Hfe Actions in Kupffer Cells Are Dispensable for Hepatic and Systemic Iron Metabolism

Abstract

Mutations in the HFE/Hfe gene cause Hereditary Hemochromatosis (HH), a highly prevalent genetic disorder characterized by elevated iron deposition in multiple tissues. HFE acts in hepatocytes to control hepcidin expression, whereas HFE actions in myeloid cells are required for cell-autonomous and systemic iron regulation in aged mice. To address the role of HFE specifically in liver-resident macrophages, we generated mice with a selective Hfe deficiency in Kupffer cells (Hfe^Clec4fCre). The analysis of the major iron parameters in this novel Hfe^Clec4fCre mouse model led us to the conclusion that HFE actions in Kupffer cells are largely dispensable for cellular, hepatic and systemic iron homeostasis.

Keywords: HFE-hemochromatosis; Kupffer cells; hepcidin; iron; liver; macrophage.

Figure 1

Successful generation of a mouse line specifically lacking Hfe in Kupffer cells: Hfe^Clec4fCre. (A) Relative Hfe/Rpl7 mRNA expression of isolated liver cells from Hfe^Clec4fCre (n = 3 mice) and control mice (n = 3–5); (B,C) Immunofluorescent staining of F4/80 positive cells in liver tissue (green) with counter staining of DNA with DAPI (blue) and Actin with Phalloidin (red) and quantification of F4/80 positive signals as mean fluorescent intensity (MFI) normalized to MFI of DAPI signals; (D,E) Relative Hfe/Rpl7 mRNA expression in different macrophage populations of Hfe^Clec4fCre (n = 3 mice), Hfe^LysMCre (n = 3 mice) and control mice (n = 3–5): Kupffer cells (KC), splenic macrophages (sMF) and bone-marrow-derived macrophages (BMDMs). Data are representative of one experimental analysis. The experiment has been performed twice with another set of Hfe^Clec4fCre (n = 3–8 mice), Hfe^LysMCre (n = 3–5 mice) and control mice (n = 3–5). Significance was tested by using Mann-Whitney-U-test (two-tailed, unpaired, unequal variance.) Significant results were indicated by * p < 0.05 or ** p < 0.01.

Figure 2

Kupffer cell Hfe in young mice is dispensable for cellular and systemic iron homeostasis. (A,B) Systemic iron levels in Hfe^Clec4fCre and Hfe^fl/fl control mice (n = 8; 5); (C) cellular iron levels in isolated primary liver cells from Hfe^Clec4fCre (n = 4) and Hfe^fl/fl control mice (n = 3); (D) Relative Hepcidin/Rpl7 mRNA expression in the liver from Hfe^Clec4fCre and Hfe^fl/fl control mice (n = 8; 5) and isolated primary liver cells obtained from Hfe^Clec4fCre (n = 6) and control mice (n = 3); (E,F) Hematological indices in Hfe^Clec4fCre and Hfe^fl/fl control mice (n = 8; 5). Significance was tested by using Mann-Whitney-U-test (two-tailed, unpaired, unequal variance). KC: Kupffer cells; liver sinusoidal endothelial cells (LSECs), hepatic stellate cells (HSCs), hepatocytes (HC).

Figure 3

Hfe in liver-resident macrophages does not contribute to age-dependent symptomatic manifestation of hemochromatosis. (A) Hepatic, splenic, duodenal non-heme iron content in aged Hfe^Clec4fCre (n = 7) and Hfe^fl/fl control mice (n = 5); (B) plasma hepcidin levels and (C) circulating plasma in aged Hfe^Clec4fCre (n = 3) and Hfe^fl/fl control mice (n = 3); (D) Cellular iron content in isolated primary Kupffer cells from Hfe^Clec4fCre (n = 3) and Hfe^fl/fl control mice (n = 3); (E,F) Hematological indices in Hfe^Clec4fCre (n = 7) and Hfe^fl/fl control mice (n = 5). Data are representative of one experimental analysis. The experiment has been performed twice with another set of Hfe^Clec4fCre (n = 4 mice) and control mice (n = 3). Significance was tested by using Mann-Whitney-U-test (two-tailed, unpaired, unequal variance). Significant results were indicated by * p < 0.05.