P093 Toll-like receptor 4 inhibition acts synergistically with G-CSF to prevent organ injury and induce liver regeneration in acute-on-chronic liver failure

Abstract

<h3>Background and Aims</h3> Acute-on-chronic liver failure (ACLF) is characterised by lack of regeneration. Granulocyte colony stimulating factor (G-CSF) carries pro-regenerative properties and has been shown to be of benefit in ACLF. However, the large trial of G-CSF (GRAFT study) in patients with ACLF showed no benefit and in certain groups mortality tended to be higher. This study was performed to define the mechanisms underlying the negative effect of G-CSF and determine whether its beneficial effect could be harnessed using a toll-like receptor 4 (TLR4) antagonist. <h3>Method</h3> Two mouse models of ACLF were used: CCL4 (0.5mg/ml,6w) to induce chronic liver injury followed by LPS i.p. (Klebsiella, 4mg/kg) (n=4–10) or Galactosamine (GalN) i.p. (1000mg/kg) as a second hit (n=8). 1h after, G-CSF 250µg/kg s.c. and/or TLR4-inhibitor TAK-242 10mg/kg i.p. were injected and continued every 24h. The treatment duration was 24h and 5d in the LPS model and 48h in the GalN model. Samples were stored and analysed for liver injury, inflammation, senescence and regeneration. <h3>Results</h3> 6w CCL4 led to bridging fibrosis, TLR4 up-regulation and infiltration of G-CSFr expressing cells. LPS increased ALT levels, cell death (TUNEL+), enhanced hepatic infiltration of neutrophils (Ly6G+), macrophages (F4/80+) and TNFa. G-CSF increased the 48h mortality from 0% to 66%, aggravated liver inflammation with macrophage and NK cell (CD45+,CD49b+,CD3-,CD19-) infiltration and IL6 expression. G-CSF+TAK-242 reduced the mortality to 0%, abrogated the liver injury (TUNEL) and liver inflammation (macrophages, neutrophils, TNFa, IL6) significantly. In the second model, GalN also induced a significant liver injury. Treatment with G-CSF+TAK-242 was significantly more effective than the individual therapies. G-CSF+TAK-242 was associated with increased liver regeneration evidenced by increased tissue expression of pSTAT3 and BCL2. CCL4+LPS induced a p53 and p16-dependent cell cycle arrest and lack of proliferation (CyclinA) in hepatocytes. G-CSF+TAK-242 mitigated senescence and significantly increased the rate of CyclinA expressing hepatocytes (figure) suggesting enhanced liver regeneration. <h3>Conclusion</h3> The present study shows that G-CSF is deleterious in LPS-associated ACLF through further activation of inflammatory pathways and immune cell infiltration. TLR4 inhibition with TAK-242 prevented G-CSF driven tissue injury and induced liver regeneration showing evidence of synergy between the two molecules thereby providing a novel therapeutic strategy for ACLF patients.