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S. Recalcati et al.
catabolism, as well as high expression of the iron exporter ferroportin (FPN).16
A tight control of iron metabolism is needed for appro- priate tissue homeostasis and healing. Excess iron, both in macrophages and in the extracellular milieu, has a delete- rious effect on tissue repair,17 and heme iron has pro-oxi- dant and pro-inflammatory properties, so that its clear- ance and degradation by M2 macrophages contributes to resolution.18 However, it is also conceivable that increased iron retention in macrophages leads to lower iron avail- ability for neighboring cells, thus compromising the trophic role of macrophages. In fact, given the necessity of iron for many essential biological functions, including cell replication,19,20 defective iron release can jeopardize iron-dependent functions essential for cutaneous home- ostasis and efficient tissue restoration. Decreased iron availability could impair the growth of fibroblasts, as well as epithelial and endothelial cells during new tissue for- mation. Moreover, the hydroxylases necessary for effi- cient collagen assembly during the repair phase are iron- dependent enzymes.21
In this study, we investigated the role of macrophage iron metabolism in tissue homeostasis and repair exploiting a mouse line with iron retention in macrophages caused by targeted FPN inactivation in cells of the myeloid lineage, thus avoiding artefactual systemic iron overload and other confounding elements, such as increased local iron accumu- lation in other cell types. Using the skin as a model tissue, we show that macrophage-dependent FPN-mediated iron release is required for hair growth in homeostatic condi- tions and for efficient wound healing, a process which is essential for survival and also clinically relevant, as non- healing wounds are a major clinical problem associated with various human diseases.22,23
Methods
Animals
The crossing of mice carrying a floxed Fpn allele (Fpnfl/fl),24 pro- vided by Dr Nancy Andrews, with mice expressing Cre under the control of the LysM promoter in the C57BL/6J background25 in order to generate mice with specific FPN-macrophage inacti- vation (Fpn1fl/flLysCre+/-) is described in detail in the Online Supplementary Material.
Procedures involving animals handling and care conformed with protocols approved by the Humanitas Clinical and Research Center in compliance with national (DL 116, GU suppl. 40, 18-2-1992; DL 26, GU 4-3-2014) and international law and policies (EEC Council Directive 2010/63/EU, OJ L 276/33, 22–09-2010; National Institutes of Health Guide for the Care and Use of Laboratory Animals, US National Research Council, 2011). The study was approved by the Italian Ministry of Health. All efforts were made to minimize the number of ani- mals used and their suffering.
Statistical analysis
Results are expressed as the mean ± standard error of mean. Statistical significance between two groups was assessed by an unpaired two-tailed Mann-Whitney test or Student t test with Prism software (GraphPad). For comparison of more than two groups, data were analyzed using one-way analysis of variance (ANOVA).
Full details of the Methods are available in the Online Supplementary Material.
Results
Ferroportin deletion in macrophages causes hair follicle alterations and alopecia
To generate mice that lack FPN in macrophages, we crossed Fpnfl/fl mice24 with LysMCre mice25 to create myeloid cell-specific FPN knockout mice. The phenotypic characterization of these Fpn1fl/flLysCre+/- mice is described in the Online Supplementary Material and illustrated in Online Supplementary Figure S1. FPN deletion in macrophages resulted in a significant decrease of hemato- logic parameters, such as hemoglobin level and hematocrit (Figure 1A,B), and red blood cell count, mean corpuscular volume and mean cell hemoglobin (Online Supplementary Table S1), at weaning (3 weeks after birth). Thereafter, both parameters rapidly returned to normal levels and remained almost unaltered until 18 weeks. In line with the mild ane- mia observed in 2-month old 129/SvEvTac mice lacking macrophage FPN,26 in weaned Fpn1fl/flLysCre+/- mice hemo- globin levels and hematocrit were lower than in Fpn1fl/flLysCre-/- mice at all time-points, although the differ- ence never reached statistical significance (Figure 1A,B). Serum iron levels and transferrin saturation showed a ten- dency to decrease with age, but were never statistically dif- ferent between Fpn1fl/flLysCre+/- mice and their control Fpn1fl/flLysCre-/- littermates. The hepatic expression of hep- cidin (HAMP), which regulates systemic iron homeostasis by inhibiting FPN,20 showed age-related variations but was not different between Fpn1fl/flLysCre+/- mice and their con- trol littermates. Similarly, we did not detect significant dif- ferences in skin HAMP mRNA levels, which were much lower than in the liver (Figure 1B), while hepcidin levels in skin lysates were undetectable. Accordingly, the expres- sion of Fam132b mRNA encoding for erythroferrone, the erythroid regulator of hepcidin,27 was unchanged in both spleen and bone marrow (Figure 1B and Online Supplementary Figure S2). Fpn1fl/flLysCre+/- mice showed dif- fuse alopecia with sparing of the head in 100% of both male and female mice until the fourth week of age (Figure 1A,C). Histological analysis in 3-week old mice showed no differences between the two genotypes in any organ eval- uated, with the exception of the increased iron accumula- tion in spleen and liver macrophages (Online Supplementary Figure S1) and a moderate/severe and diffuse/multifocal to coalescing dilatation of hair follicles, which contained rem- nants of hair shafts and keratin, and slight acanthosis of the superficial epidermis (Figure 1C). Alopecia gradually disap- peared and hair re-growth was evident starting 2 weeks after weaning (Figure 1A), but minor skin alterations were still detectable in adult Fpn1fl/flLysCre+/- mice, which had a reduced number of hair follicles, multifocal areas of hair shaft rarefaction and a thin hypodermis with an apparent increase of adipose tissue (Figure 1D). Taken together, these results indicate that targeted FPN deletion in macrophages results in severe alterations of the hair follicle and transient alopecia.
Alopecia is not related to systemic iron deficiency
In Fpn1fl/flLysCre+/- mice hair regrowth was not complete until 3 weeks after weaning, while hemoglobin levels and hematocrit had already returned to normal after 1 week (Figure 1A) and at all time-points there was no difference in serum iron availability between Fpn1fl/flLysCre+/- and control littermates. This suggested that alopecia in Fpn1fl/flLysCre+/- mice was not a local reflection of systemic iron deficiency
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haematologica | 2019; 104(1)