Polarization dictates iron handling by inflammatory and alternatively activated macrophages

“M2 cells play a pivotal role in sustaining angiogenesis, wound repair and tumor growth.(41,42) These functions are associated with their ability to provide nutrients, including iron, to the healing tissues.”

“They involve specific well-defined gene signatures, which allow polarized macrophages to exert their biological functions.(16,19) The limited ability of M1 macrophages to recycle iron is possibly involved in the ability of these cells to control bacterial pathogenicity.”

“M1 macrophages differentiate under the control of cytokines produced by Th1 cells, and in turn sustain establishment and maintenance of Th1 responses.(3,39) Here we observed that M1 cells maintained the ability to present antigens to memory T cells even after iron depletion: this feature possibly allows them to keep a…”

“They involve specific well-defined gene signatures, which allow polarized macrophages to exert their biological functions.(16,19) The limited ability of M1 macrophages to recycle iron is possibly involved in the ability of these cells to control bacterial pathogenicity.”

“Gene-expression profiling and data analysis Gene expression profiling analysis was carried out as described with minor modifications.(9) Total cellular RNA was extracted from M1 and M2 polarized macrophages using the RNeasy midi kit, following the manufacturer’s recommendations.”

“Via the HIF1α pathway hypoxia elicits a coordinated response for controlling iron metabolism and oxygen transport, mediated via hepcidin/ferroportin.(44) Our study suggests a differential expression of the transcription factor in the M1 and M2 populations, and modulations at this level are possibly involved in macroph…”

“Its overexpression causes, for example, enhanced release of iron derived from the processing of phagocytosed erythrocytes.(26) Hemin has a role in the control of Fpn transcription.”

“Iron homeostasis requires the finely tuned expression of molecules involved in iron uptake, storage, export and heme degradation.(4,5) The transcriptional and post-transcriptional control of many of the genes responsible for these functions depends on inflammatory cytokines, free radicals, and on the ability of iron r…”

“Macrophages Bone marrow precursors from C57BL/6 female mice were isolated and propagated for 7 days in α-MEM (GIBCO, Invitrogen, Carlsbad, CA, USA) containing 10% fetal bovine serum (Lonza, Basel, Switzerland) in the presence of rm-M-CSF (100 ng/mL) to generate macrophages as described elsewhere.(7) Cells were culture…”

“Moreover, they are involved in wound healing and have immunoregulatory functions.(1) Cytokines, IFNγ and IL4, in particular, drive macrophage polarization.”

“using human monocyte-derived macro phages.(46) The conservation of the pathway between mouse and human clearly indicates that a dichotomy in iron handling represents a general feature of the functional polarization of macrophages.”

“Iron homeostasis requires the finely tuned expression of molecules involved in iron uptake, storage, export and heme degradation.(4,5) The transcriptional and post-transcriptional control of many of the genes responsible for these functions depends on inflammatory cytokines, free radicals, and on the ability of iron r…”

“Quantification of the labile iron pool The labile iron pool was measured by loading cells with the iron-sensitive probe Calcein-AM (Molecular Probes) as previously described.(13) Briefly, macrophages were incubated in 48-well plates with αMEM supplemented with 1 mg/mL bovine serum albumin and 0.”

“The transcriptional and post-transcriptional control of many of the genes responsible for these functions depends on inflammatory cytokines, free radicals, and on the ability of iron regulatory proteins 1 and 2 (IRP1, IRP2) to bind to iron responsive elements (IRE) on target mRNA.(6) In this study, we verified how the…”

“Heme binds Fpn promoter(33) and the heme protoporphyrin ring effectively increases Fpn transcription in immortalized murine macrophages.(34) FtH and Fpn protein levels are under the control of the iron-dependent IRP-IRE regulation.”

“Selective qPCR failed to detect the non-IRE Fpn isoform (data not shown), suggesting that both macrophage populations expressed only the IRE form.(18) DFO treatment further enhanced TfR1 expression in M2 macrophages, but not in M1 ones (Figure 2A,B; Online Supplementary Figure S3) and reduced Fpn in both cell types.”

“M2 macrophages satisfy these requirements: they are professional scavengers of senescent and apoptotic cells.(31,32) Furthermore we found that they express high levels of membrane molecules involved in iron internalization, such as TfR1 and the CD163 hemoglobin/haptoglobin receptor, and low levels of FtH.”

“This is relevant in view of the greater importance in iron regulation of IRP2 compared to IRP1 in cultured mammalians cells.(17) Next we explored whether iron regulation of IRP1 and IRP2 is maintained in both macrophage populations (Figure 1).”

“M2 macrophages satisfy these requirements: they are professional scavengers of senescent and apoptotic cells.(31,32) Furthermore we found that they express high levels of membrane molecules involved in iron internalization, such as TfR1 and the CD163 hemoglobin/haptoglobin receptor, and low levels of FtH.”

“Limited intracellular iron availability has been clearly demonstrated to impair intracellular pathogen growth thus facilitating the microbicidal function of macrophages.(2,20,21) It is tempting to speculate that this function is preferentially associated with macrophages polarized towards an M1 phenotype, which are ch…”

“Macrophage polarization was verified by flow cytometry after staining with fluorochrome-conjugated antibodies.(8) Briefly, 100,000 macrophages were incubated with fluorescent-specific antibodies or isotype control antibodies at 4°C for 20 min (final concentration 5 μg/mL) in phosphate-buffered saline containing 10% fe…”

“Limited intracellular iron availability has been clearly demonstrated to impair intracellular pathogen growth thus facilitating the microbicidal function of macrophages.(2,20,21) It is tempting to speculate that this function is preferentially associated with macrophages polarized towards an M1 phenotype, which are ch…”

“Interestingly, low environmental iron was recently reported to result in defective production of cytokines by activated macrophages.(40) Further studies are warranted to better characterize the effect of polarized iron handling by macrophages on acquired immune functions.”

“Our study suggests a differential expression of the transcription factor in the M1 and M2 populations, and modulations at this level are possibly involved in macrophage polarization.(45) Confirmation of this possibility will require further experiments.”

“Limited intracellular iron availability has been clearly demonstrated to impair intracellular pathogen growth thus facilitating the microbicidal function of macrophages.(2,20,21) It is tempting to speculate that this function is preferentially associated with macrophages polarized towards an M1 phenotype, which are ch…”

“M2 cells play a pivotal role in sustaining angiogenesis, wound repair and tumor growth.(41,42) These functions are associated with their ability to provide nutrients, including iron, to the healing tissues.”

“To evaluate (55)Fe incorporation into ferritin (Ft), equal amounts of proteins from supernatants were analyzed by non-denaturing PAGE and visualized by autoradiography.(12) In selected experiments, to detect FtH, equal amount of proteins were separated on 7.”

“Iron that is not released is mostly incorporated into Ft, the central protein for iron storage.(29,30) Therefore, macrophages in charge of recycling iron are expected to have a relatively high ability to internalize senescent cells, to endocytose transferrin-bound iron via TfR, and to release but not to store iron.”

“Legumain and transferrin receptor are two other target receptors overexpressed on M2-TAMs compared to M1TAMs but that are also present on several cancer cell types [200][201][202].”

“M2-polarized cells are thought to be specialized for iron recycling by expressing molecules involved in iron uptake and export (Corna et al., 2010).”