In severe cases of acetaminophen (APAP) overdose, acute liver injury rapidly progresses to acute liver failure (ALF), producing life threatening complications including, hepatic encephalopathy and multi-organ failure. Systemic levels of the proinflammatory cytokine interleukin-6 (IL-6) and the anti-inflammatory cytokine interleukin-10 (IL-10) are highest in ALF patients with the poorest prognosis. However, the mechanistic basis for dysregulation of these cytokines, and their association with outcome in ALF remains poorly defined. Standard experimental settings of APAP hepatotoxicity in mice (i.e., 300 mg/kg) do not recapitulate key features of ALF in critically ill patients, including impaired hepatocyte proliferation, kidney injury, evidence of hepatic encephalopathy (HE), and cytokine dysregulation. Further, in stark contrast to ALF patients, IL-6 and IL-10 limit liver injury, increase hepatocyte proliferation, and reduce mortality under these experimental conditions. Thus, to investigate cytokine dysregulation in true ALF, we used a robust experimental setting of failed liver repair after APAP overdose in which a high dose of APAP is administered. Under these experimental conditions, we detected high serum levels of several pro- and anti-inflammatory cytokines, including IL-6 and IL-10, and observed increased levels of PD-L1 on macrophages mimicking systemic inflammatory response syndrome (SIRS) that occurs in critically ill ALF patients. Further, cerebral blood flow was markedly reduced in these mice, recapitulating a key feature of hepatic encephalopathy in ALF patients. Remarkably, neutralization of IL-6 in this setting restored cerebral blood flow, reduced mortality, and normalized levels of IL-10 and PD-L1. Furthermore, neutralization of IL-6 increased levels of Gas6, the primary ligand for the receptor Axl, which our studies revealed is essential for protection of the hepatic sinusoidal vasculature from injury after APAP overdose. In addition, our studies demonstrated that macrophage-mediated clearance of necrotic cells was prevented in mice with ALF, but was restored by neutralization of IL-10. Lastly, our studies identified Kupffer cells (KCs), the resident macrophages of the liver, as a primary source of IL-6, IL-10 and PD-L1 in APAP-induced ALF, and demonstrated further that the transcriptional regulator, NFKBIZ, may be responsible for enhanced IL-6 production in ALF. Collectively, our studies reveal that exaggerated levels of IL-6 are detrimental in APAP-induced ALF in mice, and suggest that therapies aimed at reducing IL-6 levels in patients with APAP-induced ALF may be beneficial. Additionally, as a part of this dissertation, we have developed a high-throughput assay that can detect differentiation of proinflammatory macrophages into pro-repair macrophages for use as a drug screening platform to identify chemicals/drugs that stimulate this process. Drugs identified from this screen could ultimately be used to restore macrophage function and liver repair in patients with both acute and chronic forms of liver failure.
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