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S. Recalcati et al.
but a consequence of iron sequestration in skin macrophages, ultimately resulting in impaired hair follicle growth. To further assess this issue, we analyzed hemato- logic parameters and alopecia after exposure to low iron diet according to the protocol outlined in Figure 2A. Until week 5, both Fpn1fl/flLysCre+/- and control littermates were anemic, and hemoglobin levels and other hematologic parameters were always slightly and not significantly lower in animals lacking macrophage FPN (Figure 2B,C, Online Supplementary Table S1). Serum iron levels and trans- ferrin saturation were lower in 3-week old Fpn1fl/flLysCre+/- mice than in control littermates, although not statistically significantly so, but returned to normal levels at 6 weeks without differences between the two strains (Figure 2C). Liver hepcidin expression was repressed by the iron-defi- cient diet, but was not different between Fpn1fl/flLysCre+/- mice and their control littermates (Figure 2C). HAMP mRNA levels in the skin were below the threshold of detection. A significant increase in erythroferrone expres- sion was found in Fpn1fl/flLysCre+/- pups at 3 weeks (Figure 2C and Online Supplementary Figure S2), which is indicative of higher erythropoietic activity. After the introduction of the normal diet, both hepcidin and erythroferrone expres- sion returned to normal levels (Figure 2C and Online Supplementary Figure S2). Mice with loss of macrophage FPN were grossly affected by diffuse alopecia of the trunk throughout the period of exposure to the low iron diet, whereas Fpn1fl/flLysCre-/- mice, despite low serum iron avail- ability, did not develop alopecia (Figure 2B). Remarkably, alopecia did not appear in Fpn1fl/flLysCre-/- mice even after exposure to the iron-deficient diet for 11 weeks. In Fpn1fl/flLysCre+/- mice, after reintroduction of a normal diet, hemoglobin and serum iron returned to normal levels 2 weeks before the restoration of normally haired skin (Figure 2B,C), thus indicating that alopecia and hypofer- remia/anemia are not associated. Histological analysis showed that in Fpn1fl/flLysCre+/- mice challenged with the low iron diet alopecia was associated with severe follicular keratosis with intraluminal accumulation of keratin and distorted hair shafts and subsequent dilation of the hair fol- licles. Conversely, no relevant histopathological changes were found in the haired skin of the Fpn1fl/flLysCre-/- mice maintained in the same dietary conditions (Figure 2D). Both in Fpn1fl/flLysCre+/- and Fpn1fl/flLysCre-/- mice main- tained under iron deprivation conditions for 5 weeks and subsequently fed a normal diet for 2 weeks, skin histology showed that hair follicles were in the anagen stage, but in Fpn1fl/flLysCre+/- mice hair shafts did not exit the follicular ostia and follicular keratosis/dilation, increased epidermal hyperplasia and dermal inflammation were observed (Figure 2E).
Ferroportin deletion in macrophages leads to epithelial iron deficiency and decreased proliferation in cutaneous hair follicles
Since we showed that iron released by macrophages via FPN supports in vitro cell proliferation,15 an important role for FPN in skin macrophages could be to mediate the release of sufficient iron in the microenvironment for cell multiplication. Indeed, confocal microscopy revealed a sig- nificantly lower expression of the proliferation marker Ki67 in the epithelial cells of the hair bulbs of 3-week old Fpn1fl/flLysCre+/- mice (Figure 3). Conversely, in the same cells we found a strong increase of transferrin receptor (TfR1) expression, which is indicative of cellular iron dep-
rivation (Figure 3). Notably, F4/80+ macrophages, which are abundant in the skin stroma but with no differences in number between Fpn1fl/flLysCre+/- and Fpn1fl/flLysCre-/- mice (Figure 3), expressed lower levels of both Ki67 and TfR1 but had an increased content of both the L and H subunits of the iron storage protein ferritin (Figure 3 and Online Supplementary Figure S3) as compared to epithelial cells. Qualitative analysis also showed that in Fpn1fl/flLysCre-/- mice ferritin is detectable only in epithelial cells (Online Supplementary Figure S3), whereas in Fpn1fl/flLysCre+/- mice ferritin expression is particularly strong in F4/80+ macrophages. These results suggest that iron retention in resident macrophages, by starving neighboring hair follicle cells of iron and hence inhibiting their proliferation, has detrimental effects on tissue homeostasis.
Ferroportin deletion in macrophages compromises wound healing
Resident macrophages support parenchymal cells with trophic signals, particularly under conditions character- ized by increased cell proliferation, such as during tissue repair following injury.2 To test the role of macrophage- derived iron in this context, we investigated the wound healing process after incisional skin damage during the entire time course of repair, i.e. the early-inflammatory [2 days post injury (dpi)], middle-proliferative (7 dpi), and late-remodeling phases (12 dpi). We first investigated FPN expression in FACS-sorted macrophages from wounds; in Fpn1fl/flLysCre-/- mice FPN mRNA levels progressively increased during repair (Figure 4A), suggesting a predomi- nant role of FPN in the late phases, whereas, as expected, FPN mRNA was always barely detectable in Fpn1fl/flLysCre+/- mice. The analysis of other iron-related genes showed a rise in the expression of TfR1, which medi- ates iron uptake, and a decrease of ferritin H subunit during the middle-late phase of repair in macrophages of Fpn1fl/flLysCre-/- mice, but not Fpn1fl/flLysCre+/- mice, which is evidence of iron deposition in these cells (Figure 4A). Accordingly, histological analysis showed iron accumula- tion in wound macrophages of Fpn1fl/flLysCre+/- mice (Figure 4B). Hepcidin-dependent FPN modulation should not play a role in wound healing, as no difference was seen in liver HAMP expression between Fpn1fl/flLysCre-/- and Fpn1fl/flLysCre+/- mice during wound repair (Online Supplementary Figure S4A) and HAMP expression in FACS- sorted macrophages was undetectable. Hepcidin levels in the wound lysate, which were much lower than in serum, were not different between Fpn1fl/flLysCre-/- and Fpn1fl/flLysCre+/- mice (Online Supplementary Figure S4B). Macroscopic analysis of wound size showed that the process of closure was considerably delayed in Fpn1fl/flLysCre+/- mice than in control littermates, with sig- nificantly wider lesions at all time points and a lag of 3-5 days at 3 dpi through 12 dpi (Figure 4C). Histological analy- sis performed according to the criteria described in Online Supplementary Table S2 supported this observation, as Fpn1fl/flLysCre+/- mice displayed a more prolonged inflam- matory response and delayed granulation tissue formation, associated with diminished fibroplasia, whereas mononu- clear cells and granulocytes were unchanged (Figure 4D).
Ferroportin deletion in macrophages has no impact on leukocyte recruitment and activation in the wound
Given the role of leukocytes in tissue repair,23 we evaluat- ed leukocyte recruitment in our experimental setting.
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haematologica | 2019; 104(1)