Macrophage-hepatocyte crosstalk modulates hepcidin under physiological oxygen levels

Liver-secreted hepcidin is the systemic masterswitch of iron homeostasis and its dysregulation is a key event for carcinogenic iron accumulation in most of the chronic liver diseases. Hepcidin is strongly upregulated by iron, inflammation, cytokines or H2O2 but the role of liver monocyte-derived macrophages and Kupffer cells on hepcidin regulation under (patho)physiological conditions is poorly understood. We here investigate the cellular crosstalk involved in hepatic hepcidin regulation by developing an in vitro co-culture model of hepatocytes and macrophages mimicking physiologic cell ratios and oxygen levels.

Huh7 cells and THP-1 monocytes differentiated into macrophages were directly co-cultured at a physiological (10:1) or cell ratios found in inflamed liver (4:1) under normoxic (21% O2) or hypoxic conditions (1 or 5% O2) over 24h. The incubation of Huh7 cells with macrophage conditioned medium was also investigated. Hepcidin, NOX2 and IL-1β mRNA expression was assessed by qRT-PCR and the generation of ROS during macrophage differentiation was detected by dichlorofluorescein assay. The contribution of STAT3 and BMP/SMAD signaling pathway on hepcidin regulation was analyzed by western blot and qRT-PCR. Transfection of different truncated hepcidin promoter constructs was used to identify binding sites responsible for hepcidin regulation. Furthermore, a cytokine array was carried out to assess the role of macrophage-secreted cytokines and the specific role of IL-1β was studied using IL-1 receptor antagonist (IL-1RA).

We first demonstrated that the presence of macrophages leads to significantly increased hepcidin mRNA levels. This effect was even potentiated when co-cultures were maintained under low O2 levels (1 and 5% O2). Interestingly, hepcidin was also upregulated when Huh7 cells were exposed to macrophage-conditioned medium under 1% O2 suggesting the role of a soluble and secreted factor in mediating hepatocyte hepcidin induction. Western Blot analysis, as well as studies with truncated hepcidin promoter constructs confirmed the involvement of the STAT3 but not BMP signaling pathway. A cytokine array pointed towards IL-1β and IL-8 as possible inducers of hepatic hepcidin via STAT3 signaling pathway under (patho)physiological conditions. Finally, the selective inhibition of IL-1 receptor, using IL-1RA, clearly suggested IL-1β as the key cytokine involved in the macrophage-mediated induction of hepatic hepcidin.

Our findings underscore the importance of the hepatocyte/macrophage crosstalk for hepatic hepcidin regulation including IL-1β as the prominent macrophage-released factor, which is rapidly expressed during acute or chronic liver injury. We highlight the induction of hepcidin through IL-1β-dependent activation of STAT3 signaling pathway, independent of IL-6-induced signaling.