Heme-mediated SPI-C induction promotes monocyte differentiation into iron-recycling macrophages

Abstract

Splenic red pulp macrophages (RPM) degrade senescent erythrocytes and recycle heme-associated iron. The transcription factor SPI-C is selectively expressed by RPM and is required for their development, but the physiologic stimulus inducing Spic is unknown. Here, we report that Spic also regulated the development of F4/80(+)VCAM1(+) bone marrow macrophages (BMM) and that Spic expression in BMM and RPM development was induced by heme, a metabolite of erythrocyte degradation. Pathologic hemolysis induced loss of RPM and BMM due to excess heme but induced Spic in monocytes to generate new RPM and BMM. Spic expression in monocytes was constitutively inhibited by the transcriptional repressor BACH1. Heme induced proteasome-dependent BACH1 degradation and rapid Spic derepression. Furthermore, cysteine-proline dipeptide motifs in BACH1 that mediate heme-dependent degradation were necessary for Spic induction by heme. These findings are the first example of metabolite-driven differentiation of a tissue-resident macrophage subset and provide new insights into iron homeostasis.

Figure 1. Spic is required for the development of RPM and BMM

(A) Flow cytometry with the indicated markers on Splenocytes and BM of the indicated genotypes. (B) Fixed and frozen BM and spleen sections of the indicated genotypes were stained with the indicated antibodies and DAPI (for nuclei). Right panel shows higher magnification images of indicated regions (white box). (C) Single colored histograms for the indicated markers on SPIC-EGFP^hi (red) and SPIC-EGFP⁻ (blue) gated Spic^igfp/igfp BM cells. Ly6G and Ly6C expression within the SPIC-EGFP⁻ gate represent cells additionally gated for CD11b positivity. (D) Flow cytometry with the indicated markers on Splenocytes and BM of the indicated genotypes. (E) Single colored histograms for CD169 expression in MHC-II⁺ BM cells of the indicated genotypes. (F) Flow cytometry plots showing CD71 and Ter119 expression in CD45⁻ BM cells of the indicated genotypes. The right panel represents cumulative data from 5 mice of each genotype (bar: mean +/− S.D). * indicates statistically significant difference (P < 0.05, unpaired t-test). Numbers in flow cytometry plots represent percentage of cells within the indicated gates. All data in this figure is representative of ≥ 3 mice. See also Figure S1 and S2.

Figure 2. Heme induces Spic expression in macrophages generated in vitro

(A and B) BMDM of the indicated genotypes were treated with heme or vehicle after 6–7 days in culture. Adherent cells were harvested 2 days later and stained with the indicated markers as shown in the flow cytometry plots. (C) BMDM were treated with vehicle or increasing concentrations of heme after 6 days in culture. Adherent cells were harvested 2 days later and RNA extracted. (Top) Expression of Spic (Y-axis, Log₁₀) in response to vehicle or increasing levels of heme (X-axis) measured by real time quantitative PCR (bars: mean +/− S.D). Results represent two biological replicates with three technical replicates for each sample. Spic expression was normalized to Hprt and levels are relative to the expression in vehicle treated macrophages. (Bottom) Representative gel for RT-PCR products generated with the indicated primers. (D) BMDM of the indicated genotypes were treated with Heme (10 μm), biliverdin (BV, 50 μm), or bilirubin (BR, 50 μm) at the onset of culture. Cells were harvested after 8 days in culture. Shown are the single color histograms for EGFP expression in cells gated for F4/80 expression. Numbers in the flow cytometry plots represent percentage of cells within the indicated gate. All data is representative of three or more experiments with at least two replicates per group. (E) BMDM of the indicated genotypes were treated with heme or vehicle after 5 days in culture and harvested 5 days later. Shown are the percentages of 7AAD⁻ (live) cells expressing EGFP (bars: mean with range, n=2). * indicates statistically significant (P < 0.05, unpaired t-test) differences. See also Figure S3 and S4.

Figure 3. Heme induces Spic expression in myeloid cells in vivo

(A) Splenocytes of the indicated genotypes were stained for the indicated markers as shown in the flow cytometry plots. (B) WT and Spic^igfp/igfp mice were treated intraperitoneally with 100 μl of PBS or Crmp (5 μm/kg in PBS) on three consecutive days. On day 4, splenocytes were harvested and stained for the indicated markers as shown in the flow cytometry plots. (C) WT and Spic^igfp/igfp mice were treated intraperitoneally with 200 μl PBS or PDZ (2 mg in PBS) three times 48 hours apart and sacrificed 24 hr. after the final treatment. Splenocytes were harvested and stained for the indicated markers as shown in the flow cytometry plots. (D) Fixed and frozen sections of spleen of the indicated genotypes were stained with the indicated antibodies and DAPI. Lower panel shows higher magnification of the indicated regions (white box). (E) BMDM of the indicated genotypes were treated with vehicle or increasing concentrations of heme after 7 days in culture. Cells were harvested 2 days later and stained for the indicated markers as shown in the flow cytometry plots. (F) BMDM of the indicated genotypes were treated with vehicle or high concentration of heme after 5 days in culture. Cells were harvested 24 hours later, stained for F4/80, and subsequently stained with annexin V and 7AAD. Shown are the flow cytometry plots with indicated markers on cells gated for F4/80 expression. (G) WT and Spic^igfp/igfp mice were treated intraperitoneally with 200 μl of PBS or heme (0.5 mg in PBS) on three consecutive days. Splenocytes were harvested on day 4 and stained for indicated markers as shown in the flow cytometry plots. Numbers in the flow cytometry plots represent percentage of cells within the indicated gate. Red boxes represent RPM and blue boxes pre-RPM. All data in this figure is representative of two or more experiments with at least two mice or samples per group. See also Figure S5.

Figure 4. Spic⁺ myeloid cells are intermediates between Spic⁻ monocytes and Spic^hi RPM

(A, B) Splenocytes from Spic^igfp/igfp and Ho1^−/−Spic^igfp/igfp were stained for the indicated markers. (A) Flow cytometry plots with the indicated markers in CD11b^hi EGFP⁻ (left) and CD11b^hi EGFP⁺ (right) gated cells from Ho1^−/−Spic^igfp/igfp splenocytes. (B) Single color histograms for indicated markers in CD11b^hiEGFP⁻ and CD11b^hiEGFP⁺ cells from Ho1^−/−Spic^igfp/igfp splenocytes and CD11b^loEGFP^hi (RPM) from Spic^igfp/igfp splenocytes. (C, Left) 7AAD⁻ EGFP⁻ splenocytes pooled from 6 Spic^igfp/igfp mice were selected for CD11b expression while gating out CD11c^hi F4/80^hi cells. Within these cells, monocytes (M) were sorted as Ly6G⁻Ly6C⁺ and Neutrophils (N) as Ly6G⁺Ly6C⁻ cells. (C, Right) Sorted monocytes and neutrophils were cultured with 5ng/ml GM-CSF with or without heme (40 μm), harvested 3 days later, and stained for markers as shown in the flow cytometry plots. (D) BM from CD45.2⁺ C57BL/6 donors were transferred into lethally irradiated CD45.1⁺ C57BL/6 recipients and splenocytes harvested after 8 weeks. Shown are the flow cytometry plots indicating donor and host derived F4/80^hi CD11b^lo RPM (Left) and F4/80⁺CD11b^hi pre-RPM (middle). The plot on the right shows the same for pre-RPM induced by Crmp (5 μm/kg in 150 μl PBS, twice 24 hours apart) in the BM chimeras. (E) Monocytes were purified from 129SvEv Spic^igfp/igfp splenocytes by depleting Ly6G⁺ granulocytes and enriching for CD11b⁺ cells using MACS microbeads. 129SvEv Spic^+/+ recipient mice were irradiated once with 550 rad followed by single treatment with 200 μl of intra-peritoneal PBS or PDZ (2 mg in PBS). About 2×10⁶ monocytes were injected (IV) into recipient mice 24 hours later. Some recipient mice were injected with about 4×10⁶ unfractionated 129SvEv Spic^igfp/igfp BM cells (instead of monocytes). 4 days after the cell transfers, recipient splenocytes were stained for markers as shown in the flow cytometry plots. The dual colored flow cytometry plot at the bottom displays the overlapping pattern of F4/80 and CD11b expression between BM and monocyte derived SPIC-EGFP⁺ cells in the host spleen. (F) Monocytes (CD11b^hiLy6C⁺ SPIC-EGFP ⁻), pre-RPM (CD11b^hiLy6C⁺ SPIC-EGFP ⁺), and RPM (F4/80^hiCD11b^lo SPIC-EGFP ^hi) were sorted from Spic^igfp/+ mice. RNA was extracted from the sorted cells and gene expression analysis carried out using the affymetrix expression platform. Shown is a heat map representing expression levels for selected RPM-specific genes. (G) 129 SvEv Spic^igfp/igfp mice were treated with 200 μl PDZ (2 mg in PBS). Splenocytes were harvested 48 hours later and enriched for CD11b⁺ cells using MACS magnetic microbeads. Ter119⁻Ly6G⁻F4/80⁻CD3⁻B220⁻CD11b⁺ cells from this CD11b enriched fraction were sorted into SPIC-EGFP⁺ and SPIC-EGFP⁻ cells (Left). About 2×10⁶ SPIC-EGFP⁻ and 1×10⁶ SPIC-EGFP⁺ cells were injected (IV) into 129SvEv Spic^−/− recipients. Splenocytes from the recipients were harvested 4 days later. The dual colored flow cytometry plot on right displays the overlapping pattern of TREML4 and VCAM1 expression between SPIC-EGFP⁺ and SPIC-EGFP⁻ donor derived cells. (H) Lethally irradiated CD45.1⁺ C57BL/6 mice were transplanted with CD45.2⁺ C57BL/6 BM cells and treated with two doses (48 hours apart) of 200 μl of PBS or PDZ (2 mg in PBS) 5 weeks after radiation. 4 weeks after treatment, splenocytes from the recipients were stained for the indicated markers as shown in the flow cytometry plots. Numbers in the flow cytometry plots represent percentage of cells within the indicated gate. The data is representative of two or more experiments with at least two mice per group. See also Figure S6 and S7.

Figure 5. Bach1 is a repressor of Spic expression.

(A, B) Spic^igfp/igfp BMDM were treated with heme (80 μm) or vehicle after 6 days in culture and harvested 24 hr. after treatment. RNA was extracted and microarray analysis performed using the affymetrix 1.0 platform. Shown are the expression levels (Y axis) of selected genes in vehicle (gray bars) and heme (black bars) treated cells. (C) Spic^igfp/igfp BMDM were infected with retroviruses expressing mouse Bach1 and the human Cd4 (Bach1-RV) or expressing only the human Cd4 (Empty-RV). 4 days after infection, the cells were treated with heme (40 μm) or vehicle. Cells were harvested four days later and stained for the indicated markers as shown in the flow cytometry plots. Numbers in the flow cytometry plots represent percentage of cells within the indicated gate. Data is representative of two or more experiments with at least two mice or samples per group. (D) Bach1^−/− BMDM were treated with heme (40 μm) or vehicle after 6 days in culture. RNA was extracted 48 hrs. later and quantitative RT-PCR performed for Spic transcripts. Shown is the expression value (Y-Axis, Log₁₀) relative to vehicle treated WT cells and normalized to Hprt expression (bars: mean +/− S.D). Results represent two distinct biological samples with three technical replicates for each biological sample.

Figure 6. Heme degrades Bach1 to de-repress Spic expression

(A) miR155^−/− and WT splenocytes were stained for the indicated markers as shown in the flow cytometry plots. (B) miR155^−/− and WT mice were treated IP with a single dose of 200 μl PBS or PDZ (2 mg in PBS). Splenocytes were harvested and stained for F4/80 and CD11b 48 hours or 6 weeks after treatment as shown in the flow cytometry plots. (C) miR155^−/− BMDM were treated with heme (40 μm) or vehicle after 6 days in culture. RNA was extracted 24 hours later and RT-PCR performed with Spic and Hprt specific primers as shown in the representative agarose gel (D) Spic^igfp/igfp BMDM were treated with the indicated concentrations of heme or vehicle after 7 days in culture. Cells were harvested 24 hrs. later and protein extracted. Shown are the western blots with the indicated antibodies. An anti-Bach1 reactive band appears at significantly higher molecular weight (*) in the presence of heme. (E) Spic^igfp/igfp BMDM were treated with heme (40 μM), heme with the proteasomal inhibitor MG132 (10 μm), or vehicle after 7 days in culture. 2 days after treatment the cells were stained for the indicated markers as shown in the flow cytometry plots. (F) BACH1 protein contains six heme-binding Cysteine-Proline motifs (black circle), a ‘repressive’ BTB domain (blue box) at the N-terminus, and a basic DNA binding region preceding a protein dimerization leucine zipper domain (bZIP, orange box). Mutant BACH1 were generated by mutating the 4 Carboxy-terminal CP motifs to AP (Alanine-Proline, red circles). Human 293T cell based phoenix cells were transfected with mouse WT or mutant Bach1 expression constructs. 24 hrs. later the cells were treated with heme (40 μM) with or without cycloheximide (CHX, 25 μg/ml) to block new protein synthesis as indicated. Total protein was isolated 4 hours after treatments. Shown in the bottom panel are the western blots with the indicated antibodies. (G) Spic^igfp/igfp BMDM cultures were infected with the following retroviral vectors: expressing only the human Cd4 (empty-RV, empty bar), Bach1 and human Cd4 (Bach1-RV, gray bar), and mutant Bach1 and human Cd4 (mutant Bach1-RV, black bar). 4 days after infection, cells were treated with heme (40 μm) or vehicle. 4 days after treatment, cells were harvested and stained for hCD4 and F4/80. Within individual wells, infected cells (hCD4⁺) were analyzed separately (Left) from uninfected (hCD4⁻) cells (right). Shown are the percentages (Y axis) of EGFP expressing cells within F4/80⁺ gate (N = 3, bars: mean +/− S.D). * indicates statistically significant difference (p < 0.05, unpaired t-test). (H) Splenocytes of the indicated genotypes were stained for the indicated markers as shown in the flow cytometry plots. Numbers in the flow cytometry plots represent percentage of cells within the indicated gate. All data in this figure is representative of two or more experiments with at least two mice or sample per group.

Figure 7. Bach1 inhibition by heme induces RPM and BMM development in vivo

(A) WT and Bach1^−/− BM and splenocytes were stained for the indicated markers as shown in the flow cytometry plots. (B–C) CD45.1⁺ C57BL/6 recipient mice were lethally irradiated and injected with a mix of CD45.2⁺ Bach1^−/− and CD45.2⁺/CD45.1⁺ WT BM cells or with a mix of CD45.2⁺ WT and CD45.2⁺/CD45.1⁺ WT BM cells. 7–10 weeks after transplant various organs were harvested. The Y-axis represents the ratio of the percent CD45.2⁺ donor-derived (color-coded for genotype) RPM (F4/80^hiVCAM1^hi), BMM (F4/80^hiVCAM1^hi), peritoneal macrophages (P-Mac, F4/80^hi), kidney macrophages (K-Mac, F4/80^hi), Dendritic cells (DC; CD11c^hiMHC-II^hi), Monocytes (Mono; CD11b^hiLy6G⁻Ly6C⁺ in BM), and Neutrophils (Neu; CD11b^hiLy6G⁺Ly6C⁻ in BM) to the percent CD45.2⁺ of all donor derived CD45⁺ cells (bars: mean +/− S.D). * denotes statistically significant differences (p< 0.05, n ≥ 3, Mann-Whitney test). (D–E) Bach1^−/− BMDM were treated with heme (40 μm) or vehicle after 6 days in culture. (D) RNA was extracted 24 hrs. after treatment and microarray analysis performed using the affymetrix 1.0 platform. Shown is the fold change in expression (Y axis, log₂ scale) of selected heme-inducible genes. (E) Cells were harvested 72 hrs. after treatment and stained for the indicated markers as shown in the flow cytometry plots. (F) Heme-induced development of heme-degrading macrophages.