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.     

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.     

Potentiation of caspase-1 activation by the P2X7 receptor is dependent on TLR signals and requires NF-kappaB-driven protein synthesis

The proinflammatory cytokines IL-1beta and IL-18 are inactive until cleaved by the enzyme caspase-1. Stimulation of the P2X7 receptor (P2X7R), an ATP-gated ion channel, triggers rapid activation of caspase-1. In this study we demonstrate that pretreatment of primary and Bac1 murine macrophages with TLR agonists is required for caspase-1 activation by P2X7R but it is not required for activation of the receptor itself. Caspase-1 activation by nigericin, a K+/H+ ionophore, similarly requires LPS priming. This priming by LPS is dependent on protein synthesis, given that cyclohexamide blocks the ability of LPS to prime macrophages for activation of caspase-1 by the P2X7R. This protein synthesis is likely mediated by NF-kappaB, as pretreatment of cells with the proteasome inhibitor MG132, or the IkappaB kinase inhibitor Bay 11-7085 before LPS stimulation blocks the ability of LPS to potentiate the activation of caspase-1 by the P2X7R. Thus, caspase-1 regulation in macrophages requires inflammatory stimuli that signal through the TLRs to up-regulate gene products required for activation of the caspase-1 processing machinery in response to K+-releasing stimuli such as ATP.     

Proteasomes control caspase-1 activation in anthrax lethal toxin-mediated cell killing

Activation of caspase-1 through the inflammasome protein Nalp1b controls anthrax lethal toxin (LT)-induced necrosis in murine macrophages. In this study we analyzed physiological changes controlled by caspase-1 in LT-treated murine macrophages. The caspase-1 inhibitor Boc-D-cmk blocked caspase-1 activity and membrane impairment in LT-treated cells. To determine the relationship between caspase-1 activation and membrane integrity, we added Boc-D-cmk to J774A.1 macrophages at different time points following LT exposure. Remarkably, Boc-D-cmk rescued LT-treated macrophages, even when added at the peak of caspase-1 activation. Late addition of the caspase-1 inhibitor reversed the losses of plasma membrane integrity and metabolic activity in these cells. Similar results were obtained with the proteasome inhibitor MG132, one of the most potent inhibitors of LT toxicity. LT-treated macrophages displaying evidence of membrane impairment recovered upon the addition of MG132, mirroring the Boc-D-cmk response. Strikingly, late addition of proteasome inhibitors also abrogated caspase-1 activity in LT-treated macrophages. Proteasomal control of caspase-1 activity and membrane impairment, however, was restricted to LT-induced cytolysis, because proteasome inhibitors did not block caspase-1 activation and cell death triggered by lipopolysaccharide and nigericin. Our findings indicate that proteasome inhibitors do not target caspase-1 directly but instead control an upstream event in LT-treated macrophages leading to caspase-1 activation. Taken together, caspase-1-mediated necrosis appears to be tightly controlled and differentially regulated by proteasomes depending on the source of caspase-1 induction.     

A Role for Stefin B (Cystatin B) in Inflammation and Endotoxemia

Stefin B (cystatin B) is an endogenous cysteine cathepsin inhibitor, and the loss-of-function mutations in the stefin B gene were reported in patients with Unverricht-Lundborg disease (EPM1). In this study we demonstrated that stefin B-deficient (StB KO) mice were significantly more sensitive to the lethal LPS-induced sepsis and secreted higher amounts of pro-inflammatory cytokines IL-1β and IL-18 in the serum. We further showed that increased caspase-11 gene expression and better pro-inflammatory caspase-1 and -11 activation determined in StB KO bone marrow-derived macrophages resulted in enhanced IL-1β processing. Pretreatment of macrophages with the cathepsin inhibitor E-64d did not affect secretion of IL-1β, suggesting that the increased cathepsin activity determined in StB KO bone marrow-derived macrophages is not essential for inflammasome activation. Upon LPS stimulation, stefin B was targeted into the mitochondria, and the lack of stefin B resulted in the increased destabilization of mitochondrial membrane potential and mitochondrial superoxide generation. Collectively, our study demonstrates that the LPS-induced sepsis in StB KO mice is dependent on caspase-11 and mitochondrial reactive oxygen species but is not associated with the lysosomal destabilization and increased cathepsin activity in the cytosol.     

ATP treatment of human monocytes promotes caspase-1 maturation and externalization

Mechanisms that regulate conversion of prointerleukin-1beta (pro-IL-1beta) to its mature form by the cysteine protease caspase-1 are not well understood. In this study, we demonstrate that mature caspase-1 subunits are produced when human monocytes are treated with ATP and, like mature IL-1beta, are released extracellularly. Characterization of the pharmacological sensitivity of this stimulus-coupled response revealed that some caspase-1 inhibitors allow pro-IL-1beta secretion, whereas others do not. Two nonselective alkylating agents, N-ethylmaleimide and phenylarsine oxide, also blocked maturation and release of pro-IL-1beta. Two inhibitors of anion transport, glyburide and ethacrynic acid, blocked maturation of both caspase-1 and pro-IL-1beta and prevented release of the propolypeptides. Procaspase-3 was detected in monocyte extracts, but its proteolytic activation was not efficient in the presence of ATP. Maturation of procaspase-1 and release of the mature enzyme subunits therefore accompany stimulus-coupled human monocyte IL-1 post-translational processing. Agents that appear to selectively inhibit mature caspase-1 do not prevent ATP-treated cells from releasing their cytosolic components. On the other hand, anion transport inhibitors and alkylating agents arrest ATP-treated monocytes in a state where membrane latency is maintained. The data provided support the hypothesis that stimulus-coupled IL-1 post-translational processing involves a commitment to cell death.     

Mechanisms of caspase-1 activation by P2X7 receptor-mediated K+ release

The mechanisms underlying caspase-1 activation and IL-1 processing during inflammatory activation of monocytes and macrophages are not well defined. Here, we describe an in vitro proteolytic processing assay that allows for comparison of caspase-1 regulatory components in a cell-free system separately from the confounding issue of IL-1 secretion. Analysis of in vitro IL-1 and caspase-1 processing in lysates from unstimulated Bac1 murine macrophages indicated a slow rate of basal caspase-1 activation and proteolytic maturation of IL-1. In contrast, brief (5 min) treatment of intact macrophages with extracellular ATP (as an activator of the P2X₇ receptor) or nigericin before cell lysis markedly accelerated the in vitro processing of caspase-1 and IL-1. This acceleration of in vitro processing was strictly dependent on loss of intracellular K⁺ from the intact cells. The induction of in vitro caspase-1 activation by lysis per se or by K⁺ loss before lysis was sensitive to pretreatment of intact macrophages with the tyrphostin AG-126 or bromoenol lactone, an inhibitor of Ca²⁺-independent phospholipase A₂. Caspase-1 activation and IL-1 processing in lysates from unstimulated macrophages were also accelerated by addition of recombinant ASC, a previously identified adapter protein that directly associates with caspase-1. These data indicate that increased K⁺ efflux via P2X₇ nucleotide receptor stimulation activates AG-126- and bromoenol lactone-sensitive signaling pathways in murine macrophages that result in stably maintained signals for caspase-1 regulation in cell-free assays. AG-126; ASC; bromoenol lactone; IL-1; inflammation     

Maturation modulates caspase-1-independent responses of dendritic cells to Anthrax lethal toxin

Anthrax lethal toxin (LT) contributes to the immune evasion strategy of Bacillus anthracis by impairing the function of cells of the immune system, such as macrophages and dendritic cells (DCs). Macrophages from certain inbred mice strains undergo rapid death upon LT treatment mediated by caspase-1 activation dependent on Nalp1b, an inflammasome component. Rapid LT-induced death is however, not observed in macrophages from human and many mouse strains. Here, we focused on the responses of various murine DCs to LT. Using a variety of knockout mice, we found that depending on the mouse strain, death of bone marrow-derived DCs and macrophages was mediated either by a fast Nalp1b and caspase-1-dependent, or by a slow caspase-1-independent pathway that was triggered by the impairment of MEK1/2 pathways. Caspase-1-independent death was observed in cells of different genetic backgrounds and interestingly occurred only in immature DCs. Maturation, triggered by different types of stimuli, led to full protection of DCs. These studies illustrate that the cellular damage inflicted by LT depends not only on the innate responses but also on the maturation stage of the cell, which modulates the more general caspase-1-independent responses.     

Virus infection activates IL-1 beta and IL-18 production in human macrophages by a caspase-1-dependent pathway.

Monocytes and macrophages play a significant role in host's defense system, since they produce a number of cytokines in response to microbial infections. We have studied IL-1 beta, IL-18, IFN-alpha/beta, and TNF-alpha gene expression and protein production in human primary monocytes and GM-CSF-differentiated macrophages during influenza A and Sendai virus infections. Virus-infected monocytes released only small amounts of IL-1 beta or IL-18 protein, whereas 7- and 14-day-old GM-CSF-differentiated macrophages readily produced these cytokines. Constitutive expression of proIL-18 was seen in monocytes and macrophages, and the expression of it was enhanced during monocyte/macrophage differentiation. Expression of IL-18 mRNA was clearly induced only by Sendai virus, whereas both influenza A and Sendai viruses induced IL-1 beta mRNA expression. Since caspase-1 is known to cleave proIL-1 beta and proIL-18 into their mature, active forms, we analyzed the effect of a specific caspase-1 inhibitor on virus-induced IL-1 beta and IL-18 production. The release of IL-1 beta and IL-18, but not that of IFN-alpha/beta or TNF-alpha, was clearly blocked by the inhibitor. Our results suggest that the cellular differentiation is a crucial factor that affects the capacity of monocytes/macrophages to produce IL-1 beta and IL-18 in response to virus infections. Furthermore, the virus-induced activation of caspase-1 is required for the efficient production of biologically active IL-1 beta and IL-18.     

Distinct role of p38 and c-Jun N-terminal kinases in IL-10-dependent and IL-10-independent regulation of the costimulatory molecule B7.2 in lipopolysaccharide-stimulated human monocytic cells

The costimulatory molecule B7.2 (CD86) plays a vital role in immune activation and development of Th responses. The molecular mechanisms by which B7.2 expression is regulated are not understood. We investigated the role of mitogen-activated protein kinases (MAPK) in the regulation of B7.2 expression in LPS-stimulated human monocytic cells. LPS stimulation of human monocytes resulted in the down-regulation of B7.2 expression that could be abrogated by anti-IL-10 Abs. Furthermore, SB202190, a specific inhibitor of p38 MAPK, inhibited LPS-induced IL-10 production and reversed B7.2 down-regulation, suggesting that LPS-induced B7.2 down-regulation may be mediated, at least in part, via regulation of IL-10 production by p38 MAPK. In contrast to human promonocytic THP-1 cells that are refractory to the inhibitory effects of IL-10, LPS stimulation enhanced B7.2 expression. This IL-10-independent B7.2 induction was not influenced by specific inhibitors of either p38 or p42/44 MAPK. To ascertain the role of the c-Jun N-terminal kinase (JNK) MAPK, dexamethasone, an inhibitor of JNK activation, was used, which inhibited LPS-induced B7.2 expression. Transfection of THP-1 cells with a plasmid expressing a dominant-negative stress-activated protein/extracellular signal-regulated kinase kinase 1 significantly reduced LPS-induced B7.2 expression, thus confirming the involvement of JNK. To study the signaling events downstream of JNK activation, we show that dexamethasone did not inhibit LPS-induced NF-kappaB activation in THP-1 cells, suggesting that JNK may not be involved in NF-kappaB activation leading to B7.2 expression. Taken together, our results reveal the distinct involvement of p38 in IL-10-dependent, and JNK in IL-10-independent regulation of B7.2 expression in LPS-stimulated monocytic cells.     

Anthrax lethal toxin-induced inflammasome formation and caspase-1 activation are late events dependent on ion fluxes and the proteasome

Anthrax lethal toxin (LT) is cytotoxic to macrophages from certain inbred mouse strains. The gene controlling macrophage susceptibility to LT is Nalp1b . Nalp1b forms part of the inflammasome, a multiprotein complex involved in caspase-1 activation and release of interleukin (IL)-1β and IL-18. We confirm the role of caspase-1 in LT-mediated death by showing that caspase inhibitors differentially protected cells against LT, with the degree of protection corresponding to each compound's ability to inhibit caspase-1. Caspase-1 activation and cytokine processing and release were late events inhibited by elevated levels of KCl and sucrose, by potassium channel blockers, and by proteasome inhibitors, suggesting that inflammasome formation requires a protein-degradation event and occurs downstream of LT-mediated potassium efflux. In addition, IL-18 and IL-1β release was dependent on cell death, indicating that caspase-1-mediated cytotoxicity is independent of these cytokines. Finally, inducing NALP3-inflammasome formation in LT-resistant macrophages did not sensitize cells to LT, suggesting that general caspase-1 activation cannot account for sensitivity to LT and that a Nalp1b-mediated event is specifically required for death. Our data indicate that inflammasome formation is a contributing, but not initiating, event in LT-mediated cytotoxicity and that earlier LT-mediated events leading to ion fluxes are required for death.     

Alpha 1-Antitrypsin Does Not Inhibit Human Monocyte Caspase-1

BackgroundAlpha 1-antitrypsin (A1AT) is a 52 kDa serine protease inhibitor produced largely by hepatocytes but also by mononuclear phagocytes. A1AT chiefly inhibits neutrophil elastase and proteinase-3 but has also been reported to have immune modulatory functions including the ability to inhibit caspases. Its clinical availability for infusion suggests that A1AT therapy might modulate caspase related inflammation. Here we tested the ability of A1AT to modulate caspase-1 function in human mononuclear phagocytes.MethodsPurified plasma derived A1AT was added to active caspase-1 in a cell-free system (THP-1 lysates) as well as added exogenously to cell-culture models and human whole blood models of caspase-1 activation. Functional caspase-1 activity was quantified by the cleavage of the caspase-1 specific fluorogenic tetrapeptide substrate (WEHD-afc) and the release of processed IL-18 and IL-1β.ResultsTHP-1 cell lysates generated spontaneous activation of caspase-1 both by WEHD-afc cleavage and the generation of p20 caspase-1. A1AT added to this cell free system was unable to inhibit caspase-1 activity. Release of processed IL-18 by THP-1 cells was also unaffected by the addition of exogenous A1AT prior to stimulation with LPS/ATP, a standard caspase-1 activating signal. Importantly, the A1AT exhibited potent neutrophil elastase inhibitory capacity. Furthermore, A1AT complexed to NE (and hence conformationally modified) also did not affect THP-1 cell caspase-1 activation. Finally, exogenous A1AT did not inhibit the ability of human whole blood samples to process and release IL-1β.ConclusionsA1AT does not inhibit human monocyte caspase-1.     

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.     

Cytosolic,Autocrine Alpha-1 Proteinase Inhibitor (A1PI) Inhibits Caspase-1 and Blocks IL-1β Dependent Cytokine Release in Monocytes

Rationale

Activation state-dependent secretion of alpha-1 proteinase inhibitor (A1PI) by monocytes and macrophages was first reported in 1985. Since then, monocytes and tissue macrophages have emerged as key sentinels of infection and tissue damage via activation of self-assembling pattern recognition receptors (inflammasomes), which trigger inflammation and cell death in a caspase-1 dependent process. These studies examine the relationship between A1PI expression in primary monocytes and monocytic cell lines, and inflammatory cytokine expression in response to inflammasome directed stimuli.

Methods

IL-1 β expression was examined in lung macrophages expressing wild type A1PI (A1PI-M) or disease-associated Z isoform A1PI (A1PI-Z). Inflammatory cytokine release was evaluated in THP-1 monocytic cells or THP-1 cells lacking the inflammasome adaptor ASC, transfected with expression vectors encoding A1PI-M or A1PI-Z. A1PI-M was localized within monocytes by immunoprecipitation in hypotonic cell fractions. Cell-free titration of A1PI-M was performed against recombinant active caspase-1 in vitro.

Results

IL-1 β expression was elevated in lung macrophages expressing A1PI-Z. Overexpression of A1PI-M in THP-1 monocytes reduced secretion of IL-1β and TNF-α. In contrast, overexpression of A1PI-Z enhanced IL-1β and TNF- α secretion in an ASC dependent manner. A1PI-Z-enhanced cytokine release was inhibited by a small molecule caspase-1 inhibitor but not by high levels of exogenous wtA1PI. Cytosolic localization of A1PI-M in monocytes was not diminished with microtubule-inhibiting agents. A1PI-M co-localized with caspase-1 in gel-filtered cytoplasmic THP-1 preparations, and was co-immunoprecipitated with caspase 1 from nigericin-stimulated THP-1 cell lysate. Plasma-derived A1PI inhibited recombinant caspase-1 mediated conversion of a peptide substrate in a dose dependent manner.

Conclusions

Our results suggest that monocyte/macrophage-expressed A1PI-M antagonizes IL-1β secretion possibly via caspase-1 inhibition, a function which disease-associated A1PI-Z may lack. Therapeutic approaches which limit inflammasome responses in patients with A1PI deficiency, in combination with A1PI augmentation, may provide additional respiratory tissue-sparing benefits.     

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.     

Interleukin-32-induced thymic stromal lymphopoietin plays a critical role in macrophage differentiation through the activation of caspase-1 in vitro

Introduction

Interleukin (IL)-32 is an inflammatory cytokine induced by Mycobacterium tuberculosis and Mycobacterium bovis in a variety of cell types and discovered in the synovial of patients with rheumatoid arthritis (RA). Thymic stromal lymphopoietin (TSLP) play several roles in the pathogenesis of RA. However, the role of IL-32 and TSLP in RA has not been elucidated.

Methods

We evaluated the specific mechanism of between IL-32 and TSLP in RA using human monocyte cell line, THP-1 cells.

Results

Here we documented for the first time that IL-32 highly increased TSLP production in THP-1 cells and human blood monocytes. TSLP expression was induced by IL-32 via activation of caspase-1 and nuclear factor-κB. TSLP produced by IL-32 increased differentiation of monocytes but depletion of TSLP prevented differentiation of monocytes into macrophage-like cells. Chondroprotective drugs such as chondroitin sulfate (CS) and the traditional Korean medicine, BaekJeol-Tang (BT) decrease production of TSLP and activation of caspase-1 and nuclear factor-κB. In addition, CS and BT inhibited IL-32-induced monocytes differentiation.

Conclusions

Taken together, IL-32 and TSLP are important cytokines involved in the development of RA. The effects of CS and BT were associated with the downregulation of TSLP and caspase-1 through negative regulation of IL-32 pathways in RA.     

Adenovirus membrane penetration activates the NLRP3 inflammasome

Adenovirus type 5 (Ad5) infection of macrophages results in rapid secretion of interleukin-1β (IL-1β) and is dependent on the inflammasome components NLRP3 and ASC and the catalytic activity of caspase-1. Using lentivirus-expressed short hairpin RNA (shRNA) and competitive inhibitors, we show that Ad-induced IL-1β release is dependent upon Toll-like receptor 9 (TLR9) sensing of the Ad5 double-stranded DNA (dsDNA) genome in human cell lines and primary monocyte-derived macrophages but not in mouse macrophages. Additionally, a temperature-sensitive mutant of Ad5 unable to penetrate endosomal membranes, ts1, is unable to induce IL-1β release in TLR2-primed THP-1 cells, suggesting that penetration of endosomal membranes is required for IL-1β release. Disruption of lysosomal membranes and the release of cathepsin B into the cytoplasm are required for Ad-induced NLRP3 activation. Ad5 cell entry also induces reactive oxygen species (ROS) production, and inhibitors of ROS prevent Ad-induced IL-1β release. Ad5 activation of NLRP3 also induces necrotic cell death, resulting in the release of the proinflammatory molecule HMGB1. This work further defines the mechanisms of virally induced inflammasome activation.