| Staphylococcal enterotoxin B (SEB) is a potent exotoxin produced by the Staphylococcus aureus bacterium. This toxin is classified as a superantigen because of its ability to directly bind MHC II class molecules with the receptors of T cells (TCR), leading to robust activation of a large proportion of T cells bearing specific Vbeta-TCRs. Commonly associated with classic food poisoning, SEB has also been shown to induce more fatal conditions, such as toxic shock syndrome. SEB exposure is also a major concern in regards to community-acquired diseases and nosocomial infections. Because this toxin is stable even under extreme conditions and is easily aerosolized, use of SEB as a biological warfare weapon has also become a major issue. In the present study, we assessed the ability of indole-3-carbinol (I3C) and one of its byproducts, 3,3'-diindolylmethane (DIM), to counteract the effects of SEB-induced activation of T cells in female C57BL/6 mice. Injection of SEB into the footpads of mice (10 microg/footpad) led to the expansion of T cells in popliteal lymph nodes, but treatment with either I3C or DIM (40 mg/kg) by intraperitoneal injection was able to significantly reduce these numbers. Treatment of SEB-stimulated in vitro cultures with either I3C or DIM (100 microM) revealed these compounds were able to prevent T cell activation and prevent release of several proinflammatory cytokines. In our studies we also showed how SEB activation and SEB-induced cytokine release by T cells was greatly dependent on class-I histone deacetylases (HDAC-I) through class-specific HDAC inhibition studies. This was a significant finding, as few studies have looked into the role particular classes of HDACs play in SEB stimulation. Interestingly, both I3C and DIM were effective at downregulating HDAC-I expression after activation by SEB, thereby preventing T cell activation and proinflammatory cytokine release. In addition, we also determined how effective these compounds would be in treating mice subjected to SEB-induced acute liver injury. Injection of SEB (40 microg) into D-galactosamine-sensitized female C57BL/6 mice resulted in liver injury as indicated by an increase in aspartate transaminase enzyme (AST) levels, induction of inflammatory cytokines, and massive infiltration of immune cells into the liver. Administration of I3C and DIM (40mg/kg), by intraperitonal injection, attenuated SEB-induced acute liver injury, as evidenced by decrease in levels of AST, inflammatory cytokines, and cellular infiltration in the liver. I3C and DIM triggered apoptosis in SEB-activated cells primarily through activation of the intrinsic mitochondrial pathway. In addition, inhibitor studies involving caspases revealed that I3C and DIM-mediated apoptosis in these activated cells was dependent on caspase-2, but independent of caspase-8, 9 and 3. Analysis of microRNA (miR) array samples revealed that I3C and DIM downregulated several miRs that could possibly target and suppress the expression of caspase-2. Specifically, we found that I3C and DIM downregulated miR-31, which we were able to show for the first time directly targeted caspase-2. Collectively, our data showed that I3C and DIM, through epigenetic modification and miRNA modulation, are very effective preventative interventions against SEB. |
