Inhibition of SEB Intoxication by a MyD88 Mimetic Figure 5. EM-163 targets newly expressed MyD88 and stabilizes the MyD88 complex. HEK 293T cells were co-transfected with plasmids MyD88- Flag or pCMV-HA-MyD88. Seven hours after transfection, cells were incubated for 13 h with or without 2163 (500 mM to 5 mM). (A), Cell extracts were immunoprecipitated (IP) with anti-Flag antibody, and immune-precipitated proteins were analyzed in Western blot with anti-HA. (B) Densitometry analysis of the results shown in (A). The results are representative of three independent experiments. doi:10.1371/journal.pone.0040773.g005 HEK 293 cells expressing HLA-DR and MyD88, and its inhibition by EM-163. These results clearly demonstrate that MHC class II-linked activation of MyD88 signaling [12] was also inhibited by EM-163. EM-163 Inhibits SEB-induced TNF-a, IFN-c, IL-1b, IL-2 and IL-6 Production in Human Primary Cells The results above indicated that EM-163 inhibits downstream signaling by targeting MyD88 in a transfected HEK 293 cell line, so next we asked if the change in signaling would likewise lead to a decrease in cytokine production in cultured human primary cells. To determine therapeutic potential, it was also important to examine the efficacy of EM-163 when administered after SEB exposure. To assess the inhibitory effects of EM-163 on SEB- induced cytokine production in primary culture, MNCs were treated with EM-163 for 30 min before (pre-exposure) or 30 min after (post-exposure) being exposed to SEB. Culture supernatants were collected after 16 h and analyzed for cytokines. As shown in Figure 7, pre- or post-exposure treatment with EM-163 inhibited SEB-induced TNF-a, IFN-c, IL-1b, IL-2 and IL-6 production (IC50 15 mM to 400 mM), although EM-163 was generally more effective (had a lower IC50) when applied pre-SEB exposure compared to post-exposure treatment. As predicted, EM-163 was PLoS ONE | www.plosone.org 7 July 2012 | Volume 7 | Issue 7 | e40773