IL-6 mediates hypoferremia of inflammation by inducing the synthesis of the iron regulatory hormone hepcidin

Abstract

Hypoferremia is a common response to systemic infections or generalized inflammatory disorders. In mouse models, the development of hypoferremia during inflammation requires hepcidin, an iron regulatory peptide hormone produced in the liver, but the inflammatory signals that regulate hepcidin are largely unknown. Our studies in human liver cell cultures, mice, and human volunteers indicate that IL-6 is the necessary and sufficient cytokine for the induction of hepcidin during inflammation and that the IL-6-hepcidin axis is responsible for the hypoferremia of inflammation.

Figure 1

Anti_IL-6 Ab's neutralize the induction of hepcidin mRNA. Hepcidin mRNA was analyzed relative to G3PDH mRNA by quantitative RT-PCR (qRT-PCR). P values were determined by one-way RM-ANOVA with a Tukey test. (A) Primary hepatocytes (n = 4 donors): untreated (0), treated for 24 hours with 100 ng/ml LPS (LPS), or treated with 100 ng/ml LPS + 6 ∝g/ml anti_IL-6 Ab's (LPS + IL-6 Ab's). (B) Hep3B cells: untreated (0), treated for 24 hours with 16.6% medium from monocyte-derived macrophages (n = 2 donors) or Kupffer cells (n = 2 donors) either unstimulated (M) or stimulated with 100 ng/ml LPS (M-LPS) or treated for 24 hours with the same 16.6% M-LPS + 6 μg/ml anti_IL-6 Ab's (M-LPS + IL-6 Ab's).

Figure 2

The effect of inflammation on hepcidin mRNA and serum iron in WT and IL-6 KO mice. WT (circles) and IL-6 KO (triangles) mice on an iron-depleted diet were treated with a single subcutaneous injection of turpentine. (A) Median expression of hepcidin-1 (normalized to mouse β-actin) increased 12-fold in WT mice treated with turpentine (n = 13) compared with PBS-treated controls (n = 10, P < 0.001 by Mann-Whitney rank sum test). Conversely, expression fell 6.4-fold in turpentine-treated IL-6 KO mice (n = 13) compared with controls (n = 11, P = 0.014). P < 0.001 for the comparison of WT to IL-6 KO mice (by two-way ANOVA). (B) Mean serum iron fell 49% in WT mice treated with turpentine (n = 9) compared with controls (n = 7, P = 0.015 by Student's t test), whereas there was a slight and marginally significant increase in median serum iron of IL-6 KO mice (n = 9 and n = 7, respectively; P = 0.049). The difference between iron decrease in WT and IL-6 KO mice was highly significant (P < 0.001 by two-way ANOVA).

Figure 3

Counterregulation of hepcidin mRNA expression by TNF-α. Human hepatoma Hep3B cells were treated with 20 ng/ml of the indicated cytokine(s) with or without anti_IL-6 Ab's for 24 hours. Their hepcidin mRNA was then analyzed relative to G3PDH mRNA by qRT-PCR. In each experiment, the hepcidin mRNA was expressed as a ratio to that of untreated cells (0), and the mean and standard deviation of four experiments are shown. Paired Student's t test was used to determine the significance of the effects of TNF-α.

Figure 4

IL-6 infusion increases urinary hepcidin and decreases serum iron in humans. Six subjects were infused with rhIL-6 for 3 hours at the rate of 30 ∝g/h. Urine and serum samples were collected: prior to infusion at 0 hours, at the end of the infusion (3 h inf), 2 hours after the infusion (2 h after), and 24 hours after the infusion (24 h). (A) Serum IL-6 concentrations. (B) Urinary hepcidin levels. Thick line represents the geometric mean. The increase in hepcidin at 2 hours after infusion is highly significant (P < 0.001 by one-way RM ANOVA). (C) Serum iron concentration. (D) Serum transferrin saturation. Thick lines represent the arithmetic mean. The decreases in serum iron concentration and transferrin saturation at 2 hours after infusion are both highly significant compared with preinfusion values (both P < 0.001 by one-way RM ANOVA). creat, creatinine.

Figure 5

Iron ingestion induces urinary hepcidin in humans. Five subjects collected their first morning urine for 9 days and took 65 mg of iron (as ferrous sulfate) in the morning of days 3, 4, and 5. Thick line represents the arithmetic mean. There is a significant increase in hepcidin excretion on day 4 (24 hours after the first dose of iron) compared with day 3 (P = 0.017 by paired Student's t test).

Figure 6

Iron ingestion induces hepcidin mRNA in WT and IL-6 KO mice. WT (circles) and IL-6 KO (triangles) mice on an iron-depleted diet were switched to a standard diet for 24 hours. Median hepcidin-1 mRNA expression (normalized to mouse β-actin mRNA) increased significantly in both WT (10.7-fold, P < 0.001 by Mann-Whitney rank sum test) and IL-6 KO (ninefold, P = 0.009 by Student's t test) mice. After the switch to a standard diet, there was no significant difference in hepcidin-1 mRNA levels in WT and IL-6 KO mice (P = 0.536 by two-way ANOVA on ranks).