Acute liver failure (ALF) is a fulminant complication of multiple etiologies, characterized by rapid hepatic destruction, multi-organ failure and mortality. ALF treatment is mainly limited to supportive care and liver transplantation. Here we utilize the acetaminophen (APAP) and thioacetamide (TAA) ALF models in characterizing 56,527 single-cell transcriptomes to define the mouse ALF cellular atlas. We demonstrate that unique, previously uncharacterized stellate cell, endothelial cell, Kupffer cell, monocyte and neutrophil subsets, and their intricate intercellular crosstalk, drive ALF. We unravel a common MYC-dependent transcriptional program orchestrating stellate, endothelial and Kupffer cell activation during ALF, which is regulated by the gut microbiome through Toll-like receptor (TLR) signaling. Pharmacological inhibition of MYC, upstream TLR signaling checkpoints or microbiome depletion suppress this cell-specific, MYC-dependent program, thereby attenuating ALF. In humans, we demonstrate upregulated hepatic MYC expression in ALF transplant recipients compared to healthy donors. Collectively we demonstrate that detailed cellular/genetic decoding may enable pathway-specific ALF therapeutic intervention.

Monocyte-derived macrophages (MoMF) play a pivotal role in the resolution of acetaminophen-induced liver injury (AILI). Timely termination of neutrophil activity and their clearance are essential for liver regeneration following injury. Here, we show that infiltrating Ly6Chi monocytes, their macrophage descendants, and neutrophils spatially and temporally overlap in the centrilobular necrotic areas during the necroinflammatory and resolution phases of AILI. At the necroinflammatory phase, inducible ablation of circulating Ly6Chi monocytes resulted in reduced numbers and fractions of reactive oxygen species (ROS)-producing neutrophils. In alignment with this, neutrophils sorted from monocyte-deficient livers exhibited reduced expression of NADPH oxidase 2. Moreover, human CD14+ monocytes stimulated with lipopolysaccharide or hepatocyte apoptotic bodies directly induced ROS production by cocultured neutrophils. RNA-seq-based transcriptome profiling of neutrophils from Ly6Chi monocyte-deficient versus normal livers revealed 449 genes that were differentially expressed with at least twofold change (p ≤ 0.05). In the absence of Ly6Chi monocytes, neutrophils displayed gene expression alterations associated with decreased innate immune activity and increased cell survival. At the early resolution phase, Ly6Chi monocytes differentiated into ephemeral Ly6Clo MoMF and their absence resulted in significant accumulation of late apoptotic neutrophils. Further gene expression analysis revealed the induced expression of a specific repertoire of bridging molecules and receptors involved with apoptotic cell clearance during the transition from Ly6Chi monocytes to MoMF. Collectively, our findings establish a phase-dependent task division between liver-infiltrating Ly6Chi monocytes and their MoMF descendants with the former regulating innate immune functions and cell survival of neutrophils and the later neutrophil clearance.

Etoposide (VP-16) is a topoisomerase II (topo II) inhibitor chemotherapeutic agent. Studies indicate that VP-16 enhances proinflammatory cytokines secretion from tumour cells, including IL-8, a chemokine associated with proangiogenic effects. Fluoroquinolones inhibit topo II activity in eukaryotic cells by a mechanism different from that of VP-16. The fluoroquinolone moxifloxacin (MXF) has pronounced anti-inflammatory effects in vitro and in vivo. We studied the effects of MXF and VP-16 on purified human topo II activity and further analysed their combined activity on proliferation, apoptosis and caspase-3 activity in THP-1 and Jurkat cells. Moxifloxacin alone slightly inhibited the activity of human topo II; however, in combination with VP-16 it led to a 73% reduction in enzyme activity. VP-16 inhibited cell proliferation in a time and dose-dependent manner. The addition of moxifloxacin for 72 h to low-dose VP-16 doubled its cytotoxic effect in THP-1 and Jurkat cells (1.8-and 2.6-fold decrease in cell proliferation, respectively) (Po0.004). Moxifloxacin given alone did not induce apoptosis but enhanced VP-16-induced apoptosis in THP-1 and Jurkat cells (1.8-and two-fold increase in annexin V positive cells and caspase-3 activity, respectively) (Po0.04). VP-16 induced the release of IL-8 in a time and dose-dependent manner from THP-1 cells. Moxifloxacin completely blocked the enhanced release of IL-8 induced by 0.5 and 1 mg ml À1 VP-16, and decreased IL-8 release from cells incubated for 72 h with 3 mg ml À1 VP-16 (Po0.001). VP-16 enhanced the release of IL-1b and TNF-a from THP-1 cells, whereas the addition of MXF prevented the enhanced cytokine secretion (Po0.001). We conclude that MXF significantly enhances VP-16 cytotoxicity in tumour-derived cells while preventing VP-16-induced proinflammatory cytokine release. This unique combination may have clinical benefits and cytotoxic drug 'sparing effect' and should be further studied in vivo.

Erythropoietin (EPO) is the main hormone driving mammalian erythropoiesis, with activity mediated via the surface receptor, EPO-R, on erythroid progenitor cells. Recombinant human EPO is currently used clinically for the treatment of anemia in patients with end-stage renal disease, and in certain cancer patients suffering from anemia induced either by the tumor itself or by chemotherapy. EPO-R expression is also detected in non-erythroid cells, including macrophages present in the peritoneum, spleen, and bone marrow (BM). Here we demonstrate that Kupffer cells (KCs) - the liver-resident macrophages - are EPO targets. We show that, in vitro, EPO initiated intracellular signalling and enhanced phagocytosis in a rat KC line (RKC-2) and in sorted KCs. Moreover, continuous EPO administration in mice, resulted in an increased number of KCs, up-regulation of liver EPO-R expression and elevated production of the monocyte chemoattractant CCL2, with corresponding egress of Ly6Chi monocytes from the BM. In a model of acute acetaminophen-induced liver injury, EPO administration increased the recruitment of Ly6Chi monocytes and neutrophils to the liver. Taken together, our results reveal a new role for EPO in stimulating KC proliferation and phagocytosis, and in recruiting Ly6Chi monocytes in response to liver injury.