A computational multi-scale model for the integration of paracrine stimuli activating NF-kB signalling in hepatocytes after lipopolysaccharide (LPS) treatment

Background: Bacterial lipopolysaccharide (LPS) efficiently stimulates the secretion of tumor necrosis factor (TNF)-α from non-parenchymal Kupffer cells (KCs), liver sinusoidal endothelial cells (LSECs), and hepatic stellate cells (HSCs). Paracrine-acting TNFα subsequently activates the canonical NF-kB pathway in hepatocytes, which is a central regulator of acute phase protein expression and the inflammatory response. However, the impact of individual liver cell types on this pro-inflammatory response has not been sufficiently analysed i.a. due to technical limitations. To establish a holistic view on this complex multi-scale process in a quantitative and time-resolved manner, systems biology serves as a valuable tool.

Methods: The cellular response of murine hepatocytes upon different TNFα amounts (0.1 – 50 ng/ml) was measured by quantitative Western immunoblotting (8 time points, after 0 to 240 min) using antibodies detecting p65, IkBα, p38, and MSK1 as well as the respective phosphorylated isoforms. Using primary KCs, LSECs, and HSCs the secretion of TNFα was measured after treatment with LPS (0.5 – 100 ng/ml) for up to 24 hours (Luminex). To understand the inter-cellular communication process, we utilised a multi-scale ordinary differential equation (ODE)-based mathematical model (including 33 variables, 59 reactions) combining quantitative and time-resolved data.

Results: Based on experimental results, we established a hepatocyte-specific mathematical model for TNFα-induced NF-κB signalling, which accurately described the dynamic behaviour of NF-kB pathway activation, target gene expression, protein turnover, p65/IkBα complex formation, TNFα dose-dependence, as well as activation of the p38/MSK-1 axis. Quantitative TNFα secretion profiles of KCs, LSECs, and HSCs upon LPS stimulation revealed that LSECs and KCs secreted similar high cytokine amounts (up to 0.026µM and 0.023µM, respectively), while HSC-derived TNFα was much lower (constitutive mean 0.004µM). Subsequently, NF-kB model simulations using the TNFα input derived from LSECs and/or KCs were computed individually or in combination. The results clearly illustrated that low concentrations of LSEC- and KC-derived TNFα (up to 0.12 ng/ml), which were induced by low LPS amounts (from 0.1 to 1 ng/ml), synergised to establish an adjustable and sensitive NF-kB pathway response in hepatocytes. Higher LPS concentrations (> 5 ng/ml) induced sufficient TNFα amounts (> 0.3 ng/ml) from KCs or LSECs to induce a maximal NF-kB pathway response without any additive or synergistic effects.

Conclusion: The combination of experimental data and computational multi-scale modelling illustrates that low doses of LPS and TNFα induce partial and adjustable cellular responses in non-parenchymal cells (LSECs and KCs) and hepatocytes. Under pathological conditions, high LPS/TNFα amounts stimulate non-adjustable and full-blown cellular responses. This integrative approach represents the basis for further analysis on the role of different cell types during inflammation-induced hepatic disease.

Corresponding author: Breuhahn, Kai

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