Contributions
Abstract: S230
Type: Oral Presentation
Session title: Stem cell transplantation - Preclinical/experimental
Background
Prolonged isolated thrombocytopenia(PT) is a serious complication following allogeneic hematopoietic stem cell transplantation(allo-HSCT). Our previous studies found dysfunctional megakaryopoiesis are responsible for the occurrence of PT. As an important component of the BM microenvironment, macrophages(MՓs) are heterogeneous and polarized into classically activated MՓs(M1) and alternatively activated MՓs(M2) with distinct function. In this regard, our previous study found that the aberrant M1/M2 polarization plays a critical role in regulating hematopoiesis of poor graft function patients, a clinical manifestation of pancytopenia after allo-HSCT. However, the effect of MФ polarization on regulating megakaryopoiesis and novel approach targeting aberrant MФ polarization in PT patients remain to be elucidated.
Aims
To address the polarization state and the function of BM MФs in PT patients. Moreover, to investigate the effect of M1 and M2 on megakaryopoiesis and potential therapeutic target regulating aberrant MФ polarization for PT patients.
Methods
This prospective case-control study enrolled 12 PT patients, 24 matched good graft function(GGF) patients and 12 healthy donors(HD). BM MՓs subsets were analyzed by flow cytometry and in situ histological analyses. The functions of BM MՓs were evaluated by cytokine secretion assays and coculture experiment with megakaryocytes(MKs). To determine the the effect of MՓ on megakaryopoiesis, both primary human BM MՓs and THP1 were polarized into M1 and M2 and cocultured with MKs. To further investigate the underlying mechanism of MՓ in regulating MKs, RNA-seq analyses were performed in primary MФs from patients, and polarized M1 and M2 from HD, respectively. To explore potential approach of targeting the aberrant MФ polarization, the chemical activators or inhibitors and lentivirus for PI3K-AKT pathway were administrated to the primary MФs from patients and THP1 derived MФs. BM MФ specific PI3K-knockdown mice were constructed and megakaryopoiesis were analyzed. Subsequently, subsequent changes in maturation related signaling pathways of MKs were analyzed.
Results
Compared with GGF patients and HD, aberrant BM M1/M2 polarization, characterized by increased M1 and decreased M2 and accompanied by impaired megakaryopoiesis-supporting ability, was found in PT patients. RNA-seq and western blot analysis showed PI3K-AKT pathway was significantly downregulated in 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 suppress, whereas M2 support MK maturation and platelet formation in humans. Chemical inhibition of PI3K-AKT pathway reduced megakaryopoiesis-supporting ability of M2, which could be alleviated by chemical activation of PI3K-AKT pathway. Importantly, genetic knockdown of PI3K-AKT pathway impaired the megakaryopoiesis-supporting ability of MФs both in vitro and in a MФ specific PI3K-knockdown murine model. Furthermore, mechanistic study indicated that TGF-β released by M2 may facilitate megakaryopoiesis through upregulation of the JAK2/STAT5 and MAPK/ERK pathways in MKs.
Conclusion
The current study demonstrated aberrant BM M1/M2 polarization with impaired megakaryopoiesis-supporting ability involved in the pathogenesis of PT. M2, but not M1, support megakaryopoiesis via up-regulating PI3K-AKT pathway. Although further validation is required, restoring M1/M2 MՓ polarization via up-regulating PI3K-AKT pathway might be a potential therapeutic approach for PT patients.
Keyword(s): Macrophage, Megakaryopoiesis, PI3-K/AKT, Thrombocytopenia
Abstract: S230
Type: Oral Presentation
Session title: Stem cell transplantation - Preclinical/experimental
Background
Prolonged isolated thrombocytopenia(PT) is a serious complication following allogeneic hematopoietic stem cell transplantation(allo-HSCT). Our previous studies found dysfunctional megakaryopoiesis are responsible for the occurrence of PT. As an important component of the BM microenvironment, macrophages(MՓs) are heterogeneous and polarized into classically activated MՓs(M1) and alternatively activated MՓs(M2) with distinct function. In this regard, our previous study found that the aberrant M1/M2 polarization plays a critical role in regulating hematopoiesis of poor graft function patients, a clinical manifestation of pancytopenia after allo-HSCT. However, the effect of MФ polarization on regulating megakaryopoiesis and novel approach targeting aberrant MФ polarization in PT patients remain to be elucidated.
Aims
To address the polarization state and the function of BM MФs in PT patients. Moreover, to investigate the effect of M1 and M2 on megakaryopoiesis and potential therapeutic target regulating aberrant MФ polarization for PT patients.
Methods
This prospective case-control study enrolled 12 PT patients, 24 matched good graft function(GGF) patients and 12 healthy donors(HD). BM MՓs subsets were analyzed by flow cytometry and in situ histological analyses. The functions of BM MՓs were evaluated by cytokine secretion assays and coculture experiment with megakaryocytes(MKs). To determine the the effect of MՓ on megakaryopoiesis, both primary human BM MՓs and THP1 were polarized into M1 and M2 and cocultured with MKs. To further investigate the underlying mechanism of MՓ in regulating MKs, RNA-seq analyses were performed in primary MФs from patients, and polarized M1 and M2 from HD, respectively. To explore potential approach of targeting the aberrant MФ polarization, the chemical activators or inhibitors and lentivirus for PI3K-AKT pathway were administrated to the primary MФs from patients and THP1 derived MФs. BM MФ specific PI3K-knockdown mice were constructed and megakaryopoiesis were analyzed. Subsequently, subsequent changes in maturation related signaling pathways of MKs were analyzed.
Results
Compared with GGF patients and HD, aberrant BM M1/M2 polarization, characterized by increased M1 and decreased M2 and accompanied by impaired megakaryopoiesis-supporting ability, was found in PT patients. RNA-seq and western blot analysis showed PI3K-AKT pathway was significantly downregulated in 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 suppress, whereas M2 support MK maturation and platelet formation in humans. Chemical inhibition of PI3K-AKT pathway reduced megakaryopoiesis-supporting ability of M2, which could be alleviated by chemical activation of PI3K-AKT pathway. Importantly, genetic knockdown of PI3K-AKT pathway impaired the megakaryopoiesis-supporting ability of MФs both in vitro and in a MФ specific PI3K-knockdown murine model. Furthermore, mechanistic study indicated that TGF-β released by M2 may facilitate megakaryopoiesis through upregulation of the JAK2/STAT5 and MAPK/ERK pathways in MKs.
Conclusion
The current study demonstrated aberrant BM M1/M2 polarization with impaired megakaryopoiesis-supporting ability involved in the pathogenesis of PT. M2, but not M1, support megakaryopoiesis via up-regulating PI3K-AKT pathway. Although further validation is required, restoring M1/M2 MՓ polarization via up-regulating PI3K-AKT pathway might be a potential therapeutic approach for PT patients.
Keyword(s): Macrophage, Megakaryopoiesis, PI3-K/AKT, Thrombocytopenia