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. 2018 Oct 18;2(20):2732–2743. doi: 10.1182/bloodadvances.2018019737

Figure 5.

Figure 5.

Macrophage infusion restored the Kupffer cell population and iron homeostasis in the liver of Hmox1 KO mice. (A) Immunohistochemistry with the macrophage marker F4/80 detected Kupffer cells in the livers of WT (left) but not Hmox1−/− mice (middle). Macrophage infusion completely restored the macrophage populations in Hmox1−/− mice (right). Scale bar, 100 μm. (B) Quantification of F4/80 staining intensity for each group. No difference in staining was observed between the WT and macrophage-infused WT group, whereas the macrophage-infused KO group showed fivefold higher staining than that of the WT group (4 mice for each group). (C) Hmox1 immunofluorescence was observed in the liver of WT mice (left), but not in KO mice (middle). Macrophage infusion dramatically increased Hmox1 signal (right). Scale bar, 25 μm. (D) Quantification of Hmox1 staining intensity for each group. No difference was observed between the WT and macrophage-infused WT group. The macrophage-infused KO group showed threefold higher Hmox1 levels than the WT group (4 mice for each group). (E) Perls’ Prussian Blue iron staining of paraffin-embedded liver sections. Iron-positive Kupffer cells (indicated by arrows) were observed in WT mice (left), but not in KO mice (middle panel). Macrophage infusion dramatically increased the iron-positive Kupffer cells in KO mice (right panel). Of note, the hepatocytes were positive for iron staining in Hmox1 KO mice but not in WT or macrophage-infused Hmox1 KO mice. Scale bar, 100 μm. (F) Hmox1 and (G) CD163 mRNA levels in the liver of WT and KO mice without or with macrophage infusion (n = 6). (H) The percentage of WT cells in the liver of macrophage-infused KO mice was assessed by quantification of the WT Hmox1 genomic DNA in genomic DNA of actin. Primers that target exon 3 of the Hmox1 gene, which was deleted in Hmox1 KO mice, were used for the quantification. Samples were harvested 6 weeks (W; n = 3), 12 weeks (n = 6), and 22 weeks (n = 3) after macrophage infusion. All samples except panel H were harvested 12 weeks after macrophage infusion. (B,D) The intensity was analyzed using ImageJ. (B,D,F-H) Error bars represent the SD. Statistical analyses were performed using 2-way ANOVA (multiple comparisons). The graphs were done with GraphPad Prism software. *P < .05, **P < .01, ***P < .001, ****P < .0001. a.u., arbitrary unit; DAPI, 4′,6-diamidino-2-phenylindole.