aspects of host defense. Because of the critical role of MyD88 signaling, mice deficient in MyD88 have profoundly impaired innate immune responses and are susceptible to a wide range of infectious diseases [18]. However, in regards to innate immune- response regulation, MyD88 signaling performs a delicate balanc- ing act. Perturbed regulation or excessive stimulation of the innate immune system can trigger, inflammatory signaling that can spiral out of control and lead to profound clinical syndrome [18]. For example, excess of MyD88-mediated signaling can lead to severe pathological consequences such as toxic shock syndrome (TSS) and sepsis. However, the crucial role of MyD88 in these disorders also provides a target for therapeutic intervention. Earlier results from our laboratory demonstrated that a synthetic mimetic of the BB-loop in the TIR domain of MyD88 (Compound 1) attenuated SEB-induced pro-inflammatory cytokine production in human primary cells and increased survivability of mice from toxic shock-induced death after a lethal SEB challenge [19]. It is known that the BB-loop region acts as the mediator of the homo- (adaptor-adaptor) and hetero- (receptor-adaptor) dimerization that is necessary for the function of TIR domains to induce MyD88- mediated signaling [15,16]. Seeking to improve the efficacy of Compound 1 as an inhibitor of MyD88 signaling, we synthesized a dimeric molecule in which two Compound 1 moieties were covalently linked together, reasoning that the dimeric compound would be a more potent inhibitor of protein-protein interactions. This molecule, EM-163, was tested in primary cultures of human mononuclear cells (MNCs) for inhibition of cytokine release associated with exposure to SEB. Our results provide evidence that, by targeting MyD88, EM-163 inhibited SEB-induced inflammatory cytokine production in human primary cells. Importantly, EM-163 abrogated pro-inflammatory cytokine re- sponse in vivo and completely protected mice from toxic shock- induced death regardless of whether it was administered pre- or post-exposure to a lethal SEB challenge. Materials and Methods Reagents Staphylococcal enterotoxin B (SEB) and SEA was purchased from Porton Down, Inc. (Salisbury, UK) and stored at 250uC. SEB or SEA was endotoxin free and prepared under GMP conditions. Escherichia coli LPS (055:B5) was purchased from Sigma-Aldrich (Saint Louis, MO). Pooled human AB sera were obtained from Pel-Freez (Brown Deer, WI). The BDTM cytometric bead array (CBA) human Th1/Th2 and inflammatory cytokine kit was purchased from BD Biosciences (San Diego, CA). The Meso Scale Discovery (MSD) multi spot array ultra-sensitive cytokine assay kit was purchased from MSD (Gaithersburg, MD). Magnetic bead-conjugated anti-CD14 and anti-CD3 mAbs were obtained from Miltenyi Biotech Inc. (Auburn, CA). Ficoll-Hypaque was purchased from GE Healthcare Biosciences (Piscataway, NJ). Primary anti-MyD88 antibody was obtained from AnaSpec, Inc., (San Jose, CA) and Alexis Biochemicals (San Diego, CA). Anti-b- actin antibody was purchased from Cell Signaling technology (Danvers, MA). Plasmids expressing HLA-DRa/b were a kind gift from Dr. Robert Ulrich (USAMRIID). Plasmid 12287 (pCMV- HA-MyD88) and plasmid 13093 (MyD88-flag) were purchased through an MTA agreement with Addgene (Cambridge, MA). The HEK 293 T cell line was obtained from the ATCC (Manassas, VA). MyD88 KO HEK293 cell line (HEK293-I3A) was a kind gift from G. Stark (Dept. of Molecular Genetics, Lerner Research Institute, Cleveland Clinic, OH). The transfection reagent lipofectamine was purchased from Invitrogen (Carlsbad, CA). Vectashield mounting medium containing 4-,6-diamidino-2- Inhibition of SEB Intoxication by a MyD88 Mimetic phenylindole (DAPI) was purchased from Vector Laboratories (Burlingame,CA). Mice Pathogen-free, 6–8 weeks old BALB/c and C57BL/6 mice were obtained from Charles River (NCI-Frederick, Frederick, MD). Cell Isolation and Purification Peripheral blood mononuclear cells (MNC) were obtained from consenting healthy donors in accordance with an Institutional Review Board-approved research donor protocol. MNCs were isolated by standard density gradient centrifugation with Ficoll- Hypaque, harvested from the interface, washed, and re-suspended in RPMI 1640 medium. Monocytes (CD14+) and T (CD3+) cells were isolated as previously described [19]. Isolated cell populations had .98% purity. Cytokine Analysis Cell cultures were incubated (37uC, 5% CO2) for 16 h. Cytokines in culture supernatants were measured by a CBA kit using captured beads coated with antibodies specific for cytokines. Flow cytometry was performed as described elsewhere [20]. Cytokine measurement was confirmed by dilution of culture supernatant using Inflammation and Th1/Th2 CBA kits by acquiring 1800 beads. We also used a Meso Scale Discovery (MSD) multi spot array ultra sensitive cytokine assay kit to measure cytokines in culture supernatants (according to the manufacturer’s protocol) as described [19]. Western Blot Analysis The transfected cells were chilled on ice for 5 min before being pelleted into fresh 1.5 ml centrifuge tubes. Membrane and cytoplasm separation was done by suspending the pellets in 50 ml of lysis buffer (Active Motif) in the presence of DTT, protease inhibitors and phosphatase inhibitors and incubated on ice for 30–60 min. The membrane fraction was collected by centrifuging the lysates at 140006g for 20 min. The supernatant contained the cytoplasmic fraction. Samples containing 10 mg of total cytoplasmic proteins were separated by gel electrophoresis and transferred to nitrocellulose membranes. The membranes were blocked overnight in 16 Tris-buffered saline (TBS) contain- ing 0.1% Tween-20 and 3% bovine serum albumin at 4 oC. The membranes were washed extensively with 16TBS buffer and then probed with anti-MyD88 polyclonal antibody followed by horseradish peroxide-conjugated secondary antibody (goat anti- rabbit). After additional rinsing with 16 TBS buffer, the membranes were exposed to a chemiluminescent substrate in the presence of hydrogen peroxide, using Immun-Star WesternC Chemiluminescent kit (BioRad). A VersaDoc Model 4000 (BioRad) imaging system was used to capture the image. Intracellular Staining and Confocal Microscopy Freshly isolated monocytes (CD14+) adhered to sterile culture slides (Corning Glass, Corning, NY) were incubated with either SEB (200 ng/ml), SEB+EM-163, or kept untreated in RPMI 1640 medium. Cells were gently washed with PBS (pH 7.4, 4uC) and then fixed with 1% paraformaldehyde (Tousimis Research, Rockville, MD) plus 0.1% glutaraldehyde (Sigma-Aldrich) in PBS (5 min, 20uC). The fixed cells were washed with PBS containing 0.5% bovine serum albumin (BSA) and incubated (30 min, 20uC) in a permeabilization solution (Becton Dickinson). Cells were then washed and incubated (15 min, 20uC) with PBS containing 5% BSA to block non-specific antibody binding. The PLoS ONE | www.plosone.org 2 July 2012 | Volume 7 | Issue 7 | e40773