Anthrax lethal toxin-mediated killing of human and murine dendritic cells impairs the adaptive immune response

Many pathogens have acquired strategies to combat the immune response. Bacillus anthracis interferes with host defenses by releasing anthrax lethal toxin (LT), which inactivates mitogen-activated protein kinase pathways, rendering dendritic cells (DCs) and T lymphocytes nonresponsive to immune stimulation. However, these cell types are considered resistant to killing by LT. Here we show that LT kills primary human DCs in vitro, and murine DCs in vitro and in vivo. Kinetics of LT-mediated killing of murine DCs, as well as cell death pathways induced, were dependent upon genetic background: LT triggered rapid necrosis in BALB/c-derived DCs, and slow apoptosis in C57BL/6-derived DCs. This is consistent with rapid and slow killing of LT-injected BALB/c and C57BL/6 mice, respectively. We present evidence that anthrax LT impairs adaptive immunity by specifically targeting DCs. This may represent an immune-evasion strategy of the bacterium, and contribute to anthrax disease progression. We also established that genetic background determines whether apoptosis or necrosis is induced by LT. Finally, killing of C57BL/6-derived DCs by LT mirrors that of human DCs, suggesting that C57BL/6 DCs represent a better model system for human anthrax than the prototypical BALB/c macrophages.     

Anthrax lethal toxin rapidly activates caspase-1/ICE and induces extracellular release of interleukin (IL)-1beta and IL-18

Anthrax lethal toxin (LT), a critical virulence factor for Bacillus anthracis, has been demonstrated to cleave and to inactivate mitogen-activated protein kinase kinases (MAPKKs) that propagate prosurvival signals in macrophages (1-5). Whether this action of anthrax LT leads to the production of proinflammatory cytokines by macrophages has been more controversial (6, 7). We now report that anthrax LT treatment leads to the specific extracellular release of interleukin (IL)-1beta and IL-18 by the murine macrophage cell lines, RAW264.7 and J774A.1. Studies of the processing of IL-1beta reveal that the levels of activated/cleaved IL-1beta in RAW264.7 and J774.A1 cells are increased following treatment with anthrax LT. Enhanced processing of IL-1beta directly correlates with increased levels in the activation of its upstream regulator, IL-1beta-converting enzyme/Caspase-1 (ICE). The extracellular release of IL-1beta and IL-18 in response to anthrax LT is ICE-dependent, as an ICE-specific inhibitor blocks this process. These data indicate that ICE, IL-1beta, and IL-18 are downstream effectors of anthrax LT in macrophages, providing the basis for new bioassays for anthrax LT activity and representing potential therapeutic targets.     

Anthrax lethal toxin has direct and potent inhibitory effects on B cell proliferation and immunoglobulin production

Protective host immune responses to anthrax infection in humans and animal models are characterized by the development of neutralizing Abs against the receptor-binding anthrax protective Ag (PA), which, together with the lethal factor (LF) protease, composes anthrax lethal toxin (LT). We now report that B cells, in turn, are targets for LT. Anthrax PA directly binds primary B cells, resulting in the LF-dependent cleavage of the MAPK kinases (MAPKKs) and disrupted signaling to downstream MAPK targets. Although not directly lethal to B cells, anthrax LT treatment causes severe B cell dysfunction, greatly reducing proliferative responses to IL-4-, anti-IgM-, and/or anti-CD40 stimulation. Moreover, B cells treated with anthrax LT in vitro or isolated from mice treated with anthrax LT in vivo have a markedly diminished capacity to proliferate and produce IgM in response to TLR-2 and TLR-4 ligands. The suppressive effects of anthrax LT on B cell function occur at picomolar concentrations in vitro and at sublethal doses in vivo. These results indicate that anthrax LT directly inhibits the function of B cells in vitro and in vivo, revealing a potential mechanism through which the pathogen could bypass protective immune responses.     

Rac2 is an essential regulator of neutrophil nicotinamide adenine dinucleotide phosphate oxidase activation in response to specific signaling pathways

Rac2 is a hematopoietic-specific Rho family GTPase implicated as an important constituent of the NADPH oxidase complex and shares 92% amino acid identity with the ubiquitously expressed Rac1. In bone marrow (BM) neutrophils isolated from rac2(-/-) mice generated by gene targeting, we previously reported that PMA-induced superoxide production was reduced by about 4-fold, which was partially corrected in TNF-alpha-primed BM neutrophils and in peritoneal exudate neutrophils. We investigated receptor-mediated activation of the NADPH oxidase in the current study, finding that superoxide production in rac2(-/-) BM and peritoneal exudate neutrophils was normal in response to opsonized zymosan, reduced to 22% of wild type in response to IgG-coated SRBC, and almost absent in response to fMLP. In wild-type murine BM neutrophils, phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), p38 mitogen-activated protein kinase, c-Jun N-terminal kinase, and Akt was induced by PMA or fMLP, which was decreased in rac2(-/-) neutrophils for ERK1/2 and p38. Activation of p38 by either opsonized zymosan or IgG-coated SRBC was similar in wild-type and rac2(-/-) cells. Inhibition of ERK1/2 or p38 activation using either PD98059 or SB203580, respectively, had only a modest effect on fMLP-elicited superoxide production and no effect on the PMA-induced response. These data provide genetic evidence supporting an important role for Rac2 in regulating neutrophil NADPH oxidase activation downstream of chemoattractant and Fcgamma receptors. The effect of Rac2 deficiency on superoxide production is probably exerted through multiple pathways, including those independent of mitogen-activated protein kinase activation.     

Neutrophil elastase mediates pathogenic effects of anthrax lethal toxin in the murine intestinal tract

Neutrophils isolated from BALB/c or C57BL/6 mice and treated in vitro with anthrax lethal toxin release bioactive neutrophil elastase, a proinflammatory mediator of tissue destruction. Similarly, neutrophils isolated from mice treated with anthrax lethal toxin in vivo and cultured ex vivo release greater amounts of elastase than neutrophils from vehicle-treated controls. Direct measurements from murine intestinal tissue samples demonstrate an anthrax lethal toxin-dependent increase in neutrophil elastase activity in vivo as well. These findings correlate with marked lethal toxin-induced intestinal ulceration and bleeding in neutrophil elastase(+/+) animals, but not in neutrophil elastase(-/-) animals. Moreover, neutrophil elastase(-/-) mice have a significant survival advantage over neutrophil elastase(+/+) animals following exposure to anthrax lethal toxin, thereby establishing a key role for neutrophil elastase in mediating the deleterious effects of anthrax lethal toxin.     

MicroRNA-92a Negatively Regulates Toll-like Receptor (TLR)-triggered Inflammatory Response in Macrophages by Targeting MKK4 Kinase

Toll-like receptors (TLRs) play a critical role in the initiation of immune responses against invading pathogens. MicroRNAs have been shown to be important regulators of TLR signaling. In this study, we have found that the stimulation of multiple TLRs rapidly reduced the levels of microRNA-92a (miRNA-92a) and some other members of the miRNA-92a family in macrophages. miR-92a mimics significantly decreased, whereas miR-92a knockdown increased, the activation of the JNK/c-Jun pathway and the production of inflammatory cytokines in macrophages when stimulated with ligands for TLR4. Furthermore, mitogen-activated protein kinase kinase 4 (MKK4), a kinase that activates JNK/stress-activated protein kinase, was found to be directly targeted by miR-92a. Similar to the effects of the miR-92a mimics, knockdown of MKK4 inhibited the activation of JNK/c-Jun signaling and the production of TNF-α and IL-6. In conclusion, we have demonstrated that TLR-mediated miR-92a reduction feedback enhances TLR-triggered production of inflammatory cytokines in macrophages, thus outlining new mechanisms for fine-tuning the TLR-triggered inflammatory response.     

Structural characteristics and <Emphasis Type="Italic">in vitro</Emphasis> macrophage activation of acetyl fucoidan from <Emphasis Type="Italic">Cladosiphon okamuranus</Emphasis>

We investigated a structural characteristics of acetyl fucoidan (CAF) isolated from commercially cultured Cladosiphon okamuranus. The CAF-induced macrophage activation and its signaling pathways in murine macrophage cell line, RAW 264.7 were also investigated. From the results of methylation analysis, CAF consisted of α-1→3 linked l-fucosyl residues and substituted sulfate and acetyl groups at C-4 on the main chain. CAF induced production of nitric oxide (NO), tumor necrosis factor-α and interleukin-6 in RAW 264.7 cells. Sulfate and acetyl groups of CAF involved in CAF-induced NO production. Neutralizing anti-Toll-like receptor 4 (TLR4), anti-CD14 and anti-scavenger receptor class A (SRA) but not anti-complement receptor type 3 monoclonal antibodies decreased CAF-induced NO production. The results of immunoblot analysis indicated that CAF activated mitogen-activated protein kinases (MAPKs) such as p38 MAPK, extracellular signal-regulated kinase (ERK)1/2 and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK). SB203580 (p38 MAPK inhibitor) and SP600125 (SAPK/JNK inhibitor), but not U0126 (MAPK/ERK kinase 1/2 inhibitor) decreased CAF-induced NO production. The results suggested that CAF induced macrophage activation through membrane receptors TLR4, CD14 and SRA, and MAPK signaling pathways.     

Hepatocyte resistance to oxidative stress is dependent on protein kinase C-mediated down-regulation of c-Jun/AP-1

The prevention of injury from reactive oxygen species is critical for cellular resistance to many death stimuli. Resistance to death from the superoxide generator menadione in the hepatocyte cell line RALA255-10G is dependent on down-regulation of the c-Jun N-terminal kinase (JNK)/AP-1 signaling pathway by extracellular signal-regulated kinase 1/2 (ERK1/2). Because protein kinase C (PKC) regulates both oxidant stress and JNK signaling, the ability of PKC to modulate hepatocyte death from menadione through effects on AP-1 was examined. PKC inhibition with Ro-31-8425 or bisindolylmaleimide I sensitized this cell line to death from menadione. Menadione treatment led to activation of PKCmicro, or protein kinase D (PKD), but not PKCalpha/beta, PKCzeta/lambda, or PKCdelta/. Menadione induced phosphorylation of PKD at Ser-744/748, but not Ser-916, and translocation of PKD to the nucleus. PKC inhibition blocked menadione-induced phosphorylation of PKD, and expression of a constitutively active PKD prevented death from Ro-31-8425/menadione. PKC inhibition led to a sustained overactivation of JNK and c-Jun in response to menadione as determined by in vitro kinase assay and immunoblotting for the phosphorylated forms of both proteins. Cell death from PKC inhibition and menadione treatment resulted from c-Jun activation, since death was blocked by adenoviral expression of the c-Jun dominant negative TAM67. PKC and ERK1/2 independently down-regulated JNK/c-Jun, since inhibition of either kinase failed to affect activation of the other kinase, and simultaneous inhibition of both pathways caused additive JNK/c-Jun activation and cell death. Resistance to death from superoxide therefore requires both PKC/PKD and ERK1/2 activation in order to down-regulate proapoptotic JNK/c-Jun signaling.     

Ras participates in CpG oligodeoxynucleotide signaling through association with toll-like receptor 9 and promotion of interleukin-1 receptor-associated kinase/tumor necrosis factor receptor-associated factor 6 complex formation in macrophages

CpG oligodeoxynucleotides (ODN) activate immune cells to produce immune mediators by Toll-like receptor 9 (TLR9)-mediated signal transduction, which activates mitogen-activated protein kinases (MAPKs) and nuclear factor-kappaB (NF-kappaB) through the MyD88/IRAK/TRAF6 kinases cascade. However, the precise mechanisms of CpG ODN activation of immune cells have not been fully elucidated. The small GTP-binding protein Ras mediates MAPK activation in response to a variety of stimuli. Up to now, it is not clear whether Ras plays a role in CpG ODN signaling. In the present study, we found that the dominant-negative version of Ras (RasN17) and specific Ras inhibitor, FTI-277, inhibited CpG ODN-induced nitric oxide (NO) and tumor necrosis factor-alpha (TNF-alpha) production by murine macrophage cell line RAW264.7. While overexpression of wild-type Ras enhanced CpG ODN-induced extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK), and NF-kappaB activation, overexpression of RasN17 inhibited CpG ODN-induced ERK, JNK, and NF-kappaB activation. RasN17 overexpression also inhibited CpG ODN-induced IRAK1/TRAF6 complex formation. Further studies revealed that CpG ODN activated Ras in a time- and dose-dependent manner, and Ras associated with TLR9 in a CpG ODN-dependent manner. Most interestingly, activation of Ras preceded the association of Ras with TLR9, giving rise to a possibility that Ras activation might not be dependent on the interaction between Ras and TLR9. Our data demonstrate for the first time that Ras can be activated by CpG ODN in macrophages, and Ras is involved in CpG ODN signaling as an early event by associating with TLR9 and promoting IRAK1/TRAF6 complex formation, and MAPK and NF-kappaB activation.     

Tec kinases regulate actin assembly and cytokine expression in LPS-stimulated human neutrophils via JNK activation

The acute inflammatory response involves neutrophils wherein recognition of bacterial products, such as lipopolysaccharide (LPS), activates intracellular signaling pathways. We have shown that the mitogen-activated protein kinase (MAPK) c-Jun NH₂ terminal kinase (JNK) is activated by LPS in neutrophils and plays a critical role in monocyte chemoattractant protein (MCP)-1 expression and actin assembly. As the Tec family kinases are expressed in neutrophils and regulate activation of the MAPKs in other cell systems, we hypothesized that the Tec kinases are an upstream component of the signaling pathway leading to LPS-induced MAPKs activation in neutrophils. Herein, we show that the Tec kinases are activated in LPS-stimulated human neutrophils and that inhibition of the Tec kinases, with leflunomide metabolite analog (LFM-A13), decreased LPS-induced JNK, but not p38, activity. Furthermore, LPS-induced actin polymerization as well as MCP-1, tumor necrosis factor-α, interleukin-6, and interleukin-1β expression are dependent on Tec kinase activity.     

TLR2 mediates phagocytosis and autophagy through JNK signaling pathway in Staphylococcus aureus-stimulated RAW264.7 cells

Toll-like receptor 2 (TLR2) is involved in phagocytosis and autophagy to enhance host innate immune response to bacterial infection. TLR2 has been reported to participate in the recognition of Staphylococcus aureus (S. aureus). However, the role of TLR2 in phagocytosis and autophagy in S. aureus-stimulated macrophages and the underlying mechanisms as yet remain unclear. In the present study, stimulation of mouse macrophage cell line RAW264.7 with S. aureus activated multiple signaling pathways including mitogen-activated protein kinases (MAPKs), myeloid differentiation factor 88 (MyD88), phosphatidylinositide 3-kinase (PI3K) and Rac1 and triggered autophagy process. Knockdown of TLR2 by siRNA significantly reduced phagocytosis and autophagy of macrophages upon S. aureus infection. Interestingly, TLR2 siRNA markedly attenuated S. aureus-induced phosphorylation of c-Jun N-terminal kinase (JNK) but not p38 or extracellular regulated protein kinase (ERK) in macrophages. Similarly, SP600125, a JNK inhibitor, also down-regulated phagocytosis and autophagy in S. aureus-stimulated macrophages. Furthermore, TLR2 siRNA and SP600125 simultaneous treatment showed similar phagocytosis and autophagy compared to that in TLR2 siRNA treatment alone. Collectively, our results indicate that TLR2 may be critical for phagocytosis and autophagy through JNK signaling pathway, and provide an underlying mechanistic link between innate immune receptor and induction of phagocytosis and autophagy in S. aureus-stimulated macrophages.     

Microtubule inhibitors elicit differential effects on MAP kinase (JNK, ERK, and p38) signaling pathways in human KB-3 carcinoma cells

Microtubule inhibitors are widely used in cancer chemotherapy, but the signaling mechanisms that link microtubule disarray to destructive or protective cellular responses are poorly understood. Because members of the mitogen-activated protein kinase (MAPK) family have been implicated in regulation of cell survival and cell death, we examined the extent and kinetics of activation of JNK, ERK, and p38 MAPKs in response to treatment of KB-3 carcinoma cells with several microtubule inhibitors. All four agents tested (vinblastine, vincristine, Taxol, and colchicine) caused significant (6- to 13-fold) activation of JNK, concomitant inactivation of ERK, and a reduction in basal p38 MAPK activity. JNK activation and ERK inactivation occurred prior to caspase 3 activation. The microtubule inhibitors also induced phosphorylation of Raf-1 kinase. SEK-1, upstream of JNK, was also activated and phosphorylated in response to the microtubule inhibitors, and sustained phosphorylation of three endogenous JNK substrates (c-Jun, ATF-2, and JunD) was observed. By comparison, the antitumor agent doxorubicin induced activation of JNK and p38 but had no effect on ERK activity or Raf-1. These data demonstrate that microtubule inhibitors elicit distinct and specific effects on MAPK-mediated signaling pathways and suggest in particular that coordinate and reciprocal alterations in JNK and ERK activities are important facets of the cellular response to microtubule disruption.     

Diosgenin inhibits superoxide generation in FMLP-activated mouse neutrophils via multiple pathways

Abstract

Diosgenin possesses anti-inflammatory and anticancer properties. Activated neutrophils produce high concentrations of the superoxide anion which is involved in the pathophysiology of inflammation-related diseases and cancer. In the present study, the inhibitory effect and possible mechanisms of diosgenin on superoxide generation were investigated in mouse bone marrow neutrophils. Diosgenin potently and concentration-dependently inhibited the extracellular and intracellular superoxide anion generation in Formyl-Met-Leu-Phe (FMLP)- activated neutrophils, with IC₅₀ values of 0.50 ± 0.08 μM and 0.66 ± 0.13 μM, respectively. Such inhibition was not mediated by scavenging the superoxide anion or by a cytotoxic effect. Diosgenin inhibited the phosphorylation of p47phox and membrane translocation of p47phox and p67phox, and thus blocking the assembly of nicotinamide adenine dinucleotide phosphate oxidase. Moreover, cellular cyclic adenosine monophosphate (cAMP) levels and protein kinase A (PKA) expression were also effectively increased by diosgenin. It attenuated FMLP-induced increase of phosphorylation of cytosolic phospholipase A (cPLA₂), p21-activated kinase (PAK), Akt, p38 mitogen-activated protein kinase (p38MAPK), extracellular signal-regulated kinase (ERK1/2), and c-Jun N-terminal kinase (JNK). Our data indicate that diosgenin exhibits inhibitory effects on superoxide anion production through the blockade of cAMP, PKA, cPLA₂, PAK, Akt and MAPKs signaling pathways. The results may explain the clinical implications of diosgenin in the treatment of inflammation-related disorders.     

Cleavage of mitogen-activated protein kinases in human neutrophils undergoing apoptosis: role in decreased responsiveness to inflammatory cytokines

Extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) are major signaling molecules activated in human neutrophils stimulated by cytokines. Both molecules were cleaved at the N-terminal portion in neutrophils undergoing apoptosis induced by in vitro culture alone or treatment with TNF and/or cycloheximide. The cleavage of both molecules was inhibited by G-CSF and benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone, a caspase inhibitor, both of which can inhibit neutrophil apoptosis. In a cell-free system, ERK and p38 MAPK were not cleaved by recombinant caspase-3 or caspase-8 while gelsolin was cleaved by caspase-3 under the same condition. The cleavage of both molecules appears to be specific to mature neutrophils, since it was not detected in immature cells (HL-60 and Jurkat) undergoing apoptosis, indicating that proteases responsible for the cleavage of both molecules may develop during differentiation into mature neutrophils. Concomitant with the cleavage of ERK and p38 MAPK, GM-CSF- and TNF-induced superoxide release, adherence, and phosphorylation of ERK and p38 MAPK were decreased in neutrophils undergoing apoptosis. In addition, GM-CSF- and TNF-induced superoxide release and adherence were inhibited by PD98059 MAPK/ERK kinase inhibitor) as well as SB203580 (p38 MAPK inhibitor), suggesting possible involvement of ERK and p38 MAPK in superoxide release and adherence induced by these cytokines. These findings indicate that ERK and p38 MAPK are cleaved and degraded in neutrophils undergoing apoptosis in a caspase-dependent manner and the cleavage of both molecules may be partly responsible for decreased functional responsiveness to inflammatory cytokines.     

Bz-423 superoxide signals apoptosis via selective activation of JNK, Bak, and Bax

Bz-423 is a proapoptotic 1,4-benzodiazepine with potent therapeutic properties in murine models of lupus and psoriasis. Bz-423 modulates the F(1)F(0)-ATPase, inducing the formation of superoxide within the mitochondrial respiratory chain, which then functions as a second messenger initiating apoptosis. Herein, we report the signaling pathway activated by Bz-423 in mouse embryonic fibroblasts containing knockouts of key apoptotic proteins. Bz-423-induced superoxide activates cytosolic ASK1 and its release from thioredoxin. A mitogen-activated protein kinase cascade follows, leading to the specific phosphorylation of JNK. JNK signals activation of Bax and Bak which then induces mitochondrial outer membrane permeabilization to cause the release of cytochrome c and a commitment to apoptosis. The response of these cells to Bz-423 is critically dependent on both superoxide and JNK activation as antioxidants and the JNK inhibitor SP600125 prevents Bax translocation, cytochrome c release, and cell death. These results demonstrate that superoxide generated from the mitochondrial respiratory chain as a consequence of a respiratory transition can signal a sequential and specific apoptotic response. Collectively, these data suggest that the selectivity of Bz-423 observed in vivo results from cell-type specific differences in redox balance and signaling by ASK1 and Bcl-2 proteins.     

Tollip regulates proinflammatory responses to interleukin-1 and lipopolysaccharide

Activation of interleukin-1 (IL-1) receptor (IL-1R), Toll-like receptor 2 (TLR2), and TLR4 triggers NF-kappaB and mitogen-activated protein kinase (MAPK)-dependent signaling, thereby initiating immune responses. Tollip has been implicated as a negative regulator of NF-kappaB signaling triggered by these receptors in in vitro studies. Here, deficient mice were used to determine the physiological contribution of Tollip to immunity. NF-kappaB, as well as MAPK, signaling appeared normal in Tollip-deficient cells stimulated with IL-1beta or the TLR4 ligand lipopolysaccharide (LPS). Similarly, IL-1beta- and TLR-driven activation of dendritic cells and lymphocytes was indistinguishable from wild-type cells. In contrast, the production of the proinflammatory cytokines, IL-6 and tumor necrosis factor alpha was significantly reduced after IL-1beta and LPS treatment at low doses but not at lethal doses of LPS. Tollip therefore controls the magnitude of inflammatory cytokine production in response to IL-1beta and LPS.