Functional role of macrophage subsets in acetaminophen-induced acute liver failure and therapeutic implica-tions of its modulation by chemokine pathways

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Liver damage resulting from an overdose of acetaminophen (paracetamol, APAP) is one of the leading causes of acute liver failure (ALF) in humans, a life-threatening conditions oftentimes requiring liver trans-plantation. While the initial hepatocyte necrosis due to APAP toxicity can be therapeutically ameliorated using N-acetylcysteine, the outcome of APAP injury at later stages is critically determined by a massive inflammatory reaction. In experimental models of ALF as well as in patients, different macrophage subsets, derived from resident Kupffer cells and infiltrating monocytes, can be found in the liver. The working hypothesis is that macrophage heterogeneity functionally impacts the outcome of APAP-related and non-APAP-related ALF and that interference with the chemokine-mediated recruitment can ameliorate their contribution to liver damage. In this proposal, we will investigate the dynamics and differentiation of macrophage subpopulations as well as the functional involvement of the chemokine receptor CCR2 (and of alternative pathways) in APAP-induced and other models of acute liver injury. The therapeutic potential of blocking CCL2, the main CCR2 ligand, and of CCR2 itself will be explored using innovative pharmacological inhibitors in this mouse model and thus enables translational approaches for clinical applications. Specific aims are: 1. Characterization and dynamics of macrophage subsets and CCR2-dependent inflammation in APAP-induced liver injury (using transgenic mouse strains in three acute liver injury models, intravital multiphoton microscopy imaging of liver for CCR2+ monocytes, extensive phenotyping by Nanostring-based multiplex gene analyses and functional validation by adoptive transfers); 2. exploration of alternative chemokine pathways and effects of pre-existing steatosis for monocyte recruitment and differentiation (specifically CCR1, CCR8, CCR9, CX3CR1; Western-diet steatosis model in ApoE-deficient mice); 3. molecular mechanisms of macrophage differentiation in APAP-induced liver injury (employing elaborate co-culture in vitro set-ups with primary hepatic cell subsets); 4. investigating therapeutic implications by blocking CCR2 and the CCR2 ligand CCL2 by innovative specific pharmacological inhibitors (an CCL2 inhibiting RNA-aptamer / Spiegelmer as well as a small molecular CCR2/CCR5 inhibitor; administration during progression and resolution); 5. translational approaches to the pathogenesis of human acute liver failure (by characterizing human liver samples by multicolor FACS and Nanostring-based gene expression pattern and comprehensive comparisons to the mouse model data). Collectively, we will shed light on the contribution of the diverse monocyte/macrophage populations on APAP-induced ALF and explore therapeutic implications for the treatment of acute liver failure in humans.