Rajagopalan, Govindarajan

Abstract Bacterial superantigens (BSAg) are produced by Staphylococcus aureus and Streptococcus pyogenes. BSAg bind directly to the MHC class II molecules without undergoing any processing and subsequently activate T cells in an antigen non-specific manner by interacting with the TCR Vβ region. BSAg have been implicated primarily in acute diseases such as food poisoning, toxic shock syndrome, etc. Since carrier strains of S. aureus and S. pyogenes can elaborate superantigens, there is a distinct possibility of chronic exposure to low doses of BSAg and it is imperative to study its outcome. As BSAg bind more avidly to human (HLA) than to murine (H-2) MHC class II, we delivered low concentrations (total of 10 μg/mouse) of BSAg to HLA-DR3 [staphylococcal enterotoxin (SE) B] or HLA-DQ8 [streptococcal pyrogenic exotoxin (SPE) A] transgenic mice through subcutaneously implanted miniosmotic pumps for 7 days. Continuous exposure to low concentrations of BSAg resulted in a significant protracted expansion of CD4+ T cells expressing TCR Vβ8 (for SEB); Vβ8 and Vβ14 (for SPEA) with a concomitant decrease in percentage of CD8+ T cells expressing these TCR. T cells bearing TCR Vβ6 showed an inverse correlation (slight decrease in CD4+ and increase in CD8+ compartments with SEB and SPEA). Histological examinations of major organs showed intense perivascular infiltration in lungs, kidneys and liver along with inflammatory infiltrates in the cardiac valvular endothelium in mice bearing pumps delivering BSAg but not PBS. In conclusion, we have shown the immunological outcome of chronic exposure to low levels of BSAg using the HLA class II transgenic mouse model.

Abstract Staphylococcal Enterotoxin B (SEB) is a superantigen made by S. aureus. SEB can cause food poisoning and shock and its stability and ease of preparation make it a potential agent of bioterrorism. SEB-mediated activation induces T cell proliferation and secretion of cytokines. Antibodies that block the interaction between SEB and MHC or SEB and TCR, can neutralize the activating effects of SEB and provide protection against its debilitating effects. Human IVIG, a therapy for SEB intoxication is of low potency and sometimes, short supply. To surmount we have derived of mouse monoclonal antibodies that block the binding of SEB to MHC at separate and distinct sites. Combinations of these antibodies synergistically neutralize SEB mediated T cell activation. Genes encoding the VL and VH genes of synergistically neutralizing pairs of cell lines were cloned and grafted onto genes encoding either the constant region of human IgH or Igκ and transfected into SP2/0 cells. Chimeric human-mouse antibodies expressed by the transfectants were purified. Tests of these chimeric antibodies demonstrated that they were neutralizing and in combination with each other, still capable of synergistic neutralization of SEB. Supported by NIH grant # AI057652

Abstract Menstrual or non-menstrual toxic shock syndrome (TSS), caused by the superantigen exotoxins of Staphylococcus aureus or Streptococcus pyogenes, is characterized by a robust activation of the immune system, systemic cytokine/chemokine storm, multiple organ failure and possibly death, if not treated promptly. We tested whether increasing the numbers of Treg in vivo by administration of IL-2-anti-IL-2 complexes (IL2C) could be beneficial in TSS. Administration of IL2C [prepared by incubating 1µg of IL-2 (Peprotech) and 5µg anti-IL-2 (eBioscience, clone-JES6-1A12) in a total 200 µl at 37oC for 30 min] on days 0, 1 and 2, caused a 3 to 4-fold increase in CD4+CD25+FoxP3+ Treg numbers in the spleens of HLA-DR3 transgenic mice by day 5. However, upon challenge with staphylococcal enterotoxin B (SEB, 50µg), compared to untreated HLA-DR3 transgenic mice, IL2C-treated mice produced significantly higher levels of IFN-γ (1714±1365 versus 6642±2253 pg/ml), MIP-1α (782±23 versus 2305±376 pg/ml,), MIP-1β, RANTES and TNF-α. SEB-induced mortality was also significantly increased in the IL2C treated group. While only 1/5 mice died in untreated group, 6/11 mice died in the IL2C treated group. In conclusion, even though administration of IL2C significantly increased the Treg numbers, such a treatment also increased their susceptibility to TSS. Hence, the benefits of T reg-promoting IL-2-anti-IL-2 complexes in TSS or related conditions need more stringent evaluation.

Abstract Bacterial superantigen-induced toxic shock syndrome (TSS) and sepsis are characterized by a profound systemic inflammatory response. Given the important role of NF¿B pathway in inflammation, we investigated whether a clinically approved proteasome inhibitor, Bortezomib, capable of inhibiting NF¿B pathway, be of therapeutic use in such conditions using HLA class II transgenic mice. Bortezomib significantly blocked staphylococcal enterotoxin B (SEB)-induced T cell activation and cytokine production in vitro. In SEB-induced TSS, in vivo administration of Bortezomib significantly reduced serum levels of several pro-inflammatory cytokines and chemokines. Nevertheless, 100% of SEB challenged mice treated with Bortezomib succumbed to TSS, and Bortezomib sensitized mice to TSS at concentrations of SEB which are otherwise non-lethal (i.e., as low as 2 μg/mouse), even when administered 24 hours after exposure to SEB. Sensitization to TSS by Bortezomib was not due to the pro-apoptotic effects of Bortezomib. Serum biochemical parameters and histopathological findings suggested acute liver failure as the possible cause of mortality. In sepsis model, whereas LPS alone (10 μg/mouse) was seldom lethal, it was uniformly lethal when administered along with Bortezomib. In conclusion, Bortezomib sensitizes mice to certain acute systemic inflammatory diseases. Supported by R01AI068741 and AI 72211

To understand the role of regulatory T cells in staphylococcal enterotoxin B‐induced toxic shock syndrome (TSS), we performed gene expression profiling in HLA‐DR3 transgenic mice using microarrays. We observed that at 3 hours after systemic exposure to SEB (10 μg), the splenic expression of FoxP3, a hall mark of regulatory T cells, was increased by 5 folds (p<0.005). In addition, other genes that regulate FoxP3 + Tregs were also significantly modulated by SEB. These include IL‐2R (27‐fold), IL‐2 (234‐fold), IL‐6 (689‐fold), IL‐21 (21‐fold), TGF‐B1 (0.7‐fold), IL‐17 (5.5‐fold) and IL‐22 (98‐fold). To further characterize the homeostasis of Tregs cells during superantigen‐induced TSS in vivo, HLA‐DR3 transgenic mice were first challenged with SEB and were administered BrdU in drinking water for 3 days. Mice were killed 3 days later and splenocytes analyzed by flow cytometry. We observed that in addition to FoxP3‐TCR Vβ8 + T cells, FoxP3 + TCR Vβ8 + T cells also significantly expanded in vivo as evident from their BrdU staining patterns. This indicated that SEB is able to activate Tregs as well. Depletion of Tregs using anti‐CD25 antibodies (clone PC‐61, 500 ug/mouse) 2 days prior to SEB challenge did not significantly alter serum cytokine levels as compared to control mice treated with rat IgG and SEB. Thus, T regulatory cells have a minimal role in SEB‐induced TSS.

AbstractStaphylococcal enterotoxin B (SEB), which is produced by the major human pathogen, Staphylococcus aureus, represents a powerful superantigenic toxin and is considered a bioweapon. However, the contribution of SEB to S. aureus pathogenesis has never been directly demonstrated with genetically defined mutants in clinically relevant strains. Many isolates of the predominant Asian community-associated methicillin-resistant S. aureus lineage sequence type (ST) 59 harbor seb, implying a significant role of SEB in the observed hypervirulence of this lineage. We created an isogenic seb mutant in a representative ST59 isolate and assessed its virulence potential in mouse infection models. We detected a significant contribution of seb to systemic ST59 infection that was associated with a cytokine storm. Our results directly demonstrate that seb contributes to S. aureus pathogenesis, suggesting the value of including SEB as a target in multipronged antistaphylococcal drug development strategies. Furthermore, they indicate that seb contributes to fatal exacerbation of community-associated methicillin-resistant S. aureus infection.

Abstract Mature peripheral double negative T (DNT) cells expressing αβ TCR but lacking CD4/CD8 coreceptors play protective as well as pathogenic roles. To better understand their development and functioning in vivo, we concomitantly inactivated CD4 and CD8 genes in mice with intact MHC class I and class II molecules with the hypothesis that this would enable the development of DNT cells. We also envisaged that these DNT cells could be activated by bacterial superantigens in vivo as activation of T cells by superantigens does not require CD4 and CD8 coreceptors. Because HLA class II molecules present superantigens more efficiently than murine MHC class II molecules, CD4 CD8 double knockout (DKO) mice transgenically expressing HLA-DR3 or HLA-DQ8 molecules were generated. Although thymic cellularity was comparable between wild type (WT) and DKO mice, CD3+ αβ TCR+ thymocytes were significantly reduced in DKO mice, implying defects in thymic-positive selection. Splenic CD3+ αβ TCR+ cells and Foxp3+ T regulatory cells were present in DKO mice but significantly reduced. However, the in vivo inflammatory responses and immunopathology elicited by acute challenge with the staphylococcal superantigen enterotoxin B were comparable between WT and DKO mice. Choric exposure to staphylococcal enterotoxin B precipitated a lupus-like inflammatory disease with characteristic lympho-monocytic infiltration in lungs, livers, and kidneys, along with production of anti-nuclear Abs in DKO mice as in WT mice. Overall, our results suggest that DNT cells can develop efficiently in vivo and chronic exposure to bacterial superantigens may precipitate a lupus-like autoimmune disease through activation of DNT cells.

AbstractToxic shock syndrome toxin‐1 (TSST‐1) is a potent superantigen produced by Staphylococcus aureus. In addition to menstrual and nonmenstrual toxic shock syndromes, TSST‐1 is also implicated in the immunopathogenesis of pneumonia, infective endocarditis, neonatal exanthematous disease, and atopic dermatitis among others. Superantigens first bind to major histocompatibility complex (MHC) class II molecules and then activate a large proportion of T cells by cross‐linking their T cell receptor. As binding to MHC class II molecules is a critical step in the robust activation of the immune system by TSST‐1 and other superantigens, polymorphic variations between different HLA‐DR alleles could potentially influence the magnitude of immune activation and immunopathology caused by TSST‐1. As TSST‐1 is highly toxic to humans and given that multiple variations of alleles of HLA‐DR and HLA‐DQ are expressed in each individual, it is difficult to determine how HLA‐DR polymorphisms quantitatively and qualitatively impact immune activation caused by TSST‐1 in humans. However, such investigations can be conducted on transgenic mice lacking all endogenous MHC class II molecules and expressing specific HLA class II alleles. Therefore, transgenic mice expressing different HLA‐DRB1 alleles (HLA‐DRB1*15:01, HLA‐DRB1*15:02, HLA‐DRB1*03:01, HLA‐DRB1*04:01), and sharing HLA‐A1*01:01 chain, were systemically challenged with purified TSST‐1 and multiple immune parameters were assessed. Among the HLA‐DR alleles, mice expressing HLA‐DRB1*15:01 allele elicited a significantly higher serum cytokine/chemokine response; greater splenic T cell expansion and most severe organ pathology. Our study highlights the potential utility of human leukocyte antigen (HLA) transgenic mice in understanding the impact of HLA polymorphisms on the outcomes of diseases caused by TSST‐1 and other superantigens.