Iron regulatory protein (IRP)-mediated iron homeostasis is critical for neutrophil development and differentiation in the bone marrow

Abstract

Iron is mostly devoted to the hemoglobinization of erythrocytes for oxygen transport. However, emerging evidence points to a broader role for the metal in hematopoiesis, including the formation of the immune system. Iron availability in mammalian cells is controlled by iron-regulatory protein 1 (IRP1) and IRP2. We report that global disruption of both IRP1 and IRP2 in adult mice impairs neutrophil development and differentiation in the bone marrow, yielding immature neutrophils with abnormally high glycolytic and autophagic activity, resulting in neutropenia. IRPs promote neutrophil differentiation in a cell intrinsic manner by securing cellular iron supply together with transcriptional control of neutropoiesis to facilitate differentiation to fully mature neutrophils. Unlike neutrophils, monocyte count was not affected by IRP and iron deficiency, suggesting a lineage-specific effect of iron on myeloid output. This study unveils the previously unrecognized importance of IRPs and iron metabolism in the formation of a major branch of the innate immune system.

Fig. 1.. Acute IRP ablation during adulthood causes erythropenia and myelopenia.

P1/2-CTR and P1/2-KO littermates received tamoxifen on days 1 and 3 to induce IRP ablation. Mice were analyzed on day 10. (A) Genomic polymerase chain reaction (PCR) analysis of the Irp1 (Aco1) and Irp2 (Ireb2) alleles in tissues from P1/2-KO mice. The floxed (flox) and truncated (Δ) alleles are indicated. (B) RBC parameters (P1/2-CTR, n = 16; P1/2-KO, n = 18). HGB, hemoglobin; MCV, mean corpuscular volume; MCHC, mean corpuscular hemoglobin concentration. (C) Hepatic and splenic iron levels (n = 11). (D) Serum iron parameters (n = 10). (E) RBC decay markers (n = 10). (F) Serum levels of EPO (P1/2-CTR, n = 18; P1/2-KO, n = 17) and hepcidin (P1/2-CTR, n = 18; P1/2-KO, n = 16). (G) Spleen index = √[(100 × spleen weight in milligrams per body weight in grams)] (n = 7), and reticulocyte frequency in peripheral blood (PB) (P1/2-CTR, n = 7; P1/2-KO, n = 9). A.U., arbitrary units. (H) White blood cell (WBC) and platelet counts in PB (P1/2-CTR, n = 16; P1/2-KO, n = 18). (I) Flow cytometry (FCM) analysis of major WBC populations in PB. The gating strategy is shown on contour plots on the left. The histograms display cell counts for 3 × 10⁵ events recorded (n = 8). (B to I) The results are presented as box plots (minimum to maximum values). Unpaired, two-tailed t test between P1/2-CTR and P1/2-KO. *P < 0.05; **P < 0.01; ***P < 0.001. APC, allophycocyanin; PE, phycoerythrin.

Fig. 2.. Immuno-phenotyping of BM cell populations in adult mice with acute loss of IRP function.

(A) BM cellularity (n = 8). (B) Schematic representation of the hematopoietic system (created with BioRender.com) with self-renewing multipotent LT-HSCs giving rise to ST-HSCs that progress to lineage-committed progenitors with increasingly limited differentiation and self-renewal capacity and lastly to terminally differentiated hematopoietic cells. (C to G) FCM analysis of BM cell populations: (C) LT- and ST-HSCs (P1/2-CTR, n = 25; P1/2-KO, n = 26) and MPPs (P1/2-CTR, n = 18; P1/2-KO, n = 20); (D) CMP (n = 10), CLP (P1/2-CTR, n = 5; P1/2-KO, n = 6), MEP (n = 10), and GMP (n = 10); (E) Mgk (P1/2-CTR, n = 7; P1/2-KO, n = 9); (F) T and B cells, and myeloid cells (granulocytes/monocytes) (n = 8); (G) monocytes (MON) versus neutrophils (NEU; n = 7). The gating strategy is indicated for each cell population analyzed. Box plots (minimum to maximum values) show the number of cells in the BM of both hindlimbs. The data are presented as box plots (minimum to maximum values). Unpaired, two-tailed t test between P1/2-CTR and P1/2-KO. *P < 0.05; **P < 0.01; ***P < 0.001. Genomic PCR panels below the box plots show recombination of the Irp1 (Aco1) and Irp2 (Ireb2) alleles in cell populations isolated from the BM of P1/2-KO (right) versus P1/2-CTR (left) mice by FCM-activated cell sorting (floxed allele, flox; truncated allele, Δ). FITC, fluorescein isothiocyanate.

Fig. 3.. IRP deficiency impairs neutropoiesis.

(A) FCM analysis of terminal erythroid differentiation (FSCA, forward side scatter area) in the BM (Pro: pro-erythroblasts; Baso, Poly, and Ortho: baso, poly-, and ortho-chromatic cells, respectively; Retic: reticulocytes). Box plots (minimum to maximum values, n = 5 to 6) show the number of TER119⁺ in the BM of both hindlimbs (left), the viability of TER119⁺ cells (middle), and the frequency of cells at different stages of erythroid differentiation (right). The panels below the box plots show the recombination of Irp1 (Aco1) and Irp2 (Ireb2) alleles in P1/2-KO (right) versus P1/2-CTR (left) mice. BUV, brilliant ultraviolet. (B) FCM analysis of neutrophil differentiation in the BM. c-KIT^highLY6G⁻ represent progenitor (Prog) cells; c-KIT⁻LY6G^high, polymorphonuclear (PMN) neutrophils; c-KIT⁻LY6G^low, preneutrophils (prNeu). Box plots (minimum and maximum values) show the overall frequency of Prog, prNeu, and PMN cells in the BM (left), and the corresponding fraction of Zombie dye-positive cells (right) (P1/2-CTR, n = 6; P1/2-KO, n = 7). (C and D) FCM analysis of (C) ROS (reactive oxygen species) production and (D) engulfment of pHrodo-labeled S. aureus particles. Top: Representative FCM plots. Bottom: Data obtained from five mice (box plots with minimum to maximum values). In (D), the dashed line delimits pHrodo-positive and -negative cells. (A to D) P values correspond to separate pairwise comparisons (unpaired two-tailed t test) between P1/2-CTR and P1/2-KO for each parameter and cell population analyzed (*P < 0.05; **P < 0.01; ***P < 0.001).

Fig. 4.. IRPs promote neutropoiesis in a cell-autonomous manner.

(A) Top: CD45.1⁺ BM cells from WT mice transplanted into irradiated P1/2-CTR (n = 3) versus P1/2-KO (n = 10) recipients. (B) Top: CD45.2⁺ BM cells from P1/2-CTR (n = 9) versus P1/2-KO (n = 10) mice transplanted into WT recipients. (C) Top: WT recipients transplanted with a 1:1 mixture of BM cells from WT and either P1/2-KO (n = 6) or P1/2-CTR (n = 7) mice. (A to C) Following stable engraftment, chimeras were treated with tamoxifen on days 1 and 3 and were analyzed on day 10. Representative FCM plots based on c-KIT and LY6G markers are shown (same gating strategy as in Fig. 3B). Bar graphs (means + SEM) and box plots (minimum to maximum values) display monocyte counts in the BM of both hindlimbs and the frequency of Prog, prNeu, and PMN cells. (D) LIN⁻ BM cells from tamoxifen-treated P1/2-CTR versus P1/2-KO mice were expanded ex vivo with KIT ligand (KITLG) and interleukin-3 (IL-3) and then differentiated into LY6G⁺ neutrophils with G-CSF/CSF3. Bottom: Representative FCM plot analysis of c-KIT (top) and LY6G (bottom); the dashed line delimits marker-positive and -negative cells. Box plots (minimum to maximum values, n = 7) display the median fluorescence intensity (MFI) for c-KIT and LY6G, respectively, and the percentage of cells positive for those markers. Comparisons between WT➔CTR and WT➔KO (A), P1/2-CTR(+WT)➔WT and P1/2-KO(+WT)➔WT (C), or WT➔WT(+P1/2-CTR) and WT➔WT(+P1/2-KO) (C) were made using the Mann-Whitney test. Comparisons between CTR➔WT and KO➔WT (B) were made using unpaired two-tailed t test. *P < 0.05; **P < 0.01; ***P < 0.001. (A to D) Top schemes created with BioRender.com.

Fig. 5.. Impact of IRP deficiency on gene expression dynamics during neutropoiesis.

(A to D) RNA sequencing (RNA-seq) analysis of the transcriptome of Prog, prNeu, and PMN cell populations isolated from the BM of P1/2-CTR versus P1/2-KO mice (n = 3). (A) Principal components (PC) analysis. (B) Genes differentially expressed during neutropoiesis were grouped in four main categories using k-means clustering (left). The pie charts (right) indicate (i) the total number of genes in a cluster, (ii) the number of genes found in that cluster only in P1/2-CTR (white) or in P1/2-KO (orange) cells, respectively, and (iii) the number of genes with similar expression patterns in both P1/2-CTR and P1/2-KO cells (green). (C) Heatmaps display expression profiles that differ between P1/2-CTR and P1/2-KO cells. For each cluster in P1/2-CTR cells, the expression trajectory of the corresponding genes in P1/2-KO cells is shown. (D) GO enrichment analysis of genes dysregulated during neutropoiesis in P1/2-KO cells. The dot plots show enrichment for biological processes. NF-κB, nuclear factor κB; IκB, inhibitor of nuclear factor κB; GTPase, guanosine triphosphatase. (E) Proteome analysis of whole BM LY6G⁺ cells from P1/2-KO versus P1/2-CTR mice (n = 3). The colors highlight proteins whose orthologs have been reported to be either down- or up-regulated during neutrophil differentiation in human BM (24). FC, fold change, P1/2-KO–P1/2-CTR. (F) Bar graphs: FC expression for selected granule (left) and ribosomal (right) proteins. In (E) and (F), the color code indicates the P value adjusted with the Benjamini-Hochberg method for multiple testing.

Fig. 6.. IRP deficiency causes abnormal metabolic remodeling during neutrophil development and differentiation.

(A) Expression of selected glycolysis proteins in whole BM LY6G⁺ cells (same representation as Fig. 5F). The color code indicates the P value adjusted with the Benjamini-Hochberg method for multiple testing. (B to D) FCM analysis of (B) 2-NBDG uptake, (C) mitochondrial mass (with MitoTracker green dye), and (D) mitochondrial membrane potential (MMP) (with JC-1 dye) during neutropoiesis. Top: Representative FCM plots (the dashed line separates stained from unstained cells). Bottom: Box plot with maximum to minimum MFI values [(A) and (B): P1/2-CTR, n = 5; P1/2-KO, n = 4; (D): n = 5]. Middle box plot in (D): Percentage of JC-1–positive cells (maximum to minimum values, n = 5). Prog, prNeu, and PMN cell populations were gated using the same strategy as in Fig. 2. P values (Mann-Whitney) correspond to separate pairwise comparisons between P1/2-CTR and P1/2-KO for each parameter and cell population analyzed. *P < 0.05; **P < 0.01.

Fig. 7.. IRPs support neutrophil differentiation by securing iron bioavailability.

(A) FCM analysis of CD71/TFRC expression during neutropoiesis in vivo. Top: Representative FCM plots. Bottom: Box plot with maximum to minimum MFI values (P1/2-CTR, n = 5; P1/2-KO, n = 6). Prog, prNeu, and PMN cell populations were gated using the same strategy as in Fig. 2. (B and C) Western blot analysis of ferritin (B) and FPN (C) expression in lineage negative (LIN⁻) versus LY6G⁺ cells from the BM of P1/2-CTR (CreERT2⁻) versus P1/2-KO (CreERT2⁺) mice. Loading control, ACTB (actin-β). (D) Box plot (maximum to minimum values) showing reduced LIP in BM cells of P1/2-KO versus P1/2-CTR mice during neutropoiesis in vivo (P1/2-CTR, n = 5; P1/2-KO, n = 6). (E and F) Top schemes (created with BioRender.com): Ex vivo differentiation of LIN⁻ BM cells as in Fig. 4D, in the presence of either (E) FAC or (F) DFP; control cells were treated with vehicle. Bottom: Representative FCM plots showing LY6G and c-KIT levels, together with box plots (maximum to minimum values) displaying the MFI (F) or percentage of cells positive (E and F) for either marker [(E): P1/2-CTR, n = 4; P1/2-KO, n = 5; (F): n = 5]. The dashed lines delineate marker-positive and -negative cells. Comparisons were made between P1/2-CTR and P1/2-KO (A and D), vehicle and FAC (E), or vehicle and DFP (F). P values (Mann-Whitney test), *P < 0.01; **P < 0.01; ***P < 0.001.