Faculty Bibliography

MIF is a proinflammatory cytokine that has been implicated in the pathogenesis of sepsis, arthritis, and other inflammatory diseases. Antibodies against MIF are effective in experimental models of inflammation, and there is interest in strategies to inhibit its deleterious cytokine activities. Here we identify a mechanism of inhibiting MIF pro-inflammatory activities by targeting MIF tautomerase activity. We designed small molecules to inhibit this tautomerase activity; a lead molecule, "ISO-1 ((S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester)," significantly inhibits the cytokine activity in vitro. Moreover, ISO-1 inhibits tumor necrosis factor release from macrophages isolated from LPStreated wild type mice but has no effect on cytokine release from MIFdeficient macrophages. The therapeutic importance of the MIF inhibition by ISO-1 is demonstrated by the significant protection from sepsis, induced by cecal ligation and puncture in a clinically relevant time frame. These results identify ISO-1 as the first small molecule inhibitor of MIF proinflammatory activities with therapeutic implications and indicate the potential of the MIF active site as a novel target for therapeutic interventions in human sepsis.

Despite significant advances in intensive care therapy and antibiotics, severe sepsis accounts for 9% of all deaths in the United States annually. The pathological sequelae of sepsis are characterized by a systemic inflammatory response, but experimental therapeutics that target specific early inflammatory mediators [tumor necrosis factor (TNF) and IL-1beta] have not proven efficacious in the clinic. We recently identified high mobility group box 1 (HMGB1) as a late mediator of endotoxin-induced lethality that exhibits significantly delayed kinetics relative to TNF and IL-1beta. Here, we report that serum HMGB1 levels are increased significantly in a standardized model of murine sepsis, beginning 18 h after surgical induction of peritonitis. Specific inhibition of HMGB1 activity [with either anti-HMGB1 antibody (600 microg per mouse) or the DNA-binding A box (600 microg per mouse)] beginning as late as 24 h after surgical induction of peritonitis significantly increased survival (nonimmune IgG-treated controls = 28% vs. anti-HMGB1 antibody group = 72%, P < 0.03; GST control protein = 28% vs. A box = 68%, P < 0.03). Animals treated with either HMGB1 antagonist were protected against the development of organ injury, as evidenced by improved levels of serum creatinine and blood urea nitrogen. These observations demonstrate that specific inhibition of endogenous HMGB1 therapeutically reverses lethality of established sepsis indicating that HMGB1 inhibitors can be administered in a clinically relevant time frame.

Excessive inflammation and tumour-necrosis factor (TNF) synthesis cause morbidity and mortality in diverse human diseases including endotoxaemia, sepsis, rheumatoid arthritis and inflammatory bowel disease. Highly conserved, endogenous mechanisms normally regulate the magnitude of innate immune responses and prevent excessive inflammation. The nervous system, through the vagus nerve, can inhibit significantly and rapidly the release of macrophage TNF, and attenuate systemic inflammatory responses. This physiological mechanism, termed the 'cholinergic anti-inflammatory pathway' has major implications in immunology and in therapeutics; however, the identity of the essential macrophage acetylcholine-mediated (cholinergic) receptor that responds to vagus nerve signals was previously unknown. Here we report that the nicotinic acetylcholine receptor alpha7 subunit is required for acetylcholine inhibition of macrophage TNF release. Electrical stimulation of the vagus nerve inhibits TNF synthesis in wild-type mice, but fails to inhibit TNF synthesis in alpha7-deficient mice. Thus, the nicotinic acetylcholine receptor alpha7 subunit is essential for inhibiting cytokine synthesis by the cholinergic anti-inflammatory pathway

Sepsis, a potentially fatal clinical syndrome, is mediated by an early (e.g., tumor necrosis factor and IL-1) and late [e.g., high mobility group B-1 (HMGB1)] proinflammatory cytokine response to infection. Specifically targeting early mediators has not been effective clinically, in part because peak mediator activity often has passed before therapy can be initiated. Late-acting downstream effectors, such as HMGB1, that mediate sepsis lethality may be more relevant therapeutic targets. Ethyl pyruvate (EP) recently was identified as an experimental therapeutic that significantly protects against lethal hemorrhagic shock. Here, we report that EP attenuates lethal systemic inflammation caused by either endotoxemia or sepsis even if treatment begins after the early tumor necrosis factor response. Treatment with EP initiated 24 h after cecal puncture significantly increased survival (vehicle survival = 30% vs. EP survival = 88%, P < 0.005). EP treatment significantly reduced circulating levels of HMGB1 in animals with established endotoxemia or sepsis. In macrophage cultures, EP specifically inhibited activation of p38 mitogen-activated protein kinase and NF-kappaB, two signaling pathways that are critical for cytokine release. This report describes a new strategy to pharmacologically inhibit HMGB1 release with a small molecule that is effective at clinically achievable concentrations. EP now warrants further evaluation as an experimental "rescue" therapeutic for sepsis and other potentially fatal systemic inflammatory disorders.