TFPI from Erythroblasts Drives Heme Production in Central Macrophages Promoting Erythropoiesis in Polycythemia

doi:10.21203/rs.3.rs-3202992/v1

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TFPI from Erythroblasts Drives Heme Production in Central Macrophages Promoting Erythropoiesis in Polycythemia

https://doi.org/10.21203/rs.3.rs-3202992/v1

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published 10 May, 2024

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Bleeding and thrombosis are known as common complications of polycythemia for a long time. However, the role of coagulation system in erythropoiesis is unclear. Here, we discover that an anticoagulant protein tissue factor pathway inhibitor (TFPI) plays an essential role in erythropoiesis via the control of heme biosynthesis in central macrophages. TFPI levels are elevated in erythroblasts of human erythroblastic islands with JAK2^V617F mutation and hypoxia condition. Erythroid lineage-specific knockout TFPI results in impaired erythropoiesis through decreasing ferrochelatase expression and heme biosynthesis in central macrophages. Mechanistically, the TFPI interacts with thrombomodulin to promote the downstream ERK1/2-GATA1 signaling pathway to induce heme biosynthesis in central macrophages. Furthermore, TFPI blockade impairs human erythropoiesis in vitro, and normalizes the erythroid compartment in mice with polycythemia. These results show that erythroblast-derived TFPI plays an important role in the regulation of erythropoiesis and reveal an interplay between erythroblasts and central macrophages.

Biological sciences/Developmental biology/Haematopoiesis/Erythropoiesis

Biological sciences/Cell biology

Health sciences/Diseases/Haematological diseases

Adult humans produce 2 to 3 million red blood cells (RBCs) every second in bone marrow (BM) during steady state erythropoiesis, which is challenged by multiple variables such as JAK2^V617F mutation and high altitude hypoxia^{1, 2}. The clinical course of polycythemia is marked by the high incidence of bleeding and thrombosis³. Tissue factor (TF) is best known as the primary cellular initiator of blood coagulation. TF pathway inhibitor (TFPI) is the principal inhibitor of the initiation of blood coagulation through high-affinity TF inhibition⁴. TF activity is increased in neutrophils from polycythemia vera patients⁵. These results establish abnormal coagulation as an important physiological process in polycythemia, in which the underlying molecular mechanisms affecting erythropoiesis by coagulation factors are unknown.

The process of erythropoiesis is the largest consumer of iron, which is employed by erythroid lineages to synthesize heme as a structural component of hemoglobin of erythroid precursors⁶. Intercellular heme transportation may play an important role in erythropoiesis. Macrophages express feline leukemia virus subgroup C receptor (FLVCR), a heme export protein⁷. Hematopoietic cell-specific FLVCR knockout mice display severe erythropoietic deficiency⁸. Heme released from degraded RBCs in macrophage is exported as intact heme through heme exporters⁷. Moreover, heme carrier protein 1 is responsible for exogenous heme import, which mediates erythroblasts differentiation to red cells in BM⁹, suggesting that exogenous heme may contribute to erythropoiesis. It is possible that non-erythroid lineage-derived heme contributes to erythropoiesis during steady-state or stress conditions.

Erythropoiesis requires a specific microenvironment comprised of central macrophages surrounded by developing erythroblasts¹⁰. Macrophages promote erythropoiesis via directly interacting with erythroblasts and secreting growth factors¹¹. Macrophages produce bone morphogenetic protein 4 to promote erythropoietic recovery following myeloablation¹². Furthermore, splenic macrophages degrade heme and recycle the iron for de novo erythropoiesis¹³. Macrophages express mechanosensitive piezo1 to affect ion metabolism and subsequent erythrocyte turnover¹⁴. Moreover, erythroblasts also secrete regulatory factors to affect hematopoietic progenitor cells or iron loading. Erythroblast-derived fibroblast growth factor 23 is needed to release hematopoietic progenitor cells from BM into the circulation¹⁵. The erythroid lineage-derived hormone erythroferrone is released after erythropoietic stimuli to mobilize iron for erythropoiesis¹⁶. It has been found that erythroblasts obtain mitochondria from central macrophages in stress erythropoiesis through CD47 signaling¹⁷. It is necessary to further investigate how erythroblast signaling regulates central macrophages in erythropoiesis.

TFPI is identified as a Kunitz-type serine protease inhibitor that endogenously suppresses the tissue factor pathway in coagulation system¹⁸. TFPI has been implicated in inflammation¹⁹, hematopoietic stem cell homing²⁰, and vascular development²¹. Accumulating platelets in the growing blood clot release TFPI, firstly named as lipoprotein associated coagulation inhibitor²². Furthermore, TFPI is also expressed in endothelial cells¹⁹, fibroblasts²³, and muscle cells²⁴. Here, we found that TFPI was highly expressed in erythroblasts, while lowly expressed in central macrophages. Erythroid lineage-specific TFPI deficiency led to reduced erythropoiesis in BM. Moreover, TFPI interacted with thrombomodulin (Thbd) as a functional receptor in central macrophages to inhibit heme production, which contributed to stress erythropoiesis.

TFPI knockout impairs erythropoiesis

To investigate the relevance of blood coagulation to erythropoiesis, we measured plasma TF and TFPI in healthy controls and polycythemia patients with JAK2^V617F mutation. Plasma TF was higher in JAK2^V617F-mutated patients (Fig. 1A), suggesting that erythropoiesis associated with coagulation activation. We then established in vitro erythroblastic islands with erythroblasts and macrophages derived from human cord blood cells (Fig. 1B and 1C), to explore the role of TF and TFPI in erythropoiesis. The results showed that TFPI expression of erythroblasts was upregulated in JAK2^V617F mutation, while TF expression of erythroblasts remained unchanged (Fig. 1D). Erythropoiesis occurs mostly in BM. We found that the expression of TFPI in BM was also up-regulated in Jak2^V617F-mutated mice (supplemental Fig. 1A). Moreover, Gene Expression Commons (GEXC) analysis showed that in BM, TFPI expression was highest in primitive colony-forming-unit erythroid (pCFU-E) among monocyte, megakaryocyte progenitor (MkP), hematopoietic stem cell (HSC), and pCFU-E (supplemental Fig. 1B). TFPI expression in erythroid lineages was also higher than that in macrophages (Fig. 1D and 1E). Therefore, we next generated shRNA against TFPI to gain further insights into the role of TFPI in erythropoiesis. TFPI knockdown reduced RBC numbers, hemoglobin (Hb), and hematocrit (HCT) in peripheral blood (PB, supplemental Fig. 1C). The stages (referred to here as RI, RII, RIII, RIV, and RV) of erythropoiesis were characterized by flow cytometry using the expression of Ter119, CD71, and CD44. The cell numbers of stages RIII and RIV of erythropoiesis were increased, while the cell number of RV was decreased after shTFPI treatment (supplemental Fig. 1D).

We then employed EpoR-Cre mice to prepare erythroid lineage-specific TFPI knockout mice (TFPI^f/f;EpoR, Fig. 1F). PB RBC numbers, Hb, and HCT were decreased in TFPI^f/f;EpoR mice (Fig. 1G). The cell numbers of stages RIII and RIV of erythropoiesis were increased, while the cell number of RV was decreased in TFPI^f/f;EpoR mice (Fig. 1H). Moreover, erythroid lineage-specific TFPI knockout also led to increased apoptosis in erythroblasts (Fig. 1I). The CFU-E colonies were decreased in TFPI^f/f;EpoR mice (Fig. 1J). To examine the role of TFPI in polycythemia development, we crossed Vav-iCre;Jak2^V617F/+ mice with TFPI^f/f mice and analyzed the resulting double mutant Jak2^V617F;TFPI^f/f;Vav mice. Loss of TFPI on the Jak2^V617F mutation background resulted in decreased PB RBC numbers, Hb, and HCT as well as impaired terminal differentiation (Fig. 1K and 1L). Hypoxia can also induce polycythemia which lead to an increase in erythropoiesis². RNA-seq data showed that TFPI was one of the most up-regulated secreted factor genes after 1 week hypoxia exposure (supplemental Fig. 1E). The protein level of TFPI in BM was also up-regulated in hypoxia-exposed mice (supplemental Fig. 1F). TFPI^f/f;EpoR mice showed a decrease in PB RBC numbers, Hb, and HCT under hypoxia (Fig. 1M), and also resulted in impaired terminal differentiation of erythroid cells (Fig. 1N). New RBCs will be produced rapidly under stress conditions²⁵. In stress erythropoiesis induced by phenylhydrazine (PHZ), TFPI^f/f;EpoR mice developed more severe erythropoietic impairment and had a delayed RBC recovery response compared to TFPI^f/f mice (supplemental Fig. 1G and 1H). The erythropoietic impairment in PHZ-administered TFPI^f/f;EpoR mice observed on Day 7 was preceded by an increase in stage RIII and RIV erythroblast frequency on Day 6 (supplemental Fig. 1I). The results showed that TFPI regulated erythropoiesis under both steady state and stress conditions.

Central macrophages involved in the effect of TFPI on erythropoiesis

To determine whether TFPI acts directly on erythroblasts, we measured the proliferation of Ter119⁺ CD71⁺ cells (Fig. 2A). However, TFPI shRNA treatment did not change erythroblast population proliferation in bromodeoxyuridine (BrdU) incorporation experiments (Fig. 2B). Central macrophages are the key component of erythroblastic island where definitive erythropoiesis occurs²⁶. We further employed CD169^DTR/+ mice to investigate whether central macrophages mediate the effect of TFPI on erythropoiesis (Fig. 2C). PB RBC numbers, Hb, and HCT were decreased in CD169^DTR/+ mice, while TFPI shRNA treatment had no effect on erythropoiesis in CD169^DTR/+ mice after DT treatment (Fig. 2D). Erythroid lineage-specific TFPI knockout didn’t influence erythropoiesis in mice after macrophage depletion as well (Fig. 2E and 2F). Consistently, in clodronate liposome treated groups, the cell numbers of stages RIII, RIV, and RV in BM had no substantial change in TFPI^f/f;EpoR mice (Fig. 2G). The results illustrated that the effect of TFPI on erythropoiesis was mediated by central macrophages.

We injected rTFPI to further study the effect of TFPI on macrophage-mediated erythropoiesis. The plasma TFPI concentration was higher in rTFPI-treated mice (supplemental Fig. 2A). Moreover, rTFPI treatment increased PB RBC numbers, Hb, and HCT and promoted terminal differentiation in both BM and spleen (supplemental Fig. 2B-D). In PHZ-induced stress erythropoiesis model, rTFPI treatment also resulted in increased PB RBC numbers, Hb, and HCT as expected (supplemental Fig. 2E and 2F). However, rTFPI treatment did not result in an increase in PB RBC numbers, Hb, and HCT, nor did it influence terminal differentiation in CD169^DTR/+ mice after DT treatment (supplemental Fig. 2G and 2H).

Since TFPI was slightly expressed in central macrophages (Fig. 1E), we prepared macrophage-specific TFPI knockout mice to further explore the effect of macrophage-derived TFPI on erythropoiesis (supplemental Fig. 3A). TFPI expression was ablated completely in central macrophages of TFPI^f/f;CD169 mice (supplemental Fig. 3B). We did not observe any changes in PB RBC numbers, Hb, and HCT in TFPI^f/f;CD169 mice (supplemental Fig. 3C). Cell numbers of stages RI to RV of erythropoiesis also remained unchanged in TFPI^f/f;CD169 mice (supplemental Fig. 3D). Furthermore, macrophage-specific TFPI knockout did not change erythroblast apoptosis and proliferation (supplemental Fig. 3E and 3F). Additionally, the mRNA levels of genes required for mature RBC production including HBA-A1, HBB-B1, GYPA, EPB41, and AQP1 in erythroblasts also remained unchanged (supplemental Fig. 3G). Therefore, erythroid lineage-derived, but not macrophage-derived, TFPI promoted erythropoiesis in bone marrow.

TFPI increases heme production in central macrophages

Central macrophages were isolated from TFPI^f/f or TFPI^f/f;EpoR mice (supplemental Fig. 4A), and RNA sequencing (RNA-seq) was performed to understand the molecular mechanism underlying the impairment of erythropoiesis following TFPI knockout. Analysis of RNA-seq data by Gene Ontology (GO) of differentially expressed genes showed that erythroid lineage-specific ablation of TFPI denoted down-regulation of several pathways in central macrophages including heme biosynthetic process and porphyrin-containing compound biosynthetic process, which were related to heme production (Fig. 3A and 3B). RNA-seq data revealed the down-regulation of seven enzymes in heme biosynthesis including 5-aminolevulinate synthase 2 (ALAS2), Delta-aminolevulinic acid dehydratase (ALAD), hydroxymethylbilane synthase (HMBS), uroporphyrinogen III synthase (UROS), uroporphyrinogen decarboxylase (UROD), coproporphyrinogen III oxidase (CPOX), and Ferrochelatase (Fech) at the mRNA level (Fig. 3B). Fech is the rate-limiting enzyme that incorporates ferrous iron into protoporphyrin IX (PPIX) in heme biosynthesis in final step²⁷. We found that the mRNA levels of Fech were significantly reduced in central macrophages of TFPI^f/f;EpoR mice but increased after rTFPI treatment (Fig. 3C and 3D). Fech mRNA level was decreased in central macrophages but did not change in Ter119⁺ cells of TFPI^f/f;EpoR mice (Fig. 3E). TFPI^f/f;EpoR mice showed a reduction in heme content in Ter119⁺ cells and central macrophages compared with TFPI^f/f mice (Fig. 3F and 3G). These findings supported the fact that erythroid lineage-specific TFPI knockout impaired heme synthesis in central macrophages. Moreover, Heme content of Ter119⁺ cells after macrophage-depletion was similar in TFPI shRNA and control shRNA treatment (Fig. 3H and 3I). GATA1 has been reported as an essential regulator of erythroid cell gene expression^{28, 29}. We found that the phosphorylation level of GATA1 in Ter119⁺ cells and central macrophages increased over time (Fig. 3J). Additionally, rTFPI treatment did not change the heme content in central macrophages after GATA1 shRNA treatment (Fig. 3K). These data suggested that erythroid lineage-specific TFPI knockout impaired heme biosynthesis in central macrophages.

To further investigate the role of heme in central macrophages during erythropoiesis, we first found the mRNA and protein levels of Fech in central macrophages increased in Jak2^V617F-mutated, hypoxia-exposed, and PHZ-treated mice (supplemental Fig. 4B-D). We then employed CD169-Cre mice to prepare macrophage-specific Fech knockout mice (Fech^f/f;CD169, supplemental Fig. 4E), which showed a decrease in central macrophage heme content, PB RBC numbers, Hb, and HCT (supplemental Fig. 4F and 4G). Furthermore, macrophage-specific Fech knockout prevented terminal differentiation and promoted apoptosis in erythroblasts. (supplemental Fig. 4H and 4I). After PHZ treatment, Fech^f/f;CD169 mice developed more severe erythropoietic impairment and had a delayed RBC recovery response (supplemental Fig. 4J). Additionally, rTFPI treatment failed to increase central macrophage heme content, PB RBC numbers, Hb, and HCT in Fech^f/f;CD169 mice (supplemental Fig. 4K and 4L), suggesting that TFPI promoted erythropoiesis by regulating Fech expression in central macrophages.

TFPI interacts with Thbd in central macrophages

To understand the mechanism by which TFPI promotes erythropoiesis, we aimed to identify a potential TFPI receptor. We first explored whether TF was involved in the role of TFPI in erythropoiesis. However, there was no change in TF expression in BM of Jak2^V617F-mutated or hypoxia-exposed mice (Fig. 4A). In both TFPI^f/f and TFPI^f/f;EpoR mice, TF shRNA treatment also did not change heme content and Fech expression in central macrophages as well as PB RBC numbers and Hb content (Fig. 4B-D). We then carried out a yeast two-hybrid screen to search for candidate TFPI-interacting proteins (Table S1), and confirmed the interaction between TFPI and Thbd by co-immunoprecipitation (Co-IP, Fig. 4E). To narrow down the region of Thbd that mediated its binding to TFPI, we divided the extracellular region of Thbd into two segments based on its structure. The Thbd (205–518) segment, but not Thbd (17–204) segment, interacted with TFPI (Fig. 4F and 4G). We also confirmed that TFPI interacted with Thbd in BM cells (Fig. 4H). Additionally, GST pull-down assays showed that Thbd interacted with TFPI (Fig. 4I). We next sought to determine whether Thbd was involved in TFPI-mediated promotion of heme production. The mRNA level of Fech was not increased by rTFPI treatment after Thbd shRNA treatment (Fig. 4J), but it was increased by rTFPI treatment after Thbd transfection in HEK293T cells (Fig. 4K).

TFPI-2 is another secreted factor gene homologous to TFPI, so we further investigated the interaction between TFPI-2 and Thbd. Sequence alignment showed that TFPI-2 shared low identity with TFPI (supplemental Fig. 5A). We noted a lower TFPI-2 content in BM than in plasma (supplemental Fig. 5B). Additionally, our results showed that TFPI-2 did not interact with Thbd (supplemental Fig. 5C), indicating that Thbd specifically interacted with TFPI.

Macrophage-specific Thbd knockout decreases erythropoiesis

Since CD169-cre transgene and Thbd gene are located very close on chromosome 2, Thbd^f/f;LysM mice were prepared to explore the effect of macrophage-specific Thbd knockout on erythropoiesis (Fig. 5A). Our findings revealed that Fech expression levels and heme content were reduced in central macrophages of Thbd^f/f;LysM mice (Fig. 5B and 5C), which also resulted in decreased PB RBC numbers, Hb, and HCT (Fig. 5D). Additionally, macrophage-specific Thbd knockout prevented terminal differentiation and promoted apoptosis in erythroblasts (Fig. 5E and 5F). After PHZ treatment, Thbd^f/f;LysM mice showed a delayed RBC recovery response and impaired terminal differentiation (Fig. 5G and 5H). Furthermore, rTFPI treatment failed to increase central macrophage heme content, as well as PB RBC numbers, Hb, and HCT in Thbd^f/f;LysM mice (Fig. 5I and 5J). The results suggested that Thbd in central macrophages was required for erythropoiesis.

We next employed erythroid lineage-specific Thbd knockout mice to determine whether erythroid-expressed Thbd also regulated erythropoiesis (supplemental Fig. 6A). However, in Thbd^f/f;EpoR mice, no changes were observed in PB RBC numbers, Hb, and HCT, as well as terminal differentiation (supplemental Fig. 6B and 6C). Heme content of Ter119⁺ cells also remained unchanged in Thbd^f/f;EpoR mice (supplemental Fig. 6D). Moreover, erythroid lineage-specific Thbd knockout had no effect on erythroblast apoptosis and proliferation (supplemental Fig. 6E and 6F). The mRNA levels of HBA-A1, HBB-B1, GYPA, EPB41, and AQP1 in erythroblasts also remained unchanged in Thbd^f/f;EpoR mice (supplemental Fig. 6G). the results suggested that erythroid lineage-specific Thbd knockout had no effect on erythropoiesis.

Thbd promotes the signaling pathway of heme synthesis in central macrophages

We then examined the downstream signaling pathways of the TFPI/Thbd axis. Previous studies have shown that activated protein C (aPC) plays a crucial role in Thbd signaling³⁰. We found that the mRNA levels of aPC remained unchanged in Jak2^V617F-mutated and hypoxia-exposed mice (Fig. 6A). However, aPC shRNA treatment decreased PB RBC numbers, Hb, and HCT in Thbd^f/f mice, but did not change erythropoiesis in Thbd^f/f;LysM mice (Fig. 6B), which indicated that aPC mediated the effect of Thbd on erythropoiesis. We further analyzed RNA-seq data of central macrophages in TFPI^f/f and TFPI^f/f;EpoR mice. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis showed the top 15 down-regulated signaling pathways in TFPI^f/f;EpoR mice including FoxO signaling pathway and MAPK signaling pathway (Fig. 6C). We found that the heme content in central macrophages of rTFPI-treated mice was reduced after treatment with an inhibitor of ERK1/2, SCH772984 (Fig. 6D), while the heme content in central macrophages was not changed after treatment with an inhibitor of JNK, JNK-IN-8, and an inhibitor of FOXO1, AS1842856 (Fig. 6D). SCH772984 treatment resulted in no further reduction in heme content after GATA1 shRNA treatment (Fig. 6E), indicating that ERK1/2 participates in GATA1 activation after rTFPI treatment. Moreover, Western blot analysis confirmed the up-regulation of p-ERK1/2 in rTFPI-treated mice (Fig. 6F). We then found that the protein level of p-GATA1 was decreased in Thbd^f/f;LysM or SCH772984-treated mice and could not be rescued by rTFPI (Fig. 6G and 6H). Similarly, protein levels of p-GATA1 were increased in central macrophages over time in control mice but remained unchanged after rTFPI treatment in Thbd^f/f;LysM mice (Fig. 6I). Moreover, treatment with rTFPI increased ALAS2 and Fech mRNA levels in Thbd^f/f mice, but not in the Thbd^f/f;LysM mice, and this function was abrogated by SCH772984 and GATA1 shRNA treatment. (Fig. 6J-6L). Therefore, the results suggested that the TFPI/Thbd axis promoted heme synthesis via the aPC/ERK1/2/GATA1 signaling pathway (Fig. 6M).

TFPI knockdown represses erythropoiesis in polycythemia

Since our results indicated that elevated levels of TFPI led to increased erythropoiesis, we then performed human EBI formation assay using macrophages and erythroblasts derived from human cord blood CD34⁺ cells (Fig. 7A), and examined the expression of TFPI and Thbd in erythroblasts and macrophages. The TFPI protein level was higher in erythroblasts, while the Thbd protein level was higher in macrophages (Fig. 7B and 7C). To further explore the roles of TFPI on human erythropoiesis, we pretreated erythroblasts with TFPI shRNA before co-culturing with macrophages. The Hb and heme content in erythroblasts decreased after TFPI shRNA treatment. The Fech mRNA expression and heme content in macrophages decreased after TFPI shRNA treatment (Fig. 7D). However, these effects were reversed by rTFPI (Fig. 7E).

To explore whether TFPI inhibition had therapeutic efficacy against polycythemia, we treated Jak2^V617F-mutated mice with TFPI monoclonal antibody (mAb, supplemental Fig. 7A). TFPI mAb treatment induced decreases in PB RBC numbers and Hb and central macrophage heme content in Jak2^V617F-mutated mice (supplemental Fig. 7B and 7C), while normalized HCT levels as well (supplemental Fig. 7D). Furthermore, we constructed a mouse model of hypoxia-induced polycythemia by exposing mice to hypoxia for up to 3 weeks (supplemental Fig. 7E). TFPI mAb treatment similar decreased PB RBC numbers and Hb (supplemental Fig. 7F), and reduced central macrophage heme content in hypoxia-induced polycythemia mice (supplemental Fig. 7G). This treatment also normalized HCT as expected (supplemental Fig. 7H). The results found that TFPI mAb treatment reduced erythropoiesis in polycythemia.

The erythroblastic island niche consists of central macrophages and surrounding developing erythroid cells in BM^{10, 31, 32}. Central macrophages participate in erythropoiesis by providing growth factors and iron. Here, we identified a soluble protein TFPI from erythroblasts that regulated central macrophage function in erythroblastic island niche. We found that the expression of the rate-limiting enzyme Fech for heme biosynthesis in central macrophages was down-regulated in TFPI knockout mice. Therefore, we identified a signal protein TFPI from erythroblasts to direct heme biosynthesis in central macrophages for erythropoiesis improvement in turn.

Heme, which is composed of iron and the organic molecule protoporphyrin, is the essential cofactor of hemoglobin in erythroid cells. Heme biosynthetic changes in erythroid cells result in erythropoietic disorders. Deficiency of UROS, a heme synthetic enzyme, leads to chronic hemolytic anemia in congenital erythropoietic porphyria³³. The succinyl-CoA deficiency in isocitrate dehydrogenase 1-mutant hematopoietic cells attenuates heme biosynthesis and blocks erythroid differentiation at the late erythroblast stage³⁴. Heme also participates in a variety of physiological roles in macrophages. Heme catabolism in tumor associated macrophages shapes a prometastatic tumor microenvironment to favor immunosuppression, angiogenesis and epithelial-to-mesenchymal transition³⁵. Heme oxygenase 1, an enzyme responsible for heme breakdown, is implicated in oxidative stress and inflammatory response in macrophages³⁶. Almost all mammalian cell types possess heme biosynthetic pathway except mature erythrocytes³⁷. In spite of macrophages with expressing heme biosynthetic enzymes, macrophage heme is not thought to affect erythropoiesis in BM before our work. Here, we found that heme in BM central macrophages contributed to erythropoiesis, suggesting that macrophage heme has paracrine function. Why do BM macrophages provide heme for erythropoiesis besides providing iron? In cardiomyocytes, heme degradation releases free iron to induce cardiac injury³⁸. Local iron reduces self-renewal and increases differentiation of hematopoietic stem cells in BM³⁹. This phenomenon may represent a protective mechanism by which macrophages transfer heme iron, but not free iron, to erythroblasts in BM.

TFPI forms a stable complex with TF/fVIIa to inhibit coagulation⁴⁰. Furthermore, protein S acts as a cofactor for TFPI to accelerate coagulation inhibition⁴¹. The very low density lipoprotein receptor interacts with TFPI to regulate apoptotic, antiangiogenic, and antitumor activity⁴². Here, we identified a single transmembrane receptor Thbd as a functional receptor of TFPI. Thbd was first determined as a ligand for thrombin and a critical cofactor for the major natural anticoagulant protein C system⁴³. Recently, Thbd was also implicated in inflammation, migration, angiogenesis, and leukocyte adhesion⁴³. In infiltrating macrophages of aortic aneurysm, Thbd regulates migration, matrix metalloproteinase activities, and oxidative stress⁴⁴. Here, we found that Thbd mediated heme synthesis in macrophages through the aPC/ERK1/2/GATA1 pathway, illustrating an intracellular pathway of Thbd in macrophages.

It has been known for a long time that coagulation system is activated in polycythemia⁴⁵. Thrombosis and major hemorrhage are frequent symptoms of polycythemia^{46, 47}. This finding suggests that the coagulation system plays an important role in polycythemia. TFPI was first identified as a primary inhibitor of the initiation of blood coagulation and modulates bleeding and clotting⁴⁰. Further investigation found that TFPI is a multivalent protein implicated in bacterial sepsis⁴⁸, metastatic tumor growth^{42, 49}, atherosclerosis^{50, 51}, and Clostridioides difficile infection⁵². Here, we found that TFPI knockout inhibited erythropoiesis, illustrating a regulator exists between coagulation and erythropoiesis. Furthermore, we illustrated that TFPI knockout increased the percentages of polychromatic and orthochromatic erythroblasts, while decreased the percentages of reticulocytes and RBCs, suggesting that TFPI affected the development of erythroid cells. Two antibodies Concizumab, and Marstacimab that target TFPI have been in clinical evaluation for hemophilia care because of their ability to modulate blood coagulation^{53, 54}. On the basis of the present results, the inhibition of TFPI would interfere with erythropoiesis. Inhibition of TFPI may be especially suitable to therapy polycythemia with complication of hemorrhage.

In summary, we identified TFPI as a regulator from erythroid cells to increase heme production through binding with its receptor Thbd in central macrophages of BM, which mediated erythropoiesis by providing heme. Our results reveal a signal pathway of the coagulation system that affects erythropoiesis and represents a potential therapeutic strategy for polycythemia.

Human samples

This study included 18 healthy donors (20–68 years old, mean = 40; 11 females and 7 males), and 21 JAK2^V617F-mutated patients (16–65 years old, mean = 37; 11 females and 10 males) from the Second Affiliated Hospital of Zhejiang University School of Medicine. Informed consent was obtained from all subjects. The study was approved by the Second Affiliated Hospital of Zhejiang University School of Medicine (No. 20230705). Blood samples were collected and the protein levels of TF and TFPI in plasma were measured by ELISA kit according to the manufacturer's instructions (R&D Systems, Minneapolis, MN, USA).

Animals

C57BL/6 mice were purchased from Zhejiang Provincial Laboratory Animal Center. EpoR-Cre mice were kindly provided by Stuart H. Orkin (Harvard Medical School, Boston, MA)⁵⁵. LysM-Cre (Stock# 004781) and Jak2^V617F/+ (Stock# 301658) mice were purchased from The Jackson Laboratory. Vav-iCre (Stock# C001019) and TFPI^f/f (Stock# S-CKO-06215) mice were purchased from Cyagen Biosciences Inc. (Suzhou, China). CD169-Cre (Stock# NM-KI-215032) and Thbd^f/f (Stock# NM-CKO-2101896) mice were obtained from Shanghai Model Organisms Center, Inc. (Shanghai, China). To generate erythroid lineage-specific and macrophage-specific TFPI knockout mice, EpoR-Cre and CD169-Cre mice were crossed with TFPI^f/f mice on a C57BL/6 background. LysM-Cre mice were crossed with Thbd^f/f mice to generate macrophage-specific Thbd knockout mice. CD169-DTR heterozygous (CD169^DTR/+) mice on a C57BL/6 background⁵⁶, which were generated with DTR complementary DNA (cDNA)⁵⁷, were bred in house by crossing CD169^DTR/DTR mice with C57BL/6 mice. Vav-iCre mice were crossed with Jak2^V617F/+ mice to generate Vav-iCre;Jak2^V617F/+ mice (Jak2^V617F). All mice were housed in a specific pathogen-free barrier facility. Experiments were performed on 6–8-week-old mice. The experimental conditions and procedures were approved by the Zhejiang University Institutional Animal Care and Use Committee and were consistent with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Fech gene contains 11 exons, and exon 6 was selected as a conditional knockout region. PCR-generated homology arm and conditional knockout region were used to design targeting vector. Cas9, gRNA and targeting vector were co-injected into zygotes for the generation of Fech^f/f mice. Mouse pups were genotyped by PCR and verified by sequencing. CD169-Cre mice were crossed with Fech^f/f mice to generate macrophage-specific Fech knockout mice.

Reagents

Recombinant mouse TFPI proteins (mouse rTFPI; R&D Systems) were injected intravenously (i.v.) into mice at a dose of 50 µg/kg for 5 days. mouse rTFPI and human rTFPI (R&D Systems) were added to the cell culture medium at a concentration of 200 ng/ml. Anti-TFPI monoclonal antibodies were injected i.v. into mice at a dose of 5 mg/kg. SCH772984, a ERK1/2 inhibitor, was injected intraperitoneally (i.p.) into mice at a dose of 10 mg/kg for 7 days. AS1842856, a FOXO1 inhibitor, was injected i.p. into mice at a dose of 10 mg/kg for 7 days. JNK-IN-8, a JNK inhibitor, was injected i.p. into mice at a dose of 10 mg/kg for 7 days. In BrdU incorporation assays, 1 mg of BrdU was administered to mice by i.p. injection, Ter119⁺ CD71⁺ cells were collected and processed according to the manufacturer’s instructions in the BrdU Kit (BD Biosciences, San Jose, CA, USA).

Complete blood count analysis

Mice were bled to collect ~ 25 µl via the tail vein to collect blood in EDTA-coated BD Microtainer Blood Collection Tubes (BD, San Jose, CA, USA). Blood was diluted 1:20 in PBS and complete blood counts were measured on an Automatic Blood Analyzer (Sysmex, Kobe, Japan).

Real-time quantitative polymerase chain reaction (RT-qPCR)

Total RNA from tissue and cell samples were isolated using RNAiso reagent (TaKaRa, Dalian, China). After treatment with DNase I (Roche, Basel, Switzerland), reverse transcription was performed using AMV reverse transcriptase (TaKaRa) to obtain cDNA. Primers are listed in the Table S2. 18S rRNA was used as an internal reference gene. RT-qPCR was performed using an ABI StepOne Real-Time PCR System (Applied Biosystems, Foster City, CA) with TB Green Premix Ex Taq II (TaKaRa).

Western blot and co-immunoprecipitation (Co-IP) assays

For Western blot, tissue and cell samples were homogenized in lysis buffer (20 mM HEPES, 1.5 mM MgCl₂, 0.2 mM EDTA, 100 mM NaCl, 0.2 mM dithiothreitol, 0.5 mM sodium orthovanadate, 0.4 mM PMSF, pH 7.4) containing phosphatase inhibitor (phosphatase inhibitor cocktail; Sigma-Aldrich). The soluble protein concentration was measured using the Bradford method. Proteins (20 µg of each sample) were separated by SDS-PAGE and electroporated onto polyvinylidene difluoride (PVDF) membranes. Then, non-fat milk was blocked, primary and secondary antibodies were incubated, and ECL reactions were performed.

For Co-IP, cells were washed with cold PBS and then lysed using lysis buffer for 1 h. The supernatants were collected after centrifugation 12,000 rpm for 10 min at 4°C and incubated with antibody-coupled Protein A beads or Protein G beads (Sigma-Aldrich) according to the manufacturer’s instructions. The beads were washed three times with lysis buffer, followed by Western blot analysis.

The following primary antibodies were used for Western blot. Anti-TFPI antibody (1:500, Abcam, Cambridge, UK), anti-Fech antibody (1:1000, Proteintech, Wuhan, China), anti-Thbd antibody (1:1000, Abcam, Cambridge, UK), anti-GATA1 antibody (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-p85 antibody (1:1000, Cell Signaling Technology, Boston, MA, USA), anti-Flag antibody (1:1000, Sigma-Aldrich), anti-Myc antibody (1:1000, Cell Signaling Technology), anti-GST antibody (1:1000, Cell Signaling Technology), anti-p-GATA1 antibody (1:500, Thermo Fisher Scientific, Waltham, MA, USA), anti-β-actin antibody (1:1000, Santa Cruz Biotechnology) and anti-GAPDH antibody (1:1000, Santa Cruz Biotechnology).

GST pull-down assay

GST pull-down was performed as described previously⁵⁸. For tagging sequences with Flag, the cDNA encoding the sequence of TFPI was cloned into the pcDNA3.1 vector and then transfected into HEK293T cells in a 100-mm culture dish. GST-tagged Thbd protein was expressed in E. coli. BL21, the protein was purified and the GST-tagged Thbd protein was incubated with GSH-agarose in binding buffer (50 mM Tris/HCl, 150 mM NaCl, 1 mM EDTA, 0.5% NP40, 10% glycerol, pH 7.4), and rotated at 4°C for 1 h. Then, the beads loaded with the GST-tagged Thbd protein were collected and incubated with the Flag-tagged TFPI protein at 4°C for 2 h. The beads were washed 3 times followed by Western blot.

PHZ treatment

For induction of hemolytic anemia, mice were injected i.p. with PHZ (Sigma-Aldrich) at a dose of 40 mg/kg on days 0 and 1 of the experiment. Peripheral blood was collected 4 days before the start of treatment and on days 4, 7 and 12 after treatment.

Hypoxia exposure

Mice were exposed to hypoxia simulating an altitude of 5000 m (54.02 kPa, 10.8% O₂) in a well-ventilated hypobaric chamber. Control mice were set at sea level (100.08 kPa, 20.9% O₂) in the same chamber.

Flow cytometry and cell isolation

BM cells were isolated by thoroughly flushing tibias, femurs, and humeri using a 5 ml polystyrene tube with a strainer (BD Biosciences). Spleens were mashed through a 70 µm nylon filter. Cells were labeled with fluorochrome-conjugated antibodies in staining buffer for 30 min at 4°C. Samples were analyzed on a Gallios flow cytometer (Beckman Coulter, Miami, FL, USA). The analysis was performed using FlowJo software (Tree Star, Ashland, OR, USA).

Erythroid lineages were labeled with antibodies directed at CD71, Ter119 and CD44. Central macrophages were labeled with antibodies directed at Ter119, Ly6G, F4/80, VCAM-1, and CD169.

The following fluorescently labeled antibodies (BioLegend, San Diego, USA) were used: PE-anti-TER-119/Erythroid cells (clone Ter-119, 1:100), PE/Cyanine7-anti-CD71 (clone RI7217, 1:100), APC-anti-CD44 (clone IM7, 1:100), PE-anti-Ly6G (clone 1A8, 1:100), FITC-anti-TER-119/Erythroid cells (clone Ter-119, 1:100), BV421-anti-F4/80 (clone BM8, 1:25), and APC-anti-VCAM-1 (clone 429, 1:100). Flow analysis of live cells by exclusion of dead cells using propidium iodide (PI, Sigma-Aldrich). Identification of apoptotic cells were carried out using the FITC Annexin V Apoptosis Detection kit (BioLegend). For sorting of Lin⁻ c-kit⁺ CD71⁺ cells, Ter119⁺ CD71⁺ cells, Ter119⁺ cells, erythroblasts and central macrophages, samples were processed under sterile conditions and sorted on FACS sorting with Moflo Astrios EQ (Beckman Coulter).

Colony-forming unit (CFU) assay

For CFU-E assay, the Lin⁻ c-kit⁺ CD71⁺ cells were flow sorted and plated in erythropoietin-containing methylcellulose culture medium (StemCell Technologies, Vancouver, BC, Canada) and incubated at 37°C in 5% CO₂ humidified atmosphere for 7 days. The number of colonies formed on each plate was counted using an inverted microscope.

Measurement of heme and Hb content

Intracellular heme content was determined according to fluorometric assays, as previously reported³⁴. Briefly, cells were harvested and resuspended in 2 M oxalic acid and heated at 100°C for 30 min to remove iron from heme. The resultant protoporphyrin was measured by fluorescence (400 nm excitation and 662 nm emission). Endogenous protoporphyrin content was measured by detecting fluorescence in oxalic acid-treated unheated cells. The Hb content was quantified with the Drabkin’s reagent (Sigma-Aldrich).

Cell culture and transfection

HEK293T cells were obtained from American Type Culture Collection (ATCC) and were cultured in media composed of Dulbecco's Modified Eagle's Medium (DMEM), 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Ter119⁺ CD71⁺ cells were sorted and cultured in media composed of Iscove's modified Dulbecco's medium (IMDM), 10% FBS, 1% bovine serum albumin, 0.2 mg/mL holotransferrin, 10 mg/mL insulin and erythropoietin at the different concentrations. Central macrophages were sorted and cultured in media composed of RPMI 1640, 10% FBS, 10 mM HEPES, and 10 ng/mL macrophage colony-stimulating factor (G-CSF). Cell viability was determined using trypan blue and counted with a hemocytometer. Cells were incubated at 37°C in 5% CO₂ humidified atmosphere. HEK293T cells were transfected with pcDNA3.1 vector carrying the cDNA encoding sequence of TFPI, Thbd, and GATA1.

Macrophage depletion

To deplete CD169⁺ macrophages, heterozygous CD169^DTR/+ mice were injected i.p. with diphtheria toxin (Sigma-Aldrich) at a dose of 10 µg/kg. In some experiments, macrophages were depleted by intravenous injection 200 µl of clodronate liposomes.

Lentivirus production and infection

For generation of mouse TFPI, aPC, CD71, GATA1, Thbd, TF, and human TFPI lentiviral vectors for knockdown, shRNA sequences targeting specific genes were synthesized and cloned into pLKO.1 vectors. The vectors were co-transfected into HEK293T cells with pSPAX2 (Addgene, Cambridge, MA, USA) and pMD2.G (Addgene, Cambridge, MA, USA) for packaging of lentiviral vectors. Lentiviral supernatants were collected 48 h post-transfection. For central macrophage infection, cells were transduced with lentivirus at a multiplicity of infection (MOI) of 10 and selected with puromycin (8 µg/ml) for 48 h. The knockdown efficiency was assessed by RT-qPCR and Western blot. Mice were injected with lentivirus at the dose of 6 × 10⁸ pfu by tail vein injection. Target sequences are listed in Table S3.

Enzyme-linked immunosorbent assay (ELISA)

Femoral BM was rinsed with PBS and centrifuged to obtain cell supernatant. Blood samples were treated with sodium citrate and centrifuged at 4°C to extract plasma. The protein expression of TF and TFPI in plasma were measured by TF and TFPI ELISA kit according to the manufacturer's instructions (R&D Systems). The protein expression of TFPI-2 in BM supernatant or plasma were measured by TFPI-2 ELISA kit according to the manufacturer's instructions (USCN Life Science Inc., Wuhan, China).

In vitro human EBI formation

CD34⁺ cells were sorted by CD34 MicroBeads (Miltenyi Biotec, Germany) from human cord blood. Macrophages were derived from CD34⁺ cells by culturing in IMDM medium containing 2% human peripheral blood plasma, 3% human AB serum, 3 IU/mL heparin, 10 µg/mL insulin, 10 ng/mL stem cell factor (SCF), 1 ng/mL interleukin-3 (IL-3), 100 ng/mL macrophage colony-stimulating factor (M-CSF), 50 ng/mL fms-like tyrosine kinase 3 (FLT3), and 1 × penicillin-streptomycin. Erythroblasts were also derived from CD34⁺ cells. The cell culture procedure was comprised of 3 phases and 2 phased were used in present. In day 0 to day 6, CD34⁺ cells were cultured in IMDM containing 2% human peripheral blood plasma, 3% human AB serum, 200 µg/mL holo-human transferrin, 3 IU/mL heparin, 10 µg/ mL insulin, 10 ng/mL SCF, 1 ng/mL IL-3, and 3 IU/mL erythropoietin for 6 days. In day 7 to day 11, IL-3 was omitted from the culture medium. The Day 11 erythroblasts were pretreated with TFPI shRNA or control shRNA and mixed with macrophages at a 20:1 ratio. Then cells were cultured for 12 h in an IMDM medium containing 2% human peripheral blood plasma, 3% human AB serum, 3 IU/mL heparin, 10 µg/mL insulin, 200 µg/mL holo-human transferrin, 10 IU/ml EPO, 5 mM Mg²⁺, and 5 mM Ca²⁺. 1 × 10⁵ cells were collected for cytospin analysis.

Transduction of JAK2 ^V617F mutation into CD34 ⁺ cells

The JAK2-transduced CD34⁺ cells were prepared as previously described⁵⁹. Briefly, human wild type or mutant JAK2 cDNAs were respectively cloned into the MIGR1-IRES-GFP vector (Addgene, Cambridge, MA, USA). The vectors were co-transfected with lentivirus packaging plasmids pMD.G into HEK293T cells with Lipofectamine 3000. After 48 h, the lentiviral supernatants were collected, concentrated and stored at − 80°C. For infections, CD34⁺ cells were incubated with 50 µl of viral stock for 48 h.

RNA sequencing analysis

RNA was extracted from sorted central macrophages of TFPI^f/f and TFPI^f/f;EpoR mice, or BM cells of normoxia- and hypoxia-exposed mice. RNA quality was assessed by an Agilent 2100 Bioanalyzer (Agilent, Palo Alto, CA, USA) and quantified by a Nanodrop ND-2000 Spectrophotometer (Thermo Scientific, Waltham, MN, USA) prior to sequencing. High-quality RNA samples were used to construct sequencing libraries. RNA-seq transcriptome libraries were prepared using 1 µg of total RNA using the TruSeq RNA Sample Prep Kit from Illumina (San Diego, CA, USA). Libraries were sequenced using Illumina Novaseq 6000 with 2 × 151 bp read length. Expression levels for each transcript were using the fragments per kilobase of exon per million mapped reads (FPKM) method. Secreted proteins were identified as proteins carrying a signal peptide but lacking a transmembrane region⁶⁰. Gene Oncology (GO) enrichment analysis were performed for the differentially expressed genes (DEGs) in the DAVID resource (https://david.ncifcrf.gov/). Kyoto Encyclopedia of genes and genomes (KEGG) path analysis of DEGs were carried out through clusterprofiler package in R. The ggplot2 package in R were used for Heatmap generation.

Quantification and statistical analysis

Data were shown as mean ± standard error of the mean (SEM). The biological repeats were indicated by ‘N’. Statistical analysis was performed using one-way ANOVA with SPSS (version 13.0) software. When the variances were significantly different (P < 0.05), logarithmic transformation was used to stabilize the variance. If the data did not have a normal distribution, statistical significance was evaluated using the Mann-Whitney U-test (two-tailed). P values < 0.05 was considered statistically significant.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

RNA-seq data are available at GEO under accession number GSE224993 and GSE224994. The processed data reported in this paper are provided in the Supplementary Data files. All data supporting the findings of this study are present in the article and/or its Supplementary Information files.

Acknowledgments

We thank Stuart H. Orkin for providing EpoR-Cre mice. This work was supported by the Program for the Natural Science Foundation of China (41776151), the Ten thousand plan youth talent support program of Zhejiang Province, the Zhejiang Provincial Natural Science Foundation of China (LZ23C110001).

Author contributions

Q.Z., T.B.L., and X.J.L. conceived and supervised the study. J.K.M., L.D.S., and J.R.H. collected clinical samples and data. J.K.M, L.D.S., and L.L.F. performed most of the experiments. L.P., Q.D., Y.W.L., and X.Q.C. helped with experiments. J.K.M, L.L.F., T.Y.S., X.L.Z., S.Y.C., S.B.Y., Q.Z., T.B.L., and X.J.L. performed the data analysis. J.K.M., Q.Z., T.B.L., and X.J.L. wrote the manuscript.

Competing interests

Conflict-of-interest disclosure: The authors declare no competing financial interests.

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TFPI from Erythroblasts Drives Heme Production in Central Macrophages Promoting Erythropoiesis in Polycythemia

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Abstract

Figures

Introduction

Results

TFPI knockout impairs erythropoiesis

Central macrophages involved in the effect of TFPI on erythropoiesis

TFPI increases heme production in central macrophages

TFPI interacts with Thbd in central macrophages

Macrophage-specific Thbd knockout decreases erythropoiesis

Thbd promotes the signaling pathway of heme synthesis in central macrophages

TFPI knockdown represses erythropoiesis in polycythemia

Discussion

Materials and methods

Human samples

Animals

Reagents

Complete blood count analysis

Real-time quantitative polymerase chain reaction (RT-qPCR)

Western blot and co-immunoprecipitation (Co-IP) assays

GST pull-down assay

PHZ treatment

Hypoxia exposure

Flow cytometry and cell isolation

Colony-forming unit (CFU) assay

Measurement of heme and Hb content

Cell culture and transfection

Macrophage depletion

Lentivirus production and infection

Enzyme-linked immunosorbent assay (ELISA)

RNA sequencing analysis

Quantification and statistical analysis

Declarations

References

Additional Declarations

Supplementary Files

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