C/EBPβ-Thr217 Phosphorylation Stimulates Macrophage Inflammasome Activation and Liver Injury.

Inflammation contributes to the pathogenesis of most acute and chronic liver diseases1. Excessive liver injury and inflammation associated with liver diseases induced by viral, toxic, immunologic, and metabolic diseases2 results in liver dysfunction and in chronic conditions in the potential deposition of scar tissue and the development of cirrhosis, which is in turn a major contributor to the morbidity and mortality of patients affected by chronic liver diseases2,3. We first reported that amplification of toxic liver injury is mediated by macrophages since TLR-4 ko mice were resistant to hepatotoxins and that reconstitution of bone marrow irradiated TLR-4 ko mice with TLR-4+/+ macrophages conferred susceptibility of these animals to hepatotoxins4. The role of macrophages in liver inflammation in toxic liver injury has been confirmed using macrophage ablation5, and further characterized in an experimental alcoholic liver injury model using an IL-1 receptor antagonist6, and in LPS/D-galactosamine induced liver injury using Adenosine-2A (A2A) receptor-ko mice7. Fas–mediated IL-18 secretion from macrophages causes acute liver injury in mice8, and macrophage phagocytosis removes hepatocyte debris during hepatocyte injury9. However, the signal transduction mechanisms in liver macrophages that are indispensable to amplify liver injury have been only partially characterized1. The inflammasome is a protein complex that is essential for triggering activation of inflammatory reactions in macrophages as well as the consequent macrophage activation1,10,11. The CCAAT/Enhancer Binding Protein-β (C/EBPβ)12,13,14 has been shown to be a critical signaling molecule for macrophages as expression of a dominant inhibitor of C/EBPβ DNA-binding sites15 or a targeted deletion of C/EBPβ results in impaired macrophage differentiation16. In addition, C/EBPβ expression is dramatically increased during differentiation of these cells, and is induced by macrophage modulators (LPS, IL-1, G-CSF, TGFβ, vitamin D, retinoic acid)13,17. In this context, we and others have shown that phosphorylation of C/EBPβ by Ribosomal S-Kinase-2 (RSK-2), which is activated directly by Extracellular-Regulated Kinase (ERK)-1/2 phosphorylation, plays an essential role in the ERK/ Mitogen Activated Protein Kinase (MAPK) signaling pathway regulating cell survival18,19,20,21. Relevant to macrophage activation and survival, we have reported that expression of the dominant positive, phosphorylation-mutant C/EBPβ-Glu217, which mimics phosphorylated C/EBPβ-Thr217 in biological assays22, was sufficient to rescue the impaired macrophage function and activity induced by Anthrax lethal toxin23. Knowledge of the specific signaling that targets a single amino acid within a specific phosphoacceptor domain of the mechanistic protein (in this case C/EBPβ) is necessary to understand the process of the disease and to eventually design effective targeted therapeutics that are still lacking in the treatment of human liver injury. Therefore, we investigated whether signaling through phosphorylation of C/EBPβ-Thr217, a potential novel therapeutic target, might be a major mechanism responsible for liver inflammation and injury through the activation of the inflammasome in liver macrophages. We studied the effects of C/EBPβ-Phospho-Thr217 signaling that is evolutionarily conserved (identical in human C/EBPβ-Phospho-Thr266) on macrophage inflammasome activity and liver injury induced by hepatotoxins in mice and humans.