Staphylococcal Enterotoxin B Exposure Research Articles

Staphylococcal enterotoxin B exposed to pregnant rats inhibits the hedgehog signaling pathway in thymic T lymphocytes of the offspring

The Hedgehog (Hh) signaling pathway is involved in T cell differentiation and development and plays a major regulatory part in different stages of T cell development. A previous study by us suggested that prenatal exposure to staphylococcal enterotoxin B (SEB) changed the percentages of T cell subpopulation in the offspring thymus. However, it is unclear whether prenatal SEB exposure impacts the Hh signaling pathway in thymic T cells. In the present study, pregnant rats at gestational day 16 were intravenously injected once with 15 μg SEB, and the thymi of both neonatal and adult offspring rats were aseptically acquired to scrutinize the effects of SEB on the Hh signaling pathway. It firstly found that prenatal SEB exposure clearly caused the increased expression of Shh and Dhh ligands of the Hh signaling pathway in thymus tissue of both neonatal and adult offspring rats, but significantly decreased the expression levels of membrane receptors of Ptch1 and Smo, transcription factor Gli1, as well as target genes of CyclinD1, C-myc, and N-myc in Hh signaling pathway of thymic T cells. These data suggest that prenatal SEB exposure inhibits the Hh signaling pathway in thymic T lymphocytes of the neonatal offspring, and this effect can be maintained in adult offspring via the imprinting effect.

Activation of STAT6 by intranasal allergens correlated with the development of eosinophilic chronic rhinosinusitis in a mouse model.

Eosinophilic chronic rhinosinusitis (ECRS) is a chronic inflammatory disease characterized by prominent eosinophilic infiltration along with a T-helper-2 (Th2) response. It has been well documented that signal transducer and activator of transcription 6 (STAT6) is a nuclear transcription factor that mediates Th2-type immunity and is implicatory of STAT1 and STAT3 in the pathogenesis of allergic airway diseases. However, little is known about the association between STATs and ECRS. Here, we explored the relationship between STAT1, STAT3, and/or STAT6 and eosinophilic inflammation accompanied by Th2-type immunity in a mouse model of ECRS. An ovalbumin (OVA)-staphylococcal enterotoxin B (SEB)-induced ECRS murine model was first established. The mucosal histological alterations were determined using hematoxylin and eosin staining. The number of eosinophils in peripheral blood was measured using a blood cell analyzer. The cytokine (IL-4, IL-5, IL17 A and IFN-γ) expression levels in the sinonasal mucosa and total and OVA-specific IgE from serum were measured using ELISA. Then, the protein levels of STAT1, STAT3, STAT6, phosphorylated STAT1 (p-STAT1), p-STAT3, p-STAT6, T-box expressed in T-cells (T-bet), GATA binding protein 3 (GATA-3), and retinoic acid receptor-related orphan receptor γ (RORγt) in the sinonasal mucosa were examined by immunohistochemical staining or Western blotting. Local administration of OVA combined with SEB (OVA + SEB) induced multiple polyp-like lesions, accompanied by prominent eosinophilic infiltration in the sinonasal mucosa. The OVA- and OVA+SEB-treated groups showed significantly higher eosinophil counts from peripheral blood and total and OVA-specific IgE levels from serum than those in the PBS- and SEB-treated groups. The levels of p-STAT6 were markedly increased by OVA + SEB exposure, as well as GATA-3, IL-4, and IL-5, but did not affect STAT6, p-STAT1, p-STAT3, T-bet, RORγt, IFN-γ, or IL-17A. Furthermore, an eosinophil count in the sinonasal mucosa showed a positive correlation with the level of p-STAT6 in the ECRS mouse model. Signal transducer and activator of transcription 6 signaling could be activated in the OVA+SEB-induced ECRS model and might be a crucial signal transducer in the development of Th2-skewed ECRS.

Exposure to aerosolized staphylococcal enterotoxin B potentiated by lipopolysaccharide modifies lung transcriptomes and results in lung injury in the mouse model.

Lipopolysaccharide (LPS) is one of the main constituents of the cell wall in Gram-negative bacteria. Staphylococcal enterotoxin B (SEB) is produced by the Gram-positive opportunistic pathogen, Staphylococcus aureus. Emerging evidence suggests that intraperitoneal injection of LPS combined with low-dose aerosolized SEB exposure can cause severe lung injury and even death, while SEB or LPS alone cause neither mortality nor severe pulmonary symptoms in mice. However, pulmonary effects from exposure to aerosolized SEB potentiated by LPS have not been evaluated. This study investigates the global transcriptome profile of lung tissue in mice after exposure to aerosolized SEB potentiated by LPS or LPS alone. A mouse model of intratracheal exposure to LPS-potentiated aerosolized SEB is established and described through histological examination. Transcriptome analysis revealed LPS-potentiated aerosolized SEB affected mouse lungs within 72 h post-SEB inhalation, gradually causing lung injury starting from 24 h post inhalation. Hub genes leading to lung injury at 48 h post inhalation have been identified. Flow cytometry revealed that LPS potentiation of low-dose SEB produces a superantigen response that T cells expressing a particular T cell receptor Vβ induces a proliferation response by 72 h post inhalation in the lungs of mice. This study represents the first research to investigate pulmonary transcriptional responses of LPS-potentiated aerosolized low-dose SEB exposure. This research helps to elucidate the molecular mechanisms underlying the process by which the two bacterial components combined to produce lung damage and provides an insight into potential treatments for alleviating inflammation of the lung when coinfection is present.

Exposure of pregnant rats to staphylococcal enterotoxin B increases offspring splenic Treg number and function via decreasing FoxP3 methylation

Staphylococcus aureus is an infectious pathogen that is relatively common, but that can cause severe disease in pregnant women and their fetus. We previously demonstrated that exposing pregnant rats to staphylococcal enterotoxin B (SEB) altered splenic CD4/CD8 T cell frequencies in their offspring. Whether prenatal SEB exposure impacts Tregs in these offspring, however, remains to be determined. As such, in this study, we intravenously injected pregnant rats with 15 μg of SEB on gestational day 16. Splenic tissue was then harvested from 1-, 3-, and 5-day-old neonatal rats and analyzed via flow cytometry to assess Treg numbers. In addition, FoxP3 expression levels were assessed via qPCR and western blotting, while FoxP3 methylation status was evaluated via methyl-DNA immunoprecipitation qPCR. Immunosuppression assays were additionally used to gauge Treg suppressive functionality. We found that exposing pregnant rats to SEB resulted in a significant increase in Treg numbers, FoxP3 expression, and Treg suppressive capacity in the spleens of both neonatal and adult offspring. In addition, total T cell, CD4+T cell, and non-Treg CD4+ T cell numbers were elevated in the spleens of offspring following prenatal SEB exposure. We additionally determined that SEB exposure resulted in a significant reduction in FoxP3 DNA methylation. Together, our results indicate that prenatal SEB exposure can markedly enhance offspring splenic Treg numbers and functionality at least in part by decreasing FoxP3 methylation.

Airways exposure of bacterial superantigen SEB enhances bone marrow eosinophil population and facilitates its egress to blood and lung tissue

BackgroundDifferentiation of bone marrow eosinophils (BM-EO) and its trafficking to peripheral blood and respiratory mucosa are a hallmark of inflammatory diseases. Staphylococcal enterotoxin B (SEB) has been shown to aggravate airways eosinophilic inflammation. This study aimed to investigate the effects of mouse airways SEB exposure on BM-EO population, as well as its adhesive properties and release of cytokines/chemokines that orchestrate BM-EO trafficking to lungs. MethodsMale BALB/c mice were intranasally exposed to SEB (1 μg), and at 4, 16, 24 and 48 h thereafter, bone marrow (BM), circulating blood and bronchoalveolar lavage (BAL) fluid were collected. Levels of cytokines/chemokines and expressions of VLA-4 and CCR3 in BM were evaluated. Adhesion of BM to ICAM-1 and VCAM-1 were also evaluated. ResultsSEB exposure promoted a marked eosinophil influx to BAL at 16 and 24 h after exposure, which was accompanied by significant increases in counts of immature (16 h) and mature (4 to 48 h) forms of eosinophil in BM, along with blood eosinophilia (16 h). In BM, higher levels of eotaxin, IL-5, IL-4, IL-3 and IL-7 were detected at 16 to 48 h. SEB also significantly increased CCR3 expression and calcium levels in BM-EO, and enhanced the cell adhesion to ICAM-1 (24 h) and ICAM-1 (48 h). ConclusionAirways SEB exposure increases the number of eosinophils in BM by mechanisms involving a network of cytokine and chemokine release, facilitating the BM-EO adhesion to ICAM-1 and VCAM-1 to gain access to the peripheral blood and lung tissues.

MiR-222 inhibition alleviates Staphylococcal Enterotoxin B-induced inflammatory acute lung injury by targeting Foxo3

Acute lung injury (ALI) is a common acute and severe disease in clinical practice. Staphylococcal Enterotoxin B (SEB) is a superantigen that can cause inflammatory ALI. MiR-222 has been demonstrated to be upregulated in SEB-induced inflammatory ALI, but its exact roles and functions remain ill-defined. In this study, SEB exposure led to inflammatory ALI and high expression of miR-222 in model mice and lung infiltrating mononuclear cells, but the inflammatory response and high expression of miR-222 were restored in miR-222-/- mice. Moreover, we investigated the roles of miR-222 in vitro and observed that the concentrations of inflammatory cytokines and the expression of miR-222 were all elevated in SEB-activated splenocytes and miR-222 inhibition reversed the effects. Foxo3 was confirmed as a direct target of miR-222. Interestingly, SEB exposure led to a decrease of Foxo3 expression, and Foxo3 knockdown partially reversed the promotion of Foxo3 and the inhibition of inflammatory cytokines induced by miR-222 inhibitor in SEB-activated splenocytes. Our data indicated that miR-222 inhibition could alleviate SEB-induced inflammatory ALI by directly targeting Foxo3, shedding light on the potential therapeutic of miR-222 for SEB-induced inflammation in the lung.

Prenatal exposure of staphylococcal enterotoxin B attenuates the development and function of blood regulatory T cells to repeated staphylococcal enterotoxin B exposure in adult offspring rats.

Introduction Staphylococcal enterotoxin B (SEB) is an extensively studied super-antigen. A previous study by us suggested that SEB exposure during pregnancy could alter the percentage of CD4+ and CD8+ T cells in the peripheral blood of neonatal offspring rats.Aim It is unknown whether SEB exposure during pregnancy can influence the development of regulatory T cells (Tregs) in the peripheral blood of neonatal offspring rats.Methodology Pregnant rats at gestational day 16 were intravenously injected with 15 µg SEB. Peripheral blood was acquired from neonatal offspring rats on days 1, 3 and 5 after delivery and from adult offspring rats for determination of Treg number by cytometry, cytokines by ELISA, and FoxP3 expression by real-time PCR and western blot.Results SEB given to pregnant rats significantly increased the absolute number of Tregs and the expression levels of FoxP3, IL-10 and TGF-β (P<0.05, P<0.01) in the peripheral blood of not only neonatal but also adult offspring rats. Furthermore, repeated SEB exposure in adult offspring rats significantly decreased the absolute number of Tregs (P<0.01), and the expression levels of FoxP3, IL-10 and TGF-β (P<0.05, P<0.01) in their peripheral blood.Conclusion Prenatal SEB exposure attenuates the development and function of Tregs to repeated SEB exposure in the peripheral blood of adult offspring rats.

Attenuation of Staphylococcal enterotoxin-B (SEB)-induced lung injury (ALI) by resveratrol results from miR-193a down-regulation and alterations in metabolic profile

Abstract Resveratrol, (RES), a phytoalexin, is well-known for its anti-inflammatory and anti-oxidant properties. SEB, a superantigen, is known to trigger ALI and cause mortality. In the current study, we tested the effect of RES in a dual-dose model of SEB exposure that triggers ALI and causes 100% mortality in C3H/HeJ-mice. The data revealed RES attenuated SEB-induced ALI and prevented mortality. Forty eight hours post-SEB exposure, lung-infiltrating mononuclear cells were tested for microRNA expression profile to determine the epigenetic regulation by resveratrol. SEB-activated splenocytes were pre-treated with 50 μM of RES or vehicle for metabolic profile analysis by measuring oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). We also noted significant decline in miR-193a in the lungs of RES-treated SEB group, which targeted and caused an increase in TGFβ2 and TGFβR3, potent inhibitors of T-cell proliferation, by using RT-PCR and validation by transfection studies. RES-induced downregulation of miR-193a also influenced the activity of mechanistic target of rapamycin (mTOR) as well as pyruvate kinase muscle isozyme2 (PKM2) genes, and caused RES-treated SEB-activated T cells to be quiescent metabolically in comparison to the energetic vehicle-treated SEB-activated T cells. Together, RES caused inhibition in the proliferation of SEB-activated T-cells by alterations in miR expression and metabolic profiles. (Supported by NIH grants P01AT003961, R01AT006888, R01ES019313, R01MH094755, P20GM103641 and R01AI129788).

The Akt Pathway Inhibitor Degeulin Prevents Staphylococcal Enterotoxin B Induced Splenocyte Proliferation and Inflammation

Staphylococcal Enterotoxin B (SEB) is considered a potential biological weapon. It is toxic by both inhalation and ingestion. Effects of ingestion include fever, vomiting and diarrhoea, while inhalation may additionally result in chest pain, dyspnoea, pulmonary oedema and respiratory failure. Severe exposure may be fatal and treatment relies on symptomatic support. At a cellular level, SEB up-regulates T-cell proliferation leading to a pathological inflammatory response. Deguelin, a rotenoid isolated from the African plant Mundulea sericea (Leguminosae), has been shown to reduce cellular proliferation by inhibiting the phosphoinositide 3-kinase/Akt (PI3K/Akt) signalling pathway. Using isolated murine splenocytes, we have demonstrated that treatment with deguelin reduces SEB inducing T cell proliferation by 60%. Deguelin treatment also decreased IL-2 and CCL2 secretion by splenocytes exposed to SEB. We demonstrate that targeting cellular proliferation can significantly reduce inflammation after SEB exposure and suggest that anti-proliferatives may have a role as potential generic medical counter measures if superantigens are used as biological weapons.

Stress-caused anergy of leukocytes towards Staphylococcal enterotoxin B and exposure transcriptome signatures.

Leucocytes from soldiers exposed to battlefield-like stress (RASP: Rangers Assessment and Selection Program) were exposed in vitro to Staphylococcal enterotoxin B (SEB). We assayed SEB-induced regulation of gene expression, both in the presence and absence of severe stress, to generate two sets of gene profiles. One set of transcripts and microRNAs were specific to post-RASP SEB exposure, and another set were signatures of SEB exposure common to both the pre- and post-RASP leucocytes. Pathways and upstream regulatory analyses indicated that the post-RASP SEB-signature transcripts were manifestation of the anergic state of post-RASP leucocytes. These were further verified using expression-based predictions of cellular processes and literature searches. Specificity of the second set of transcripts to SEB exposure was verified using machine-learning algorithms on our and four other (Gene Expression Omnibus) data sets. Cell adhesion, coagulation, hypoxia and vascular endothelial growth factor-mediated vascular leakage were SEB-specific pathways even under the background of severe stress. Hsa-miR-155-3p was the top SEB exposure predictor in our data set, and C-X-C motif chemokine ligand 9 was SEB specific in all the analyzed data sets. The SEB-signature transcripts (which also showed distinct expression signatures from Yersinia pestis and dengue virus) may serve as potential biomarkers of SEB exposure even under the background of stress.

Staphylococcal enterotoxin B induced expression of IL-17A in nasal epithelial cells and its association with pathogenesis of nasal polyposis

Interleukin (IL)-17A is a highly inflammatory cytokine and is known to be produced by Th17 cells. The importance of IL-17A expression in nasal epithelial cells is not well understood. The goal of this study is to explore the expression of IL-17A in nasal epithelial cells in vivo and in vitro. IL-17A and staphylococcal enterotoxin B (SEB) were detected by immunofluorescence (IF) in nasal epithelial cells of control mucosa (n = 10) and nasal polyps (n = 20). Expression of IL-17A, RORC, IL-6, and TGF-β1 was also measured by RT-PCR in the tissue of control nasal mucosa (n = 10) and nasal polyps (n = 20). IL-17A expression was evaluated in the human nasal epithelial cells after SEB stimulation. Finally, IL-17A expression was demonstrated by immunohistochemistry and IF following intranasal SEB instillation in mice. Expression of IL-17A in nasal epithelial cells was higher in nasal polyps compared to control mucosa. There was a significant correlation between IL-17A and SEB detection in nasal polyps using IF. SEB increased IL-17A expression in human nasal epithelial cells, and in epithelial cells of SEB instilled mice. In conclusion, SEB exposure of nasal epithelial cells induces the enhanced expression of IL-17A. SEB may be involved in pathogenesis of nasal polyps by enhancing IL-17A expression in epithelial cells in nasal polyps.

Transcriptome and microRNA Signatures of Staphylococcal enterotoxin B Exposure under the Background of Battlefieldlike Stress

Leucocytes isolated from pre‐ and post‐training whole blood samples of US Army Ranger Cadets were exposed in vitro to Staphylococcal enterotoxin B (SEB) to analyze SEB‐induced regulation of gene expressions both in the presence and absence of severe‐stress. Gene profiling data showed suppression of immune response genes that are important in pattern recognition receptors, phagocytosis, antigen presentation, chemotaxis and activation of lymphocytes. Immune response genes that were suppressed by the training stressors remained suppressed after SEB exposure. Post‐training leucocytes seemed to be anergic towards SEB. Stress‐regulated microRNAs were identified as potential up‐stream regulators of many transcripts important in chemotaxis, antigen presentation, inflammation, lymphocyte activations and transcription factors important in immune response. Despite the complications due to suppressed immune responses seen at the end of the intensive Army Ranger Training, we have also identified other groups of genes that showed consistent differential expression towards SEB both in pre‐ and post‐training leukocytes. Some of these genes were SEB‐specific and have the potential to serve as diagnostic markers of SEB exposure irrespective of other pathophysiologic conditions.

Multiple anti‐inflammatory pathways triggered by resveratrol lead to amelioration of staphylococcal enterotoxin B‐induced lung injury

Inhalation of the superantigen,staphylococcal enterotoxin B (SEB), leads to the activation of the host T and invariant natural killer (iNK) T cells, thereby resulting in acute lung inflammation and respiratory failure but the underlying mechanism(s) of disease remain elusive, with limited treatment options. In this study, we investigated the therapeutic effectiveness of resveratrol, a plant polyphenol, during SEB-induced lung inflammation. C57BL/6 mice were exposed to SEB (50 µg·per mouse), administered intranasally, and were treated with resveratrol (100 mg·kg(-1)) before or after SEB exposure. Lung injury was studied by measuring vascular permeability, histopathological examination, nature of infiltrating cells, inflammatory cytokine induction in the bronchoalveolar fluid (BALF), apoptosis in SEB-activated T cells and regulation of SIRT1 and NF-κB signalling pathways. Pretreatment and post-treatment with resveratrol significantly reduced SEB-induced pulmonary vascular permeability, and inflammation. Resveratrol significantly reduced lung infiltrating cells and attenuated the cytokine storm in SEB-exposed mice, which correlated with increased caspase-8-dependent apoptosis in SEB-activated T cells. Resveratrol treatment also markedly up-regulated Cd11b+ and Gr1+ myeloid-derived suppressor cells (MDSCs) that inhibited SEB-mediated T cell activation in vitro. In addition, resveratrol treatment was accompanied by up-regulation of SIRT1 and down-regulation of NF-κB in the inflammatory cells of the lungs. The current study demonstrates that resveratrol may constitute a novel therapeutic modality to prevent and treat SEB-induced lung inflammation inasmuch because it acts through several pathways to reduce pulmonary inflammation.

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.

Therapeutic Inhibition of Pro-Inflammatory Signaling and Toxicity to Staphylococcal Enterotoxin B by a Synthetic Dimeric BB-Loop Mimetic of MyD88

Staphylococcal enterotoxin B (SEB) exposure triggers an exaggerated pro-inflammatory cytokine response that often leads to toxic shock syndrome (TSS) associated with organ failure and death. MyD88 mediates pro-inflammatory cytokine signaling induced by SEB exposure and MyD88−/− mice are resistant to SEB intoxication, suggesting that MyD88 may be a potential target for therapeutic intervention. We targeted the BB loop region of the Toll/IL-1 receptor (TIR) domain of MyD88 to develop small-molecule therapeutics. Here, we report that a synthetic compound (EM-163), mimic to dimeric form of BB-loop of MyD88 attenuated tumor necrosis factor (TNF)- α, interferon (IFN)-γ, interleukin (IL)-1β, IL-2 and IL-6 production in human primary cells, whether administered pre- or post-SEB exposure. Results from a direct binding assay, and from MyD88 co-transfection/co-immunoprecipitation experiments, suggest that EM-163 inhibits TIR-TIR domain interaction. Additional results indicate that EM-163 prevents MyD88 from mediating downstream signaling. In an NF-kB-driven reporter assay of lipopolysaccharide-stimulated MyD88 signaling, EM-163 demonstrated a dose-dependent inhibition of reporter activity as well as TNF-α and IL-1β production. Importantly, administration of EM-163 pre- or post exposure to a lethal dose of SEB abrogated pro-inflammatory cytokine responses and protected mice from toxic shock-induced death. Taken together, our results suggest that EM-163 exhibits a potential for therapeutic use against SEB intoxication.

CD44 as a novel target for treatment of staphylococcal enterotoxin B-induced acute inflammatory lung injury

Exposure to bacterial superantigens, such as staphylococcal enterotoxin B (SEB), can lead to the induction of acute lung injury/acute respiratory distress syndrome (ALI/ARDS). In the current study, we investigated the role of CD44 in ALI/ARDS. Intranasal exposure of CD44 wild-type mice to SEB led to a significant increase in the expression of CD44 on lung mononuclear cells. CD44 knockout mice developed significantly reduced SEB-induced ALI/ARDS, through reduced inflammatory cytokine production and reduced lung inflammatory cells, compared to similarly treated CD44 wild-type mice. Mechanistically, deletion of CD44 altered SEB-induced cytokine production in the lungs and reduced the ability of SEB-exposed leukocytes to bind to lung epithelial cells. Finally, treatment of SEB-exposed mice with anti-CD44 mAbs led to significant reduction in vascular permeability, reduction in cytokine production, and prevented inflammatory cell infiltration in the lungs. Together, these results suggest the possibility of targeting CD44 for the treatment of SEB-induced ALI/ARDS.

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. We evaluated the efficacy of intranasal administration of rapamycin in a lethal mouse model of SEB‐induced shock. The results show that intranasal rapamycin delivered as late as 17 h after SEB still protected 100% of mice from lethality. Additionally, rapamycin diminished the weight loss and temperature fluctuations elicited by SEB. Furthermore, only 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.Source of support: Defense Threat Reduction Agency

CD1d-Independent Activation of Invariant Natural Killer T Cells by Staphylococcal Enterotoxin B through Major Histocompatibility Complex Class II/T Cell Receptor Interaction Results in Acute Lung Injury

There are two important mechanisms of activation of invariant natural killer T cells (iNKT cells) by microbes: direct activation of the invariant T-cell receptor (TCR) by microbial glycolipids presented by CD1d and indirect activation, mediated by the responses of antigen-presenting cells to microbes. In this study, we provide evidence for a novel CD1d-independent direct activation of iNKT cells involving a microbial protein superantigen presented in the context of major histocompatibility complex class II (MHC-II), which plays a critical role in pathogenesis, thereby redefining the role of iNKT cells. Intranasal exposure to staphylococcal enterotoxin B (SEB) in C57BL/6 wild-type mice caused acute lung injury (ALI) characterized by vascular leak, cytokine storm, and infiltration of mononuclear cells in the lungs. In contrast, the vascular leak and inflammation were decreased by ~50% in NKT cell-deficient Jα18(-/-) and CD1d(-/-) mice following SEB exposure, which was reversed following adoptive transfer of iNKT cells into CD1d(-/-) mice. In vitro, SEB could directly stimulate iNKT cells in a CD1d-independent manner via MHC-II/TCR interaction, specifically involving Vβ8. These studies not only demonstrate that iNKT cells can be activated directly by a bacterial protein superantigen independent of CD1d but also indicate that in addition to the conventional T cells, iNKT cells play a critical role in SEB-mediated ALI.

Generation and characterization of high affinity human monoclonal antibodies that neutralize staphylococcal enterotoxin B

BackgroundStaphylococcal enterotoxins are considered potential biowarfare agents that can be spread through ingestion or inhalation. Staphylococcal enterotoxin B (SEB) is a widely studied superantigen that can directly stimulate T-cells to release a massive amount of proinflammatory cytokines by bridging the MHC II molecules on an antigen presenting cell (APC) and the Vβ chains of the T-cell receptor (TCR). This potentially can lead to toxic, debilitating and lethal effects. Currently, there are no preventative measures for SEB exposure, only supportive therapies.MethodsTo develop a potential therapeutic candidate to combat SEB exposure, we have generated three human B-cell hybridomas that produce human monoclonal antibodies (HuMAbs) to SEB. These HuMAbs were screened for specificity, affinity and the ability to block SEB activity in vitro as well as its lethal effect in vivo.ResultsThe high-affinity HuMAbs, as determined by BiaCore analysis, were specific to SEB with minimal crossreactivity to related toxins by ELISA. In an immunoblotting experiment, our HuMAbs bound SEB mixed in a cell lysate and did not bind any of the lysate proteins. In an in vitro cell-based assay, these HuMAbs could inhibit SEB-induced secretion of the proinflammatory cytokines (INF-γ and TNF-α) by primary human lymphocytes with high potency. In an in vivo LPS-potentiated mouse model, our lead antibody, HuMAb-154, was capable of neutralizing up to 100 μg of SEB challenge equivalent to 500 times over the reported LD50 (0.2 μg) , protecting mice from death. Extended survival was also observed when HuMAb-154 was administered after SEB challenge.ConclusionWe have generated high-affinity SEB-specific antibodies capable of neutralizing SEB in vitro as well as in vivo in a mouse model. Taken together, these results suggest that our antibodies hold the potential as passive immunotherapies for both prophylactic and therapeutic countermeasures of SEB exposure.

The Cellular and Molecular Immune Response of the Weanling Piglet to Staphylococcal Enterotoxin B

Staphylococcal enterotoxin B (SEB) is a biothreat agent, etiologic agent of food poisoning, and potent inducer of toxic shock syndrome. This heat-stable exoprotein is thought to act as a superantigen to induce T cell-specific pathology. Most animal models do not accurately map the clinical syndrome of human SEB exposure. Previously, we have demonstrated the utility of the weanling piglet model of SEB intoxication. Here, we analyze gross and histopathologic specimens from lymphoid tissue of these animals. Hematological testing was completed to observe changes in circulating leukocytes. Further, these leukocytes were differentiated and the subsets were subsequently analyzed using flow cytometry. Cytokine mRNA was quantified in lymphoid tissue and peripheral blood cells and compared to actual protein concentration using ELISA. The mRNA expression levels for several cell markers implicated in T and B cell differentiation were quantified and compared to control animals, as were levels for apoptosis-related genes. Lymphadenopathy was constantly seen post mortem. SEB-exposed animals had a leukocytosis which increased linearly over the time course. Monocyte levels increased over time, while lymphocyte levels peaked at 6h and then returned to baseline. Most cytokines had mRNA levels that were upregulated after exposure. Detection of serum cytokine changes was accomplished; however, these patterns did not always follow those seen in the differentially expressed genes. Both pro- and anti-apoptotic genes were differentially expressed in exposed animals. This paper reports, for the first time, the immunological findings in the weanling piglet model of SEB intoxication. From this work it is clear that there is not one absolute cell-mediated pathway contributing to the pathology these animals exhibit as a result of SEB exposure.