Inflammasomes, Autophagy, and Cell Death: The Trinity of Innate Host Defense against Intracellular Bacteria.

Inflammasome activation is an innate host defense mechanism initiated upon sensing pathogens or danger in the cytosol. Both autophagy and cell death are cell autonomous processes important in development, as well as in host defense against intracellular bacteria. Inflammasome, autophagy, and cell death pathways can be activated by pathogens, pathogen-associated molecular patterns (PAMPs), cell stress, and host-derived damage-associated molecular patterns (DAMPs). Phagocytosis and toll-like receptor (TLR) signaling induce reactive oxygen species (ROS), type I IFN, NFκB activation of proinflammatory cytokines, and the mitogen-activated protein kinase cascade. ROS and IFNγ are also prominent inducers of autophagy. Pathogens, PAMPs, and DAMPs activate TLRs and intracellular inflammasomes, inducing apoptotic and inflammatory caspases in a context-dependent manner to promote various forms of cell death to eliminate pathogens. Common downstream signaling molecules of inflammasomes, autophagy, and cell death pathways interact to initiate appropriate measures against pathogens and determine host survival as well as pathological consequences of infection. The integration of inflammasome activation, autophagy, and cell death is central to pathogen clearance. Various pathogens produce virulence factors to control inflammasomes, subvert autophagy, and modulate host cell death in order to evade host defense. This review highlights the interaction of inflammasomes, autophagy, and host cell death pathways in counteracting Burkholderia pseudomallei, the causative agent of melioidosis. Contrasting evasion strategies used by B. pseudomallei, Mycobacterium tuberculosis, and Legionella pneumophila to avoid and dampen these innate immune responses will be discussed.

Determination of C5a in murine models of staphylococcal enterotoxin B-induced toxic shock.

Robust host innate immune response to staphylococcal enterotoxin B (SEB) and structurally related superantigens causes toxic shock and various autoimmune diseases. While proinflammatory cytokines are known for mediating SEB-induced toxicity, the role of complement C5a in SEB-mediated shock is less well-understood. An ELISA was developed to measure the complement activation product, C5a, in different murine models of toxic shock. This assay provides easy, quantifiable data for complement activation and its role in various SEB-induced toxic shock models.

Comparative virulence of three different strains of Burkholderia pseudomallei in an aerosol non-human primate model.

Melioidosis, caused by the Gram-negative bacterium Burkholderia pseudomallei, is a major cause of sepsis and mortality in endemic regions of Southeast Asia and Northern Australia. B. pseudomallei is a potential bioterrorism agent due to its high infectivity, especially via inhalation, and its inherent resistance to antimicrobials. There is currently no vaccine for melioidosis and antibiotic treatment can fail due to innate drug resistance, delayed diagnosis and treatment, or insufficient duration of treatment. A well-characterized animal model that mimics human melioidosis is needed for the development of new medical countermeasures. This study first characterized the disease progression of melioidosis in the African green monkey (AGM) and rhesus macaque (RM) for non-human primate model down-selection. All AGMs developed acute lethal disease similar to that described in human acute infection following exposure to aerosolized B. pseudomallei strain HBPUB10134a. Only 20% of RMs succumbed to acute disease. Disease progression, immune response and pathology of two other strains of B. pseudomallei, K96243 and MSHR5855, were also compared using AGMs. These three B. pseudomallei strains represent a highly virulent strain from Thailand (HBPUB101034a), a highly virulent strains from Australia (MSHR5855), and a commonly used laboratory strains originating from Thailand (K96243). Animals were observed for clinical signs of infection and blood samples were analyzed for cytokine responses, blood chemistry and leukocyte changes in order to characterize bacterial infection. AGMs experienced fever after exposure to aerosolized B. pseudomallei at the onset of acute disease. Inflammation, abscesses and/or pyogranulomas were observed in lung with all three strains of B. pseudomallei. Inflammation, abscesses and/or pyogranulomas were observed in lymph nodes, spleen, liver and/or kidney with B. pseudomallei, HBPUB10134a and K96243. Additionally, the Australian strain MSHR5855 induced brain lesions in one AGM similar to clinical cases of melioidosis seen in Australia. Elevated serum levels of IL-1ß, IL-1 receptor antagonist, IL-6, MCP-1, G-CSF, HGF, IFNγ, MIG, I-TAC, and MIP-1ß at terminal end points can be significantly correlated with non-survivors with B. pseudomallei infection in AGM. The AGM model represents an acute model of B. pseudomallei infection for all three strains from two geographical locations and will be useful for efficacy testing of vaccines and therapeutics against melioidosis. In summary, a dysregulated immune response leading to excessive persistent inflammation and inflammatory cell death is the key driver of acute melioidosis. Early intervention in these pathways will be necessary to counter B. pseudomallei and mitigate the pathological consequences of melioidosis.

Rapamycin protects mice from staphylococcal enterotoxin B-induced toxic shock and blocks cytokine release in vitro and in vivo.

Staphylococcal enterotoxins are potent activators for human T cells and cause lethal toxic shock. Rapamycin, an immunosuppressant, was tested for its ability to inhibit staphylococcal enterotoxin B (SEB)-induced activation of human peripheral blood mononuclear cells (PBMC) in vitro and toxin-mediated shock in mice. Stimulation of PMBC by SEB was effectively blocked by rapamycin as evidenced by the inhibition of tumor necrosis factor alpha (TNF-alpha), interleukin 1beta (IL-1beta), IL-6, IL-2, gamma interferon (IFN-gamma), monocyte chemoattractant protein 1 (MCP-1), macrophage inflammatory protein 1alpha (MIP-1alpha), MIP-1beta, and T-cell proliferation. In vivo, rapamycin protected 100% of mice from lethal shock, even when administered 24 h after intranasal SEB challenge. The serum levels of MCP-1 and IL-6, after intranasal exposure to SEB, were significantly reduced in mice given rapamycin versus controls. Additionally, rapamycin diminished the weight loss and temperature fluctuations elicited by SEB.

Proinflammatory mediators of toxic shock and their correlation to lethality.

Bacterial exotoxins and endotoxins both stimulate proinflammatory mediators but the contribution of each individual toxin in the release of mediators causing lethal shock is incompletely understood. This study examines the cytokine response and lethality of mice exposed to varying doses of staphylococcal enterotoxin B (SEB) or lipopolysaccharide (LPS) and their combinations. In vivo, SEB alone induced moderate levels of IL-2 and MCP-1 and all mice survived even with a high dose of SEB (100 microg/mouse). LPS (80 microg/mouse) caused 48% lethality and induced high levels of IL-6 and MCP-1. SEB induced low levels of TNFalpha, IL-1, IFNgamma, MIP-2, and LPS synergized with SEB in the expression of these cytokines and that of IL-6 and MCP-1. Importantly, the synergistic action of SEB and LPS resulted in lethal shock and hypothermia. ANOVA of cytokine levels by survival status of SEB-plus-LPS groups revealed significantly higher levels of TNFalpha, IL-6, MIP-2, and MCP-1 in nonsurvivors measured at 8 hours. Significantly higher levels of IFNgamma and IL-2 were observed at 21 hours in nonsurvivors of toxic shock compared to those in survivors. Overall, synergistic action of SEB and LPS resulted in higher and prolonged levels of these key cytokines leading to toxic shock.

Murine models of staphylococcal enterotoxin B-induced toxic shock.

Staphylococcal enterotoxin B (SEB) is a member of a large family of structurally related exotoxins produced by Staphylococcus aureus, which is the etiological agent responsible for toxic shock and staphylococcal food poisoning. SEB binds directly to the major histocompatibility complex (MHC) class II molecules on antigen-presenting cells and T-cell receptors on T cells triggering T-cell proliferation and mediator release. SEB is a biothreat agent because of its ability to potently activate cells of the immune system. In vivo animal models are critical in the development of therapeutics against SEB-induced shock. Our results show that three different mouse strains with different susceptibility to SEB can be used to study SEB-induced shock without the use of potentiating agents. The hypothermic response, weight loss, and induction of serum monocyte chemoattractant protein 1 (MCP-1), interleukin 2 (IL-2), and IL-6 correlated with mortality in all three models.

Central roles for IL-2 and MCP-1 following intranasal exposure to SEB: a new mouse model.

Murine models for bacterial superantigens like staphylococcal enterotoxin B (SEB) have to date been rather cumbersome. The reasons include: (1) necessary use of potentiating agents such as actinomycin D, d-galactosamine, lipopolysaccharide (LPS), or viruses; (2) high toxin amounts required to elicit effects; and/or (3) generation of phenotypic-stable transgenic animals. Our study employed readily available C3H/HeJ (TLR4 negative, LPS-nonresponsive) mice with intranasal and intraperitoneal administration of low microgram quantities of SEB. These animals responded to SEB with severe lung inflammation and hypothermia, culminating in death. A survey of cytokines/chemokines in sera and lungs after lethal intoxication revealed that monocyte chemoattractant protein-1 and interleukin-2 were associated with effects in this model. In contrast, SEB had minimal effects upon congenic (TLR4 positive, LPS-responsive) C3H/OuJ mice. Lethality of SEB in C3H/HeJ mice was neutralized with SEB-specific antibodies, suggesting potential utility of this model for future therapeutic studies.

Staphylococcal Superantigens: Pyrogenic Toxins Induce Toxic Shock.

Staphylococcal enterotoxin B (SEB) and related superantigenic toxins produced by Staphylococcus aureus are potent activators of the immune system. These protein toxins bind to major histocompatibility complex (MHC) class II molecules and specific Vß regions of T-cell receptors (TCRs), resulting in the activation of both monocytes/macrophages and T lymphocytes. The bridging of TCRs with MHC class II molecules by superantigens triggers an early "cytokine storm" and massive polyclonal T-cell proliferation. Proinflammatory cytokines, tumor necrosis factor α, interleukin 1 (IL-1), IL-2, interferon γ (IFNγ), and macrophage chemoattractant protein 1 elicit fever, inflammation, multiple organ injury, hypotension, and lethal shock. Upon MHC/TCR ligation, superantigens induce signaling pathways, including mitogen-activated protein kinase cascades and cytokine receptor signaling, which results in NFκB activation and the phosphoinositide 3-kinase/mammalian target of rapamycin pathways. In addition, gene profiling studies have revealed the essential roles of innate antimicrobial defense genes in the pathogenesis of SEB. The genes expressed in a murine model of SEB-induced shock include intracellular DNA/RNA sensors, apoptosis/DNA damage-related molecules, endoplasmic reticulum/mitochondrial stress responses, immunoproteasome components, and IFN-stimulated genes. This review focuses on the signaling pathways induced by superantigens that lead to the activation of inflammation and damage response genes. The induction of these damage response genes provides evidence that SEB induces danger signals in host cells, resulting in multiorgan injury and toxic shock. Therapeutics targeting both host inflammatory and cell death pathways can potentially mitigate the toxic effects of staphylococcal superantigens.

The potency of anti-oxidants in attenuating superantigen-induced proinflammatory cytokines correlates with inactivation of NF-kappaB.

Excessive release of proinflammatory cytokines and chemokines mediates the toxic effects of superantigenic staphylococcal exotoxins (SE). We evaluated the potency of two anti-oxidants, N-acetyl-cysteine (NAC) and pyrrolidine dithiocarbamate (PDTC) in inhibiting the staphylococcal enterotoxin B and staphylococcal toxic shock syndrome-1-induced activation of human peripheral blood mononuclear cells (PBMC). Both NAC and PDTC dose-dependently inhibited SE-stimulated T-cell proliferation (by 98%), production of cytokines and chemokines by PBMC and expression of SE-induced cell surface activation markers. The potency of both NAC and PDTC corresponded to their ability to inhibit NF-kappaB activation. Our results suggest that anti-oxidants might be useful to mitigate the pathogenic effects of SE by blocking transcriptional signaling activated by superantigens.

Living dangerously: Burkholderia pseudomallei modulates phagocyte cell death to survive.

Melioidosis, caused by the Gram-negative bacterium Burkholderia pseudomallei, is a major cause of sepsis and mortality in endemic regions of Southeast Asia and Northern Australia. As a facultative intracellular pathogen, B. pseudomallei produces virulence factors to evade innate host response and survive within host cells. Neutrophils and macrophages are phagocytes that play critical roles in host defense against pathogens by their ability to detect and eliminate microbes. Host defense processes against B. pseudomallei including phagocytosis, oxidative burst, autophagy, apoptosis, and proinflammatory cytokine release are all initiated by these two phagocytes in the fight against this bacterium. In vitro studies with mouse macrophage cell lines revealed multiple evasion strategies used by B. pseudomallei to counteract these innate processes. B. pseudomallei invades and replicates in neutrophils but little is known regarding its evasion mechanisms. The bidirectional interaction of neutrophils and macrophages in controlling B. pseudomallei infection has also been overlooked. Here the hypothesis that B. pseudomallei hijacks neutrophils and uses them to transport and infect new phagocytes is proposed as an evasion strategy to survive and persist in host phagocytes. This two-pronged approach by B. pseudomallei to replicate in two different types of phagocytes and to modulate their cell death modes is effective in promoting persistence and survival of the bacterium.

Superantigen-induced cytokine release from whole-blood cell culture as a functional measure of drug efficacy after oral dosing in nonhuman primates.

Evaluation of drug efficacy for human diseases is routinely performed in animal models for efficiency and in accordance with FDA regulations. Rhesus macaques have been used as models for various lethal diseases and correlates of immunity, as nonhuman primates (NHP) closely resemble humans. We examined the ex vivo cytokine response of superantigen-stimulated whole-blood cells as a first step to therapeutic efficacy testing for bacterial superantigen-induced shock in NHP after oral dosing of pentoxifylline. Doses of 120mg/kg of pentoxifylline effectively attenuated staphylococcal enterotoxin B-induced tumor necrosis factor alpha (TNFalpha), gamma interferon (IFNgamma) and interleukin 2 (IL-2) in ex vivo culture of NHP whole-blood cells by 88%, 81%, and 76%, respectively, whereas lower doses of 48 or 72mg/kg had no inhibitory effect. Thus cytokine release of stimulated peripheral blood cells provides a convenient biological measurement of the anti-inflammatory potency of pentoxifylline and has the advantage of assessing functional responses to a specific biotoxin of interest.

FDA-approved immunosuppressants targeting staphylococcal superantigens: mechanisms and insights.

Immunostimulating staphylococcal enterotoxin B (SEB) and related superantigenic toxins cause diseases in human beings and laboratory animals by hyperactivating cells of the immune system. These protein toxins bind to the major histocompatibility complex class II (MHC II) molecules and specific Vß regions of T-cell receptors (TCRs), resulting in the stimulation of both monocytes/macrophages and T lymphocytes. The bridging of TCR with MHC II molecules by superantigens triggers intracellular signaling cascades, resulting in excessive release of proinflammatory mediators and massive polyclonal T-cell proliferation. The early induction of tumor necrosis factor α, interleukin 1 (IL-1), interleukin 2 (IL-2), interferon gamma (IFNγ), and macrophage chemoattractant protein 1 promotes fever, inflammation, and multiple organ injury. The signal transduction pathways for staphylococcal superantigen-induced toxicity downstream from TCR/major histocompatibility complex (MHC) ligation and interaction of cell surface co-stimulatory molecules include the mitogen-activated protein kinase cascades and cytokine receptor signaling, activating nuclear factor κB (NFκB) and the phosphoinositide 3-kinase/mammalian target of rapamycin pathways. Knowledge of host regulation within these activated pathways and molecules initiated by SEB and other superantigens enables the selection of US Food and Drug Administration (FDA)-approved drugs to interrupt and prevent superantigen-induced shock in animal models. This review focuses on the use of FDA-approved immunosuppressants in targeting the signaling pathways induced by staphylococcal superantigens.

Staphylococcal Superantigens Spark Host-Mediated Danger Signals.

Staphylococcal enterotoxin B (SEB) of Staphylococcus aureus, and related superantigenic toxins produced by myriad microbes, are potent stimulators of the immune system causing a variety of human diseases from transient food poisoning to lethal toxic shock. These protein toxins bind directly to specific Vß regions of T-cell receptors (TCR) and major histocompatibility complex (MHC) class II on antigen-presenting cells, resulting in hyperactivation of T lymphocytes and monocytes/macrophages. Activated host cells produce excessive amounts of proinflammatory cytokines and chemokines, especially tumor necrosis factor α, interleukin 1 (IL-1), IL-2, interferon γ (IFNγ), and macrophage chemoattractant protein 1 causing clinical symptoms of fever, hypotension, and shock. Because of superantigen-induced T cells skewed toward TH1 helper cells, and the induction of proinflammatory cytokines, superantigens can exacerbate autoimmune diseases. Upon TCR/MHC ligation, pathways induced by superantigens include the mitogen-activated protein kinase cascades and cytokine receptor signaling, resulting in activation of NFκB and the phosphoinositide 3-kinase/mammalian target of rapamycin pathways. Various mouse models exist to study SEB-induced shock including those with potentiating agents, transgenic mice and an "SEB-only" model. However, therapeutics to treat toxic shock remain elusive as host response genes central to pathogenesis of superantigens have only been identified recently. Gene profiling of a murine model for SEB-induced shock reveals novel molecules upregulated in multiple organs not previously associated with SEB-induced responses. The pivotal genes include intracellular DNA/RNA sensors, apoptosis/DNA damage-related molecules, immunoproteasome components, as well as antiviral and IFN-stimulated genes. The host-wide induction of these, and other, antimicrobial defense genes provide evidence that SEB elicits danger signals resulting in multi-organ damage and toxic shock. Ultimately, these discoveries might lead to novel therapeutics for various superantigen-based diseases.

Inflammasome, mTORC1 activation, and metabolic derangement contribute to the susceptibility of diabetics to infections.

The mechanisms leading to higher risks of infection in diabetics remain unknown despite recent advances in the understanding of associated immunological and metabolic aberrations. Hyperglycemia and hyperlipidemia in diabetics not only contribute to altered metabolism but glucose and free fatty acids can directly activate inflammation and the production of the proinflammatory cytokine interleukin 1ß (IL-1ß). Long-chain saturated fatty acids activate toll-like receptor 4 (TLR4), generating diacylglycerol and activating protein kinase C to upregulate the Akt/mammalian target of rapamycin complex 1 (mTORC1) pathway. High glucose uptake switches cell metabolism from oxidative phosphorylation to glycolysis and deactivates AMP-activated protein kinase (AMPK), a critical sensor of nutrient and cellular energy, leading to mTORC1 activation. A deleterious consequence of mTORC1 activation is the suppression of autophagy which is a catabolic process for the lysosomal degradation of damaged organelles, protein aggregates and intracellular pathogens. In addition, high glucose concentration and fatty acids independently activate inflammasome, an intracellular multi-protein complex that promotes the proteolytic activation of caspase 1, leading to the processing and secretion of IL-1ß. Other caspases induced by inflammasome can trigger apoptotic cell death. A common upstream signal for the activation of inflammasome and mTORC1 is oxidative stress, which generates reactive oxygen species (ROS) from dysregulated mitochondria. Increased flux of glucose and lipids activates stress kinases, enhances electron transport, and generates ROS in mitochondria. Mitochondrial stress arising from increased mitochondrial respiration and permeability damages mitochondria, activates caspases, which then induce apoptosis via the intrinsic cell death pathway releasing mitochondrial DNA. Normally apoptosis is down-regulated by autophagy as autophagy removes damaged organelles as a result of danger and stress signals. However, in diabetics, hyperactivation of mTORC1 disrupts the host autophagic degradation of microbes and damaged mitochondria which in turn exacerbates inflammasome activation and alters cell resistance to infection. Recognition of viral lipids and bacterial components by host cell pattern recognition receptors including TLR activates NFκB and stress kinase c-jun N-terminal kinase (JNK) signaling. The transcription factor NFκB and JNK independently induce inflammatory cytokines, chemokines, and further activate inflammasome. The convergence of inflammasome and mTORC1 activation with metabolic stress and vascular dysfunction in diabetics prevents pathogen clearance and contributes to an increased risk of infection.

Sulfasalazine attenuates staphylococcal enterotoxin B-induced immune responses.

Staphylococcal enterotoxin B (SEB) and related exotoxins are important virulence factors produced by Staphylococcus aureus as they cause human diseases such as food poisoning and toxic shock. These toxins bind directly to cells of the immune system resulting in hyperactivation of both T lymphocytes and monocytes/macrophages. The excessive release of proinflammatory cytokines from these cells mediates the toxic effects of SEB. This study examined the inhibitory activities of an anti-inflammatory drug, sulfasalazine, on SEB-stimulated human peripheral blood mononuclear cells (PBMC). Sulfasalazine dose-dependently inhibited tumor necrosis factor α, interleukin 1 (IL-1) ß, IL-2, IL-6, interferon γ (IFNγ), and various chemotactic cytokines from SEB-stimulated human PBMC. Sulfasalazine also potently blocked SEB-induced T cell proliferation and NFκB activation. These results suggest that sulfasalazine might be useful in mitigating the toxic effects of SEB by blocking SEB-induced host inflammatory cascade and signaling pathways.

Molecular therapeutic targets in inflammation: cyclooxygenase and NF-kappaB.

Inflammation is the host response to infection and injury. Inflammatory cells respond to foreign substances and inflammatory stimulus by producing bioactive mediators such as prostanoids, cytokines and chemokines. These mediators have complex, pleiotropic effects and interact with many cell types to amplify the inflammatory response. Dysregulation of these processes can lead to acute and chronic inflammatory diseases and pharmacological intervention is necessary to attenuate cellular inflammation pathways. Cyclooxygenase-2, the key inducible enzyme responsible for producing prostanoids, and the nuclear factor-kappa B (NF-kappaB) activation pathway, which regulates the transcription of inflammatory genes, represent attractive targets for developing anti-inflammatory therapeutics, as both pathways are activated by diverse inflammatory stimuli. This article reviews recent advances in anti-inflammatory drug development in both of these areas. Selective inhibitors of inflammation including cyclooxygenase inhibitors, antibodies against inflammatory cytokines, cytokine receptor antagonist, antibodies against adhesion molecules and therapeutics directed against the NF-kappaB activation pathway will be discussed.

Effects of Shuanghuanglian and Qingkailing, two multi-components of traditional Chinese medicinal preparations, on human leukocyte function.

Qingkailing (QKL) and Shuanghuanglian (SHHL) are two commonly used Chinese herbal preparations with reported antiinflammatory activity. The effects of these two preparations on the capacity of staphylococcal toxic shock syndrome toxin 1 (TSST-1) to stimulate the production of cytokines (IL-1beta, IL-6, TNF-alpha, IFN-gamma) and chemokines (MIP-1alpha, MIP-1beta and MCP-1) by peripheral blood mononuclear cell (PBMC) was tested. We also evaluated their effect on LPS-stimulated NF-kappaB transcriptional activity in a THP-1 cell line, and on human monocyte chemotactic response to chemoattractants. Non-cytotoxic concentrations of QKL (0.1 to approximately 2%) and SHHL (6 to approximately 120 microg) significantly inhibited production of cytokines and chemokines in a dose-dependent manner (P < 0.05). Both, QKL at 1:100 and SHHL at 60 microg/ml, markedly inhibited RANTES, MIP-1alpha, SDF-1alpha and fMLP induced human monocyte migration (P < 0.05 or 0.01). QKL (1%) did not inhibit monocyte chemotaxis induced by super-or sub-optimal concentrations of fMLP (10(-5), 10(-6) and 10(-10) M), but only inhibited chemotaxis induced by optimal concentrations of fMLP at 10(-7), 10(-8) and 10(-9) M. QKL (0.1% or 1%) and SHHL (6 or 60 microg/ml) markedly inhibited LPS-induced NF-kappaB activity in THP-1 cells. The results suggested that the pharmacological basis for the antiinflammatory effects of QKL and SHHL is the result of suppression of NF-kappaB regulated gene transcription, leading to suppressed production of proinflammatory cytokine and chemokine. Interference with leukocyte chemotaxis also contributes to the antiinflammatory and immunomodulating effects of these medicinals. Identification of the responsible components in these two herbal preparations may yield compounds suitable for structural modification into potent novel drugs.

Yin zi huang, an injectable multicomponent chinese herbal medicine, is a potent inhibitor of T-cell activation.

OBJECTIVES: The clinical efficacy of many multiherbal Traditional Chinese Medicines (TCM) is partially attributable to their immunoregulatory properties. In this study we evaluated the effect of eight commonly used, commercially available multiherbal Chinese medicines on T-cell activation. We focused on Yin Zhi Huang (YZH, an injectable herbal medicine commonly used for the treatment of liver diseases in China), because it was the most potent inhibitor of T-cell activation in our experimental system. The effects of 10 ingredient components of YZH were also evaluated. METHODS: [3H] thymidine incorporation assay was used to assess mouse T-cell proliferation after stimulation with latex beads coated with anti-CD3/CD28 antibodies. CD25, CD69, PD-1, and I-COS expression by purified mouse CD4+ T cells treated with plate-bound anti-CD3 antibody and soluble anti-CD28 antibody was analyzed by fluorescent-activated cell sorter (FACS). Cytokine/chemokine production by human peripheral blood mononuclear cells (PBMC) stimulated with staphylococcal enterotoxin B (SEB) was determined by enzyme-linked immunosorbent assay (ELISA). RESULTS: Among tested herbal medicines, YZH was the most potent inhibitor of T-cell activation. In splenocyte proliferation assays, the inhibitory effect of YZH was dose-dependent, with a 50% inhibition concentration (IC50) of 1:3200-1:1600. Ten (10) purified compounds found in YZH were evaluated for their activity. Among them, ursolic acid (1-10 micromol), luteolin (1-10 micromol), baicalein (1-10 micromol), scopran (5-50 micromol), and crocin (5-50 micromol), exhibited dose-dependent inhibition. YZH also inhibited CD25, CD69, PD-1, and ICOS expression by stimulated mouse CD4+ T cells. In human PBMCs, YZH inhibited SEB-stimulated cytokine (interleukin [IL]-1, IL-2, IL-6, tumor necrosis factor[TNF]-alpha, interferon [IFN]-gamma) and chemokine (IP-10, MCP-1, MIP-1alpha and MIP-1beta) production in a dose-dependent manner. CONCLUSION: Our data show for the first time that YZH is a potent inhibitor of T-cell activation, and this property may be the major mechanism underlying the clinical efficacy of YZH. Our experimental results pave the way for identification of active component(s) and/or analysis of synergistic/additive effect of a YZH ingredient in future studies.

Late multiple organ surge in interferon-regulated target genes characterizes staphylococcal enterotoxin B lethality.

BACKGROUND: Bacterial superantigens are virulence factors that cause toxic shock syndrome. Here, the genome-wide, temporal response of mice to lethal intranasal staphylococcal enterotoxin B (SEB) challenge was investigated in six tissues. RESULTS: The earliest responses and largest number of affected genes occurred in peripheral blood mononuclear cells (PBMC), spleen, and lung tissues with the highest content of both T-cells and monocyte/macrophages, the direct cellular targets of SEB. In contrast, the response of liver, kidney, and heart was delayed and involved fewer genes, but revealed a dominant genetic program that was seen in all 6 tissues. Many of the 85 uniquely annotated transcripts participating in this shared genomic response have not been previously linked to SEB. Nine of the 85 genes were subsequently confirmed by RT-PCR in every tissue/organ at 24 h. These 85 transcripts, up-regulated in all tissues, annotated to the interferon (IFN)/antiviral-response and included genes belonging to the DNA/RNA sensing system, DNA damage repair, the immunoproteasome, and the ER/metabolic stress-response and apoptosis pathways. Overall, this shared program was identified as a type I and II interferon (IFN)-response and the promoters of these genes were highly enriched for IFN regulatory matrices. Several genes whose secreted products induce the IFN pathway were up-regulated at early time points in PBMCs, spleen, and/or lung. Furthermore, IFN regulatory factors including Irf1, Irf7 and Irf8, and Zbp1, a DNA sensor/transcription factor that can directly elicit an IFN innate immune response, participated in this host-wide SEB signature. CONCLUSION: Global gene-expression changes across multiple organs implicated a host-wide IFN-response in SEB-induced death. Therapies aimed at IFN-associated innate immunity may improve outcome in toxic shock syndromes.

Update on staphylococcal superantigen-induced signaling pathways and therapeutic interventions.

Staphylococcal enterotoxin B (SEB) and related bacterial toxins cause diseases in humans and laboratory animals ranging from food poisoning, acute lung injury to toxic shock. These superantigens bind directly to the major histocompatibility complex class II molecules on antigen-presenting cells and specific Vß regions of T-cell receptors (TCR), resulting in rapid hyper-activation of the host immune system. In addition to TCR and co-stimulatory signals, proinflammatory mediators activate signaling pathways culminating in cell-stress response, activation of NFκB and mammalian target of rapamycin (mTOR). This article presents a concise review of superantigen-activated signaling pathways and focuses on the therapeutic challenges against bacterial superantigens.