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Macrophage ferroportin is essential for stromal cell proliferation in wound healing

Iron recycling by macrophages is essential for erythropoiesis, but may also be relevant for iron redistribution to neighboring cells at the local tissue level. Using mice with iron retention in macrophages due to targeted inactivation of the iron exporter ferroportin, we investigated the role of macrophage iron release in hair follicle cycling and wound healing, a complex process leading to major clinical problems, if impaired. Genetic deletion of ferroportin in macrophages resulted in iron deficiency and decreased proliferation in epithelial cells, which consequently impaired hair follicle growth and caused transient alopecia. Hair loss was not related to systemic iron deficiency or anemia, thus indicating the necessity of local iron release from macrophages. Inactivation of macrophage ferroportin also led to delayed skin wound healing with defective granulation tissue formation and diminished fibroplasia. Iron retention in macrophages had no impact on the inflammatory processes accompanying wound healing, but affected stromal cell proliferation, blood and lymphatic vessel formation, and fibrogenesis. Our findings reveal that iron/ferroportin plays a largely underestimated role in macrophage trophic function in skin homeostasis and repair.


Immunoblot analysis.
For the preparation of membrane extracts, BMDM and PEC were homogenized in 10 mM Tris-HCl, pH 7.0, 1 mM MgCl2 and the postnuclear supernatant fraction obtained by centrifugation at 2000g for 10 minutes was ultracentrifuged at 150000g for 20 minutes to pellet crude membrane fractions (5).
Equal amounts of protein extracts were electrophoresed in acrylamide-SDS gels, electroblotted to Hybond membranes (Amersham Biosciences, Euroclone) and incubated with antibodies against FPN (1:250; Alpha Diagnostic, DBA, Segrate, Italy). After incubation with the secondary antibody, the protein was detected by means of chemiluminescence (ECL Plus; Amersham Biosciences, Euroclone). Signals were detected using the ChemiDoc Touch system (BioRad, Segrate, Italy) and signal intensity was quantified using ImageLab 5.2.1 software with the values being calculated after normalisation to amido black-stained proteins.

Histology and assessment of tissue sections.
The mice were received as whole body fixed. Organs and tissues were examined for gross lesions, trimmed after complete fixation and processed for paraffin blocks embedding. From each mouse 5 blocks/sections were obtained containing a scheduled list of organs/tissues. For histopathological analysis, 4 μm-thick tissue sections obtained from the paraffin blocks were stained with hematoxylin and eosin. For iron detection, 4 μm sections from each sample were stained with Perls' Prussian blue stain and semi-quantitatively evaluated under a light microscope as follows: 0 = absence of iron laden cells; 1 = rare iron laden cells; 2 = small number of iron laden cells; 3 = moderate numbers of iron laden cells; 4 = large number of iron laden cells.

Skin wound healing model.
To generate a punch wound, the back of anesthetized mice was shaved and exposed skin was cleaned with 70% ethanol. A full-thickness wound was created with a disposable 8-mm-diam biopsy punch by excising the skin and the underlying panniculus carnosus (6). Gentamicin was immediately applied and upon histological analysis no gross infection was apparent through day 14. Wound sites of each mouse were digitally photographed and measured every day in the course of wound healing.
The edges of the wounds were daily traced onto a transparency and the areas of open wounds were calculated using ImageJ software (National Institutes of Health). Changes of wound area over time were expressed as percentage of the initial wound areas. The animals were sacrificed at different times and skin biopsies were fixed with 4% paraformaldehyde, and embedded in paraffin or dehydrated with a sucrose gradient and embedded in OCT compound (Diapath, Bergamo, Italy) and stored at -80°C for histology or confocal microscopy analysis, respectively. Alternatively, skin biopsies were directly used for mRNA extraction, FACS and ELISA.

Blood analysis.
Blood was collected by cheek puncture. Hemoglobin, and hematocrit were measured using the Hemo Vet Instrument (Infratec, Portomaggiore, Italy) following manufacturer's instructions; red blood cells, MCV and MHC by using the Sysmex KX-21 automated analyser (Sysmex Italy Cornaredo).
Serum iron determination: blood was collected by cardiac puncture and serum iron was measured using standard laboratory procedures. Transferrin was measured using the mouse transferrin ELISA kit (ab157724, Abcam DBA, Italy) and transferrin saturation was calculated as serum iron concentration (µg/dL) x 100 / [serum transferrin (mg/dL) x 1.42].

ELISA assay.
Whole wounds were collected at 2, 7, and 12 days after wounding and homogenized in 50 mM Tris-HCl, pH 7.5, containing 2 mM EGTA, 1 mM PMSF, 100 KU aprotinin, 1% Triton X-100 (all from Sigma-Aldrich, Milano, Italy), and complete protease inhibitor cocktail (Roche, Monza, Italy). Total proteins were measured by DC Protein Assay, according to manufacturer's instructions (Bio-Rad Laboratories, Segrate, Italy). Cytokine levels were measured in accordance with the manufacturer's instructions (R&D Systems, Space, Milano, Italy). Hepcidin levels were measured using a specific kit (Cloud-Clone Co, Houston, TX, USA) according to the manufacturer's instructions.

Quantitative real-time polymerase chain reaction (qRT-PCR).
Total RNA isolated from liver, spleen, normal skin, wounds, bone marrow, PEC or BMDM using TRI reagent® (Sigma-Aldrich) was reverse transcribed into cDNA with Proto Script M-MuLV First Strand cDNA Synthesis Kit (New England Biolabs, Euroclone) and the obtained cDNA served as a template for Real-Time PCR, based on the TaqMan methodology (Life Technologies, Thermo Fisher). Thermal cycling parameters were 40 cycle at 95° C for 15s and 60° C for 1 min. Each sample was amplified in triplicate using the primers reported in Table S2     Erythroferrone (Fam132b) mRNA levels in the bone marrow of mice fed the normal or the iron-deficient diet at different age. mRNA levels were measured by quantitative RT-PCR and normalized to the housekeeping gene 18S RNA. Data are presented as mean ± SEM. n = 10 mice for each group. *** p < 0.0001.  CCL2, CXCL1, CXCL2, IL-1 , TGF , TNF and VEGF protein levels in wound lysates were assessed by ELISA at different times after skin injury. At the time points not shown, the levels of the respective cytokine were below the detection limit of the assay. The histograms show the mean ± SEM. n = 10 mice for each group.