Staphylococcal Enterotoxin B Stimulation Research Articles

Targeting staphylococcal enterotoxin B binding to CD28 as a new strategy for dampening superantigen-mediated intestinal epithelial barrier dysfunctions.

Staphylococcus aureus is a gram-positive bacterium that may cause intestinal inflammation by secreting enterotoxins, which commonly cause food-poisoning and gastrointestinal injuries. Staphylococcal enterotoxin B (SEB) acts as a superantigen (SAg) by binding in a bivalent manner the T-cell receptor (TCR) and the costimulatory receptor CD28, thus stimulating T cells to produce large amounts of inflammatory cytokines, which may affect intestinal epithelial barrier integrity and functions. However, the role of T cell-mediated SEB inflammatory activity remains unknown. Here we show that inflammatory cytokines produced by T cells following SEB stimulation induce dysfunctions in Caco-2 intestinal epithelial cells by promoting actin cytoskeleton remodelling and epithelial cell-cell junction down-regulation. We also found that SEB-activated inflammatory T cells promote the up-regulation of epithelial-mesenchymal transition transcription factors (EMT-TFs) in a nuclear factor-κB (NF-κB)- and STAT3-dependent manner. Finally, by using a structure-based design approach, we identified a SEB mimetic peptide (pSEB116-132) that, by blocking the binding of SEB to CD28, dampens inflammatory-mediated dysregulation of intestinal epithelial barrier.

Helminth species-specific effects on IFN-γ producing T cells during active and latent tuberculosis.

Interferon-γ (IFN-γ) is a key cytokine inducing protective immune responses during tuberculosis (TB) infection. Helminth-induced immune responses may affect IFN-γ production by T cells, although its connection with disease severity and immune recovery during treatment is unexplored. We investigated the species-specific effect of helminths on the IFN-γ production by T cells in relation to disease severity during active and latent TB infection (LTBI). In this study, 69 active pulmonary TB patients (PTB), 28 with LTBI and 66 healthy controls were included. Active TB was diagnosed using GenXpert MTB/RIF while QuantiFERON test (QFT) was used for the screening of healthy community controls (CCs) and for the diagnosis of LTBI. Helminth infection was identified by routine diagnosis whereas clinical disease severity was evaluated by the TB score. Intracellular IFN-γ production of T cells in stimulated peripheral blood mononuclear cells (PBMCs) was analyzed by flow cytometry using TB antigens (PPD), the polyclonal T cell activator staphylococcal enterotoxin B (SEB), or medium as unstimulated control. Helminth infected CCs and LTBI subjects showed a significant reduction of IFN-γ+ CD4+ T cells by PPD-stimulation compared to non-helminth infected control groups. The significant reduction in the frequency of IFN-γ+ T cells in both latent and active PTB patients following SEB stimulation was mostly attributed to Schistosoma mansoni infection, whereas Ascaris lumbricoides, Schistosoma mansoni, and hookworm infection contributed equally in CCs. Following anti-helminthic and anti-TB treatment for 2 months, the frequency of IFN-γ+ CD4 T cells in helminth coinfected PTB was restored to levels of helminth negative PTB before treatment. Helminth coinfected PTB patients with an intermediate and severe clinical course had reduced capacity for production of IFN-γ+ T cells compared to the corresponding non-helminth infected PTB. We found a reduction in IFN-γ producing T cells by helminth coinfection which was restored following anti-helminthic treatment. This reduction was helminth species-dependent in an exploratory sub-analysis and correlated to increased disease severity.

Abstract 1984: Baseline T-cell function predicts response to SBRT and immunotherapy in NSCLC

Abstract It is increasingly understood that cancers can be recognized by the immune system and inflammation relates to response. Combining stereotactic radiotherapy (SBRT), to increase release of cancer cell antigens, with an anti-CTLA-4 and PD-L1 inhibitor may lead to increased response rates. No solid biomarker in predicting treatment response is available. We set out to study feasibility of combined durvalumab (anti-PD-L1) and tremelimumab (anti-CTLA-4) with SBRT on the primary tumor. The extend of tumor-infiltrating cells has shown to correlate to improved prognosis using checkpoint inhibitor (CPI) treatment of various cancers. We hypothesized that the peripheral blood T-cell activation status may be a predictor of response, progression free survival (PFS) and overall survival (OS). Methods: Three immunotherapy regimes were combined with SBRT in sequential cohorts as ≥2nd line in CPI naïve patients with NSCLC stage IIIB/IV. One week after the 1st dose of CPI, patients were irradiated on the primary tumor (1x 20Gy on 9cc). The 1st cohort (n=3) received durvalumab, the 2nd and 3rd cohort (both n=6) a combination of durvalumab and tremelimumab, followed by durvalumab monotherapy. Exploratory endpoint was the correlation of response with T-cell function. T-cell function was assessed using flow cytometric analysis of peripheral blood T-cells and compared to simultaneously included healthy controls. In short, whole blood was activated ex vivo using Staphylococcal enterotoxin B (SEB) after which the T-cell activation status was assesses by expression of CD69 and cytokines (IL-2, IFN-γ, TNF-α). Patients were divided in above (responders, R) and below median PFS (non-responders, NR). We compared intracellular cytokine production in both CD4+ and CD8+ T-lymphocytes between both groups and with healthy controls (HC) using the Mann-Whitney U test. Findings: Fifteen patients were included as described above. Median PFS was 2 months, OS 10 months (immature). Baseline characteristics of both groups were comparable. T-cell function was assessed in 14 patients and HC at baseline. Data are shown as median (P-value) for R vs NR below. R had a significant higher percentage activated CD8+ T-cells upon SEB stimulation than NR: CD8+CD69+ 15.8 vs 3.5 (0.008) and IL-2+CD8+CD69+ 8.8 vs 2.9 (0.02). There was a trend in TNF-α+CD8+CD69+ 19.8 vs 8.0 (0.11) and IFN-ɣ+CD8+CD69+ 21.4 vs 5.5 (0.22). These differences were all significant for R compared to HC (p<0.03), but not for NR (P>0.66). Interpretation: With a mild antigen independent stimulus (SEB), the CD8+ T-cells of responders were significantly more activated at baseline compared to non-responders and healthy controls. This suggests an elevated pre-treatment T-cell activation status in patients that responded to the treatment and the possibility to use this as a potential biomarker to predict the response to CPI treatment. Disclosure: This study was sponsored by a research grant from AstraZeneca. Citation Format: Hanneke Kievit, M. Benthe Muntinghe-Wagenaar, Lucie B.M. Hijmering-Kappelle, Birgitta I. Hiddinga, J Fred Ubbels, Robin Wijsman, Bart-Jan Kroesen, Marcel J. van der Leij, A. Rutgers, Harry J. Groen, Huib A. Kerstjens, Anthonie J. van de Wekken, T Jeroen Hiltermann. Baseline T-cell function predicts response to SBRT and immunotherapy in NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1984.

Attenuation of GARP expression on regulatory T cells by protein transport inhibitors

An integrated understanding of the functional capacities of cells in the context of their physical parameters and molecular markers is increasingly demanded in immunologic studies. Regulatory T cells (Tregs) are a subpopulation of T cells involved in immune response modulation and mediating tolerance to self-antigen with their absence leading to a loss of tolerance. Glycoprotein repetitions A predominant (GARP) is a key marker for activated Tregs, but its detection may also be useful in determining the functional capacities of the cell. This study aims to deduce the optimal stimulation period and the impact of protein transport inhibitors (PTIs), commonly used in the detection of intracellular cytokines, on GARP detection. Through flow cytometric analysis we analyzed different cell culture conditions for optimal GARP expression on activated Tregs. Healthy donor PBMCs were stimulated with either Staphylococcal Enterotoxin B (SEB) or PMA/Ionomycin (PMA/Iono), in the presence and absence of PTIs monensin and/or brefeldin A (BFA) and GARP expression was assessed on CD4+ CD25+ FOXP3+ Tregs. The optimal stimulation period for the detection of GARP was highest at 24-h. Furthermore, we determined that GARP expression on Tregs is significantly reduced when cells are treated with the PTIs monensin and/or BFA following PMA/Iono stimulation. This effect was not seen following SEB stimulation. Therefore, due to the effects of PTIs, alternative methods should be considered when performing simultaneous analysis for cytokine expression and GARP expression on Tregs.

Differences Between Pediatric and Adult T Cell Responses to In Vitro Staphylococcal Enterotoxin B Stimulation.

Toxic shock syndrome (TSS) is capable of inducing life-threatening fever, rash, and systemic organ failure, though the specific mechanisms behind these symptoms remain poorly understood. Staphylococcal enterotoxin B (SEB) and other superantigens have shown to be important factors in TSS, capable of promoting cross-linking between T cell receptors and major histocompatibility complexes which results in overwhelming T cell activation, proliferation, and cytokine production. The resulting proinflammatory cytokine cascade, often referred to as the “cytokine storm,” seems to be critical to the development of disease. Interestingly, clinical studies have shown that children exhibit less severe TSS-associated morbidity than adults, though the mechanism behind this phenomenon has not been addressed. Indeed, despite the fact that most novel antigen exposure occurs early in life, be it from environmentally acquired pathogens or routine vaccination, normal pediatric T cell immune functions remain critically underexplored. This is largely due to difficulty in obtaining enough samples to explore more than a narrow sliver of the cell-mediated immune compartment. To address this limitation, we optimized a T effector (Teff)/circulating T follicular helper (cTFH) cell mass cytometry panel which allowed us to analyze a wide array of T cell populations and effector functions following in vitro SEB stimulation. We show that T cell activation—as measured by CD69 expression—following SEB stimulation is lower in pediatric participants, increasing throughout childhood, and reaching adult levels by around 15 years old. Further, while individual CD4+ effector memory T cell (TEM) effector molecules show limited age-associated differences following SEB stimulation, multifunctional CD4+ TEM are shown to positively correlate with increasing age through adolescence. Individual CD8+ TEM effectors and multifunctional phenotypes also show very strong age-associated increases following SEB stimulation. SEB stimulation has little impact on cTFH activation or functional cellular markers, regardless of age. These results, coupled with the fact that a robust proinflammatory cytokine response seems critical to developing severe TSS, suggest a possible connection between the significantly reduced T cell activation and multifunctional populations following in vitro SEB stimulation in our pediatric participants and clinical observations relating to reduced TSS mortality in children.

Abstract 1639: Combination of PD1 blockade and T cell costimulation by bispecific antibodies promotes human T cell activation and proliferation

Abstract Tumor infiltrating lymphocytes (TILs) express multiple checkpoint receptors, in contrast to lymphocytes found in the periphery (Matsuzaki et al PNAS 2010, Fourcade et al Cancer Res 2012, Gros et al JCI 2014). Checkpoint blockade has demonstrated increased clinical response rates relative to other treatment options; however, many patients fail to achieve a response to checkpoint blockade. We sought to identify an additional therapeutic modality to stack with checkpoint blockade that could increase patient response rate. We hypothesized that engagement of T cell costimulatory receptors in combination with checkpoint blockade could further increase T cell activation and proliferation. The combination of checkpoint blockade with costimulation could be accomplished using a bispecific antibody format, with the potential benefits of reduced cost and more selective targeting of TILs to improve safety. Antibodies binding to immune checkpoint PD1 and a T cell costimulatory receptor were assembled in a bispecific antibody platform with substitutions in the Fc domain to suppress effector function. PD1 x costimulation (PD1 x costim) bispecific antibodies were evaluated in vitro by measuring antibody binding and de-repression of Staphylococcal enterotoxin B (SEB) stimulated PBMCs. IL2 and IFNγ production was measured by immunoassay. In vivo activity was evaluated using a mouse model in which human PBMCs are engrafted into NSG mice (huPBMC-NSG) and the extent of T cell engraftment is monitored by flow cytometry. We produced PD1 x costim bispecific antibodies that bound PD1 and a T cell costimulatory receptor monovalently. The bispecifics bound to SEB-stimulated T cells more tightly than monovalent controls, indicating that a single bispecific molecule was capable of avid simultaneous co-engagement of both PD1 and a costimulatory receptor. The bispecifics enhanced IL2 and IFNγ production in an in vitro SEB stimulation assay relative to control (p < 0.001, n = 19 donors). IL2 and IFNγ production was superior to anti-PD1 or anti-costim antibodies alone (p < 0.001), indicating productive and synergistic combination of checkpoint blockade plus costimulation. Treatment of huPBMC-NSG mice with checkpoint bispecifics promoted enhanced T cell engraftment relative to control. Engraftment levels promoted by bispecifics were superior to those found for anti-PD1 treatment alone. For example, in one run of the model, while anti-PD1 treatment alone promoted a 2-fold increase in human CD45+ cells, a PD1 x costim bispecific antibody induced a 16-fold increase. Combination of checkpoint blockade and costimulation with bispecific antibodies is feasible and promotes strong T cell activation in vitro and in vivo. Compelling activity suggests clinical development is warranted for the treatment of human malignancies. Citation Format: Gregory L. Moore, Michael Hedvat, Matthew J. Bernett, Rajat Varma, Suzanne Schubbert, Christine Bonzon, Kendra N. Avery, Rumana Rashid, Alex Nisthal, Liz Bogaert, Irene W. Leung, Seung Y. Chu, Umesh S. Muchhal, John R. Desjarlais. Combination of PD1 blockade and T cell costimulation by bispecific antibodies promotes human T cell activation and proliferation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1639. doi:10.1158/1538-7445.AM2017-1639

Interleukin-18 correlates with interleukin-4 but not interferon-γ production in lymphocyte cultures from atopic dermatitis patients after staphylococcal enterotoxin B stimulation.

Staphylococcus aureus (S. aureus) triggers exacerbation of atopic dermatitis (AD) and causes chronic inflammation through the action of various proteins such as staphylococcal enterotoxin B (SEB). SEB has a role in activating interleukin (IL)-18, an important regulator of interferon (IFN)-γ and IL-4, in regards to a therapeutic strategy. To determine the correlation of IL-18 level with the IL-4 and IFN-γ level in lymphocyte cultures from AD patients following SEB stimulation. Twenty patients with AD based on the Hanifin and Rajka criteria and 20 healthy subjects as a control group were selected. A 5 ml blood sample from each subject was taken for lymphocyte culture. The culture was stimulated with SEB for two days and the outcomes were assessed by enzyme-linked immunosorbent assays (ELISA) to evaluate the levels of IL-18, IL-4, and IFN-γ. In the AD group, the levels of IL-18, IL-4, and IFN-γ in lymphocyte cultures with SEB were significantly increased compared with non-SEB exposed cells (each p<0.001); similar results were found in the control group. The level of IL-18 was significantly elevated in lymphocyte cultures with SEB stimulation in AD vs. control (p<0.05) and without SEB in AD vs. control (p<0.05). Furthermore, IL-18 levels were significantly correlated with IL-4 levels and score atopic dermatitis (SCORAD) values in AD patients with SEB (r=0.41, p<0.05; and r=0.70, p<0.05, respectively); on the in contrary, there was no correlation between IL-18 and IFN-γ levels (p=0.469). Our results suggest that IL-18 is correlated with increased of IL-4 levels in SEB-stimulated AD lymphocyte cultures.

Increased frequency of circulating Tc22/Th22 cells and polyfunctional CD38(-) T cells in HIV-exposed uninfected subjects.

Some individuals are resistant to HIV-1 infection despite repeated exposure to the virus, suggesting the presence of a complex antiviral response. Innate factors like IL-22 exert gut mucosal protection and polyfunctional T cells have been associated with low progression in HIV infection; therefore, we evaluated the frequencies of CD4+ and CD8+ T cell-secreting cytokines, including Tc22/Th22 cells and polyfunctional T cells in HIV-1-exposed uninfected individuals (EUs), their HIV-1-infected partners and healthy controls. EUs exhibited an increased frequency of p15 Gag CD4+ IL-22+ secreting T cells, whereas HIV-infected partners demonstrated a high frequency of CD4+ IL-17+ T cells in response to p24. Similar responses of Th22 and Tc22 cells to Gag peptides and Staphylococcal enterotoxin B (SEB) stimulation were detected in the serodiscordant couples. However, polyfunctionality in HIV subjects was associated with an HIV Gag response of CD38+ T cells, whereas polyfunctionality for EUs was induced upon SEB stimulation by CD38- T cells. EUs demonstrated the presence of Tc22/Th22 cells and polyfunctional CD38- T cells with a low activation profile. These data suggest that SEB-induced polyfunctional CD4+ and CD8+ T cells together with Tc22/Th22 cells in EU individuals can provide an immunological advantage in the response to pathogens such as HIV-1.

Staphylococcal Exotoxins Induce Interleukin 22 in Human Th22 Cells

Background: We have shown previously that T cells from atopic dermatitis (AD) patients produce more IL-22 upon staphylococcal exotoxin stimulation compared to psoriasis patients and healthy controls. The role of staphylococcal exotoxins on polarized memory T helper (Th)22 cells which are enriched in inflamed AD skin remains elusive. Our aim was to investigate IL-22 production in response to staphylococcal enterotoxin B (SEB) and α-toxin stimulation in human memory T cells and polarized Th22 cells. Methods: IL-22 induction was investigated in human peripheral blood-derived CD4+CD45RO+CD45RA- T cells and polarized Th22 cells after SEB and sublytic α-toxin stimulation in a time-dependent manner at the mRNA and protein (ELISA) levels. Results: Th22 cells secreted more IL-22 compared to freshly isolated peripheral blood-derived memory T cells. SEB and α-toxin induced IL-22 in memory T cells as well as in Th22 cells. More IL-22 was induced by SEB and α-toxin in freshly isolated peripheral blood memory T cells compared to Th22 cells derived from memory T cells in long-term cell culture without polarization and Th22 cells under Th22-promoting conditions with IL-6 and TNF-α. No differences in IL-22 induction by staphylococcal exotoxins were observed between cells from AD compared to psoriasis patients and healthy controls. Conclusions: Increased IL-22 secretion can promptly be induced by staphylococcal exotoxins in skin infiltrating CD4+CD45RO+CD45RA- memory T cells and can potentially amplify chronic skin inflammation in AD in the context of bacterial colonization and infection. This should be investigated further in detail in lesional skin of AD and psoriasis patients.

In-utero exposure to maternal HIV infection alters T-cell immune responses to vaccination in HIV-uninfected infants.

In sub-Saharan Africa, HIV-exposed uninfected (HEU) infants have higher morbidity and mortality than HIV-unexposed infants. To evaluate whether immune dysfunction contributes to this vulnerability of HEU infants, we conducted a longitudinal, observational cohort study to assess T-cell immune responses to infant vaccines (Mycobacterium bovis BCG and acellular pertussis) and staphylococcal enterotoxin B (SEB). In total, 46 HEU and 46 HIV-unexposed infants were recruited from Khayelitsha, Cape Town. Vaccine-specific T-cell proliferation (Ki67 expression) and intracellular expression of four cytokines [interferon-γ, interleukin (IL)-2, IL-13 and IL-17] were measured after whole blood stimulation with antigens at 6 and 14 weeks of age. HEU infants demonstrated elevated BCG-specific CD4 and CD8 T-cell proliferative responses at 14 weeks (P = 0.041 and 0.002, respectively). These responses were significantly increased even after adjusting for birth weight, feeding mode and gestational age. Similar to BCG, increased CD4 and CD8 T-cell proliferation was evident in response to SEB stimulation (P = 0.004 and 0.002, respectively), although pertussis-specific T cells proliferated comparably between the two groups. Within HEU infants, maternal CD4 cell count and length of antenatal antiretroviral exposure had no effect on T-cell proliferation to BCG or SEB. HIV exposure significantly diminished measurable cytokine polyfunctionality in response to BCG, Bordetella pertussis and SEB stimulation. These data show for the first time, when adjusting for confounders, that exposure to HIV in utero is associated with significant alterations to CD4 and CD8T-cell immune responses in infants to vaccines and nonspecific antigens.

Role of Staphylococcal Enterotoxin B on the Differentiation of Regulatory T Cells in Nasal Polyposis

The pathogenesis of nasal polyposis has not been fully understood. Recent studies indicate that there is a subset of CD4(+)CD25(high)FoxP3(+)T cells (regulatory T cells [Tregs]) that express retinoic acid receptor related orphan receptor C (RORC) or IL-17, and these cells might be new proinflammatory cells because of the expression of IL-17 with loss of their suppressive function. The goals of this study were to localize Th17-like Tregs (Th17-like Tregs or RORC(+)Tregs) in nasal polyps and to investigate the role of staphylococcal enterotoxin B (SEB) on the differentiation of Tregs to RORC(+)Tregs in vitro. A total of 60 patients were enrolled in this study. Of the 60 patients, 40 had chronic rhinosinusitis with nasal polyps (CRSwNPs), and 20 subjects who were undergoing septoplasty were enrolled as control subjects. The nasal polyps of CRSwNP patients were subclassified as either eosinophilic polyp (EP) and noneosinophilic polyp (NEP) according to the result of hematoxylin and eosin stain. Tissues and whole blood were collected from all subjects. Double immunofluorescent staining and reverse-transcription polymerase chain reaction for RORC and FOXP3 were conducted on the tissues. RORC expressions of Tregs were measured in the tissue using flow cytometry. The proportions of RORC(+)Tregs subsets and cytokines profiles from the supernatant were measured using flow cytometry after stimulation with SEB. The cells that express both RORC and FOXP3 and RORC(+)Tregs were significantly higher in the nasal polyps, especially in EPs compared with NEPs, and control mucosa. RORC(+)Tregs in peripheral blood mononuclear cells significantly increased in patients with EPs 24 hours after SEB stimulation in vitro. The results indicate that SEB may be involved in the differentiation of Tregs to RORC(+)Tregs, and these cells may be involved in the pathogenesis of eosinophilic nasal polyposis.

Staphylococcal enterotoxin B influences the DNA methylation pattern in nasal polyp tissue: a preliminary study

Staphylococcal enterotoxins may influence the pro-inflammatory pattern of chronic sinus diseases via epigenetic events. This work intended to investigate the potential of staphylococcal enterotoxin B (SEB) to induce changes in the DNA methylation pattern. Nasal polyp tissue explants were cultured in the presence and absence of SEB; genomic DNA was then isolated and used for whole genome methylation analysis. Results showed that SEB stimulation altered the methylation pattern of gene regions when compared with non stimulated tissue. Data enrichment analysis highlighted two genes: the IKBKB and STAT-5B, both playing a crucial role in T- cell maturation/activation and immune response.

Assessment of the Performance of Three Multiplex Array Panels for the Detection of Circulating Cytokines and Chemokines in Naive, LPS, and SEB-treated Cynomolgus Macaques

To assess relative sensitivity for detection of cytokines and chemokines in cynomolgus serum samples, we tested three commercially available multiplex array kits using the Luminex® platform with sera from animals exposed by intravenous injection to 150 μg/kg staphylococcal enterotoxin B (SEB) or 20 μg/kg lipopolysaccharide (LPS). Each of these kits detected similar patterns of changes in circulating cytokines/chemokines in response to SEB or LPS stimulation, especially the induction of high amounts of interleukin (IL)-2 and interferon-gamma (IFN-γ) in response to SEB but not LPS. However, there were clear differences in sensitivity for particular analytes, especially for IL-10. Additional experiments that focused on one multiplex array kit demonstrated very low or undetectable levels of cytokines in naive cynomolgus macaques, except for highly variable background levels of IL-8, monocyte chemoattractant protein-1, and macrophage inflammatory protein-1β. Therefore, multiplex array analysis of circulating cytokine/chemokine patterns was capable of detection of systemic activation of diverse immune cell subsets.

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.

Unexpected heterogeneity of multifunctional T cells in response to superantigen stimulation in humans

Toxic shock syndrome (TSS) is a potentially life threatening condition characterized by fever, rash, shock, and multi-organ failure. Staphylococcal enterotoxin B (SEB) is a well characterized superantigen that has been shown to play an important role in TSS. Although the precise mechanisms by which SEB and other superantigens cause TSS are unknown, induction of a pro-inflammatory cytokine cascade appears central to this phenomenon. We show that CD4+ and CD8+ Teffector/memory (T(EM)) and other subsets produce IL-17A following SEB stimulation. We also show that IL-17A is co-produced with other pro-inflammatory cytokines (i.e., IL-2, IFN-γ and TNF-α). These responses are significantly different than those elicited by mitogenic stimulation. Multifunctional IL-17A producing cells possess markers typical of the T(H)17/T(C)17 and T(H)1 subsets, including CCR6, IL-22, and transcription factors retinoic acid receptor-related orphan nuclear receptor (ROR)-γt and T-bet. These results suggest a possible role for IL-17A-producing multifunctional T cells in the pathogenesis of TSS.

COX/PGE2 axis critically regulates effects of LPS on eosinophilia‐associated cytokine production in nasal polyps

Lipopolysaccharide (LPS) has shown heterogeneous effects on eosinophilic inflammation in airways. However, little is known about how LPS regulates pathogenesis of chronic rhinosinusitis with nasal polyps, a major form of eosinophilic inflammation in the upper airway. We sought to investigate the effect of LPS on cytokine production by dispersed nasal polyp cells (DNPCs). Either diclofenac-treated or untreated DNPCs were cultured with or without staphylococcal enterotoxin B (SEB) in the presence or absence of LPS, after which the levels of IL-5, IL-13, IL-17A and IFN-γ within the supernatant were measured. The effects of PGE(2) on LPS-induced responses by diclofenac-treated DNPCs were also examined. LPS-induced PGE(2) production and mRNA expression of COX-1, COX-2 and microsomal PGE(2) synthase-1 (m-PGES-1) were measured. Staphylococcal enterotoxin B induced IL-5, IL-13, IL-17A and IFN-γ production by DNPCs. Pre-treatment with LPS prior to SEB stimulation inhibited production of these cytokines. After stimulation with LPS, PGE(2) production and expression of COX-2 and m-PGES-1 mRNA by DNPCs increased significantly. In the presence of diclofenac, the suppressive effects of LPS were eliminated. LPS pre-treatment enhanced SEB-induced IL-5, IL-13 and IL-17A production in diclofenac-treated DNPCs, while addition of PGE(2) inhibited IL-5, IL-13 and IFN-γ production. LPS alone induced IL-5, IL-13 and IFN- γ production by diclofenac-treated DNPCs, while the addition of EP2 and EP4 receptor-selective agonists, as well as PGE(2) itself, inhibited IL-5 and IL-13 production. These results suggest that the regulatory effects of LPS on eosinophilic airway inflammation are controlled via the COX-2/PGE(2) axis. For clinical implications, indiscreet use of non-steroidal anti-inflammatory drugs should be avoided in patients with chronic rhinosinusitis with nasal polyps.

Skin‐homing CD4+ Foxp3+ T cells exert Th2‐like function after staphylococcal superantigen stimulation in atopic dermatitis patients

The effect of staphylococcal superantigens (SsAgs) on cutaneous lymphocyte-associated antigen (CLA)(+) CD4(+) Foxp3(+) T cells of atopic dermatitis (AD) patients is unknown. To compare the effects of SsAgs on the ratio, function, and apoptosis of CCR6(+) subtype and CCR6(-) subtype of CLA(+) CD4(+) Foxp3(+) T cells among AD patients, asthma/allergic rhinitis (AR) patients without AD, and healthy subjects. Using immunofluorescence staining followed by flow cytometric analysis, we analysed peripheral blood mononuclear cells cultured with or without staphylococcal enterotoxin B (SEB) stimulation in 20 AD patients, 20 asthma/AR patients without AD, and 20 healthy subjects. SEB decreased CCR6(+) /CCR6(-) ratio in CLA(+) CD4(+) Foxp3(+) T cells from AD patients and increased CCR6(+) /CCR6(-) ratio in those from healthy subjects. SEB induced the production of type 2 T helper cell (Th2) cytokine interleukin (IL)-5 in CCR6(-) subtype and anti-inflammatory cytokine IL-10 in CCR6(+) subtype of CLA(+) CD4(+) Foxp3(+) T cells. CLA(+) CD4(+) Foxp3(+) T cells from AD patients produced more IL-5 and less IL-10 after SEB stimulation than those from healthy subjects. CCR6(-) subtype of CLA(+) CD4(+) Foxp3(+) T cells from AD patients and CCR6(+) subtype of those cells from healthy subjects were more resistant to SEB-induced caspase-3 activation than the other subtype and those from other subjects. Despite a phenotype of regulatory T cells, skin-homing CD4(+) Foxp3(+) T cells of AD patients exert effector Th2-like function after SsAgs stimulation, which may aggravate allergic skin inflammation.

Staphylococcal exotoxins are strong inducers of IL-22: A potential role in atopic dermatitis

Patients with atopic dermatitis (AD) and psoriasis are frequently colonized with Staphylococcus aureus that produces staphylococcal enterotoxin B (SEB) and α-toxin. In patients with AD, S aureus colonization is positively correlated with the severity of their eczema. Moreover, IL-22-producing cells have been shown to accumulate in AD skin and to correlate with disease severity. To assess IL-22 production in response to SEB and sublytic α-toxin stimulation in patients with AD and psoriasis compared with healthy controls. IL-22 induction was investigated in PBMCs, T cells, and autologous cocultures of keratinocytes and T cells on SEB and α-toxin stimulation in a time-dependent and dose-dependent manner at the mRNA and protein (ELISA and flow cytometry) level. Anti-IL-1 receptor or anti-IL-6 antibodies were used in blocking experiments. Staphylococcal enterotoxin B and sublytic α-toxin concentrations induced IL-22 production in PBMCs and isolated CD4(+) T cells. IL-22 secretion was enhanced by α-toxin stimulation in autologous cocultures of keratinocytes and T cells. In T cells and PBMCs from patients with AD, IL-22 secretion was significantly enhanced on α-toxin stimulation compared with patients with psoriasis and healthy controls. Increased IL-22 secretion induced by staphylococcal exotoxins in the skin partially explains how skin colonization and infection with S aureus can contribute to chronic skin inflammation in AD.

Cytotoxic activity of V beta 8+ T cells in Crohn's disease: the role of bacterial superantigens.

In Crohn's disease, disease-related stimuli could alter the T cell receptor (TCR) repertoire. To examine the possibility that changes in function may occur in T cell subsets without obvious changes in expression of TCR, we analysed the TCR repertoire of cytotoxic T lymphocytes in Crohn's disease peripheral blood. Furthermore, we examined the effect of bacterial superantigens, staphylococcal enterotoxin B (SEB) and E (SEE) on the cytotoxic function of T cell subsets bearing different TCR V genes using MoAbs specific for CD3 and TCR V gene products in a redirected cytotoxicity assay. There was no difference between patients and controls in the cytotoxicity measured in concanavalin A (Con A)-stimulated peripheral blood mononuclear cells (PBMC) with anti-CD3 or with six of seven anti-TCR V gene MoAbs. However, the cytotoxicity of V beta 8 T cells was decreased in Crohn's disease patients. This was not due to a decrease in total or CD8+ T cells expressing V beta 8. Furthermore, in normal subjects, PBMC stimulation with SEE and SEB selectively expanded and increased the cytotoxicity of V beta 8 and V beta 12 T cells, respectively. In Crohn's disease, although SEB stimulation increased the number and cytolytic function of the V beta 12 subset, SEE stimulation failed to increase cytolytic activity of V beta 8+ T cells in spite of the expansion of V beta 8+ T cells. These results suggest that the changes in cytotoxic function observed in V beta 8 T cells in Crohn's patients may reflect previous exposure to a V beta 8-selective superantigen.

In vitro and clinical immunomodulatory effects of a novel Pentaherbs concoction for atopic dermatitis

Our group recently reported a randomized and placebo-controlled clinical trial on the efficacy of a twice-daily concoction of five herbal ingredients (Pentaherbs formulation, PHF) in treating children with atopic dermatitis (AD). To investigate the immunomodulatory effects that may be induced by PHF treatment. We investigated the effects of PHF on cytotoxicity and proliferation of phytohaemagglutinin (PHA)- and staphylococcal enterotoxin B (SEB)-stimulated peripheral blood mononuclear cells (PBMC) isolated from buffy coat of blood donors. PHF-induced immunomodulation for five inflammatory mediators in cultured PBMC was measured by reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay. The effects of a 3-month, open-label study of PHF on circulating inflammatory mediators in children with AD were also assessed. PHF at up to 1 mg mL(-1) dose-dependently suppressed PBMC proliferation. The addition of PHF to cultured PBMC reduced supernatant concentrations of brain-derived neurotrophic factor (BDNF), interferon (IFN)-gamma and tumour necrosis factor (TNF)-alpha in response to PHA, and BDNF and thymus and activation-regulated chemokine (TARC) following SEB stimulation. PHF increased epithelial cell-derived neutrophil activating peptide-78 levels in culture supernatants. At the RNA level, PHF suppressed the transcription of BDNF, TARC, IFN-gamma and TNF-alpha. Twenty-eight children with AD were treated with PHF for 3 months, and their mean plasma concentrations of BDNF and TARC decreased significantly from 1798 pg mL(-1) and 824 pg mL(-1) at baseline to 1378 pg mL(-1) and 492 pg mL(-1) (P = 0.002 and 0.013, respectively) upon study completion. PHF possesses in vitro and in vivo immunomodulatory properties that may mediate the clinical efficacy observed in AD treatment.