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. 2023 Jan 31;12:e79196. doi: 10.7554/eLife.79196

Figure 1. Iron-reduced diet normalizes body iron parameters during aging, diminishes iron retention in RPMs, and prevents oxidative stress.

(A) Splenic and (B) liver non-heme iron content was determined in young, aged, and aged IR mice. (C) Plasma transferrin saturation was determined in young, aged, and aged IR mice. (D) Relative mRNA expression of hepcidin (Hamp) in the liver of young, aged, and aged IR mice was determined by qPCR. (E) Serum IL-6 protein levels in young, aged, and aged IR mice were measured by Mouse IL-6 Quantikine ELISA Kit. (F) Expression of ferroportin (FPN) on the cell membrane of young, aged, and aged IR RPMs was assessed by flow cytometry. (G) Intracellular H-Ferritin protein levels in young, aged, and aged IR RPMs were quantified by flow cytometry. (H) Cytosolic ferrous iron (Fe2+) levels in young, aged, and aged IR RPMs were measured using FerroOrange with flow cytometry. (I) The total intracellular iron content in young, aged, and aged IR magnetically-sorted RPMs was assessed using the Iron Assay Kit. (J) The cytosolic ROS levels in young, aged, and aged IR RPMs were assessed by determining CellROX Deep Red fluorescence intensity with flow cytometry. (K) The percentage of RPMs from CD45 + live cells present in the spleen of young, aged, and aged IR mice was assessed by flow cytometry. (L) Erythrophagocytosis capacity in young, aged, and aged IR RPMs was determined using flow cytometry by measuring the percentage of RPMs that phagocytosed transfused PKH67-labeled temperature-stressed RBCs. (M) Lysosomal activity of young, aged, and aged IR RPMs was determined using Lysosomal Intracellular Activity Assay Kit with flow cytometry. (N) Erythrophagocytosis capacity in young, aged, and aged IR Kupffer cells (KCs) was determined using flow cytometry by measuring the percentage of KCs that phagocytosed transfused PKH67-labeled temperature-stressed RBCs. (O) Percentages of KCs in total live cells in the livers of young, aged, and aged IR mice and representative flow cytometry plots of KCs. Each dot represents one mouse. Data are represented as mean ± SEM. Statistical significance among the three groups was determined by One-Way ANOVA test with Tukey’s Multiple Comparison test. ns p>0.05, *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001.

Figure 1—source data 1. Related to Figure 1A–O.

Figure 1.

Figure 1—figure supplement 1. RPMs of aged mice show increased labile iron levels, oxidative stress and diminished iron-recycling functions.

Figure 1—figure supplement 1.

(A–D) Spleen, muscle and heart non-heme iron content was determined in young and aged mice. (E) Plasma transferrin saturation and (F) blood hemoglobin (Hgb) concention were determined in young and aged mice. (G) Intracellular H-Ferritin and (H) L-Ferritin expression levels were quantified in young and aged RPMs with flow cytometry. (I) Cytosolic ferrous iron (Fe2+) and (J) ROS levels in young and aged RPMs were quantified using the FerroOrange and CellROX Deep Red probe, respectively, with flow cytometry. (K) The phagocytosis capacity for PKH67-labeled temperature-stressed RBCs and (L) zymosan A fluorescent particles by young and aged RPMs were analyzed ex vivo by flow cytometry. The percentage of cargo-positive RPMs is indicated. (M) Lysosomal activity in young and aged RPMs was determined using Lysosomal Intracellular Activity Assay Kit with flow cytometry. (N) Mitochondrial activity in young and aged RPMs was determined using the TMRE probe with flow cytometry. Each dot represents one mouse. Data are represented as mean ± SEM. Welch’s unpaired t-test determined statistical significance between the two groups. ns p>0.05, *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001.
Figure 1—figure supplement 1—source data 1. Related to Figure 1—figure supplement 1A–N.
Figure 1—figure supplement 2. Gating strategy for RPMs.

Figure 1—figure supplement 2.

(A) Non-permeabilized cells are shown, including an FMO control. (B) Permeabilized cells are shown.
Figure 1—figure supplement 3. Validation of the antibodies against H and L ferritin.

Figure 1—figure supplement 3.

(A) Shown is the intracellular staining and flow cytometric analysis of H-ferritin in bone marrow-derived macrophages treated with an iron chelator DFO for 18 hr. An expected drop in fluorescence intensity was observed. (B) Shown is the intracellular staining and flow cytometric analysis of L-ferritin in endothelial cells treated with Holo-ferritin for 9 hr (blue) compared to untreated cells (red). The uptake of ferritin, which was independently confirmed with other methods, is observed. Each dot represents one independent cell-based experiment. Data are represented as mean ± SEM. Welch’s unpaired t-test determined statistical significance between the two groups. **p<0.01.
Figure 1—figure supplement 3—source data 1. Related to Figure 1—figure supplement 3A.
Figure 1—figure supplement 4. Peritoneal macrophages in aged mice do not show functional impairments.

Figure 1—figure supplement 4.

(A) Gating strategy for peritoneal macrophages. (B) The cytosolic ROS levels in young and aged peritoneal macrophages were quantified using CellRox Deep Red with flow cytometry. (C) Lysosomal activity in young and aged peritoneal macrophages was determined using Lysosomal Intracellular Activity Assay Kit with flow cytometry. (D) Mitochondrial activity in young and aged peritoneal macrophages was determined using the TMRE probe with flow cytometry. Each dot represents one mouse. Data are represented as mean ± SEM. Welch’s unpaired t-test determined the statistical significance between the two groups. ns p>0.05.
Figure 1—figure supplement 4—source data 1. Related to Figure 1—figure supplement 4B–D.
Figure 1—figure supplement 5. Hematological parameters and erythropoietic activity in aging mice.

Figure 1—figure supplement 5.

(A) Blood hemoglobin (Hgb) concentration, (B) hematocrit (HCT), (C) RBC counts, (D) mean corpuscular volume (MCV) and (E) mean corpuscular hemoglobin (MCH) were determined in young, aged, and aged IR mice. (F) Shown is the percentage of erythroid progenitor cells (EPCs, TER119+ CD71+) present in the bone marrow and (G) the spleen of young, aged, and aged IR mice. (H) EPO concentration in the plasma of young, aged, and aged IR mice was measured by Mouse Erythropoietin/EPO Quantikine ELISA Kit. Each dot represents one mouse. Data are represented as mean ± SEM. Statistical significance among the three groups was determined by the One-Way ANOVA test with Tukey’s Multiple Comparison test. ns p>0.05, *p<0.05, **p<0.01.
Figure 1—figure supplement 5—source data 1. Related to Figure 1—figure supplement 5A–H.
Figure 1—figure supplement 6. Gating strategy for erythroid progenitor cells in the spleen (A) and the bone marrow (B).

Figure 1—figure supplement 6.

Figure 1—figure supplement 7. Validation of the ferroportin antibody for flow cytometry.

Figure 1—figure supplement 7.

Shown is the cell membrane staining and flow cytometric analysis of ferroportin (FPN) in bone marrow-derived macrophages treated with ferric ammonium citrate (FAC, 50 µM, 24 hr) an iron chelator DFO (100 µM, 18 hr). Expected changes in fluorescence intensity were observed. Each dot represents one mouse. Data are represented as mean ± SEM. Statistical significance among the three groups was determined by the One-Way ANOVA test with Tukey’s Multiple Comparison test. **p<0.01 and ***p<0.001.
Figure 1—figure supplement 7—source data 1. Related to Figure 1—figure supplement 7.
Figure 1—figure supplement 8. RPMs derived from aged IR versus aged mice show higher proliferation capacity.

Figure 1—figure supplement 8.

(A) Heat map representing 55 differentially regulated genes between aged and aged IR RPMs. Genes positively involved in proliferation control are marked in green, and those that negatively regulate proliferation are highlighted in red. (B) Intracellular Ki-67 protein levels in RPMs derived from aged, and aged IR mice were assessed by flow cytometry. Each dot in (B) represents one mouse. Data are represented as mean ± SEM. Welch’s unpaired t-test determined the statistical significance between the two groups. *p<0.05.
Figure 1—figure supplement 8—source data 1. Related to Figure 1—figure supplement 8B.
Figure 1—figure supplement 9. RPM depletion in aged mice affects embryonically-derived RPMs and is not a consequence of diminished recruitment from monocytes.

Figure 1—figure supplement 9.

(A) Percentage of the total, monocyte-derived (M), and embryonically-derived (E) RPMs from CD45+ live cells present in the spleen of 8-, 20-, and 36-week-old mice. The results represent a re-analysis of the data from Liu et al., 2019. (B) Percentage of splenic monocytes (CD11b-high, F4/80-low) from CD45+ cells. (C) Percentage of splenic pre-RPMs (CD11b-high, F4/80 high) from CD45+ cells. (D) Percentage of splenic granulocytes (Gr1-high) from CD45+ cells. Each dot represents one mouse. Data are represented as mean ± SEM. Statistical significance among the three groups was determined by the One-Way ANOVA test with Tukey’s Multiple Comparison test; Welch’s unpaired t-test determined statistical significance between the two groups . ns p>0.05, *p<0.05, **p<0.01.
Figure 1—figure supplement 9—source data 1. Related to Figure 1—figure supplement 9A–D.