Cytokine driven hepatocyte proliferation and survival under necrotic stress

: insights into the IL-4/STAT6 pathway

Abstract

Liver regeneration is a highly coordinated process regulated by interactions between inflammatory cues, metabolic stress and cytokine-driven signalling networks. Although interleukin-4 (IL-4) is classically recognised for its immunomodulatory functions, its role in hepatocyte proliferative signalling within necrosis-associated microenvironments remains insufficiently characterised. This thesis investigated the effects of selected interleukins on HepG2 cell proliferation under basal and necrotic conditions before focusing on IL-4 and its associated STAT6 signalling pathway.
HepG2 cells were exposed to IL-4, IL-6, IL-10, IL-10, IL-12, IL-23 and IL-1β across a range of concentrations in the absence and presence of necrotic conditioning. Proliferative responses were initially assessed using CFSE dilution, revealing a dose and context-dependent hierarchy of interleukin activity that was reshaped by necrotic stress. IL-4 emerged as a prominent mitogenic interleukin and retained relative proliferative activity at low concentrations under necrotic conditions, leading to its selection for further investigation. IL-4 mediated responses were subsequently evaluated using Ki67 immunolabelling and MTT assays to examine cell-cycle activity and metabolic capacity respectively. Intracellular phosphorylated STAT6 (pSTAT6; Y641) was quantified by flow while soluble IL-4 receptor alpha (sIL-4Rα) was assessed to investigate an additional extracellular regulatory mechanism.
Necrotic conditioning imposed a dominant constraint on cellular responses while modifying rather than uniformly suppresing cytokine mediated proliferation. IL-4 retained context-dependent proliferative activity despite reduced metabolic capacity under necrotic conditions. Necrotic conditioning also reduced the proportion of pSTAT6-positive cells and altered IL-4 dose-response patterns, while responsive cells retained measurable signalling intensity, indicating heterogeneity in STAT6 signalling competence. Furthermore, IL-4 and necrotic conditioning altered sIL-4Rα levels in dose-dependent manner suggesting an additional extracellular mechanism through which IL-4 availability may be regulated.
These findings identify necrotic stress as a key contextual modifier of cytokine responsiveness in HepG2 cells and demonstrate that IL-4/STAT6 signalling is regulated across multiple levels. The findings support a model in which necrotic conditions reshape extracellular IL-4 regulation, restrict STAT6 signalling competence and constrain downstream proliferative and metabolic responses rather than completely eliminating IL-4 responsiveness.

Date of Award	2026
Original language	English
Awarding Institution	Canterbury Christ Church University