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Stromal cells need macrophage iron
mesenchymal and stromal cells from receiving the iron supply necessary for growth/differentiation. In line with the higher FPN expression in M2 than in M1 macrophages,15,37 the lack of macrophage FPN exerts its major effects in the middle-late phase of repair when the M1 to M2 switch occurs. Indeed, in the late stages, nor- mal M2 skin macrophages export iron through enhanced FPN expression, whereas FPN-deficient macrophages accumulate iron with concomitant induction of ferritin and repression of TfR1 (Figure 4A). The lower fibrosis score (Figure 4D) and the decreased expression of colla- gen-1 and αSMA (Figure 6) show that the stromal compo- nent is compromised, as the absence of macrophage FPN resulted in iron deprivation and impaired proliferation of stromal cells (Figure 6). In this context, fibroblasts may not receive enough iron, which can be among the paracrine factors secreted by M2 macrophages to favor cell multiplication.38 A detrimental effect on collagen syn- thesis and assembly, which require iron-dependent prolyl hydroxylases,21 or other iron-dependent functions such as dihydroxy-docosahexaenoic acid production,39 may con- tribute to defective repair.
Our results also show that macrophage iron is essential for the development of the vascular network during tis- sue healing, as both lymphatic and blood vessels were reduced (Figure 6). Although the decrease of vascular structure caused by macrophage depletion was previous- ly ascribed to the reduced production of vascular endothelial growth factor and transforming growth fac- tor-β,9 the latter being also involved in extracellular matrix deposition and αSMA expression,40 in our experimental model the levels of these growth factors were unchanged (Online Supplementary Figure S5). Similarly, given that hemoglobin levels of adult Fpn1fl/flLysCre+/- mice were normal, defective oxygenation as a possible factor involved in impaired vascularization can be ruled out. Therefore, our results showing reduced neovessel densi- ty, reduced granulation tissue formation and decreased fibrosis in the absence of macrophage iron release, in the face of unchanged levels of prominent angiogenic and fibrogenic factors, such as vascular endothelial growth factor and transforming growth factor-β, support the rel- evance of the trophic role of macrophage-derived iron in the wound milieu.
Understanding the role of iron in macrophage produc- tion of inflammatory molecules has been hampered by contradictory findings. An increased inflammatory response was found in iron-depleted macrophages,41 but not in equally iron-deficient macrophages from HFE-/- mice,42 and other studies showed that iron levels correlate positively with the synthesis of pro-inflammatory cytokines.26,43 In addition, a pro-inflammatory state has been shown in macrophages and macrophage/microglia cells exposed to heme or iron28,29 and in hemorrhagic areas within tumors.44 Similarly, decreased iron release from
macrophages, associated with pro-inflammatory activa- tion and defective M2 polarization, impaired wound healing in chronic venous leg ulcers.17 Conversely, we found that iron retention in macrophages has no impact on leukocyte recruitment and activation as well as macrophage polarization (Figure 5 and Online Supplementary Figure S5). Moreover, in vitro polarized bone marrow-derived macrophages from the two mouse lines did not show differential expression of M1 and M2 mark- ers (Figure 5B). Therefore, in our model, iron accumula- tion does not exacerbate the pro-inflammatory pheno- type of wound healing-associated macrophages, in keep- ing with a recent study showing that iron did not increase M1 polarization of RAW264.7 macrophages.45 The con- flicting results may be related to the different experimen- tal models, the heterogeneity of macrophages and the exposure to different iron sources, such as heme iron which is highly toxic.46 In the absence of FPN, macrophages from Fpn1fl/flLysCre+/- mice accumulate iron in ferritin, which increases less than 2-fold (Figure 4A), but iron deposition seems less massive than in conditions such as chronic ulcers,17 in which iron content may increase 20-fold,47 or hemolysis.29,44 In our experimental setting iron accumulation may, therefore, be insufficient to interfere with the M1/M2 switch and favor a pro- inflammatory state. A recent study demonstrated that FPN downregulation in macrophages impaired skeletal muscle regeneration after injury,48 but the effect of increased iron accumulation on the inflammatory profile of macrophages was not addressed.
In conclusion, the results of our study indicate that local macrophage FPN, by supplying iron to cells in the microenvironment, affects both the physiological context of follicular anagen and the pathophysiological context of wound healing. In its absence, stromal cells are iron-defi- cient and their proliferation is impaired (Figures 3 and 6). The importance of local iron recycling is underlined by the lack of changes in hepatic and skin hepcidin. A similar requirement for iron provided locally by macrophages has been described for the repair of skeletal muscle cells, in which iron retention in macrophages, by impairing myoblast proliferation, results in smaller myofibers.48 Iron should, therefore, be added to the list of trophic media- tors produced locally by macrophages that stimulate the growth, differentiation and activity of neighboring parenchymal and stromal cells in order to maintain tissue homeostasis or repair.
Acknowledgments
This work was supported by grants from the Italian Association for Cancer Research (AIRC-IG 2016 #19213 to ML) and MIUR (COFIN to GC). The authors would like to thank Nancy Andrews for providing Fpnfl/fl mice, Alberto Mantovani for support and helpful comments, and Eugenio Scanziani and Camilla Recordati for their help with the histological analysis.
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