647 TLR7 Signaling As Potential Therapeutic Target for Mouse Model of Alcoholic Hepatitis

Abstract

Background: The activation of classical complement pathway contributes to ethanol-induced liver injury. Ethanol feeding in mice activates complement pathway via C1q binding to apoptotic cells in the liver and increases downstream activation of C3b. However, the mechanism by which ethanol activates C1q is unclear. This knowledge is critical to understanding liver injury-repair mechanisms. Molecules targeting the C1q pathway to decrease inflammation would be expected to preserve complement activation via the lectin and alternative pathways, thus maintaining the role of complement in resistance to infections. Secretory IgM (sIgM) is the primary isotype of natural antibodies that arise in the absence of exposure to foreign antigens. In addition to the anti-microbial functions, there is accumulating evidence that sIgM has critical housekeeping functions by facilitating clearance of apoptotic cells through activation of C1q. Therefore, we hypothesized that ethanol exposure activates C1q through sIgM binding to apoptotic hepatocytes. The presence of sIgM-C1q complex further exacerbates apoptosis in a feed forward mechanism. Further, C1INH (C1 esterase inhibitor) targeted to decrease the sIgM-C1q activation may prevent complement activation and decrease apoptosis in ethanol-induced liver injury.Methods: Two experimental protocols were used: 1) Female C57BL/6J wild type mice and sIgM-/mice were fed ethanol containing diets for 2 weeks, at a maximal concentration of 4% (vol/vol) ethanol (22% calories) or pair-fed control diets. 2) C57BL/6 mice were fed ethanol containing diets for 4 days, at a maximal concentration of 6%(vol/vol) ethanol (32% of calories). Mice were treated with C1INH or vehicle via tail vein injection at 24 and 48 hours prior to euthanasia. Results: Ethanol exposure increased hepatic IgM, C3b and C1q deposition as measured by IHC. C1q co-localized with IgM in liver. sIgM-/mice had decreased hepatic C1q, C3b deposition and TUNEL positive nuclei after ethanol exposure. AST/ALT, as well as MCP-1 mRNA, was decreased in sIgM-/mice compared to wild type mice after ethanol feeding. Importantly, C1INH decreased hepatic C3b deposition and TUNEL positive nuclei after ethanol feeding. Conclusion: The data indicate that sIgM contributes to activation of classical complement pathway in ethanol-induced liver injury via binding of sIgM to C1q. The presence of sIgMC1q complex is associated with cell death and may further enhance cell death and injury. Blocking of the activation via C1INH decreased C3b deposition and apoptosis. Taken together, the data suggest that sIgM is critical in ethanol-induced complement activation and that therapies targeting the classical complement pathway may be beneficial in ethanolinduced liver injury. The study is supported in part by ABMRF/The Foundation for Alcohol Research to DJC and NIH grant 5R37 AA011876 to LEN.