Extracellular vesicle-dependent crosstalk between hepatic stellate cells and Kupffer cells promotes their mutual activation

BACKGROUND & AIMS: Hepatic fibrosis results from hepatic stellate cell (HSC) activation and excessive extracellular matrix (ECM) deposition, driven by chronic inflammation. Kupffer cells (KCs) play a central role in HSC activation. We previously showed that HSC-secreted factors, particularly extracellular vesicles (EVs), activate KCs. However, the reciprocal effects of activated KCs on HSCs remain poorly understood. This study investigates the bidirectional crosstalk between HSCs and KCs, focusing on the role of KC-derived EVs in regulating HSC activation and fibrosis progression. METHODS: Primary HSCs and KCs were isolated from male Wistar rats. HSCs were co-cultured with KCs for 24 h to assess inflammatory and activation markers. LPS-stimulated KC-derived EVs and controls were administered to HSCs on day 1. LPS and the Toll-like receptor 4 (TLR4) inhibitor TAK-242 were used to investigate the intercellular communication in detail. RESULTS: Co-cultured HSCs and KCs showed mutual activation, demonstrated by elevated inflammatory markers in both cell types and enhanced HSC pro-fibrotic activation. Pro-inflammatory (LPS)-activated KCs amplified HSC activation in a TLR4-dependent fashion. Part of this augmented HSC activation was attributed to EVs. CONCLUSIONS: In co-culture, KCs and HSCs show mutual activation in a TLR4-dependent fashion. This bidirectional activation is augmented by pro-inflammatory mediators. KC-derived EVs (partially) activate HSCs, which might contribute to progression of liver fibrosis in vivo. Modulating KC activation, such as by blocking TLR4 signaling, may alter EV secretion or cargo composition, reducing HSC activation and fibrosis progression. Targeting this EV-mediated crosstalk could provide novel therapeutic strategies for liver fibrosis.