M2 macrophages, but not M1 macrophages, support megakaryopoiesis by upregulating PI3K-AKT pathway activity

Dysfunctional megakaryopoiesis hampers platelet production, which is closely associated with thrombocytopenia (PT). Macrophages (MФs) are crucial cellular components in the bone marrow (BM) microenvironment. However, the specific effects of M1 MФs or M2 MФs on regulating megakaryocytes (MKs) are largely unknown. In the current study, aberrant BM-M1/M2 MФ polarization, characterized by increased M1 MФs and decreased M2 MФs and accompanied by impaired megakaryopoiesis-supporting abilities, was found in patients with PT post-allotransplant. RNA-seq and western blot analysis showed that the PI3K-AKT pathway was downregulated in the BM MФs of PT patients. Moreover, in vitro treatment with PI3K-AKT activators restored the impaired megakaryopoiesis-supporting ability of MФs from PT patients. Furthermore, we found M1 MФs suppress, whereas M2 MФs support MK maturation and platelet formation in humans. Chemical inhibition of PI3K-AKT pathway reduced megakaryopoiesis-supporting ability of M2 MФs, as indicated by decreased MK count, colony-forming unit number, high-ploidy distribution, and platelet count. Importantly, genetic knockdown of the PI3K-AKT pathway impaired the megakaryopoiesis-supporting ability of MФs both in vitro and in a MФ-specific PI3K-knockdown murine model, indicating a critical role of PI3K-AKT pathway in regulating the megakaryopoiesis-supporting ability of M2 MФs. Furthermore, our preliminary data indicated that TGF-β released by M2 MФs may facilitate megakaryopoiesis through upregulation of the JAK2/STAT5 and MAPK/ERK pathways in MKs. Taken together, our data reveal that M1 and M2 MФs have opposing effects on MKs in a PI3K-AKT pathway-dependent manner, which may lead to new insights into the pathogenesis of thrombocytopenia and provide a potential therapeutic strategy to promote megakaryopoiesis.


INTRODUCTION
[9][10] Moreover, in vitro and in vivo treatments for restoring the impaired BM microenvironment promoted MK maturation in PT patients, [8][9][10] further confirming the vital role of the BM microenvironment in supporting megakaryopoiesis.
6][17] In patients with immune thrombocytopenia (ITP) who underwent intravenous immunoglobulin therapy, BM MΦs were found to closely contact MKs and were associated with increased platelet counts, suggesting that MΦs positively regulate megakaryopoiesis. 15However, Alves-Rosa et al. reported that depleting BM MΦs enhanced both megakaryopoiesis and platelet production in an ITP mouse model, which indicates that MΦs negatively regulate megakaryopoiesis. 16,17Typically, MΦs can be polarized into classically activated (M1) MΦs and alternatively activated (M2) MΦs, with distinct phenotypic and unique functional characteristics. 18M1 MΦs mediate resistance to intracellular pathogens and tissue destruction, whereas M2 MΦs are generally oriented to tissue remodeling and repair. 19,20Recently, a confocal laser scanning microscopy study revealed an increased number of M1 MΦs and a decreased number of M2 MΦs in the spleens of ITP patients compared with non-ITP control patients, providing clues that M1 MΦs and M2 MΦs have different roles in regulating thrombopoiesis. 21However, the specific effects of M1 or M2 MΦs on regulating megakaryopoiesis and approaches for regulating the supportive function of MΦs in megakaryopoiesis need to be further elucidated.
The PI3K-AKT pathway is a central signaling pathway for cellular growth and survival.Animal studies using knockout mice or small interfering RNAs demonstrated that the PI3K-AKT pathway is involved in the regulation of M1/M2 MФ polarization, especially with regard to the activation of M2 MФs. 22,23Furthermore, considerable evidence has implicated the PI3K-AKT pathway in the survival, proliferation, and metabolism of M2 MФs.Munugalavadla et al. 24 reported that MФs from PI3K-deficient mice exhibit significantly reduced growth and migration abilities.Chang et al. 25 found that BM MФs maintain their survival by upregulating glucose uptake, which is dependent on the PI3K-AKT pathway.Although accumulating evidence indicates that the PI3K-AKT pathway plays a crucial role in promoting M2 polarization to reprogram MФ function, the effect of the PI3K-AKT pathway on regulating the megakaryopoiesis-supporting ability of MФs has not been studied in MФ subtypes.
Therefore, the current study was performed to address the roles of M1 MФs and M2 MФs in regulating megakaryopoiesis.The effect of the PI3K-AKT pathway on regulating the megakaryopoiesis-supporting ability of MФs was investigated in vitro and in a MΦ-specific PI3K-knockdown murine model.Moreover, the role of M1/M2 MФ polarization in regulating megakaryopoiesis and pharmacological interventions of the PI3K-AKT pathway were evaluated in PT patients, a clinical model of thrombocytopenia after allo-HSCT.We aimed to identify a potential therapeutic strategy for patients with dysfunctional MK maturation and thrombocytopenia.

Downregulation of the PI3K-AKT pathway is involved in the aberrant BM-M1/M2 MФ polarization of PT patients
To verify the MФ polarization status with another more comprehensive method, the M1 and M2 score, which represents M1 and M2 phenotype, was estimated by bulk RNA sequencing (RNA-seq) data of the cultivated BM MФs (the calculation details and concept definitions are provided in the "Materials and methods" section).Consistent with our flow cytometry data, RNA-seq revealed that PT MФs were dominated by M1 MФs, whereas GGF MФs were dominant by M2 MФs (Fig. 3a, b).M1 score was significantly higher in PT MФs than that in GGF MФs (Fig. 3c; 0.60 ± 0.05 vs. 0.12 ± 0.07, P = 0.008).Conversely, the M2 score was significantly higher in GGF MФs than that in PT MФs (Fig. 3d; 0.88 ± 0.07 vs. 0.40 ± 0.05, P = 0.008).Thus, the ratio of M1 to M2 score was higher in PT MФs than that in GGF MФs (Fig. 3e; 1.57 ± 0.38 vs. 0.15 ± 0.10, P = 0.056).RNA-seq data supported that PT MФs were imbalanced polarized to M1 MФ type.Gene ontology (GO) analysis indicated that the upregulated differentially expressed genes in GGF MФs are particularly enriched in megakaryopoiesis-associated biological processes (Fig. 3f).Next, the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway gene set data were analyzed.Compared with PT MФs, GGF MФs showed significant upregulation of the PI3K-AKT pathway (Fig. 3g).Moreover, AKT1 was highly expressed in GGF MФs than in PT MФs (Fig. 3h; 1.87 ± 0.28 vs. 0.52 ± 0.40, P = 0.01).Consistent with RNAseq results, a lower level of p-AKT was observed in PT MФs compared to that in GGF MФs by western blot analysis (Fig. 3i).To further confirm whether the PI3K-AKT pathway potentially upregulated in M2 MФs, M1 MФs, and M2 MФs from HD BM were investigated using RNA-seq (Fig. 3j).As shown in Fig. 3k, KEGG enrichment analysis showed that upregulated genes were mainly enriched in PI3K-AKT pathway in the M2 MФs.Taken together, these data indicate that the PI3K-AKT pathway may be a target to improve the megakaryopoiesis-supporting ability of PT MФs.
Taken together, these results suggest that M2 MФs support MK maturation and platelet formation, whereas M1 MФs suppress the processes in both direct-contact and indirect-contact conditions.However, the effects of BM-M1 or BM-M2 on megakaryopoiesis were more significant in direct-contact than that in indirectcontact coculture conditions.
In vitro treatment with PI3K-AKT pathway inhibitor, LY294002 or MK2206 significantly decreased the megakaryopoiesis-supporting ability of THP1-M2 compared with untreated THP1-M2, as shown by decreases in MK count, number of CFU-MK, platelet count, and number of high-ploidy MKs (Supplementary Fig. S3b-f).These findings indicated that inhibiting the PI3K-AKT pathway can functionally impair M2 MФs and reduce their megakaryopoiesissupporting ability.

M2 MФs, which exhibited high TGF-β level and low TNF-α level, enhanced phospho-STAT5 and phospho-ERK activity in cocultured MKs
To study the potential mechanism underlying the enhanced megakaryopoiesis-supporting ability of M2 MФs, the media from the cultivated BM MФs were analyzed using ELISAs.Increased level of TGF-β (Fig. 7a; 8078 ± 503.09 pg/mL vs. 500.2± 167.08 pg/ mL, P = 0.008) was secreted by BM-M2 than by BM-M1.TNF-α level (Fig. 7b; 509 ± 172.04 pg/mL vs. 7611 ± 889.09 pg/mL, P = 0.008) was remarkably lower in the supernatants of BM-M2 than in that of BM-M1.Moreover, the level of TGF-β (Fig. 7c; 2300 ± 316.07 pg/ mL vs. 777 ± 195 pg/mL, P = 0.0001) was significantly higher in BM plasma of GGF patients than that in PT patients.To explore the subsequent changes in maturation-related signaling pathways of the MKs after cocultured with the different subtypes of MФs, the intracellular levels of phosphor(p)-STAT5 and p-ERK were analyzed in MKs.Notably higher levels of p-STAT5 (Fig. 7d; 3590 ± 417.09 vs. 1599 ± 196.02, P = 0.0002) and p-ERK (Fig. 7e; 2448 ± 273.09 vs. 628.05± 72.86, P = 0.0002) were detected in MKs cocultured with Growing evidence supports that M1/M2 MФ polarization governs the protective and pathogenic roles of MФs in normal and pathological states. 262][13][14] Recently, we reported that increased M1 and decreased M2 MФ polarization reduced the hematopoiesis-supporting ability of MФs in poor graft function patients, a clinical manifestation of pancytopenia after allo-HSCT, suggesting an imbalance in M1/M2 MФ polarization plays an important role in pathological states. 27Anna-Rita Migliaccio et al. 28 reported that peripheral blood mononuclear cells derived CD14 + CD163 + MФs increase the numbers of CD34 + hematopoietic stem and progenitor cells (HSPCs) during coculture, suggesting proliferation and/or survival of the CD34 + HSPCs are affected by CD14 + CD163 + MФs.Moreover, Luo et al. 29 found that M2 MФs promote but M1 MФs inhibit HSC self-renewal, and coculture of human umbilical cord blood CD34 + cells with M2 MФs resulted in a significant expansion of CD34 + cells and long-term SCID mice- repopulating ability, demonstrating that M1 and M2 MФs play different roles in regulating hematopoiesis.Moreover, M1 MФs were reported to engage in higher rates of phagocytosis of platelets than M2 MФs, suggesting that M1 and M2 MФs may exert distinct regulatory functions in platelets. 30However, the precise roles of M1 and M2 MФs or MФ polarization in the regulation of MKs and platelet formation are largely unknown.In the current study, we revealed that M1 MФs suppress megakaryopoiesis and platelet formation in humans whereas M2 MФs support these processes.More importantly, we provided further evidence that M1 and M2 MФs play different roles in regulating megakaryopoiesis in patients with PT, which is characterized by megakaryopoiesis failure after allo-HSCT.Based on our previous work and the current study, we speculate that aberrant BM-M1/M2 MФ polarization, especially increased M1 MФs and decreased M2 MФs in BM, hinders megakaryopoiesis, ultimately leading to failed MK maturation and thrombocytopenia.Therefore, modulating M1/M2 MФ polarization may be a potential therapeutic approach to enhance megakaryopoiesis in patients with thrombocytopenia such as PT after allo-HSCT.
2][33] Luyendyk et al. 32 reported that activation or overexpression of PI3K or AKT kinases resulted in reduced M1 MФ polarization.Ruckerl et al. 33 showed that intact PI3K-AKT signaling is an important factor for driving M2 polarization and proliferation in vitro and in vivo.In agreement with previous reports, upregulation of PI3K-AKT pathway activity was observed in M2 MФs in humans, which leads to increased megakaryopoiesis-supporting ability.In vitro treatment with inhibitors of the PI3K-AKT pathway or Akt1 knockdown disrupted the ability of M2 MФs to support megakaryopoiesis and platelet formation.Moreover, the BM MФ specific PI3K-knockdown mice demonstrated a dramatic drop in megakaryopoiesis and platelet production in vivo.Our data provide further evidence that the PI3K-AKT pathway plays a critical role in regulating the megakaryopoiesis-supporting ability of M2 MФs.Consistently, we observed that activation of the PI3K-AKT pathway improved the impaired ability of MФs from PT patients to support megakaryopoiesis in vitro.Therefore, our data indicate that improving M2 MФ polarization by activating the PI3K-AKT pathway may be a potential therapeutic target for patients with thrombocytopenia.
M2 MФs are even heterogeneous, which can be further subdivided into M2a, M2b, M2c, and M2d subsets. 18,19Our data indicated that M2a MФs, which are typically induced by IL-4 and IL-13, 19 supported MK maturation and platelet release.Besides M1/M2 MФs, other BM-derived MФ subtypes also exert regulatory functions in megakaryopoiesis.For example, Xia et al. 34 showed that mesenchymal stem cells-reprogrammed BM resident MФs, with arginase 1 positive phenotype and tissue-repair features, improved thrombopoiesis in leukemia-bearing mice.Activation of JAK2/STAT5 and MAPK/ERK pathways play crucial roles in MK maturation. 2,35Our preliminary data indicated that TGF-β released by M2 MФs may facilitate megakaryopoiesis through upregulation of the JAK2/STAT5 and MAPK/ERK pathways in MKs.However, we are aware that the precise molecular regulatory mechanism on how MФs mediate megakaryopoiesis needs to be further explored by performing RNA-seq using M1 and M2 MФs directly isolated from BM and tracking phenotypes of both MФs and MKs during coculture in the future.
In summary, the current study demonstrated that M1 MФs and M2 MФs exert opposing effects on megakaryopoiesis and the PI3K-AKT pathway is essential for M2 MФs to support megakaryopoiesis.Defective M2 MФ polarization in BM may be responsible for the occurrence of PT.Although further validation is required, our data may provide new insights into the underlying mechanism and potential therapeutic strategies for patients with megakaryopoiesis failure and thrombocytopenia.

MATERIALS AND METHODS
PT patients and their matched controls A prospective case-control study was conducted to evaluate the polarization of BM MФs from PT patients and their matched patients with GGF.Transplant recipients were identified from patients who received an allotransplant between March 1, 2018 and August 10, 2020 at the Peking University Institute of Hematology.A total of 17 patients who developed PT were eligible.For each PT patient enrolled, transplant recipients with GGF (N = 34) were selected from the same cohort after matching for age, pretransplant disease state and posttransplant interval (risk-set sampling).None of the clinical characteristics, such as the transplanted CD34 + cell dose, history of graft versus host disease (GvHD), or cytomegalovirus (CMV) infection, showed significant differences between PT patients and GGF patients (Table 1).7][38][39] BM samples from HD (N = 30) were normal controls.
This study was approved by the Ethics Committee of Peking University People's Hospital, and written informed consent was obtained from all subjects in compliance with the Declaration of Helsinki.

Definition of PT and GGF
As previously reported, 10 PT was defined as engraftment of all peripheral blood cell lines (absolute neutrophil cells >0.5 × 10 9 /L and hemoglobin >70 g/L without transfusion support) other than a platelet count <20 × 10 9 /L or dependence on platelet transfusions for more than 60 days following allo-HSCT in the presence of complete donor chimerism.]27,40 Patients with evidence of poor graft function, severe GvHD or hematologic relapse following allo-HSCT were excluded.
Cultivation and functional analyses of human primary BM MФs Primary human MФs from HD or patients were generated as described previously. 27BM mononuclear cells (BMMNCs) were isolated by density gradient centrifugation using a lymphocyte separation medium (GE Healthcare, Milwaukee, WI, USA).Briefly, monocytes isolated from BMMNCs of patients with PT, GGF or HD were differentiated into the cultivated PT MФs, GGF MФs or HD MФs for 7 days in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1% penicillin/ streptomycin, and 100 ng/mL macrophage colony-stimulating factor (PeproTech, Rocky Hill, NJ, USA).The resulting primary HD MФs were then polarized into M1 (BM-M1) or M2 (BM-M2) MФs by culturing for 24 hours in RPMI 1640 medium supplemented with either 100 ng/mL LPS and 20 ng/mL IFN-γ or 20 ng/mL IL-4 and 20 ng/mL IL-13, respectively.The cultivated BM MФs were evaluated with phagocytosis and migration assays as previously described. 27The supernatant of MФs and the BM plasma of patients with PT or GGF were harvested.The levels of TNF-α and TGF-β were measured using ELISA kit (Abcam, Cambridge, UK) following the manufacturer's instructions.
Coculture of BM CD34 + cells with MФs BM CD34 + cells were isolated from BMMNCs of HD using a CD34 MicroBead kit (Miltenyi Biotec, Bergisch Gladbach, Germany) as previously described. 7,8,27,40,43The treated MФs were carefully washed three times with 1 mL 1 × PBS to remove exogenous cytokines and chemical reagents.Subsequently, CD34 + cells were added and direct-contact cocultured with the treated MФs for another 7 days in StemSpanTM SFEM (Stem Cell Technologies, Vancouver, BC, Canada) containing 100 ng/mL SCF, 100 ng/mL TPO, and 10 ng/mL IL-3 (PeproTech) to promote MK differentiation. 7,8,43,44Indirect-contact coculture assay was conducted simultaneously where CD34 + cells were plated on the upper chamber of a Transwell (Corning Incorporated, NY, USA) suspended above the MФs.Appropriate controls of CD34 + cells alone and unstimulated MФs cultures were included in coculture experiments.The CFU-MKs were analyzed by a commercially available kit (MegaCult-C; Stem Cell Technologies).A total of 5 × 10 4 CD34 + cells were plated in each chamber slide for 12 days, and MK colonies were defined as groups of 3 or more glycoprotein IIb/IIIa-positive cells.Quantification of the MK count, MK polyploidy distribution, and platelet production in the coculture system was performed as previously described. 7,8,43,44To measure intracellular protein levels in MKs after 12 days of coculture, the MKs were identified by labeling with CD41a and then fixed, permeabilized, and incubated with p-STAT5 and p-ERK (Cell Signaling Technology, Danvers, MA, USA).The intracellular protein levels were evaluated by LSRFortessa software (Becton Dickinson) and expressed as the MFI (mean ± SEM).
Immunofluorescence staining and image analysis As previously described, 27 the immunofluorescence staining of the BMBs were performed with the mouse anti-human CD68 (Abcam, MA, USA) and rabbit anti-human CD163 (Abcam) antibodies.4′,6-Diamidino-2-phenylindole (DAPI) was applied to stain the nuclei, and the slides were analyzed under a Leica TCS SP8 microscope (Leica Microsystems, Wetzlar, Germany).Positive cells per HPF were quantified in a semiautomated way using ImageJ software.

RNA sequencing and data analysis
The cultivated BM MФs samples, including the primary MФs from PT and GGF patients, and the polarized BM-M1 and BM-M2 from HD, were analyzed using RNA-seq.Briefly speaking, nextgeneration RNA-seq libraries were constructed with qualified RNA samples using the NEBNext® Ultra™ RNA Library Prep Kit for Illumina.The eligible libraries were sequenced on an Illumina HiSeq XTen platform (150bp paired-end reads), yielding ~6G raw data per sample.Low-quality reads and adapter sequences were removed and the remained clean reads were quantified against an Ensembl catalog (GRCh38) at the transcript level by Salmon software and aggregated at the gene level using the R package "tximport". 45Finally, the transcripts per kilobase million (TPM) and count value were used for the following analysis.Differentially expressed genes (DEGs) were calculated with R package "DESeq2" and "IHW".With the DEGs, GO enrichment analysis and their hierarchy relation analysis were conducted with the R package **The continuous variables were compared using the Mann-Whitney Utest, and the differences in frequency between the two groups were compared using the chi-square test.The criterion for statistical significance was P < 0.05 a Group matching criteria included age at HSCT (±1years), pre-HSCT cycles of chemotherapy (±1cycle), disease status at HSCT, and BM microenvironment evaluated time after HSCT (±5 days).For each case, two GGF control was randomly selected from the same cohort at which the PT occurred (risk-set sampling) "topGO".KEGG pathway analyses were performed with the R package "clusterProfiler".To determine the MФ phenotype scores from the samples of patients, a deconvolution approach was performed using an R-based version of CIBERSORT with gene list "immunoState". 46The MФ phenotype scores include M1 and M2 phenotype, which named after M1 and M2 score, respectively.To some extent, the score integrated a more comprehensive phenotype of MФs compared with flow cytometry data.
Western blot analysis Total proteins obtained from cell lysates were subjected to SDS-PAGE and transferred onto a polyvinylidene difluoride membrane (Bio-Rad Laboratories, Hercules, CA, USA).For experiments involving signaling pathways, antibodies against PI3K p110α, AKT, and p-AKT (Ser473) (Cell Signaling Technology) were used.Membranes were probed with primary antibodies at 4 °C overnight, followed by incubation of anti-rabbit or anti-mouse secondary antibodies.The membranes were developed with ECL reagents (Millipore, Bedford, MA) before protein bands were observed on X-ray films.To control for total protein loading, membranes were stripped of the primary antibodies and reprobed with anti-GAPDH antibody (Sigma Chemical Co., St. Louis, MO, USA).Quantification of the band intensities was performed using GeneTools software (Syngene).
The GFP-positive cells in BM were measured by flow cytometry.To evaluate the MФ subsets in the BM of mice, femurs and tibias of each mouse were collected to make single-cell suspensions, and then stained with anti-CD11b-APC-Cy7, anti-F4/80-BV421, anti-CD86-PE, and anti-CD163-APC (BioLegend) and analyzed by flow cytometry.BM MK polyploidy distribution were evaluated after labeling with CD41a and then fixed and incubated with PI staining buffer.
For histopathological analysis of BM, three mice per group were sacrificed at 3 weeks and five mice per group were sacrificed at 4 weeks after injection, and the femurs were harvested and fixed in 4% paraformaldehyde for hematoxylin and eosin (H&E) staining.

Statistical analyses
Statistical analyses were performed using one-way analysis of variance (ANOVA) for comparisons among groups.Subject variables were compared using a chi-squared test for categorical variables and a Mann-Whitney U-test for continuous variables.Wilcoxon's test for paired data was used to identify the drug effects.Analyses were performed with SPSS version 25.0 (SPSS Inc, IBM, Chicago, IL, USA) and GraphPad Prism 6.0 (GraphPad Software, La Jolla, CA, USA), and P-values < 0.05 were considered significant.Data are presented as the means ± SEM.

Fig. 1
Fig. 1 Altered distribution of monocyte-macrophage subsets in the bone marrow of PT patients.Percentage of a classical monocytes, b intermediate monocytes, c non-classical monocytes, d M1 MФs, and e M2 MФs in the BM of PT patients, GGF patients and HDs as indicated by flow cytometry.f The M1/M2 ratio was calculated to represent the MФ polarization status in PT patients, GGF patients, and HD.Positive area of g CD68 + M1 MФs and h CD163 + M2 MФs per high-power field[HPF] in the BM trephine biopsies of PT patients, GGF patients, and HD as quantified by ImageJ.i M1 (CD68 + , red) and M2 (CD163 + , green) MФs in BM trephine biopsies from PT patients, GGF patients, and HD were visualized by immunofluorescence.Nuclei were counterstained with DAPI (blue).An overlay of the three colors reveals the number of M1 and M2 MФs in the BM trephine biopsies from the three groups (scale bars represent 50 μm).The images (right panel) are enlarged from the labeled square areas of the left images.Data are presented as the means ± SEM (*P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001)

Fig. 2
Fig. 2 Increased M1 macrophages and decreased M2 macrophages lead to a reduced megakaryopoiesis-supporting ability in the bone marrow of PT patients.a Representative images (left panel, scale bars represent 50 μm) and quantification (right panel) of the phagocytosis assays for the cultivated BM MФs from PT patients, GGF patients and HD after 7 days in culture.b Representative images (left panel, scale bars represent 50 μm) and quantification (right panel) of the transwell migration assays for the cultivated BM MФs from PT patients, GGF patients, and HD after 7 days in culture.The intracellular levels of c TNF-ɑ and d TGF-β were analyzed in BM MФs from PT patients and GGF patients after 7 days of culture.MK production and maturation, colony-forming unit MK (CFU-MK) plating efficiencies, and platelet release were analyzed after coculture with the cultivated BM MФs from PT patients and GGF patients.The e MK count, f CFU-MK count (left panel: representative images, scale bars represent 50 μm; right panel: quantification), g platelet count and h MK ploidy distribution were analyzed after 12 days of coculture.Data are presented as the means ± SEM (*P ≤ 0.05, **P ≤ 0.01)

Fig. 3
Fig. 3 Activating the PI3K-AKT pathway enhanced the megakaryopoiesis-supporting ability of macrophages from PT patients.a Schematic diagram of the study design on RNA-seq of the cultivated BM MФs from PT and GGF patients.b Heatmaps showed M1 and M2 scores (calculated by CIBERSORT algorithm) in PT MФs and GGF MФs (scaled by row).Box plot depicting c CIBERSORT score of M1 MФs, d CIBERSORT score of M2 MФs and e CIBERSORT score of M1/M2 in PT MФs and GGF MФs.f GO enrichment analysis showed the top ten terms enriched by the upregulated genes of GGF MФs and PT MФs.The size of each circle indicates the ratio of DEGs counts and the gene counts of the term.g KEGG enrichment analysis showed the top five pathways enriched by the upregulated genes of GGF MФs and PT MФs.h Heatmap showed expression of AKT1 gene in GGF MФs and PT MФs.i Representative western blots of p-AKT, AKT, and GAPDH expression in the cultivated BM MФs from PT patients and GGF patients.j Schematic diagram of the study design on RNA-seq of BM-M1 and BM-M2 from HD. k KEGG enrichment analysis showed the top five pathways enriched by the upregulated genes of M1 MФs and M2 MФs.The size of each circle indicates the scaled ratio of DEGs counts and the gene counts of the pathway.MK production and maturation, CFU-MK plating efficiencies, and platelet release were analyzed after coculture with BM MФs that were cultivated from PT patients and GGF patients and were subjected to various treatments.The l MK count, m MK ploidy distribution, n representative CFU-MK images (scale bars represent 50 μm), o CFU-MK count, and p platelet count were analyzed after 12 days of coculture.Data are presented as the means ± SEM (*P ≤ 0.05, ** P ≤ 0.01)

Fig. 5 Fig. 6
Fig. 5 The megakaryopoiesis-supporting ability of macrophages was improved by activating the PI3K-AKT pathway.a Representative fluorescence microscopy images of THP1-derived MФs infected with lentivirus (scale bars represent 50 μm).b Representative western blots of AKT, PI3K and GAPDH in THP1-derived MФs infected with lentivirus.MK production and maturation, CFU-MK plating efficiencies, and platelet release were analyzed after coculture with the infected MФs.The c MK count, d CFU-MK count, e platelet count, f representative CFU-MK images (scale bars represent 50 μm), and g MK ploidy distribution were analyzed after 12 days of coculture.Data are presented as the means ± SEM (*P ≤ 0.05, ** P ≤ 0.01)

Fig. 7
Fig. 7 M2 macrophages enhanced phospho-STAT5 and phospho-ERK activity in cocultured megakaryocytes.The ELISA analysis of a TGF-β and b TNF-α levels in the supernatants of BM MФs.The ELISA analysis of c TGF-β levels in the BM plasma of PT or GGF patients.Flow cytometry revealed the intracellular d phosphor(p)-STAT5 and e p-ERK levels in the MKs after cocultured with the different subtypes of MФs.Data are presented as the means ± SEM (*P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001).f Graphical abstract of the current study.Schematic illustration of the different effects of M1 MФs and M2 MФs on megakaryopoiesis

Table 1 .
Characteristics of allo-HSCT patients with PT and GGF