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S. Recalcati et al.
derived macrophages exposed in vitro to polarization stimuli, but again no difference was observed in markers for both M1 (iNOS and TNFα) and M2 (Arg1 and YM1) macrophages (Figure 5B). The expression of iron-related genes in bone marrow-derived macrophages of Fpn1fl/flLysCre-/- mice mirrored the pattern previously observed in human polarized macrophages,15 with elevat- ed expression of FPN, TfR1 and the hemoglobin/hapto- globin complex receptor CD163 in M2 macrophages. This finding is in line with the prominent expression of FPN in macrophages during the late phase of repair, when the M2 cell infiltrate is increased (Figures 4A and 5A). Deletion of macrophage FPN resulted in lower expression of TfR1 and CD163 transcript levels in M2 macrophages (Figure 5B), possibly as a consequence of iron accumula- tion.
Ferroportin deletion in macrophages affects stromal cells during wound healing
Since FPN deletion in macrophages significantly affect- ed the wound healing process but iron accumulation in FPN-deficient macrophages did not alter the inflammato- ry processes associated with wound healing, we evaluat- ed whether the defective iron release by FPN-deficient macrophages affected the biology of surrounding stromal cells in the wound tissue. Confocal analysis at 7 dpi showed reduced expression in Fpn1fl/flLysCre+/- mice, as compared to control littermates, of blood (CD31) and lymph vessel (Lyve-1) endothelium markers (Figure 6). This was accompanied by decreased expression of platelet-derived growth factor receptor-α, a marker of mesenchymal cells, and lower levels of collagen I and alpha smooth muscle actin (αSMA), which are markers of activated fibroblasts and myofibroblasts, respectively (Figure 6). Moreover, in the absence of macrophage FPN, surrounding stromal cells were iron-deficient, as indicat- ed by upregulation of TfR1 expression, and proliferated less than control counterparts, as shown by decreased Ki67 expression (Figure 6). Taken together, these results indicate that the iron retention in macrophages caused by FPN deletion impairs blood vessel formation and stromal cell proliferation, leading to delayed skin repair.
Discussion
The role of erythrophagocytic macrophages as a source of iron for erythropoiesis is well established.16 However, macrophages may also be involved in iron redistribution at a local tissue level, thus affecting neighboring cells. We previously showed that FPN-mediated iron release from human macrophages supports in vitro cell proliferation.15 In the present study, we showed that a steady supply of iron released by macrophage FPN is essential for tissue homeostasis in two conditions, follicular development and wound healing, which share many similarities, including fast cell growth rate.6 Our study, therefore, underlines a new iron-related function of macrophages in tissue homeostasis and regeneration, in line with the increasing recognition of these cells’ considerable func- tional polyvalence and trophic role, in addition to estab- lished immunological functions.30 We did not address the effect of FPN gene deletion in other myeloid cells affected in the LysM conditional model here adopted as their con- tribution to iron storage and release is negligible com-
pared to that of macrophages.11,13
Our findings showing impaired hair follicle growth in
mice with FPN deficiency in macrophages are in line with the report of similar hair and skin lesions in mice with altered expression of other proteins of iron metabolism,31- 33 although in these other settings the presence of systemic iron deficiency/anemia did not allow the relative contribu- tions of circulating iron versus local availability of macrophage-derived iron to be distinguished. We showed that the alopecia in mice with loss of macrophage FPN was not related to limited systemic iron availability, as evi- denced by the lack of differences in serum iron availability and the similar hepatic and local hepcidin expression (Figure 1). Evidence that local iron release from macrophages, which are abundant in skin tissue (Figure 3), is more important than systemic iron levels was also pro- vided by the persistence of alopecia after the return of nor- mal hemoglobin and body iron levels (Figures 1 and 2), and by the absence of hair loss in hypoferremic and ane- mic Fpn1fl/flLysCre-/- mice (Figure 2), even when fed an iron-deficient diet for a long time. In line with the alopecia and delayed entry of the hair follicle into anagen exhibited by mice overexpressing H ferritin,34 we provide evidence that iron release from macrophages is required to sustain the rapid multiplication of hair follicle cells (Figure 3). In the absence of macrophage FPN, follicle epithelial cells are iron-deficient, as demonstrated by the increased expres- sion of TfR1, and have a lower replication rate, as indicat- ed by reduced Ki67 levels. The discrepancy with the lack of alopecia in a similar model of macrophage-specific FPN inactivation reported by Zhang and colleagues26 could be explained by the different iron content of the standard diet used (157 ppm versus 232, respectively) and by the differ- ent genetic backgrounds of the mice. Indeed, the role of dietary iron absorption, which is more important in mice than in humans,35 in correcting the alopecia was also indi- cated by the effect of switching to chow diet at weaning. Alopecia may result from insufficient iron availability caused by decreased local iron release (this study), Matriptase-dependent severe systemic iron deficiency32,33 (although the role of local FPN was not addressed in those studies) and iron sequestration in ferritin.34 The absence of alopecia in Fpn1fl/flLysCre-/- mice kept on an iron-deficient diet for almost 3 months suggests that local iron release may provide iron more directly in a paracrine fashion when circulating iron levels fall. We conclude that the essential role of macrophages in hair follicle cycling5 is not only related to their production of growth stimulators, such as Wnt,7 but also to the ability to supply the growing tissue with iron. Macrophages are part of the nurturing niche of stem cells in various tissues,36 including tumors. The results reported here in skin hair follicles raise the possibility that macrophage-dependent iron provision has a more general role in different stem cell niches.
We also report here a similar role for macrophage- derived iron during skin wound healing, a complex tissue repair process consisting of overlapping phases of inflam- mation and tissue remodeling in which macrophages play a key role.2 The use of mice lacking FPN selectively in cells of the myeloid lineage allowed us to define the role of macrophage iron in wound healing in the absence of the systemic iron overload and large local iron accumula- tion present in other models.17,28 In the present setting, the disruption of iron export from local macrophages delayed wound healing, apparently by preventing neighboring
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haematologica | 2019; 104(1)