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. 2021 Jan 1;106(1):259-263.
doi: 10.3324/haematol.2019.235630.

Macrophage-HFE controls iron metabolism and immune responses in aged mice

Naveen Kumar Tangudu  1 , Dilay Yilmaz  1 , Katharina Wörle  2 , Andreas Gruber  3 , Silvia Colucci  4 , Kerstin Leopold  3 , Martina U Muckenthaler  4 , Maja Vujic Spasic  1
Affiliations

Macrophage-HFE controls iron metabolism and immune responses in aged mice

Naveen Kumar Tangudu et al. Haematologica. .
No abstract available

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Figures

Figure 1.
Figure 1.
Lack of macrophage-Hfe in 45-week old mice contributes to moderate systemic iron deficiency in contrast to physiological iron homeostasis present in 12-week old HfeLysMCre mice. (A) Non-heme iron levels in the liver, spleen and duodenum from 45-week old HfeLysMCre mutant and Hfeflox control mice, and from mice kept on an iron-deficient diet (IDD). n indicates the number of livers (n=19, 15, 5), spleens (n=19, 15, 5) and duodenums (n=6, 6, 5) isolated from HfeLysMCre, Hfeflox and IDD mice, respectively. (B) Perls staining for iron deposits in the liver, spleen and duodenum of 45-week old HfeLysMCre mutant and control mice. Scale bar 50 μm (liver) and 100 μm (spleen and duodenum). Representative stainings of three sections are shown. (C) Intracellular total iron levels measured by total-reflection x-ray fluorescence in primary hepatocytes (HC), Kupffer cells (KC), liver sinusoidal endothelial cells (LSEC), and hepatic stellate cells (HSC) from 45-week old HfeLysMCre and Hfeflox mice (n=3-5). (D) Serum hepcidin, iron and erythropoietin (EPO) levels, and (E) hematologic parameters including red blood cell (RBC) count, hemoglobin (Hgb) concentration, hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), and the percentages of white blood cells (WBC) including lymphocytes (Lym), monocytes (Mo), and granulocytes (Gra) in the blood of 45-week old HfeLysMCre mutant (n=6) and Hfeflox control mice (n=6). (F) Relative mRNA expression of iron-related genes in the liver of 45-week old HfeLysMCre mutant (n=6) and Hfeflox control mice (n=9) determined by real-time polymerase chain reaction (PCR). (G, H) Representative immunoblot analysis of pSMAD1, transferrin receptor 1 (TfR1) and ferroportin (FPN), relative to bactin levels (shown in histograms on the right), in the livers and spleens of HfeLysMCre mutant (n=8) and control mice (n=8). (I) Relative mRNA expression of iron transporters in the duodenum of 45-week old HfeLysMCre mutant (n=4) and Hfeflox control mice (n=8) measured by real-time PCR. (J-L) Non-heme iron levels (J), serum iron and hepcidin levels (K), and relative mRNA expression of hepcidin (Hamp1) and Bmp6 (L) in the livers of 12-week old HfeLysMCre mutant (n=5) and Hfeflox control mice (n=6). (M) Representative immunoblot analysis of TfR1 and FPN, relative to b-actin levels (shown in histograms on the right), in the spleens of 12-week old HfeLysMCre mutant (n=5) and Hfeflox control mice (n=6). M: Prestained Protein Marker PageRuler. n indicates the number of mice used in the analysis. Data are shown as mean ± standard error of mean. Statistically significant differences are indicated as *P<0.05, **P<0.005, ***P<0.0005.
Figure 2.
Figure 2.
Lack of macrophage-Hfe in 45-week old mice is dispensable during parenteral iron overload but contributes to better survival of HfeLysMCre mutant mice during lipopolysaccharide-induced endotoxin shock. (A, B) Non-heme and plasma iron levels (A), and hepcidin mRNA expression (B) in 45-week old HfeLysMCre mutant (n=6) and control mice (n=6) following parenteral iron overload. Data are shown as the mean ± standard error of mean. Statistically significant differences are indicated as *P<0.05, **P<0.005, ***P<0.0005, ****P<0.0001. n indicates the number of mice used in the analysis. (C-F) Survival of 45-week old HfeLysMCre mutant and control mice (n=11, 11) (C), 12-week old HfeLysMCre mutant and control mice (n=10, 10) (D), 45-week old Hfe-/- and control mice, and mice maintained on an iron-deficient diet for 4 weeks (IDD) (n=13, 13, 12) (E) and 12-week old HfeAlfpCre, Hfe-/- and control mice (n=9, 9, 9), which were injected with lipopolysaccharide (10 mg/kg) and monitored every 6 h for up to 4 days. The percentage survival rate was expressed by Kaplan-Meier curves. Statistical analyses were performed using the log-rank test and the results were considered statistically significant when *P<0.05. ns: not statistically significant; Fe-ip: intraperitoneal iron injections; LPS: lipopolysaccharide.
Figure 3.
Figure 3.
Hfe in macrophages controls cellular iron homeostasis via ferroportin. (A) Intracellular total iron levels in bone marrow-derived macrophages (BMDM) from 45-week old Hfe-/-, HfeLysMCre and HfeAlfpCre mutant mice (n=3, 4). (B) Representative immunoblot analysis of transferrin receptor 1 (TfR1) and ferroportin (FPN) relative to b-actin levels (shown in histograms below) in 45-week old Hfe-/-, HfeLysMCre and HfeAlfpCre mutant mice. (C) Relative mRNA quantification of iron-related genes, inflammatory cytokines and toll-like receptors genes by real-time polymerase chain reaction in BMDM derived from 45-week old HfeLysMCre mutant and control mice (n=6, 6). (D, E) Intracellular total iron levels (D) and immunoblot analysis (E) of TfR1 and FPN, relative to b-actin levels (shown in histograms on the right), in BMDM from 12-week old HfeLysMCre mutant and Hfeflox control mice (n=5, 4). M: Prestained Protein Marker PageRuler (Thermo Scientific). (F) Schematic illustration of the Hfe cDNA construct cloned into the pcDNA6.2 vector under the control of the cytomegalovirus (CMV) promoter, tagged with V5 epitope at the C-terminal end and a poly-A tail. (G) Relative mRNA expression of Hfe and Fpn, and (H) intracellular total iron levels in transiently transfected BMDM with vector carrying the Hfe cDNA construct (indicated as ‘o/e Hfe’) or empty vector (‘mock’) (n=4, 5). (I) Representative immunoblot analysis, from two independent experiments, of FPN, TfR1, ferritin and relative quantification to β-actin levels (shown in histograms on the right) in BMDM transiently transfected with a vector carrying Hfe cDNA or an empty vector (‘mock’) (n=4, 6). (J) Phosphorylation status of STAT3(Ser727) in the BMDM from HfeLysMCre mutant and Hfeflox control mice (n=7, 7) and in BMDM transiently transfected with a vector carrying Hfe cDNA or an empty vector (‘mock’) (n=9, 9) measured by Bio-Plex Pro Cell Signaling MAPK Panel. AU: arbitrary units. Data are shown as mean ± standard error of mean. Statistically significant differences are indicated as *P<0.05, **P<0.005. n indicates the number of mice used in the analysis.
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