Role of NOX1 on hepcidin signaling in the crosstalk between macrophages and hepatocytes

Background and Aims:

Liver-secreted hepcidin is the systemic master switch of iron homeostasis and its dysregulation leads to iron accumulation in most of chronic liver diseases. Hepcidin is regulated by iron, inflammation or H2O2, but the role of NOX1 and its products ROS/H2O2 in monocyte-derived macrophages on hepcidin regulation under (patho)physiological conditions is poorly understood. We here investigate the role of NOX1 on regulating hepcidin and cytokines in inflammatory macrophages and subsequent effects on hepatocytes mimicking (patho)physiological conditions (cell ratios, oxygen levels, and inflammation).

Methods:

THP-1 monocytes were differentiated into macrophages and co-cultured with Huh7 cells at (patho)physiological cell ratios (4:1) and treated with different LPS concentrations (10 ng/ml and 100 ng/ml) under normoxia (21% O2) or hypoxia (1% O2). The exposure of Huh7 cells to macrophage-conditioned medium with LPS was also investigated. Hepcidin, IL-1β, IL-6, C/EBPδ, and SMAD6 mRNA levels were assessed by qRT-PCR and the expression of NOX1, p-STAT3, STAT3 and p-SMAD1/5/8 proteins were analyzed by western blot.

Results:

LPS significantly increased NOX1, p-STAT3, IL-1βand IL-6 levels in THP-1 macrophages, but decreased STAT3 expression in a concentration-dependent manner under 21% and 1% O2. Interestingly, 10 ng/ml LPS increased the expression of hepcidin whereas 100 ng/ml LPS decreased the expression of hepcidin under 21% O2. In contrast, both LPS concentrations decreased the expression of hepcidin 1% O2 in THP-1 macrophages. In addition, LPS decreased SMAD6, p-SMAD1/5/8 and CEBPδin THP-1 macrophages under 21% O2. Notably, the treatment of Huh7 cells with LPS had no effect on the expression of IL-6, IL-1β, CEBPδ and hepcidin in Huh7 cells under 21% O2. Using inflammatory macrophage/hepatocyte co-cultures with direct cell-cell interactions under 21% O2 increased IL-6, IL-1β, CEBPδ, and hepcidin expression level in a concentration-dependent manner but did not further increase hepcidin in direct macrophage/hepatocyte co-cultures under 1% O2.

Conclusion:

Our findings underscore a possible role of NOX1 and subsequent ROS/H2O2 concentrations on hepcidin regulation and induction of cytokine production in inflammatory macrophages involving the STAT3 signaling pathway. In the future, we aim at studying in detail hepcidin signaling by using WT and truncated hepcidin promoter constructs and siRNA-mediated knockdown of TLR4, NOX1, STAT3 or C/EBPδ.