Major Histocompatibility Complex Class II-Restricted, CD4+ T Cell-Dependent and -Independent Mechanisms Are Required for Vaccine-Induced Protective Immunity against Coxiella burnetii.

To understand the role of major histocompatibility complex class I (MHC-I) and MHC-II in vaccine-mediated protection against Coxiella burnetii, we evaluated the protective efficacy of a formalin-inactivated C. burnetii Nine Mile phase I vaccine (PIV) in ß2-microglobulin-deficient (B2m KO) and MHC-II-deficient (MHC-II KO) mice. Vaccination reduced disease severity in wild-type (WT) and B2m KO mice but failed to reduce bacterial burden in MHC-II KO mice. This suggests that the MHC-II antigen presentation pathway is required for PIV-mediated protection against C. burnetii infection. MHC-I and MHC-II affect antibody isotype switching, since both PIV-vaccinated B2m KO and MHC-II KO mice produced less Coxiella-specific IgG than PIV-vaccinated WT mice. Interestingly, MHC-II and CD4 deficiencies were not equivalent in terms of splenomegaly and bacterial clearance. This demonstrates a partial role for CD4+ T cells while revealing MHC-II-restricted, CD4-independent mechanisms. Adoptive transfer of CD4+ T cells from PIV-vaccinated WT mice to naive CD4-deficient (CD4 KO) mice demonstrated that antigen-experienced CD4+ T cells are sufficient to generate protection. Conversely, transfer of naive CD4+ T cells to PIV-vaccinated CD4 KO mice exacerbates disease. Using Tbet-deficient (Tbet KO) mice, we showed a partial role for Th1 subset CD4+ T cells in vaccine protection. Furthermore, Th1-independent roles for Tbet were suggested by significant differences in disease between PIV-vaccinated Tbet KO and CD4 KO mice. Interferon gamma was shown to contribute to the host inflammatory response but not bacterial clearance. Collectively, these findings suggest that vaccine-induced protective immunity against a murine model of experimental Q fever requires MHC-II-restricted, CD4+ T cell-dependent and -independent mechanisms that can be exploited for a new-generation human Q fever vaccine.

Eosinophils Affect Antibody Isotype Switching and May Partially Contribute to Early Vaccine-Induced Immunity against Coxiella burnetii.

Coxiella burnetii is an obligate intracellular Gram-negative bacterium which causes human Q fever. An acidified citrate cysteine medium (ACCM-2) has been developed which mimics the intracellular replicative niche of C. burnetii and allows axenic growth of the bacteria. To determine if C. burnetii cultured in ACCM-2 retains immunogenicity, we compared the protective efficacies of formalin-inactivated C. burnetii Nine Mile phase I (PIV) and phase II (PIIV) vaccines derived from axenic culture 7, 14, and 28 days postvaccination. PIV conferred significant protection against virulent C. burnetii as early as 7 days postvaccination, which suggests that ACCM-2-derived PIV retains immunogenicity and protectivity. We analyzed the cellular immune response in spleens from PIV- and PIIV-vaccinated mice by flow cytometry at 7 and 14 days postvaccination and found significantly more granulocytes in PIV-vaccinated mice than in PIIV-vaccinated mice. Interestingly, we found these infiltrating granulocytes to be SSChigh CD11b+ CD125+ Siglec-F+ (where SSChigh indicates a high side scatter phenotype) eosinophils. There was no change in the number of eosinophils in PIV-vaccinated CD4-deficient mice compared to the level in controls, which suggests that eosinophil accumulation is CD4+ T cell dependent. To evaluate the importance of eosinophils in PIV-mediated protection, we vaccinated and challenged eosinophil-deficient ΔdblGATA mice. ΔdblGATA mice had significantly worse disease than their wild-type counterparts when challenged 7 days postvaccination, while no significant difference was seen at 28 days postvaccination. Nevertheless, ΔdblGATA mice had elevated serum IgM with decreased IgG1 and IgG2a whether mice were challenged at 7 or 28 days postvaccination. These results suggest that eosinophils may play a role in early vaccine protection against C. burnetii and contribute to antibody isotype switching.

Coxiella burnetii Inhibits Neutrophil Apoptosis by Exploiting Survival Pathways and Antiapoptotic Protein Mcl-1.

Our previous study demonstrated that neutrophils play an important role in host defense against Coxiella burnetii infection in mice. In this study, avirulent strain C. burnetii Nine Mile phase II (NMII) was used to examine if C. burnetii can modulate mouse bone marrow-derived neutrophil apoptosis. The results indicated that NMII can inhibit neutrophil apoptosis. Western blotting demonstrated that caspase-3 cleavage was decreased in NMII-infected neutrophils, while phosphorylated mitogen-activated protein kinase (MAPK) p38 and extracellular signal-regulated kinase 1 (Erk1) were increased. Additionally, p38, Erk1/2, phosphoinositide 3-kinase (PI3K), or NF-κB inhibitors reduced the ability of NMII to inhibit neutrophil apoptosis. These results suggest that NMII-mediated inhibition of neutrophil apoptosis depends on its ability to activate neutrophil MAPK pathways. Antiapoptotic protein myeloid cell leukemia-1 (Mcl-1) was significantly increased in NMII-infected neutrophils, and an Mcl-1 inhibitor significantly reduced the ability of NMII to inhibit neutrophil apoptosis. Mcl-1 protein stability was enhanced by phosphorylation at Thr-163 by Erk, and the protein levels were regulated by p38, Erk, PI3K, and NF-κB. Furthermore, the observation that a type IV secretion system (T4SS)-deficient dotA mutant showed a significantly reduced ability to inhibit neutrophil apoptosis compared to wild-type (WT) NMII suggests that T4SS-secreted factors may be involved in NMII-induced inhibition of neutrophil apoptosis. Collectively, these results demonstrate that NMII inhibits neutrophil apoptosis through inhibition of caspase-3 cleavage and activation of MAPK survival pathways with subsequent expression and stabilization of antiapoptotic protein Mcl-1, a process that may partially require the T4SS.

Both Major Histocompatibility Complex Class I (MHC-I) and MHC-II Molecules Are Required, while MHC-I Appears To Play a Critical Role in Host Defense against Primary Coxiella burnetii Infection.

To understand the role of class I major histocompatibility complex (MHC-I) and class II MHC (MHC-II) antigen presentation pathways in host defense against Coxiella burnetii infection, we examined whether MHC-I or MHC-II deficiency in mice would significantly influence their susceptibility to virulent C. burnetii Nine Mile phase I (NMI) infection. The results indicate that NMI infection induced more severe disease in both MHC-I-deficient and MHC-II-deficient mice than in wild-type (WT) mice, while only MHC-I-deficient mice developed a severe persistent infection and were unable to control bacterial replication. These results suggest that both MHC-I-restricted CD8+ T cells and MHC-II-restricted CD4+ T cells contribute to host defense against primary C. burnetii infection, while MHC-I-restricted CD8+ T cells appear to play a more critical role in controlling bacterial replication. Additionally, although NMI infection induced more severe disease in TAP1-deficient mice than in their WT counterparts, TAP1 deficiency in mice did not significantly influence their ability to eliminate C. burnetii This suggests that C. burnetii antigen presentation to CD8+ T cells by the MHC-I classical pathway may depend only partially on TAP1. Furthermore, granzyme B deficiency in mice did not significantly alter their susceptibility to C. burnetii infection, but perforin-deficient mice were unable to control host inflammatory responses during primary C. burnetii infection. These results suggest that perforin, but not granzyme B, is required for C. burnetii antigen-specific cytotoxic CD8+ T cells to control primary C. burnetii infection.

Coxiella burnetii Avirulent Nine Mile Phase II Induces Caspase-1-Dependent Pyroptosis in Murine Peritoneal B1a B Cells.

Our recent study demonstrated that virulent Coxiella burnetii Nine Mile phase I (NMI) is capable of infecting and replicating within peritoneal B1a cells and that B1a cells play an important role in host defense against C. burnetii infection in mice. However, it remains unknown if avirulent Nine Mile phase II (NMII) can infect and replicate in B1a cells and whether NMI and NMII can differentially interact with B1a cells. In this study, we examined if NMI and NMII can differentially modulate host cell apoptotic signaling in B1a cells. The results showed that NMII induced dose-dependent cell death in murine peritoneal B1a cells but NMI did not, suggesting that NMI and NMII may differentially activate host cell apoptotic signaling in B1a cells. Western blotting indicated that NMII-induced B1a cell death was not dependent on either caspase-3 or PARP-1 cleavage, but cleavage of caspase-1 was detected in NMII-infected B1a cells. In addition, inhibition or deficiency of caspase-1 activity blocked NMII-induced B1a cell death. These results suggest that NMII induces a caspase-1-dependent pyroptosis in murine peritoneal B1a cells. We also found that heat-killed NMII and type 4 secretion system (T4SS) mutant NMII were unable to induce B1a cell death and that NMII infection did not induce cell death in peritoneal B1a cells from Toll-like receptor 2 (TLR-2)- or NLRP3 inflammasome-deficient mice. These data suggest that NMII-induced caspase-1-dependent pyroptosis may require its T4SS and activation of the TLR-2 and NLRP3 signaling pathways.

Distinct renal pathology and a chemotactic phenotype after enterohemorrhagic Escherichia coli shiga toxins in non-human primate models of hemolytic uremic syndrome.

Enterohemorrhagic Escherichia coli cause approximately 1.5 million infections globally with 176,000 cases occurring in the United States annually from ingesting contaminated food, most frequently E. coli O157:H7 in ground beef or fresh produce. In severe cases, the painful prodromal hemorrhagic colitis is complicated by potentially lethal hemolytic uremic syndrome (HUS), particularly in children. Bacterial Shiga-like toxins (Stx1, Stx2) are primarily responsible for HUS and the kidney and neurologic damage that ensue. Small animal models are hampered by the inability to reproduce HUS with thrombotic microangiopathy, hemolytic anemia, and acute kidney injury. Earlier, we showed that nonhuman primates (Papio) recapitulated clinical HUS after Stx challenge and that novel therapeutic intervention rescued the animals. Here, we present detailed light and electron microscopic pathology examination of the kidneys from these Stx studies. Stx1 challenge resulted in more severe glomerular endothelial injury, whereas the glomerular injury after Stx2 also included prominent mesangiolysis and an eosinophilic inflammatory infiltration. Both toxins induced glomerular platelet-rich thrombi, interstitial hemorrhage, and tubular injury. Analysis of kidney and other organs for inflammation biomarkers showed a striking chemotactic profile, with extremely high mRNA levels for IL-8, monocyte chemoattractant protein 1, and macrophage inflammatory protein 1α and elevated urine chemokines at 48 hours after challenge. These observations give unique insight into the pathologic consequences of each toxin in a near human setting and present potential pathways for therapeutic intervention.

Regulation of cytokine and chemokine expression by the ribotoxic stress response elicited by Shiga toxin type 1 in human macrophage-like THP-1 cells.

Shiga toxins (Stxs) are cytotoxins produced by the enteric pathogens Shigella dysenteriae serotype 1 and Shiga toxin-producing Escherichia coli (STEC). Stxs bind to a membrane glycolipid receptor, enter cells, and undergo retrograde transport to ultimately reach the cytosol, where the toxins exert their protein synthesis-inhibitory activity by depurination of a single adenine residue from the 28S rRNA component of eukaryotic ribosomes. The depurination reaction activates the ribotoxic stress response, leading to signaling via the mitogen-activated protein kinase (MAPK) pathways (Jun N-terminal protein kinase [JNK], p38, and extracellular signal-regulated kinase [ERK]) in human epithelial, endothelial, and myeloid cells. We previously showed that treatment of human macrophage-like THP-1 cells with Stxs resulted in increased cytokine and chemokine expression. In the present study, we show that individual inactivation of ERK, JNK, and p38 MAPKs using pharmacological inhibitors in the presence of Stx1 resulted in differential regulation of the cytokines tumor necrosis factor alpha and interleukin-1ß (IL-1ß) and chemokines IL-8, growth-regulated protein-ß, macrophage inflammatory protein-1α (MIP-1α), and MIP-1ß. THP-1 cells exposed to Stx1 upregulate the expression of select dual-specificity phosphatases (DUSPs), enzymes that dephosphorylate and inactivate MAPKs in mammalian cells. In this study, we confirmed DUSP1 protein production by THP-1 cells treated with Stx1. DUSP1 inhibition by triptolide showed that ERK and p38 phosphorylation is regulated by DUSP1, while JNK phosphorylation is not. Inhibition of p38 MAPK signaling blocked the ability of Stx1 to induce DUSP1 mRNA expression, suggesting that an autoregulatory signaling loop may be activated by Stxs. Thus, Stxs appear to be capable of eliciting signals which both activate and deactivate signaling for increased cytokine/chemokine production in human macrophage-like cells.

Shiga toxins induce autophagy leading to differential signalling pathways in toxin-sensitive and toxin-resistant human cells.

The bacterial virulence factors Shiga toxins (Stxs) are expressed by Shigella dysenteriae serotype 1 and certain Escherichia coli strains. Stxs are protein synthesis inhibitors and induce apoptosis in many cell types. Stxs induce apoptosis via prolonged endoplasmic reticulum stress signalling to activate both extrinsic and intrinsic pathways in human myeloid cells. Studies have shown that autophagy, a lysosome-dependent catabolic process, may be associated with activation of pro-survival or death processes. It is currently unknown if autophagy contributes to apoptosis or protects cells from Stxs. To study cellular responses to Stxs, we intoxicated toxin-sensitive cells (THP-1 and HK-2 cells), and toxin-resistant cells (primary human monocyte-derived macrophages) and examined toxin intracellular trafficking and autophagosome formation. Stxs translocated to different cell compartments in toxin-resistant versus toxin-sensitive cells. Confocal microscopy revealed autophagosome formation in both toxin-resistant and toxin-sensitive cells. Proteolytic cleavage of Atg5 and Beclin-1 plays pivotal roles in switching non-cytotoxic autophagy to cell death signalling. We detected cleaved forms of Atg5 and Beclin-1 in Stx-treated toxin-sensitive cells, while cleaved caspases, calpains, Atg5 and Beclin-1 were not detected in toxin-resistant primary human monocytes and macrophages. These findings suggest that toxin sensitivity correlates with caspase and calpain activation, leading to Atg5 and Beclin-1 cleavage.

Differential response of the human renal proximal tubular epithelial cell line HK-2 to Shiga toxin types 1 and 2.

Shiga toxins (Stxs) are expressed by the enteric pathogens Shigella dysenteriae serotype 1 and certain serotypes of Escherichia coli. Stx-producing bacteria cause bloody diarrhea with the potential to progress to acute renal failure. Stxs are potent protein synthesis inhibitors and are the primary virulence factors responsible for renal damage that may follow diarrheal disease. We explored the use of the immortalized human proximal tubule epithelial cell line HK-2 as an in vitro model of Stx-induced renal damage. We showed that these cells express abundant membrane Gb(3) and are differentially susceptible to the cytotoxic action of Stxs, being more sensitive to Shiga toxin type 1 (Stx1) than to Stx2. At early time points (24 h), HK-2 cells were significantly more sensitive to Stxs than Vero cells; however, by 72 h, Vero cell monolayers were completely destroyed while some HK-2 cells survived toxin challenge, suggesting that a subpopulation of HK-2 cells are relatively toxin resistant. Fluorescently labeled Stx1 B subunits localized to both lysosomal and endoplasmic reticulum (ER) compartments in HK-2 cells, suggesting that differences in intracellular trafficking may play a role in susceptibility to Stx-mediated cytotoxicity. Although proinflammatory cytokines were not upregulated by toxin challenge, Stx2 selectively induced the expression of two chemokines, macrophage inflammatory protein-1α (MIP-1α) and MIP-1ß. Stx1 and Stx2 differentially activated components of the ER stress response in HK-2 cells. Finally, we demonstrated significant poly(ADP-ribose) polymerase (PARP) cleavage after exposure to Stx1 or Stx2. However, procaspase 3 cleavage was undetectable, suggesting that HK-2 cells may undergo apoptosis in response to Stxs in a caspase 3-independent manner.

Role of tumor necrosis factor alpha in disease using a mouse model of Shiga toxin-mediated renal damage.

Mice have been extensively employed as an animal model of renal damage caused by Shiga toxins. In this study, we examined the role of the proinflammatory cytokine tumor necrosis factor alpha (TNF-alpha) in the development of toxin-mediated renal disease in mice. Mice pretreated with TNF-alpha and challenged with Shiga toxin type 1 (Stx1) showed increased survival compared to that of mice treated with Stx1 alone. Conversely, mice treated with Stx1 before TNF-alpha administration succumbed more quickly than mice given Stx1 alone. Increased lethality in mice treated with Stx1 followed by TNF-alpha was associated with evidence of glomerular damage and the loss of renal function. No differences in renal histopathology were noted between animals treated with Stx1 alone and the TNF-alpha pretreatment group, although we noted a sparing of renal function when TNF-alpha was administered before toxin. Compared to that of treatment with Stx1 alone, treatment with TNF-alpha after toxin altered the renal cytokine profile so that the expression of proinflammatory cytokines TNF-alpha and interleukin-1beta (IL-1beta) increased, and the expression of the anti-inflammatory cytokine IL-10 decreased. Increased lethality in mice treated with Stx1 followed by TNF-alpha was associated with higher numbers of dUTP-biotin nick end labeling-positive renal tubule cells, suggesting that increased lethality involved enhanced apoptosis. These data suggest that the early administration of TNF-alpha is a candidate interventional strategy blocking disease progression, while TNF-alpha production after intoxication exacerbates disease.

Signaling through C/EBP homologous protein and death receptor 5 and calpain activation differentially regulate THP-1 cell maturation-dependent apoptosis induced by Shiga toxin type 1.

Shiga toxins (Stxs) induce apoptosis via activation of the intrinsic and extrinsic pathways in many cell types. Toxin-mediated activation of the endoplasmic reticulum (ER) stress response was shown to be instrumental in initiating apoptosis in THP-1 myeloid leukemia cells. THP-1 cells responded to Shiga toxin type 1 (Stx1) in a cell maturation-dependent manner, undergoing rapid apoptosis in the undifferentiated state but reduced and delayed apoptosis in differentiated cells. The onset of apoptosis was associated with calpain activation and changes in expression of C/EBP homologous protein (CHOP), Bcl-2 family members, and death receptor 5 (DR5). Ligation of DR5 by tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) activates the extrinsic pathway of apoptosis. We show here that expression of TRAIL and DR5 is increased by Stx1 treatment. Addition of exogenous TRAIL enhances, and anti-TRAIL antibodies inhibit, Stx1-induced apoptosis of THP-1 cells. Silencing of CHOP or DR5 expression selectively prevented caspase activation, loss of mitochondrial membrane potential, and Stx1-induced apoptosis of macrophage-like THP-1 cells. In contrast, the rapid kinetics of apoptosis induction in monocytic THP-1 cells correlated with rates of calpain cleavage. The results suggest that CHOP-DR5 signaling and calpain activation differentially contribute to cell maturation-dependent Stx1-induced apoptosis. Inhibition of these signaling pathways may protect cells from Stx cytotoxicity.

Global transcriptional response of macrophage-like THP-1 cells to Shiga toxin type 1.

Shiga toxins (Stxs) are bacterial cytotoxins produced by the enteric pathogens Shigella dysenteriae serotype 1 and some serotypes of Escherichia coli that cause bacillary dysentery and hemorrhagic colitis, respectively. To date, approaches to studying the capacity of Stxs to alter gene expression in intoxicated cells have been limited to individual genes. However, it is known that many of the signaling pathways activated by Stxs regulate the expression of multiple genes in mammalian cells. To expand the scope of analysis of gene expression and to better understand the underlying mechanisms for the various effects of Stxs on host cell functions, we carried out comparative microarray analyses to characterize the global transcriptional response of human macrophage-like THP-1 cells to Shiga toxin type 1 (Stx1) and lipopolysaccharides. The data were analyzed by using a rigorous combinatorial approach with three separate statistical algorithms. A total of 36 genes met the criteria of upregulated expression in response to Stx1 treatment, with 14 genes uniquely upregulated by Stx1. Microarray data were validated by real-time reverse transcriptase PCR for genes encoding early growth response 1 (Egr-1) (transcriptional regulator), cyclooxygenase 2 (COX-2; inflammation), and dual specificity phosphatase 1 (DUSP1), DUSP5, and DUSP10 (regulation of mitogen-activated protein kinase signaling). Stx1-mediated signaling through extracellular signal-regulated kinase 1/2 and Egr-1 appears to be involved in the increased expression and production of the proinflammatory mediator tumor necrosis factor alpha. Activation of COX-2 is associated with the increased production of proinflammatory and vasoactive eicosanoids. However, the capacity of Stx1 to increase the expression of genes encoding phosphatases suggests that mechanisms to dampen the macrophage proinflammatory response may be built into host response to the toxins.

Shiga toxins: intracellular trafficking to the ER leading to activation of host cell stress responses.

Despite efforts to improve hygenic conditions and regulate food and drinking water safety, the enteric pathogens, Shiga toxin-producing Escherichia coli (STEC) and Shigella dysenteriae serotype 1 remain major public health concerns due to widespread outbreaks and the severity of extra-intestinal diseases they cause, including acute renal failure and central nervous system complications. Shiga toxins are the key virulence factors expressed by these pathogens mediating extra-intestinal disease. Delivery of the toxins to the endoplasmic reticulum (ER) results in host cell protein synthesis inhibition, activation of the ribotoxic stress response, the ER stress response, and in some cases, the induction of apoptosis. Intrinsic and/or extrinsic apoptosis inducing pathways are involved in executing cell death following intoxication. In this review we provide an overview of the current understanding Shiga toxin intracellular trafficking, host cellular responses to the toxin and ER stress-induced apoptosis with an emphasis on recent findings.

Bcl-2 regulates the onset of shiga toxin 1-induced apoptosis in THP-1 cells.

Shiga toxins (Stxs), which are proteins expressed by the enteric pathogens Shigella dysenteriae serotype 1 and some serotypes of Escherichia coli, are potent protein synthesis inhibitors. Stx-producing organisms cause bloody diarrhea with the potential to progress to acute renal failure and central nervous system complications. Studies using animal models of these diseases have shown that Stxs are major virulence factors, and purified toxins have been shown to be capable of killing many types of cells in vitro. We showed that Stx type 1 (Stx1) rapidly induced apoptosis in undifferentiated, monocytic THP-1 cells through a mechanism involving the endoplasmic reticulum (ER) stress response. Rapid apoptosis correlated with increased expression of C/EBP homologous protein (CHOP), TRAIL, and DR5, while expression of the antiapoptotic factor Bcl-2 was downregulated. Stx1 treatment of differentiated, macrophage-like THP-1 cells was associated with cytokine production and delayed apoptosis. The mechanisms contributing to cell maturation-dependent differences in responses to Stx1 are unknown. We show here that in macrophage-like cells, Stx1 activated the proximal ER stress sensors RNA-dependent protein kinase-like ER kinase and inositol-requiring ER signal kinase 1alpha but did not activate activating transcription factor 6. Proapoptotic signaling pathways mediated by CHOP and by Bax and Bak were activated by Stx1. However, the toxin also activated prosurvival signaling through increased expression, mitochondrial translocation, and alternative phosphorylation of Bcl-2.

Shiga toxin 1-induced proinflammatory cytokine production is regulated by the phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin signaling pathway.

Shiga toxin 1 (Stx1) transiently increases the expression of proinflammatory cytokines by macrophage-like THP-1 cells in vitro. Increased cytokine production is partly due to activation of the translation initiation factor eIF4E through a mitogen-activated protein kinase (MAPK)- and Mnk1-dependent pathway. eIF4E availability for translation initiation is regulated by association with eIF4E binding proteins (4E-BP). In this study, we showed that Stx1 transiently induced 4E-BP hyperphosphorylation, which may release eIF4E for translation initiation. Phosphorylation of 4E-BP at priming sites T37 and T46 was not altered by Stx1 but was transiently increased at S65, concomitant with increased cytokine expression. Using kinase inhibitors, we showed that 4E-BP phosphorylation was dependent on phosphatidylinositol 3-kinase (PI3K), Akt, and mammalian target of rapamycin (mTOR) activation but did not require MAPKs. Stx1 treatment resulted in increased levels of cytosolic Ca(2+). PI3K and Akt activation led to the phosphorylation and inactivation of the positive cytokine regulator glycogen synthase kinase 3alpha/beta (GSK-3alpha/beta). PI3K, Akt, and mTOR inhibitors and small interfering RNA knockdown of Akt expression all increased, whereas a GSK-3alpha/beta inhibitor decreased, Stx1-induced soluble tumor necrosis factor alpha and interleukin-1beta production. Overall, these findings suggest that despite transient activation of 4E-BP, the PI3K/Akt/mTOR pathway negatively influences cytokine induction by inactivating the positive regulator GSK-3alpha/beta.

Shiga toxin 1 induces apoptosis through the endoplasmic reticulum stress response in human monocytic cells.

Shiga toxins (Stxs) expressed by the enteric pathogens Shigella dysenteriae 1 and enterohaemorrhagic Escherichia coli are potent protein synthesis inhibitors. Shiga toxins have also been shown to induce apoptosis in epithelial, endothelial and monocytic cells. The precise relationship between protein synthesis inhibition and induction of apoptosis is not known. We show that stimulation of the myelogenous leukaemia cell line THP-1 with purified Stx1 induced the endoplasmic reticulum (ER) stress response. Stx1 treatment increased activation of the ER stress sensors IRE1, PERK and ATF6. Toxin treatment increased expression of the transcriptional regulator CHOP and the death domain-containing receptor DR5 at mRNA and protein levels. Following Stx1 intoxication, levels of the survival factor Bcl-2 decreased, while secretion of the death-inducing ligand TRAIL increased. Stx1 enzymatic activity was required for optimal activation of PERK and ATF6, but not IRE1. ER stress elicited by Stx1 increased the release of Ca(2+) from ER stores and the activation of the protease calpain. Inhibition of calpain activity led to reductions in Stx1-induced cleavage of procaspase-8 and apoptosis. Collectively, these data suggest that Shiga toxins trigger monocytic cell apoptosis through the ER stress response, the increased expression of DR5 and TRAIL, and activation of caspase-8 via a calpain-dependent mechanism.

Simultaneous induction of apoptotic and survival signaling pathways in macrophage-like THP-1 cells by Shiga toxin 1.

Shiga toxins have been shown to induce apoptosis in many cell types. However, Shiga toxin 1 (Stx1) induced only limited apoptosis of macrophage-like THP-1 cells in vitro. The mechanisms regulating macrophage death or survival following toxin challenge are unknown. Differentiated THP-1 cells expressed tumor necrosis factor receptors and membrane-associated tumor necrosis factor alpha (TNF-alpha) and produced soluble TNF-alpha after exposure to Stx1. However, the cells were refractory to apoptosis induced by TNF-alpha, although the cytokine modestly increased apoptosis in the presence of Stx1. Despite the partial resistance of macrophage-like THP-1 cells to Stx1-mediated killing, treatment of these cells with Stx1 activated a broad array of caspases, disrupted the mitochondrial membrane potential (DeltaPsi(m)), and released cytochrome c into the cytoplasm. The DeltaPsi(m) values were greatest in cells that had detached from plastic surfaces. Specific caspase inhibitors revealed that caspase-3, caspase-6, caspase-8, and caspase-9 were primarily involved in apoptosis induction. The antiapoptotic factors involved in macrophage survival following toxin challenge include inhibitors of apoptosis proteins and X-linked inhibitor of apoptosis protein. NF-kappaB and JNK mitogen-activated protein kinases (MAPKs) appeared to activate survival pathways, while p38 MAPK was involved in proapoptotic signaling. The JNK and p38 MAPKs were shown to be upstream signaling pathways which may regulate caspase activation. Finally, the protein synthesis inhibitors Stx1 and anisomycin triggered limited apoptosis and prolonged JNK and p38 MAPK activation, while macrophage-like cells treated with cycloheximide remained viable and showed transient activation of MAPKs. Collectively, these data suggest that Stx1 activates both apoptotic and cell survival signaling pathways in macrophage-like THP-1 cells.

Shiga toxin 1-induced cytokine production is mediated by MAP kinase pathways and translation initiation factor eIF4E in the macrophage-like THP-1 cell line.

Upon binding to the glycolipid receptor globotriaosylceramide, Shiga toxins (Stxs) undergo retrograde transport to reach ribosomes, cleave 28S rRNA, and inhibit protein synthesis. Stxs induce the ribotoxic stress response and cytokine and chemokine expression in some cell types. Signaling mechanisms necessary for cytokine expression in the face of toxin-mediated protein synthesis inhibition are not well characterized. Stxs may regulate cytokine expression via multiple mechanisms involving increased gene transcription, mRNA transcript stabilization, and/or increased translation initiation efficiency. We show that treatment of differentiated THP-1 cells with purified Stx1 resulted in prolonged activation of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) cascades, and lipopolysaccharides (LPS) rapidly triggered transient activation of JNK and p38 and prolonged activation of extracellular signal-regulated kinase cascades. Simultaneous treatment with Stx1 + LPS mediated prolonged p38 MAPK activation. Stx1 increased eukaryotic translation initiation factor 4E (eIF4E) activation by 4.3-fold within 4-6 h, and LPS or Stx1 + LPS treatment increased eIF4E activation by 7.8- and 11-fold, respectively, within 1 h. eIF4E activation required Stx1 enzymatic activity and was mediated by anisomycin, another ribotoxic stress inducer. A combination of MAPK inhibitors or a MAPK-interacting kinase 1 (Mnk1)-specific inhibitor blocked eIF4E activation by all stimulants. Mnk1 inhibition blocked the transient increase in total protein synthesis detected in Stx1-treated cells but failed to block long-term protein synthesis inhibition. The MAPK inhibitors or Mnk1 inhibitor blocked soluble interleukin (IL)-1beta and IL-8 production or release by 73-96%. These data suggest that Stxs may regulate cytokine expression in part through activation of MAPK cascades, activation of Mnk1, and phosphorylation of eIF4E.

Shiga toxin 1 induces apoptosis in the human myelogenous leukemia cell line THP-1 by a caspase-8-dependent, tumor necrosis factor receptor-independent mechanism.

Shiga toxins (Stxs) induce apoptosis in a variety of cell types. Here, we show that Stx1 induces apoptosis in the undifferentiated myelogenous leukemia cell line THP-1 in the absence of tumor necrosis factor alpha (TNF-alpha) or death receptor (TNF receptor or Fas) expression. Caspase-8 and -3 inhibitors blocked, and caspase-6 and -9 inhibitors partially blocked, Stx1-induced apoptosis. Stx1 induced the mitochondrial pathway of apoptosis, as activation of caspase-8 triggered the (i) cleavage of Bid, (ii) disruption of mitochondrial membrane potential, and (iii) release of cytochrome c into the cytoplasm. Caspase-8, -9, and -3 cleavage and functional activities began 4 h after toxin exposure and peaked after 8 h of treatment. Caspase-6 may also contribute to Stx1-induced apoptosis by directly acting on caspase-8. It appears that functional Stx1 holotoxins must be transported to the endoplasmic reticulum to initiate apoptotic signaling through the ribotoxic stress response. These data suggest that Stxs may activate monocyte apoptosis via a novel caspase-8-dependent, death receptor-independent mechanism.

Comparative evaluation of apoptosis induced by Shiga toxin 1 and/or lipopolysaccharides in human monocytic and macrophage-like cells.

The enteric pathogens Shigella dysenteriae serotype 1 and Shiga toxin-producing Escherichia coli share the property of expressing the structurally and functionally related cytotoxins that comprise the Shiga toxin (Stx) family. Stx-producing bacteria are causative agents of bloody diarrheal diseases that may progress to life threatening complications involving the destruction of blood vessels in the kidneys and the central nervous system (CNS). The precise mechanisms of toxin transport across the gut epithelial barrier, and the role of innate immunity in the development of systemic complications, remain to be fully characterized. Earlier studies suggested that Stxs and lipopolysaccharides (LPS) induce the expression of proinflammatory cytokines from differentiated (macrophage-like) THP-1 cells. These cytokines may exacerbate vascular damage by up-regulating the expression of toxin receptors on endothelial cells. Purified Stxs have also been shown to induce apoptosis of epithelial and endothelial cells in vitro, but a comparative evaluation of Stx-induced apoptosis of monocytes and macrophages has not been reported. We used FACS, TUNEL, and DNA laddering analyses to show that Shiga toxin-1 (Stx1) and LPS induce apoptosis in undifferentiated and differentiated THP-1 cells, although the kinetics and extent of apoptosis induction differ between monocytic and macrophage-like cells. Stx1-induced apoptosis is A-subunit-dependent. Stx1 and LPS trigger DNA fragmentation and caspase-3 activation, as evidenced by the cleavage of poly(ADP-ribose) polymerase (PARP). Induction of apoptosis in response to Stx1 and/or LPS treatment occurs without the widespread transcriptional activation of apoptosis-related genes. Finally, we present a model of the role of macrophages and monocytes in the pathogenesis of disease caused by Stxs.