type cells. In response to ATP, however, pro-IL-1 beta produced by the P2X(7)R(-/-) cells is not externalized or activated by caspase-1. Nigericin, an alternate secretion stimulus, promotes release of 17-kDa IL-1 beta from P2X(7)R(-/-) macrophages. In response to in vivo lipopolysaccharide injection, both wild-type and P2X(7)R(-/-) animals display increases in peritoneal lavage IL-6 levels but no detectable IL-1. Subsequent ATP injection to wild-type animals promotes an increase in IL-1, which in turn leads to additional IL-6 production; similar increases did not occur in ATP-treated, LPS-primed P2X(7)R(-/-) animals. Absence of the P2X(7)R thus leads to an inability of peritoneal macrophages to release IL-1 in response to ATP. As a result of the IL-1 deficiency, in vivo cytokine signaling cascades are impaired in P2X(7)R-deficient animals. Together these results demonstrate that P2X(7)R activation can provide a signal that leads to maturation and release of IL-1 beta and initiation of a cytokine cascade.     

Chemotherapeutic Drugs Induce ATP Release via Caspase-gated Pannexin-1 Channels and a Caspase/Pannexin-1-independent Mechanism

Anti-tumor immune responses have been linked to the regulated release of ATP from apoptotic cancer cells to engage P2 purinergic receptor signaling cascades in nearby leukocytes. We used the Jurkat T cell acute lymphocytic leukemia model to characterize the role of pannexin-1 (Panx1) channels in the release of nucleotides during chemotherapeutic drug-induced apoptosis. Diverse pro-apoptotic drugs, including topoisomerase II inhibitors, kinase inhibitors, and proteosome inhibitors, induced functional activation of Panx1 channels via caspase-3-mediated cleavage of the Panx1 autoinhibitory C-terminal domain. The caspase-activated Panx1 channels mediated efflux of ATP, but also ADP and AMP, with the latter two comprising >90% of the released adenine nucleotide pool as cells transitioned from the early to late stages of apoptosis. Chemotherapeutic drugs also activated an alternative caspase- and Panx1-independent pathway for ATP release from Jurkat cells in the presence of benzyloxycarbonyl-VAD, a pan-caspase inhibitor. Comparison of Panx1 levels indicated much higher expression in leukemic T lymphocytes than in normal, untransformed T lymphoblasts. This suggests that signaling roles for Panx1 may be amplified in leukemic leukocytes. Together, these results identify chemotherapy-activated pannexin-1 channels and ATP release as possible mediators of paracrine interaction between dying tumor cells and the effector leukocytes that mediate immunogenic anti-tumor responses.     

Intracytosolic Listeria monocytogenes induces cell death through caspase-1 activation in murine macrophages

Listeria monocytogenes induces apoptosis in vitro and in vivo in a variety of cell types. However, the mechanism of cell death in L. monocytogenes -infected macrophages was initially reported to be distinct from apoptosis. Here, we studied the mechanism of L. monocytogenes -induced cell death using sensitive fluorescent techniques. We found that caspase-1 activation preceded cell death of macrophages infected with L. monocytogenes , using fluorogenic substrates. Caspase-1 activation was diminished after infection with wild-type L. monocytogenes when cells were treated with NH₄Cl, or if they were infected with a listeriolysin mutant that cannot escape from the phagolysosome. Mitochondrial membrane integrity was preserved during the infection. A particular mechanism of cell death, recently termed 'pyroptosis', is associated with infection by intracellular microorganisms, and has an inherent pro-inflammatory character, due to involvement of caspase-1 activation with consequent IL-1β and IL-18 production. Cell death through caspase-1 activation would constitute a defence mechanism of macrophages which induces cell death to eliminate the bacteria's intracytosolic niche and recruits early host's defences through the secretion of inflammatory cytokines.     

Critical role for cathepsin B in mediating caspase-1-dependent interleukin-18 maturation and caspase-1-independent necrosis triggered by the microbial toxin nigericin

The potassium ionophore nigericin induces cell death and promotes the maturation and release of IL-1beta in lipopolysaccharide (LPS)-primed monocytes and macrophages, the latter depending on caspase-1 activation by an unknown mechanism. Here, we investigate the pathway that triggers cell death and activates caspase-1. We show that without LPS priming, nigericin alone triggered caspase-1 activation and IL-18 generation in THP-1 monocytic cells. Simultaneously, nigericin induced caspase-1-independent necrotic cell death, which was blocked by the cathepsin B inhibitor CA-074-Me and other cathepsin inhibitors. Cathepsin B activation after nigericin treatment was determined biochemically and corroborated by rapid lysosomal leakage and translocation of cathepsin B to the cytoplasm. IL-18 maturation was prevented by both caspase-1 and cathepsin B inhibitors in THP-1 cells, primary mouse macrophages and human blood monocytes. Moreover, IL-18 generation was reduced in THP-1 cells stably transformed either with cystatin A (an endogenous cathepsin inhibitor) or antisense cathepsin B cDNA. Collectively, our study establishes a critical role for cathepsin B in nigericin-induced caspase-1-dependent IL-18 maturation and caspase-1-independent necrosis.     

Apaf-1 and caspase-9 accelerate apoptosis, but do not determine whether factor-deprived or drug-treated cells die

Apoptosis after growth factor withdrawal or drug treatment is associated with mitochondrial cytochrome c release and activation of Apaf-1 and caspase-9. To determine whether loss of Apaf-1, caspase-2, and caspase-9 prevented death of factor-starved cells, allowing them to proliferate when growth factor was returned, we generated IL-3-dependent myeloid lines from gene-deleted mice. Long after growth factor removal, cells lacking Apaf-1, caspase-9 or both caspase-9 and caspase-2 appeared healthy, retained intact plasma membranes, and did not expose phosphatidylserine. However, release of cytochrome c still occurred, and they failed to form clones when IL-3 was restored. Cells lacking caspase-2 alone had no survival advantage. Therefore, Apaf-1, caspase-2, and caspase-9 are not required for programmed cell death of factor-dependent cells, but merely affect its rate. In contrast, transfection with Bcl-2 provided long-term, clonogenic protection, and could act independently of the apoptosome. Unlike expression of Bcl-2, loss of Apaf-1, caspase-2, or caspase-9 would therefore be unlikely to enhance the survival of cancer cells.     

JNK2 is required for efficient T-cell activation and apoptosis but not for normal lymphocyte development

BACKGROUND: The Jun N-terminal kinase (JNK) signaling pathway has been implicated in cell proliferation and apoptosis, but its function seems to depend on the cell type and inducing signal. In T cells, JNK has been implicated in both antigen-induced activation and apoptosis. RESULTS: We generated mice lacking the JNK2 isozymes. The mutant mice were healthy and fertile but defective in peripheral T-cell activation induced by antibody to the CD3 component of the T-cell receptor (TCR) complex - proliferation and production of interleukin-2 (IL-2), IL-4 and interferon-gamma (IFN-gamma) were reduced. The proliferation defect was restored by exogenous IL-2. B-cell activation was normal in the absence of JNK2. Activation-induced peripheral T-cell apoptosis was comparable between mutant and wild-type mice, but immature (CD4(+) CD8(+)) thymocytes lacking JNK2 were resistant to apoptosis induced by administration of anti-CD3 antibody in vivo. The lack of JNK2 also resulted in partial resistance of thymocytes to anti-CD3 antibody in vitro, but had little or no effect on apoptosis induced by anti-Fas antibody, dexamethasone or ultraviolet-C (UVC) radiation. CONCLUSIONS: JNK2 is essential for efficient activation of peripheral T cells but not B cells. Peripheral T-cell activation is probably required indirectly for induction of thymocyte apoptosis resulting from administration of anti-CD3 antibody in vivo. JNK2 functions in a cell-type-specific and stimulus-dependent manner, being required for apoptosis of immature thymocytes induced by anti-CD3 antibody but not for apoptosis induced by anti-Fas antibody, UVC or dexamethasone. JNK2 is not required for activation-induced cell death of mature T cells.     

Bordetella pertussis adenylate cyclase toxin modulates innate and adaptive immune responses: distinct roles for acylation and enzymatic activity in immunomodulation and cell death

Adenylate cyclase toxin (CyaA) of Bordetella pertussis belongs to the repeat in toxin family of pore-forming toxins, which require posttranslational acylation to lyse eukaryotic cells. CyaA modulates dendritic cell (DC) and macrophage function upon stimulation with LPS. In this study, we examined the roles of acylation and enzymatic activity in the immunomodulatory and lytic effects of CyaA. The adenylate cyclase activity of CyaA was necessary for its modulatory effects on murine innate immune cells. In contrast, acylation was not essential for the immunomodulatory function of CyaA, but was required for maximal caspase-3 activation and cytotoxic activity. The wild-type acylated toxin (A-CyaA) and nonacylated CyaA (NA-CyaA), but not CyaA with an inactive adenylate cyclase domain (iAC-CyaA), enhanced TLR-ligand-induced IL-10 and inhibited IL-12, TNF-alpha, and CCL3 production by macrophages and DC. In addition, both A-CyaA and NA-CyaA, but not iAC-CyaA, enhanced surface expression of CD80 and decreased CpG-stimulated CD40 and ICAM-1 expression on immature DC. Furthermore, both A-CyaA and NA-CyaA promoted the induction of murine IgG1 Abs, Th2, and regulatory T cells against coadministered Ags in vivo, whereas iAC-CyaA had more limited adjuvant activity. In contrast, A-CyaA and iAC-CyaA induced caspase-3 activation and cell death in macrophages, but these effects were considerably reduced or absent with NA-CyaA. Our findings demonstrate that the enzymatic activity plays a critical role in the immunomodulatory effects of CyaA, whereas acylation facilitates the induction of apoptosis and cell lysis, and as such, NA-CyaA has considerable potential as a nontoxic therapeutic molecule with potent anti-inflammatory properties.     

YopJ-induced caspase-1 activation in Yersinia-infected macrophages: independent of apoptosis, linked to necrosis, dispensable for innate host defense

Yersinia outer protein J (YopJ) is a type III secretion system (T3SS) effector of pathogenic Yersinia (Yersinia pestis, Yersinia enterocolitica and Yersinia pseudotuberculosis) that is secreted into host cells. YopJ inhibits survival response pathways in macrophages, causing cell death. Allelic variation of YopJ is responsible for differential cytotoxicity in Yersinia strains. YopJ isoforms in Y. enterocolitica O:8 (YopP) and Y. pestis KIM (YopJ(KIM)) strains have high cytotoxic activity. In addition, YopJ(KIM)-induced macrophage death is associated with caspase-1 activation and interleukin-1β (IL-1β secretion. Here, the mechanism of YopJ(KIM)-induced cell death, caspase-1 activation, and IL-1β secretion in primary murine macrophages was examined. Caspase-3/7 activity was low and the caspase-3 substrate poly (ADP-ribose) polymerase (PARP) was not cleaved in Y. pestis KIM5-infected macrophages. In addition, cytotoxicity and IL-1β secretion were not reduced in the presence of a caspase-8 inhibitor, or in B-cell lymphoma 2 (Bcl-2)-associated X protein (Bax)/Bcl-2 homologous antagonist/killer (Bak) knockout macrophages, showing that YopJ(KIM)-mediated cell death and caspase-1 activation occur independent of mitochondrial-directed apoptosis. KIM5-infected macrophages released high mobility group protein B1 (HMGB1), a marker of necrosis, and microscopic analysis revealed that necrotic cells contained active caspase-1, indicating that caspase-1 activation is associated with necrosis. Inhibitor studies showed that receptor interacting protein 1 (RIP1) kinase and reactive oxygen species (ROS) were not required for cytotoxicity or IL-β release in KIM5-infected macrophages. IL-1β secretion was reduced in the presence of cathepsin B inhibitors, suggesting that activation of caspase-1 requires cathepsin B activity. Ectopically-expressed YopP caused higher cytotoxicity and secretion of IL-1β in Y. pseudotuberculosis-infected macrophages than YopJ(KIM). Wild-type and congenic caspase 1 knockout C57BL/6 mice were equally susceptible to lethal infection with Y. pseudotuberculosis ectopically expressing YopP. These data suggest that YopJ-induced caspase-1 activation in Yersinia-infected macrophages is a downstream consequence of necrotic cell death and is dispensable for innate host resistance to a strain with enhanced cytotoxicity.     

Caspase-1 Is an Apical Caspase Leading to Caspase-3 Cleavage in the AIM2 Inflammasome Response,Independent of Caspase-8

Canonical inflammasomes are multiprotein complexes that can activate both caspase-1 and caspase-8. Caspase-1 drives rapid lysis of cells by pyroptosis and maturation of interleukin (IL)-1β and IL-18. In caspase-1-deficient cells, inflammasome formation still leads to caspase-3 activation and slower apoptotic death, dependent on caspase-8 as an apical caspase. A role for caspase-8 directly upstream of caspase-1 has also been suggested, but here we show that caspase-8-deficient macrophages have no defect in AIM2 inflammasome-mediated caspase-1 activation, pyroptosis, and IL-1β cleavage. In investigating the inflammasome-induced apoptotic pathway, we previously demonstrated that activated caspase-8 is essential for caspase-3 cleavage and apoptosis in caspase-1-deficient cells. However, here we found that AIM2 inflammasome-initiated caspase-3 cleavage was maintained in Ripk3^?/? Casp8^?/? macrophages. Gene knockdown showed that caspase-1 was required for the caspase-3 cleavage. Thus inflammasomes activate a network of caspases that can promote both pyroptotic and apoptotic cell death. In cells where rapid pyroptosis is blocked, delayed inflammasome-dependent cell death could still occur due to both caspase-1- and caspase-8-dependent apoptosis. Initiation of redundant cell death pathways is likely to be a strategy for coping with pathogen interference in death processes.     

Different roles of spermine in glucocorticoid- and Fas-induced apoptosis

Two experimental systems representative of the mitochondrial and death receptor apoptotic pathways are the dexamethasone-induced programmed cell death in mouse thymocytes and the antibody-mediated cross-ligation of the Fas receptor in the human leukemic T-cell line Jurkat, respectively. In both cell systems, caspase-9, -8, and -3 were activated upon induction of apoptosis and a sub-G(1) peak appeared as a sign of ongoing DNA fragmentation. Addition of 1 mM spermine together with dexamethasone inhibited caspase activation and the appearance of the sub-G(1) peak in mouse thymocytes. In contrast, Fas-induced cell death was totally unaffected by spermine addition. Spermine addition significantly elevated the spermine concentration in both thymocytes and Jurkat cells. Thus, spermine per se did not inhibit the caspases but rather their activation. The fact that spermine inhibited caspase activation only in the thymocytes implies that spermine inhibited dexamethasone-induced apoptosis upstream of caspase-9 activation.