12, 15, 20, 25 Interestingly, the effect of the altered cytokine milieu generated by LPS-treated TK−/− Kupffer cells was more toxic to mouse hepatocytes in vitro compared to TK+/+ Kupffer compound screening assay cells. This may be due to higher TNF-α levels, the composition of cytokines, or the presence of an untested cytokine in the conditioned media. Moreover, it is possible that the kinetics of cytokine expression is as important as the composition of cytokines produced. TK−/− hepatocytes are protected compared to TK+/+ hepatocytes from TNF-α/ActD initiated cell death over the range of 0.5 to 5 ng/mL of TNF-α, which are similar to the serum TNF-α levels observed in the previously reported in vivo experiment.16

Whether the observed differences in hepatocyte viability are sufficient to explain the in vivo observations by Leonis et al.16 is not discernable by this assay. Hepatocytes plated ex vivo are without feedback mechanisms to Kupffer cells and other hepatic cell types and, thus, the magnitude of the effect of TNF-α ex vivo may not completely mimic what is observed in vivo. However, the results from the conditional deletion of Ron selectively in hepatocytes support the significance of our

check details ex vivo culture conditions and prior in vivo experiments. Previously, we demonstrated that the protected liver injury response to treatment with LPS/GalN in Ron TK−/− mice was associated with a 1- to 2-hour delay in the progression to death based on survival analyses.16 The lack of a more significant discrepancy in the time to mortality may be multifactorial and confounded by the sensitivity of the Ron TK−/− mice to LPS alone and by the severe necrosis and endothelial damage that is observed in the model. Despite the modest overall survival benefit of the TK−/− mice compared to controls, less liver injury was observed in the TK−/− mice as judged by liver histopathology, ALT levels, hepatocyte TUNEL staining, and the extent of hepatic apoptosis. Similar protected Nintedanib (BIBF 1120) phenotypes were observed in the Alb-Cre Ron TKfl/fl mice as the Ron

TK−/− mice compared to control mice. This is based on a reduction in liver histopathology and significantly decreased ALT levels and TUNEL staining in the Alb-Cre Ron TKfl/fl mice compared to controls. Interestingly, a 1- to 2-hour increase in survival time in the Alb-Cre Ron TKfl/fl mice was also observed compared to controls. Therefore, the data are consistent with a protected liver phenotype in the Alb-Cre Ron TKfl/fl mice compared to controls. Our data also show a significant decrease in survival of the LysCre mice and associated worsened liver phenotypes in these mice compared to Alb-Cre Ron TKfl/fl and wildtype mice and supports the premise that Ron functions in both cellular compartments in vivo. Our results show increased NF-κB activation in hepatocytes that lack Ron signaling.