model of SEB toxicity in mice [5,10,11]. BALB/c mice (n = 6) were injected intraperitoneally (i.p.) with varying amounts of EM- 163 (0.21 mg/mouse, 0.42 mg/mouse, and 0.86 mg/mouse) and 30 min later they were treated with SEB (0.5 mg/mouse, 1LD50), followed by LPS another 2 h later. All animals in the study not treated with EM-163 succumbed by 48 h. In contrast, treatment with 0.21 mg/mouse of EM-163 delayed death. Mice that were treated with EM-163 at concentrations of 0.42 mg/mouse or 0.86 mg/mouse were completely protected (p = 0.05) (Figure 8A ). When mice were exposed to a higher lethal dose of SEB (5 mg/ mouse, 10 LD50), all mice not treated with EM-163 succumbed by 30 h. In contrast, EM-163 pre-treatment with 0.86 mg/mouse delayed death and pre-treatment with 1.7 mg/mouse protected completely [Figure 8A]. Using the same experimental paradigm as for the results shown in Figure 8A, we determined the pro- inflammatory serum cytokine profile at the 4 h time point. The results shown in Figure 8B indicated that the cytokine response was abrogated in mice treated with EM-163, suggesting that cytokine responses are correlated with the survival of the mice. In addition to pre-treatment, our results also showed that mice treated with EM-163 (0.86 mg/mouse) 30 min after SEB exposure (0.5 mg/mouse, 1LD50) were completely protected. With a higher challenge dose of SEB (5 mg/mouse, 10LD50) EM-163 still delayed the death of mice (p = 0.012, log-rank pairwise comparisons tests) (Figure 8C). As for BALB/c mice challenged with SEB, C57BL/6 mice pretreated with EM-163 and challenged with a lethal dose of SEA likewise showed inhibition of cytokine responses and were protected (Figure S4). These results suggest that EM-163 effectively inhibited pro-inflammatory cytokine responses and protected mice from toxic shock-induced death from lethal SEB or SEA challenge. Taken together, the results indicate that EM-163 has substantial therapeutic potential against SEB intoxication. Discussion Our previous studies showed that MyD88-mediated pro-in- flammatory cytokine signaling is critical to SEB toxicity [11,12]. This indicated that MyD88 was a valid therapeutic target and subsequent preliminary work using a synthetic mimetic of the BB- loop of the Toll/IL-1 receptor (TIR) domain of MyD88, provided proof of concept for this strategy to treat SEB-induced toxic shock syndrome [19]. Here, we have shown that synthesis of a BB-loop mimetic with dual functional groups greatly enhanced the biological activity and target specificity of the mimetic, a large improvement over earlier work. The ‘‘dimeric’’ compound EM- 163 attenuated TNF-a, IFN-c, IL-1b, IL-2 and IL-6 production (IC50 15–400 mM) in human primary cells when applied either pre- or post-exposure to SEB. Our biochemical and cell-based reporter assay data suggest that EM-163, by targeting MyD88, inhibits pro-inflammatory cytokine signaling. Most importantly, administration of EM-163 abrogated pro-inflammatory cytokine production and completely protected mice from the toxic shock induced-death of a lethal SEB challenge. Recent reports suggest that MyD88-mediated pro-inflammatory cytokine signaling is not limited to the TLR/IL-1R, but rather is generally shared by other receptors such as MHC receptors [13]. The role for MHC class II molecules in TLR signaling was first suggested by studies that showed macrophages with no or low expression of MHC class II had a defective inflammatory response to LPS-stimulation [28]. It has also been reported that MHC class II molecules enhance toll-like receptor mediated innate immune responses [29]. In line with these observations, our recent results demonstrated that SEB, which binds to MHC class II receptors, activates MyD88-mediated pro-inflammatory signaling [12]. Inhibition of SEB Intoxication by a MyD88 Mimetic Thus, in addition to their classical function in antigen pre- sentation, MHC class II molecules are now shown to promote MyD88 signaling. As the MHC class II molecule lacks any known signaling motifs in its short cytoplasmic tail, it has been proposed that it might use associated signaling molecules at the membrane. These signaling molecules include cell receptors (such as CD20, CD79, CD23) and members of the immunoglobulin family (such as CD19), the tetraspan family (such as CD81 and CD82), and the TNF receptor family (such as CD40). All of these receptor families have been shown to associate with MHC class II molecules, and thus may likely be involved in initiating intracellular signaling [13,30]. Whether these signaling proteins are functionally linked, either directly or indirectly, to MHC class II molecules and adaptor protein MyD88 is a question that is currently under investigation in our laboratory. Nevertheless, results from our laboratory and others have demonstrated that TLR and MHC- mediated responses both engage the adaptor molecule MyD88 [14,31]. Likewise, SEB engagement of MHC class II molecules has been shown to up-regulate both TLR4 [32] and MyD88 (Kissner et al. 2011, unpublished observation) on human monocytes. Previous work from our laboratory showed that LPS potentiates SEB-induced lethal shock in mice [5,11,12]. LPS primarily interacts with CD14 receptors on macrophages, while SEB triggers MHC class II-positive cells and T-cells to release pro- inflammatory cytokines. Therefore, it is likely that MHC class II- SEB binding up regulates MyD88 and TLR4, and subsequent LPS stimulation may synergistically trigger interaction between the respective TIR domains of MyD88 and TLR/IL-1R1 receptors. This causes activation of several signaling cascade pathways that include nuclear factor NF-kB and transcription factor AP-1, as well as various stress-associated kinases, resulting in release of pathological levels of pro-inflammatory cytokines. Although the underlying mechanism by which MHC class II signals recruit MyD88 is currently under investigation, the adaptor protein is known to integrate signals through TIR domain-TIR domain interactions [16,33]. In this study, our results indicated that the compound EM-163 (designed to be structurally similar to the BB-loop in the TIR domain of MyD88) inhibited the MyD88- mediated signaling initiated by exposure to SEB. In addition, a preliminary result from our laboratory indicated that EM-163 was capable of inhibiting MyD88-mediated pro-inflammatory cytokine responses with exposure to Francisella tularensis LPS or irradiated Burkholderia mallei (unpublished observation). As signaling through MyD88 appears to be a common link in many of the host- directed inflammatory process, it is likely that EM-163 may have a potential for broader use against exposure to other bio-threat agents. The most potent microbial products implicated in the patho- genesis of septic shock are gram-positive-derived superantigenic exotoxins and gram-negative endotoxins. Both types of toxin induce comparable pro-inflammatory cytokines from human mononuclear cells in vitro, cause lethal shock in vivo, and have been identified in the bloodstream of critically ill patients [8,9]. The combination of endotoxins and superantigens has been shown to have particularly severe consequences in several animal models. For example, it has been reported that SEB potentiates LPS- induced hepatic dysfunction and cytokine responses in chronically catheterized rats [34]. Results from our laboratory also firmly established that LPS and SEB synergize to produce a dose dependent toxicity in mice that is several fold higher than the toxicity of SEB or LPS alone. Our results raise the possibility that recognition of SEB by MHC class II receptors may exacerbate the pro-inflammatory response of monocytes to gram-negative in- fection or endotoxin through activation of a common MyD88- PLoS ONE | www.plosone.org 9 July 2012 | Volume 7 | Issue 7 | e40773