Prolonged Inhibition of the MEK1/2-ERK Signaling Axis Primes Interleukin-1 Beta Expression through Histone 3 Lysine 9 Demethylation in Murine Macrophages.

Macrophages undergo different cellular states upon activation that can be hyporesponsive (tolerated) or hyperresponsive (primed or trained) to subsequent stimuli. Epigenetic modifications are known to play key roles in determining these cellular states. However, little is known about the role of signaling pathways that lead to these epigenetic modifications. Here, we examined the effects of various inhibitors targeting key signaling pathways induced by lipopolysaccharide (LPS) on tolerance and priming in murine macrophages. We found that a prolonged inhibition (>18 h) of the mitogen-activated protein kinase (MEK)1/2-extracellular signal-regulated kinase (ERK)1/2 signaling axis reversed tolerance and primed cells in expressing interleukin (IL)-1ß and other inflammatory cytokines such as IL-6, tumor necrosis factor (TNF)α, and CXCL10. The ectopic expression of catalytically active and inactive MEK1 mutants suppressed and enhanced IL-1ß expression, respectively. A transcriptomic analysis showed that cells primed by the MEK1/2 inhibitor U0126 expressed higher levels of gene sets associated with immune responses and cytokine/chemokine production, but expressed lower levels of genes with cell cycle progression, chromosome organization, and heterochromatin formation than non-primed cells. Of interest, the mRNA expressions of the histone 3 lysine 9 (H3K9) methyltransferase Suv39h1 and the H3K9 methylation reader Cbx5 were substantially suppressed, whereas the H3K9 demethylase Kdm7a was enhanced, suggesting a role of the MEK1/2-ERK signaling axis in H3K9 demethylation. The H3K9 trimethylation levels in the genomic regions of IL-1ß, TNFα, and CXCL10 were decreased by U0126. Also, the H3K9 methyltransferase inhibitor BIX01294 mimicked the U0126 training effects and the overexpression of chromobox homolog (CBX)5 prevented the U0126 training effects in both RAW264.7 cells and bone-marrow-derived macrophages. Collectively, these data suggest that the prolonged inhibition of the MEK1/2-ERK signaling axis reverses tolerance and primed macrophages likely through decreasing the H3K9 methylation levels.

The transcription factor PU.1 mediates enhancer-promoter looping that is required for IL-1ß eRNA and mRNA transcription in mouse melanoma and macrophage cell lines.

The DNA-binding protein PU.1 is a myeloid lineage-determining and pioneering transcription factor due to its ability to bind "closed" genomic sites and maintain "open" chromatin state for myeloid lineage-specific genes. The precise mechanism of PU.1 in cell type-specific programming is yet to be elucidated. The melanoma cell line B16BL6, although it is nonmyeloid lineage, expressed Toll-like receptors and activated the transcription factor NF-κB upon stimulation by the bacterial cell wall component lipopolysaccharide. However, it did not produce cytokines, such as IL-1ß mRNA. Ectopic PU.1 expression induced remodeling of a novel distal enhancer (located ∼10 kbp upstream of the IL-1ß transcription start site), marked by nucleosome depletion, enhancer-promoter looping, and histone H3 lysine 27 acetylation (H3K27ac). PU.1 induced enhancer-promoter looping and H3K27ac through two distinct PU.1 regions. These PU.1-dependent events were independently required for subsequent signal-dependent and co-dependent events: NF-κB recruitment and further H3K27ac, both of which were required for enhancer RNA (eRNA) transcription. In murine macrophage RAW264.7 cells, these PU.1-dependent events were constitutively established and readily expressed eRNA and subsequently IL-1ß mRNA by lipopolysaccharide stimulation. In summary, this study showed a sequence of epigenetic events in programming IL-1ß transcription by the distal enhancer priming and eRNA production mediated by PU.1 and the signal-dependent transcription factor NF-κB.

Inhibition of Interleukin 1ß (IL-1ß) Expression by Anthrax Lethal Toxin (LeTx) Is Reversed by Histone Deacetylase 8 (HDAC8) Inhibition in Murine Macrophages.

Many pathogenic microbes often release toxins that subvert the host's immune responses to render the environment suitable for their survival and proliferation. LeTx is one of the toxins causing immune paralysis by cleaving and inactivating the mitogen-activated protein kinase (MAPK) kinases (MEKs). Here, we show that inhibition of the histone deacetylase 8 (HDAC8) by either the HDAC8-specific inhibitor PCI-34051 or small interference (si)RNAs rendered LeTx-exposed murine macrophages responsive to LPS in pro-IL-1ß production. HDAC8 selectively targeted acetylated histone H3 lysine 27 (H3K27Ac), which is known to associate with active enhancers. LeTx induced HDAC8 expression, in part through inhibiting p38 MAPK, which resulted in a decrease of H3K27Ac levels. Inhibition of HDAC8 increased H3K27Ac levels and enhanced NF-κB-mediated pro-IL-1ß enhancer and messenger RNA production in LeTx-exposed macrophages. Collectively, this study demonstrates a novel role of HDAC8 in LeTx immunotoxicity and regulation of pro-IL-1ß production likely through eRNAs. Targeting HDAC8 could be a strategy for enhancing immune responses in macrophages exposed to LeTx or other toxins that inhibit MAPKs.

HDAC8-mediated epigenetic reprogramming plays a key role in resistance to anthrax lethal toxin-induced pyroptosis in macrophages.

Macrophages pre-exposed to a sublethal dose of anthrax lethal toxin (LeTx) are refractory to subsequent high cytolytic doses of LeTx, termed toxin-induced resistance (TIR). A small population of TIR cells (2-4%) retains TIR characteristics for up to 5-6 wk. Through studying these long-term TIR cells, we found that a high level of histone deacetylase (HDAC)8 expression was crucial for TIR. Knocking down or inhibition of HDAC8 by small interfering RNAs or the HDAC8-specific inhibitor PCI-34051, respectively, induced expression of the mitochondrial death genes Bcl2 adenovirus E1B 19 kDa-interacting protein 3 (BNIP3), BNIP3-like and metastatic lymph node 64, and resensitized TIR cells to LeTx. Among multiple histone acetylations, histone H3 lysine 27 (H3K27) acetylation was most significantly decreased in TIR cells in an HDAC8-dependent manner, and the association of H3K27 acetylation with the genomic regions of BNIP3 and metastatic lymph node 64, where HDAC8 was recruited to, was diminished in TIR cells. Furthermore, overexpression of HDAC8 or knocking down the histone acetyltransferase CREB-binding protein/p300, known to target H3K27, rendered wild-type cells resistant to LeTx. As in RAW264.7 cells, primary bone marrow-derived macrophages exposed to a sublethal dose of LeTx were resistant to LeTx in an HDAC8-dependent manner. Collectively, this study demonstrates that epigenetic reprogramming mediated by HDAC8 plays a key role in determining the susceptibility of LeTx-induced pyroptosis in macrophages.

Cathepsin B-mediated autophagy flux facilitates the anthrax toxin receptor 2-mediated delivery of anthrax lethal factor into the cytoplasm.

Anthrax lethal toxin (LeTx) is a virulence factor secreted by Bacillus anthracis and has direct cytotoxic effects on most cells once released into the cytoplasm. The cytoplasmic delivery of the proteolytically active component of LeTx, lethal factor (LF), is carried out by the transporter component, protective antigen, which interacts with either of two known surface receptors known as anthrax toxin receptor (ANTXR) 1 and 2. We found that the cytoplasmic delivery of LF by ANTXR2 was mediated by cathepsin B (CTSB) and required lysosomal fusion with LeTx-containing endosomes. Also, binding of protective antigen to ANXTR1 or -2 triggered autophagy, which facilitated the cytoplasmic delivery of ANTXR2-associated LF. We found that whereas cells treated with the membrane-permeable CTSB inhibitor CA074-Me- or CTSB-deficient cells had no defect in fusion of LC3-containing autophagic vacuoles with lysosomes, autophagic flux was significantly delayed. These results suggested that the ANTXR2-mediated cytoplasmic delivery of LF was enhanced by CTSB-dependent autophagic flux.

HDAC8 Activates AKT through Upregulating PLCB1 and Suppressing DESC1 Expression in MEK1/2 Inhibition-Resistant Cells.

Inhibition of the RAF-MEK1/2-ERK signaling pathway is an ideal strategy for treating cancers with NRAS or BRAF mutations. However, the development of resistance due to incomplete inhibition of the pathway and activation of compensatory cell proliferation pathways is a major impediment of the targeted therapy. The anthrax lethal toxin (LT), which cleaves and inactivates MEKs, is a modifiable biomolecule that can be delivered selectively to tumor cells and potently kills various tumor cells. However, resistance to LT and the mechanism involved are yet to be explored. Here, we show that LT, through inhibiting MEK1/2-ERK activation, inhibits the proliferation of cancer cells with NRAS/BRAF mutations. Among them, the human colorectal tumor HT-29 and murine melanoma B16-BL6 cells developed resistance to LT in 2 to 3 days of treatment. These resistant cells activated AKT through a histone deacetylase (HDAC) 8-dependent pathway. Using an Affymetrix microarray, followed by qPCR validation, we identified that the differential expression of the phospholipase C-ß1 (PLCB1) and squamous cell carcinoma-1 (DESC1) played an important role in HDAC8-mediated AKT activation and resistance to MEK1/2-ERK inhibition. By using inhibitors, small interference RNAs and/or expression vectors, we found that the inhibition of HDAC8 suppressed PLCB1 expression and induced DESC1 expression in the resistant cells, which led to the inhibition of AKT and re-sensitization to LT and MEK1/2 inhibition. These results suggest that targeting PLCB1 and DESC1 is a novel strategy for inhibiting the resistance to MEK1/2 inhibition.

Histone deacetylase 8 protects human proximal tubular epithelial cells from hypoxia-mimetic cobalt- and hypoxia/reoxygenation-induced mitochondrial fission and cytotoxicity.

Cell death by hypoxia followed by reoxygenation (H/R) is responsible for tissue injury in multiple pathological conditions. Recent studies found that epigenetic reprogramming mediated by histone deacetylases (HDACs) is implicated in H/R-induced cell death. However, among 18 different isoforms comprising 4 classes (I-IV), the role of each HDAC in cell death is largely unknown. This study examined the role of HDAC8, which is the most distinct isoform of class I, in the hypoxia mimetic cobalt- and H/R-induced cytotoxicity of human proximal tubular HK-2 cells. Using the HDAC8-specific activator TM-2-51 (TM) and inhibitor PCI34051, we found that HDAC8 played a protective role in cytotoxicity. TM or overexpression of wild-type HDAC8, but not a deacetylase-defective HDAC8 mutant, prevented mitochondrial fission, loss of mitochondrial transmembrane potential and release of cytochrome C into the cytoplasm. TM suppressed expression of dynamin-related protein 1 (DRP1) which is a key factor required for mitochondrial fission. Suppression of DRP1 by HDAC8 was likely mediated by decreasing the level of acetylated histone H3 lysine 27 (a hallmark of active promoters) at the DRP1 promoter. Collectively, this study shows that HDAC8 inhibits cytotoxicity induced by cobalt and H/R, in part, through suppressing DRP1 expression and mitochondrial fission.

Cathepsin B plays a key role in optimal production of the influenza A virus.

BACKGROUND: Influenza A virus (IAV) is the etiologic agent of the febrile respiratory illness, commonly referred to as 'flu'. The lysosomal protease cathepsin B (CTSB) has shown to be involved in the lifecycle of various viruses. Here, we examined the role of CTSB in the IAV lifecycle. METHODS: CTSB-deficient (CTSB-/-) macrophages and the human lung epithelial cell line A549 cells treated with CA-074Me were infected with the A/Puerto Rico/8/34 strain of IAV (IAV-PR8). Viral entry and propagation were measured through quantitative real-time RT-PCR; production and localization of hemagglutinin (HA) protein in the infected host cells were analysed by Western blots, flow cytometry and confocal microscopy; production of progeny viruses were measured by a hemagglutination assay. RESULTS: CTSB-/- macrophages and CA-074Me-treated A549 cells had no defects in incorporating IAV-PR8 virions and permitting viral RNA synthesis. However, these cells produced significantly lower amounts of HA protein and progeny virions than wild-type or untreated cells. CONCLUSION: These data indicate that CTSB is involved in the expression of IAV-PR8 HA protein and subsequent optimal production of IAV-PR8 progeny virions. Targeting CTSB can be a novel therapeutic strategy for treating IAV infection.

Mutagenesis by retroviral insertion in chemical mutagen-generated quasi-haploid mammalian cells.

Diploidy is a major obstacle to the mutagenic analysis of function in cultured mammalian cells. Here, we show that 6-8 rounds of chemical mutagenesis generates quasi-haploid cells that can be used as targets for insertional mutagenesis using a specially designed retroviral vector that permits rapid identification of disrupted genes in each cell that bears a phenotype of interest. The utility of combined chemical and insertional mutagenesis is illustrated by the identification of novel host genes that are required for macrophage sensitivity to anthrax lethal factor.

HDAC8 Prevents Anthrax Lethal Toxin-induced Cell Cycle Arrest through Silencing PTEN in Human Monocytic THP-1 Cells.

Anthrax lethal toxin (LeTx) is a cytotoxic virulence factor that causes cell cycle arrest and cell death in various cell types. However, susceptibility to the cytotoxic effects varies depending on cell types. In proliferating monocytes, LeTx has only transient cytotoxic effects due to activation of the phosphoinositide 3-kinase (PI3K)-AKT-mediated adaptive responses. To date, the mechanism of LeTx in activating PI3K-AKT signaling axis is unknown. This study shows that the histone deacetylase 8 (HDAC8) is involved in activating PI3K-AKT signaling axis through down-regulating the phosphatase and tensin homolog 1 (PTEN) in human monocytic THP-1 cells. The HDAC8-specific activator TM-2-51 and inhibitor PCI-34051 enhanced and prevented, respectively, AKT activation and cell cycle progression in LeTx-treated cells. Furthermore, HDAC8 induced tri-methylation of histone H3 lysine 27 (H3K27me3), which is known to suppress PTEN expression, through at least in part down-regulating the H3K27me3 eraser Jumonji Domain Containing (JMJD) 3. Importantly, the JMJD3-specific inhibitor GSK-J4 induced AKT activation and protected cell cycle arrest in LeTx-treated cells, regardless the presence of HDAC8 activity. Collectively, this study for the first time demonstrated that HDAC8 activity determines susceptibility to cell cycle arrest induced by LeTx, through regulating the PI3K-PTEN-AKT signaling axis.

p62/SQSTM1 enhances NOD2-mediated signaling and cytokine production through stabilizing NOD2 oligomerization.

NOD2 is a cytosolic pattern-recognition receptor that senses muramyl dipeptide of peptidoglycan that constitutes the bacterial cell wall, and plays an important role in maintaining immunological homeostasis in the intestine. To date, multiple molecules have shown to be involved in regulating NOD2 signaling cascades. p62 (sequestosome-1; SQSTM1) is a multifaceted scaffolding protein involved in trafficking molecules to autophagy, and regulating signal cascades activated by Toll-like receptors, inflammasomes and several cytokine receptors. Here, we show that p62 positively regulates NOD2-induced NF-κB activation and p38 MAPK, and subsequent production of cytokines IL-1ß and TNF-α. p62 associated with the nucleotide binding domain of NOD2 through a bi-directional interaction mediated by either TRAF6-binding or ubiquitin-associated domains. NOD2 formed a large complex with p62 in an electron-dense area of the cytoplasm, which increased its signaling cascade likely through preventing its degradation. This study for the first time demonstrates a novel role of p62 in enhancing NOD2 signaling effects.

Inhibition or deficiency of cathepsin B leads defects in HIV-1 Gag pseudoparticle release in macrophages and HEK293T cells.

Human immunodeficiency virus type 1 (HIV-1) egresses from infected cells through utilizing the host membrane budding mechanisms. Assembly of HIV-1 Gag particles occurs on membranes where the Gag multimers subsequently bud off and form enveloped viral particles. In certain cell types such as macrophages, HIV-1 Gag particles have shown to be released into intracellular virus containing compartments (VCC) such as late endosomes, multivesicular bodies (MVBs) or invaginated plasma membrane pockets. Here, we showed that macrophages or HEK293T cells treated with the cathepsin B (CTSB)-specific inhibitor CA-074Me or cells deficient in CTSB failed to release HIV-1 Gag pseudoparticles into the extracellular environment. Based on immunofluorescence and electron microscopy, these cells retained the pseudoparticles in heterogeneous intracellular VCC. CA-074Me was also able to inhibit propagation of two enveloped viruses, herpes simplex virus and influenza A virus, but not non-enveloped enterovirus. These results suggest that CTSB is required for the efficient release of HIV-1 Gag pseudoparticles and targeting CTSB can be a new therapeutic strategy for inhibiting egress of HIV-1 and other enveloped viruses.

Cellular adaptation to anthrax lethal toxin-induced mitochondrial cholesterol enrichment, hyperpolarization, and reactive oxygen species generation through downregulating MLN64 in macrophages.

Cellular adaptation to different stresses related to survival and function has been demonstrated in several cell types. Anthrax lethal toxin (LeTx) induces rapid cell death, termed "pyroptosis," by activating NLRP1b/caspase-1 in murine macrophages. We and others (S. D. Ha et al., J. Biol. Chem. 282:26275-26283, 2007; I. I. Salles et al., Proc. Natl. Acad. Sci. U. S. A. 100:12426 -12431, 2003) have shown that RAW264.7 cells preexposed to sublethal doses of LeTx become resistant to subsequent high cytolytic doses of LeTx, termed toxin-induced resistance (TIR). To date, the cellular mechanisms of pyroptosis and TIR are largely unknown. We found that LeTx caused NLRP1b/caspase-1-dependent mitochondrial dysfunction, including hyperpolarization and generation of reactive oxygen species, which was distinct from that induced by stimuli such as NLRP3-activating ATP. In TIR cells, these mitochondrial events were not detected, although caspase-1 was activated, in response to LeTx. We identified that downregulation of the late endosomal cholesterol-transferring protein MLN64 in TIR cells was involved in TIR. The downregulation of MLN64 in TIR cells was at least in part due to DNA methyltransferase 1-mediated DNA methylation. In wild-type RAW264.7 cells and primary bone marrow-derived macrophages, LeTx caused NLRP1b/caspase-1-dependent mitochondrial translocation of MLN64, resulting in cholesterol enrichment, membrane hyperpolarization, reactive oxygen species (ROS) generation, and depletion of free glutathione (GSH). This study demonstrates for the first time that MLN64 plays a key role in LeTx/caspase-1-induced mitochondrial dysfunction.

Phenotype based functional gene screening using retrovirus-mediated gene trapping in quasi-haploid RAW 264.7 cells.

In vitro random mutagenesis, followed by phenotype screening, provides a rapid and convenient tool for identifying novel genes involved in the phenotype of interest. However, the forward mutagenic approach in mammalian somatic cells is seriously limited by the diploidic nature of the genome. To overcome this impediment, we developed a method that allows functional screening for both haploid insufficient and sufficient genes involved in the phenotype of interest, utilizing a retrovirus gene trap mutagenesis in chemical mutagen-generated quasi-haploid cells. This method was used to identify novel host genes that are required for macrophage sensitivity to anthrax lethal toxin.

Cathepsin B is involved in the trafficking of TNF-alpha-containing vesicles to the plasma membrane in macrophages.

TNF-alpha is a potent proinflammatory cytokine, essential for initiating innate immune responses against invading microbes and a key mediator involved in the pathogenesis of acute and chronic inflammatory diseases. To identify molecules involved in the production of TNF-alpha, we used a functional gene identification method using retroviral integration-mediated mutagenesis, followed by LPS-stimulated TNF-alpha production analysis in macrophages. We found that cathepsin B, a lysosomal cysteine proteinase, was required for optimal posttranslational processing of TNF-alpha in response to the bacterial cell wall component LPS. Mouse bone marrow-derived macrophages from cathepsin B-deficient mice and macrophages treated with the cathepsin B-specific chemical inhibitor CA074 methyl ester or small interfering RNA against cathepsin B secreted significantly less TNF-alpha than wild-type or nontreated macrophages. We further showed that the inhibition of cathepsin B caused accumulation of 26-kDa pro-TNF-containing vesicles. Ectopic expression of GFP-conjugated pro-TNF further suggests that pro-TNF failed to reach the plasma membrane without intracellular cathepsin B activity. Altogether, these data suggest that intracellular cathepsin B activity is involved in the TNF-alpha-containing vesicle trafficking to the plasma membrane.

Hydrophilicity dependent budding and secretion of chimeric HIV Gag-V3 virus-like particles.

Virus-like particles (VLPs) of numerous viruses have been considered as possible candidates for vaccine development. We have constructed HIV chimeric genes by coupling the gag gene of HIV-2 with the V3 domain of the gp120 gene of either HIV-1 or HIV-2 and expressed the chimeric genes in SF21 cells using the recombinant baculovirus expression system. Although the level of expression of the chimeric HIV-2 gag gene with the V3 domain of either HIV-1 gp120 (gagC-1V3) or HIV-2 gp120 (gagC-2V3) was high, the VLP assembly and extracellular release of GagC-1V3 was very poor. In contrast, GagC-2V3 chimeric proteins formed VLPs and released efficiently. We have constructed substitution mutants to investigate the effects of the hydrophobic region of the V3 domain of HIV-1 Gp120 (1V3) in VLP assembly and release. The substitution mutant analyses revealed that in replacing the hydrophobic region of the 1V3 in GagC-1V3 with the hydrophilic sequence of the V3 domain of HIV-2 Gp120 (2V3) enhanced the extracellular VLP. We demonstrate here that disruption of the hydrophobic character of the C-terminus of the chimeric protein improves assembly and release of the VLPs. Our results suggest that the poor GagC-1V3 VLP release was attributed to the hydrophobic region in the V3 sequence of the chimeric protein, and that not only the N-terminal myristylation and positively charged domain of the Gag protein functioned as a targeting signal to direct membrane binding, but also that the C-terminal hydrophobic region affected release of chimeric VLPs.

Mitochondrial proteins Bnip3 and Bnip3L are involved in anthrax lethal toxin-induced macrophage cell death.

Anthrax lethal toxin (LeTx) induces rapid cell death of RAW246.7 macrophages. We recently found that a small population of these macrophages is spontaneously and temporally refractory to LeTx-induced cytotoxicity. Analysis of genome-wide transcripts of a resistant clone before and after regaining LeTx sensitivity revealed that a reduction of two closely related mitochondrial proteins, Bcl-2/adenovirus E1B 19-kDa interacting protein 3 (Bnip3) and Bnip3-like (Bnip3L), correlates with LeTx resistance. Down-regulation of Bnip3 and Bnip3L was also found in "toxin-induced resistance" whereby sublethal doses of LeTx induce resistance to subsequent exposure to cytolytic toxin doses. The role of Bnip3 and Bnip3L in LeTx-induced cell death was confirmed by showing that overexpression of either Bnip3 or Bnip3L rendered the resistant cells susceptible to LeTx, whereas down-regulation of Bnip3 and Bnip3L in wild-type macrophages conferred resistance. The down-regulation of Bnip3 and Bnip3L mRNAs by LeTx occurred at both transcriptional and mRNA stability levels. Inhibition of the p38 pathway by lethal factor was responsible for the destabilization of Bnip3/Bnip3L mRNAs as confirmed by showing that p38 inhibitors stabilized Bnip3 and Bnip3L mRNAs and conferred resistance to LeTx cytotoxicity. Therefore, Bnip3/Bnip3L play a crucial role in LeTx-induced cytotoxicity, and down-regulation of Bnip3/Bnip3L is a mechanism of spontaneous or toxin-induced resistance of macrophages.

Critical role of the phosphatidylinositol 3-kinase/Akt/glycogen synthase kinase-3 signaling pathway in recovery from anthrax lethal toxin-induced cell cycle arrest and MEK cleavage in macrophages.

Anthrax lethal toxin (LeTx) is a virulence factor causing immune suppression and toxic shock of Bacillus anthracis infected host. It inhibits cytokine production and cell proliferation/differentiation in various immune cells. This study showed that a brief exposure of LeTx caused a continual MEK1 cleavage and prevented tumor necrosis factor-alpha (TNF) production in response to lipopolysaccharide (LPS) in non-proliferating cells such as human peripheral blood mononuclear cells or mouse primary peritoneal macrophages. In human monocytic cell lines U-937 and THP-1, LeTx induced cell cycle arrest in G0-G1 phase by rapid down-regulation of cyclin D1/D2 and checkpoint kinase 1 through MEK1 inhibition. However, THP-1 cells adaptively adjusted to LeTx and overrode cell cycle arrest by activating the phosphatidylinositol 3-kinase/Akt signaling pathway. Inhibitory Ser-9 phosphorylation of glycogen synthase kinase 3beta (GSK3beta) by Akt prevented proteasome-mediated cyclin D1 degradation and induced cell cycle progress in LeTx-intoxicated THP-1 cells. Recovery from cell cycle arrest was required before recovering from on-going MEK1 cleavage and suppression of TNF production. Furthermore, pretreatment with LeTx or the GSK3-specific inhibitor SB-216763, or transfection with dominant active mutant Akt or degradation-defected mutant cyclin D1 protected cells from LeTx-induced cell cycle arrest, on-going MEK1 cleavage and suppression of TNF production. These results indicate that modulation of phosphatidylinositol 3-kinase/Akt/GSK3beta signaling cascades can be beneficial for protecting or facilitating recovery from cellular LeTx intoxication in cells that depend on basal MEK1 activity for proliferation.

G-CSF-mediated inhibition of JNK is a key mechanism for Lactobacillus rhamnosus-induced suppression of TNF production in macrophages.

Lactobacillus rhamnosus is a human commensal with known immunomodulatory properties. To date the mechanism of these immunomodulatory effects is not well understood. To unravel the immunomodulatory signalling mechanism, we investigated the effects of two strains of L. rhamnosus, L. rhamnosus GG and GR-1, in modulating production of tumour necrosis factor-alpha (TNF) in human monocytic cell line THP-1 and mouse macrophages. Live L. rhamnosus GG and GR-1 or their spent culture supernatant induced minuscule amounts of TNF production but large quantities of granulocyte-colony stimulating factor (G-CSF) in macrophages compared with those induced by pathogenic Escherichia coli GR-12 and Enterococcus faecalis. By using neutralizing antibodies and G-CSF receptor knockout mice, we demonstrated that G-CSF secreted from L. rhamnosus GG- and GR-1-exposed macrophages suppressed TNF production induced by E. coli- or lipopolysaccharide-activated macrophages through a paracrine route. The suppression of TNF production by G-CSF was mediated through activation of STAT3 and subsequent inhibition of c-Jun-N-terminal kinases (JNKs). The inhibition of JNK activation required STAT3alpha-mediated de novo protein synthesis. This demonstrates a novel role of G-CSF in L. rhamnosus-triggered anti-inflammatory effects and its mechanism in the suppression of TNF production in macrophages.