A human hepatic in vitro co-culture system for the analysis of DILI related signaling

The interaction between immune cells and hepatocytes during a sterile inflammation has come into focus in Drug-Induced Liver Injury (DILI) research during recent years. Therefore, this crosstalk was analysed in a novel indirect co-culture system with HepG2 and differentiated THP-1 cells using the antifungal drug ketoconazole as a known hepatotoxic model compound. The metabolism of ketoconazole, changes in the proteome, cytokine expression and secretion upon treatment in single- and co-culture were analysed.

HepG2 metabolised ketoconazole to several metabolites which differed from those found in THP-1 cells both in single and co-culture. In the supernatant, the ketoconazole concentration decreased time-dependently while the concentration of several metabolites increased.

The global proteomics analysis of HepG2 cells in co-culture identified the activation of the “Nrf2 mediated stress response” and the “Integrin linked kinase signalling” pathways after treatment with ketoconazole. Further upregulated proteins belonged to the NFκB, CXCL8 and sterol pathway. ELISA and qPCR assays revealed the upregulation of several pro-inflammatory cytokines including CXCL8, TNF-α and CCL3 in treated, co-cultured but not single-cultured HepG2 cells.

The advantage of this indirect system with inserts is that both cell lines can be separated and analysed individually after co-culture and the results can be compared to single-cultured cells. The activation of signalling pathways related to the hepatotoxicity of ketoconazole occurred in the co-culture at lower concentrations, compared to the single-culture. In conclusion, the novel indirect co-culture system represents a promising new tool to study hepatotoxic drug effects in vitro.

Corresponding author: Wewering, Franziska

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