Search

Staphylococcal enterotoxin B (SEB) exposure triggers an exaggerated pro-inflammatory cytokine response that often leads to toxic shock syndrome (TSS) associated with organ failure and death. MyD88 mediates pro-inflammatory cytokine signaling induced by SEB exposure and MyD88-/- mice are resistant to SEB intoxication, suggesting that MyD88 may be a potential target for therapeutic intervention. We targeted the BB loop region of the Toll/IL-1 receptor (TIR) domain of MyD88 to develop small-molecule therapeutics. Here, we report that a synthetic compound (EM-163), mimic to dimeric form of BB-loop of MyD88 attenuated tumor necrosis factor (TNF)-alpha, interferon (IFN)-gamma, interleukin (IL)-1Beta, IL-2 and IL-6 production in human primary cells, whether administered pre- or post-SEB exposure. Results from a direct binding assay and from MyD88 co-transfection/co-immunoprecipitation experiments, suggest that EM-163 inhibits TIR-TIR domain interaction. Additional results indicate that EM-163 prevents MyD88 from mediating downstream signaling. In an NF-kB-driven reporter assay of lipopolysaccharide-stimulated MyD88 signaling, EM-163 demonstrated a dose-dependent inhibition of reporter activity as well as TNF-alpha and IL-1Beta production. Importantly, administration of EM-163 pre- or post exposure to a lethal dose of SEB abrogated pro-inflammatory cytokine responses and protected mice from toxic shock-induced death. Taken together, our results suggest that EM-163 exhibits a potential for therapeutic use against SEB intoxication., Published in PLoS ONE, v7 n7 e40773, July 27, 2012. The original document contains color images.

Toxic shock syndrome (TSS) is a clinical consequence of the profound amplification of host pro-inflammatory cytokine signaling that results from staphylococcal enterotoxin (SE) exposure. We recently reported that MyD88-/- mice were resistant to SEA or SEB toxic shock and displayed reduced levels of pro-inflammatory cytokines in their serum. Here we report that SEB stimulation of total mononuclear cells up-regulated MyD88 in monocytes and T cells. Further, MyD88 gene silencing in primary human cells using siRNA prevented SEB or SEB plus lipopolyaccharide (LPS) induction of interleukin-1 Beta (IL-1 Beta) transcriptional activation, suggesting that MyD88-mediated signaling is an essential component of SEB toxicity. We synthesized small molecules that mimic the conserved BBloop in the Toll/IL-1 receptor (TIR) domain of MyD88. In primary human cells, these mimetics attenuated SEB-induced proinflammatory cytokine production. SEB stimulation of primary cells with mimetic affected newly synthesized MyD88 and downstream signaling components. Furthermore, LPS-induced MyD88 signaling was likewise inhibited in a cell-based reporter assay. More importantly, administration of mimetic reduced cytokine responses and increased survivability in a murine SEB challenge model. Collectively, these results suggest that MyD88 BB-loop mimetics interfere with SEB-induced proinflammatory signaling and toxicity, thus offering a potential approach in the therapy of toxic shock., Published in Journal of Biological Chemistry, 21 June 2011.

Ligands binding to Toll-like receptor (TLR), interleukin 1 receptor (IL-1R), or IFN-cR1 are known to trigger MyD88-mediated signaling, which activates pro-inflammatory cytokine responses. Recently we reported that staphylococcal enterotoxins (SEA or SEB), which bind to MHC class II molecules on APCs and cross link T cell receptors, activate MyD88-mediated proinflammatory cytokine responses. We also reported that MyD882/2 mice were resistant to SE- induced toxic shock and had reduced levels of serum cytokines. In this study, we investigated whether MHC class II- SE interaction by itself is sufficient to activate MyD88 in MHC class II+ cells and induce downstream pro-inflammatory signaling and production of cytokines such as TNF-a and IL-1b. Here we report that human monocytes treated with SEA, SEB, or anti-MHC class II monoclonal antibodies up regulated MyD88 expression, induced activation of NF-kB, and increased expression of IL-1R1 accessory protein, TNF-a and IL-1b. MyD88 immunoprecipitated from cell extracts after SEB stimulation showed a greater proportion of MyD88 phosphorylation compared to unstimulated cells indicating that MyD88 was a component of intracellular signaling. MyD88 downstream proteins such as IRAK4 and TRAF6 were also up regulated in monocytes after SEB stimulation. In addition to monocytes, primary B cells up regulated MyD88 in response to SEA or SEB stimulation. Importantly, in contrast to primary B cells, MHC class II deficient T2 cells had no change of MyD88 after SEA or SEB stimulation, whereas MHC class II-independent activation of MyD88 was elicited by CpG or LPS. Collectively, these results demonstrate that MHC class II utilizes a MyD88-mediated signaling mechanism when in contact with ligands such as SEs to induce pro-inflammatory cytokines., Published in PLoS ONE, v6 n1 p1-12, 20 Jan 2011.

Interleukin-15 (IL-15) is generally considered to be a growth factor for natural killer cells and for sustaining T-cell memory. Previous data from our laboratory demonstrated that IL-15 is also an important factor for developing human dendritic cells. In this study, we investigated the effect of IL-15 on antibody responses to a recombinant staphylococcal enterotoxin B (SEB) vaccine (STEBVax), in a pre-clinical model of toxic-shock syndrome induced by SEB. We observed that mouse spleen cells treated with IL-15 in ex vivo culture gained a dendritic cell-like phenotype. Administration of IL-15 to mice also resulted in an increased number of mature CD11c+ dendritic cells in mouse spleens. A significant, IL-15 dose-dependent increase in antigen-specific antibody was observed after co-administration with vaccine and an aluminum-based adjuvant (alhydrogel). Furthermore, the co-administration of IL-15 with STEBVax and alhydrogel also protected mice from lethal toxic shock above the levels that obtained without IL-15. Thus, the vaccine response enhanced by IL-15 appears to be mediated by mature dendritic cells, and results in prevalent sero-conversion to Th2-dependent antibodies. This suggests a potential use of IL-15 as an adjuvant for antibody-dependent responses to vaccines., The original document contains color images. Pub. in Jnl. of Clinical and Vaccine Immunology, v15 n1 p131-137, Jan 2008.