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.

Efficacy of two FDA-approved drug combination in a mouse model of staphylococcal enterotoxin B-induced shock.

Staphylococcal enterotoxin B (SEB) causes lethal shock by potently stimulating the host immune response. Dexamethasone and N-acetyl cysteine (NAC) are anti-inflammatory and antioxidative drugs, respectively, which can independently modulate immune function. Dexamethasone was previously shown to be effective in preventing SEB-induced shock models only if administered early and in multiple doses for a long duration. In this study, dexamethasone and NAC were used in tandem and protected mice (75%) against SEB-induced lethal shock. Hypothermia and weight loss elicited by SEB were also diminished by this novel combination treatment. The levels of monocyte chemoattractant protein-1, interleukin-2, interleukin-6, and mouse gamma interferon in lung tissue after intranasal exposure to SEB were also significantly reduced in mice given a combination of dexamethasone and NAC versus controls.

Intranasal rapamycin rescues mice from staphylococcal enterotoxin B-induced shock.

Staphylococcal enterotoxin B (SEB) and related exotoxins produced by Staphylococcus aureus are potent activators of the immune system and cause toxic shock in humans. Currently there is no effective treatment except for the use of intravenous immunoglobulins administered shortly after SEB exposure. Intranasal SEB induces long-lasting lung injury which requires prolonged drug treatment. We investigated the effects of rapamycin, an immunosuppressive drug used to prevent graft rejection, by intranasal administration in a lethal mouse model of SEB-induced shock. The results show that intranasal rapamycin alone delivered as late as 17 h after SEB protected 100% of mice from lethal shock. Additionally, rapamycin diminished the weight loss and temperature fluctuations elicited by SEB. Intranasal rapamycin attenuated lung MCP-1, IL-2, IL-6, and IFNγ by 70%, 30%, 64%, and 68% respectively. Furthermore, short courses (three doses) of rapamycin were sufficient to block SEB-induced shock. Intranasal rapamycin represents a novel use of an immunosuppressant targeting directly to site of toxin exposure, reducing dosages needed and allowing a wider therapeutic window.

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.

Antibody to the E3 glycoprotein protects mice against lethal venezuelan equine encephalitis virus infection.

Six monoclonal antibodies were isolated that exhibited specificity for a furin cleavage site deletion mutant (V3526) of Venezuelan equine encephalitis virus (VEEV). These antibodies comprise a single competition group and bound the E3 glycoprotein of VEEV subtype I viruses but failed to bind the E3 glycoprotein of other alphaviruses. These antibodies neutralized V3526 virus infectivity but did not neutralize the parental strain of Trinidad donkey (TrD) VEEV. However, the E3-specific antibodies did inhibit the production of virus from VEEV TrD-infected cells. In addition, passive immunization of mice demonstrated that antibody to the E3 glycoprotein provided protection against lethal VEEV TrD challenge. This is the first recognition of a protective epitope in the E3 glycoprotein. Furthermore, these results indicate that E3 plays a critical role late in the morphogenesis of progeny virus after E3 appears on the surfaces of infected cells.

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.

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.

Critical timing, location and duration of glucocorticoid administration rescue mice from superantigen-induced shock and attenuate lung injury.

Bacterial superantigens, such as staphylococcal enterotoxin B (SEB), are major virulence factors implicated in the pathogenesis of toxic shock. In this study we investigated the efficacy of glucocorticoid therapy in preventing SEB-induced lethal shock initiated through the respiratory route in mice. Dexamethasone, a potent anti-inflammatory steroid, administrated intranasally on the first day, followed by intraperitoneal doses on the subsequent 4 days, was effective in attenuating SEB-induced hypothermia, and reduction in systemic and pulmonary proinflammatory mediator release. This optimal dosing and schedule of glucocorticoid treatment mitigated lung inflammation and resulted in 100% survival in this intranasal mouse model of SEB-mediated shock.

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.

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.

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.

Dexamethasone attenuates staphylococcal enterotoxin B-induced hypothermic response and protects mice from superantigen-induced toxic shock.

The superantigenic staphylococcal enterotoxins are important virulence factors and contribute to various diseases, including food poisoning and toxic shock. Dexamethasone, an anti-inflammatory agent, attenuated staphylococcal enterotoxin B (SEB)-induced hypothermia and serum proinflammatory cytokines and improved survival from 0% to 86% in a lethal mouse model of SEB-mediated shock.

Doxycycline is anti-inflammatory and inhibits staphylococcal exotoxin-induced cytokines and chemokines.

Proinflammatory cytokines mediate the toxic effect of superantigenic staphylococcal exotoxins (SE). Doxycycline inhibited SE-stimulated T-cell proliferation and production of cytokines and chemokines by human peripheral blood mononuclear cells. These results suggest that the antibiotic doxycycline has anti-inflammatory effects and is therapeutically useful for mitigating the pathogenic effects of SE.