IL-1 beta-mediated macrophage-hepatocyte crosstalk upregulates hepcidin under physiological low oxygen levels

Abstract

In mammals, the iron masterswitch hepcidin efficiently controls iron recycling by the macrophage-liver axis but the exact interplay between macrophages and hepatocytes remains poorly understood. We here study hepcidin response during macrophage differentiation as well as the macrophage-hepatocyte crosstalk and its subsequent effects on hepatocyte hepcidin using an in vitro co-culture model that mimics the physiological liver microenvironment. We show that macrophage differentiation strongly induces hepcidin by 60-fold both in THP1 macrophages and primary isolated monocyte-derived macrophages. Removal of H₂O₂ by catalase or inhibition of NOX2 efficiently blocked hepcidin induction. After differentiation, macrophage hepcidin accounted for 10% of total hepatocyte hepcidin and did not respond to low oxygen levels. In contrast, co-culture of differentiated macrophages with Huh7 cells significantly induced hepatocyte hepcidin, which was further potentiated under low oxygen levels. Hepatocyte hepcidin was also upregulated when Huh7 cells were solely exposed to macrophage-conditioned hypoxic medium. A cytokine screen identified macrophage secreted IL-1β as major inducer of hepcidin in hepatocytes. In confirmation, treatment of Huh7 cells with the IL-1 receptor antagonist (anakinra) completely blunted macrophage-mediated hepcidin transcription in hepatocytes. Finally, detailed analysis of potentially involved signaling pathways points toward STAT3 and CEBPδ-mediated hepcidin induction independent of IL-6. In conclusion, our study demonstrates a strong NOX2-mediated hepcidin induction during macrophage differentiation. These differentiated macrophages are able to efficiently induce hepatocyte hepcidin mainly through secretion of IL-1β. Our data highlight a hitherto unrecognized role of macrophage-hepatocyte crosstalk for a joint and oxygen-dependent hepcidin production through STAT3 and CEBPδ.

Keywords: Cytokines; Hydrogen peroxide; Hypoxia; Iron metabolism/hepcidin; NADPH oxidase; STAT3.

Graphical abstract

Fig. 1

Differentiation of monocytes into macrophages leads to transcriptional activation of NOX2 and hepcidin. A) PMA-mediated differentiation of THP-1 monocytes into macrophages caused a significant increase of NOX2 and B) hepcidin mRNA expression during 24 h. C) Differentiation of primary isolated human monocytes into monocyte derived macrophages (MDM) using M-CSF for 7 days causes a significant increase of hepcidin mRNA expression. NOX2 and hepcidin mRNA was quantified by quantitative real-time PCR and the results are represented as mean of mRNA levels normalized to β2-microglobulin ± SD. Significant differences are marked by asterisks (   ***, P < 0.001).

Fig. 2

Differentiation-induced NOX2 leads to enhanced ROS production, IL-1β release and hepcidin upregulation. A) The release of H₂O₂ by THP-1 cells was highly increased within 60 min of PMA-induced differentiation and was efficiently blocked by co-incubation with the pan NOX inhibitor VAS2870. Extracellular H₂O₂ levels were measured by HRPO-enhanced luminescence (relative light units; RLU). B) Increased intracellular ROS accumulation could be lowered by VAS2870 during the first 24 h of differentiation with PMA. THP-1 monocytes were pre-loaded with dihydrofluorescein diacetate (DHF) and differentiated with PMA for 24 h. Intracellular ROS were detected by measuring the fluorescence at 540 nm and the mean fluorescence intensity is given in relative fluorescence units (RFU) after subtraction of the control in the absence of PMA (RFU ± SD). C) Treatment of THP-1 cells with VAS2870 while PMA-mediated differentiation significantly inhibited hepcidin, and D) IL-1β mRNA induction during 24 h. E) siRNA-mediated silencing of NOX2 while differentiation significantly decreased hepcidin transcription but had no effect on IL-1β mRNA expression. NOX2, IL-1β and hepcidin mRNA was quantified by quantitative real-time PCR and the results are the mean of mRNA levels normalized to β2-microglobulin ± SD. Significant differences are marked by asterisks (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

Fig. 3

Induction of hepatocellular hepcidin in macrophage/hepatocyte co-cultures under low oxygen levels is mediated by STAT3 but not BMP signaling pathway. A) Hepcidin mRNA as well as Stat3 protein levels are increased in hepatocyte/macrophage co-cultures using different (patho-)physiologic cell ratios and low oxygen levels. B) Smad7 mRNA as well as Smad1/5/8 protein levels are not significantly changed in hepatocyte/macrophage co-cultures using different (patho-)physiologic cell ratios and low oxygen levels. qRT-PCR results are presented as mean of target mRNAs normalized to β2-microglobulin ± SD. Western Blots are representatives of three independent experiments. Significant differences are marked by asterisks (***, P < 0.001).

Fig. 4

Macrophage-secreted factors induce hepatocellular hepcidin via STAT3 signaling pathway. A) Hepcidin mRNA as well as pStat3 protein levels were significantly increased in hepatocytes treated with medium of macrophages conditioned to 1% O₂ and further cultivated under 21% and 1% O₂. B) Macrophage-conditioned hypoxic medium induced hepatocellular hepcidin transcription as well as phosphorylation of Stat3 is completely blocked by co-treatment with the pan NOX inhibitor VAS2870. Hepcidin mRNA levels were quantified by qRT-PCR and the results are presented as mean of hepcidin mRNAs normalized to β2-microglobulin ± SD. C) The deletion of the STAT3-binding site completely blocked the induction on hepcidin promoter activity after incubation with macrophage-conditioned medium. Huh7 cells were transfected with hepcidin promoter constructs including the wild type promoter (WT), promoter regions with decreased length of the 5‘-flanking region (WT 1 kb and WT 165bp) or the WT with specific deletions of transcription factors binding sites (STAT3-binding site (STAT3bs del) and the BMP-responsive elements 1–3 (bmp-RE1 to -3 del)). Huh7 cells were transfected for 48 h and then cultivated with macrophage-conditioned hypoxic medium for 24 h. Renilla plasmid was used as control for expression and transfection. The results are expressed as fold induction ± SD of firefly/Renilla luciferase activity relatively to the normoxic control of each construct. (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

Fig. 5

IL-1β is the main cytokine involved in the macrophage-mediated induction of hepatocellular hepcidin transcription. A) The levels of 25 different cytokines were determined in the supernatant from PMA-differentiated THP-1 cells cultivated under 21% and 1% O₂ using a cytokine protein array. The level of cytokines were quantified using ImageJ software and expressed as normalized density units. IL-1β was induced under hypoxic conditions and is known to regulate hepcidin. B) Induction of hepcidin transcription in hepatocytes by MCHM was completely abolished by IL-1 receptor antagonist treatment (IL-1RA; Anakinra), whereas IL-8 inhibition by Reparixin had no significant effect. C) IL-1RA treatment also inhibited the MCHM-induced phosphorylation of Stat3 in hepatocytes. Representative data of at least three independent experiments are shown. Hepcidin mRNA levels were quantified by RT-PCR and presented as mean Hepcidin mRNA normalized to β2-microglobulin ± SD. Significant differences in relation to the respective controls are marked by asterisks (*, P < 0.05; **, P < 0.01; ***, P < 0.001) and significant differences to the macrophage-conditioned hypoxic medium control by hash tags (^#, P < 0.05; ^##, P < 0.01; ^###, P < 0.001).

Fig. 6

Low physiologic concentrations of IL-1β secreted by macrophages induce hepatocellular hepcidin independently of IL-6. A) Hepcidin as well as IL-6 mRNA are significantly induced in Huh7 cells using artificially high IL-1β concentrations (10 ng/ml recombinant IL-1β). B) Under 1% O₂ IL-1β (>0.1 ng/ml) significantly increases hepcidin C) but not IL-6 mRNA expression. CEBPδ mRNA is highly upregulated under 1% O₂ by IL-1β concentrations starting from 0.05 ng/ml. D) Under 1% O₂ IL-1β also significantly increases STAT3 phosphorylation. E) MCHM also significantly induces CEBPδ mRNA expression without affecting IL-6 mRNA in Huh7 cells. Treatment of Huh7 cells with IL-1 receptor antagonist (IL-1RA; Anakinra) or F) co-treatment of macrophages with VAS2870 completely inhibited the effect of MCHM on CEBPδ transcription (###, P < 0.001). Hepcidin, IL-6 and CEBPδ mRNA levels were quantified by RT-PCR and presented as mean mRNA normalized to β2-microglobulin ± SD. Significant differences in relation to the respective controls are marked by asterisks (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

Fig. 7

STAT3 is an essential link between macrophage-induced IL-1β signaling and hepatocellular hepcidin upregulation. A) siRNA-mediated knockdown of STAT3 or CEBPδ significantly inhibits basal MCHM-induced hepcidin expression in Huh7 cells. B) CEBPδ upregulation by MCHM is significantly blocked by siRNA against CEBPδ or STAT3. C) siRNA-mediated silencing of CEBPδ decreases the induction of pSTAT3 protein expression after treatment with MCHM. D) Induction of hepcidin promoter activity by MCHM is inhibited by transfection with siRNA against CEBPδ. Huh7 cells were co-transfected with hepcidin wild type promoter (WT) or WT promoter with specific deletion of STAT3-binding site (STAT3bs del) and CEBPδ siRNA or control siRNA for 48 h and then cultivated with MCHM for 24 h. Renilla plasmid was used as control for expression and transfection. The results are expressed as fold induction ± SD of firefly/Renilla luciferase activity relatively to the normoxic control of each construct. Representative data of at least three independent experiments are shown. Hepcidin and CEBPδ mRNA levels were quantified by RT-PCR and presented as mean mRNA normalized to β2-microglobulin ± SD. Significant differences in relation to the respective controls are marked by asterisks (**, P < 0.01; ***, P < 0.001) and significant differences to the macrophage-conditioned medium control by hash tags (^##, P < 0.01; ^###, P < 0.001).