Macrophage polarization in placenta accreta and macrophage-trophoblast interactions.

PROBLEM: Placenta accreta (PA) is defined by an abnormal invasion of placental trophoblasts into the myometrium, which can lead to serious postpartum complications. Macrophages play an important role in the regulation of trophoblast function. Both granulocyte colony-stimulating factor (G-CSF) and its receptor (granulocyte colony-stimulating factor receptor, G-CSFR) have effects on trophoblast invasion. However, the current understanding of G-CSF secretion, G-CSFR expression, abnormal polarization of decidual macrophages (dMϕ) in PA and the abnormal invasion of placental trophoblasts into the myometrium are limited. METHOD OF STUDY: The polarization of dMϕ in PA was analyzed by flow cytometry (FCM), and the expression of G-CSFR in placental trophoblasts in PA was evaluated by immunohistochemistry. In an in vitro co-culture model, we investigated the effects of HTR-8/SVneo trophoblasts cell line (HTR-8) on macrophage human monocyte cell line (THP-1) polarization and G-CSF secretion, and we also analyzed the effects of THP-1 cells, especially M2-like subtype, on primary trophoblasts and HTR-8 proliferation, invasion, and adhesion. FCM, transwell assays, adhesion assays, and proliferation assays were used in the above model. RESULTS: Compared with controls (n = 9), dMϕ showed significantly lower levels of M1 markers CD80 and CD86 and higher levels of the M2 markers CD163 and CD206, and G-CSFR expression of placental trophoblasts was increased in PA (n = 5). In vitro experiments showed that the trophoblast HTR-8 cell line induced polarization of THP-1 cells to an M2-like subtype and increased their secretion of G-CSF. Furthermore, IL-4/IL-13-induced M2-like THP-1 macrophages were able to increase the expression of G-CSFR, proliferation, invasion and adhesion of both primary trophoblasts and HTR-8 trophoblasts. CONCLUSIONS: There is an altered immune imbalance at the maternal-fetal interface in PA, which further may lead to abnormal trophoblast function. G-CSF and its receptors may play important roles in abnormal polarization of macrophages and abnormal invasion of trophoblasts.

Granulocyte Colony-Stimulating Factor Mediated Regulation of Early Myeloid Cells in Zebrafish.

BACKGROUND: Colony-stimulating factor 3 (CSF3), more commonly known as granulocyte colony-stimulating factor (G-CSF), acts via a specific cell surface receptor CSF3R (or G-CSFR) to regulate hematopoiesis, with a particularly key role in the myeloid cell lineage where it impacts the development and function of neutrophilic granulocytes. Zebrafish possess a conserved CSF3R homologue, Csf3r, which is involved in both steady-state and emergency myelopoiesis, as well as regulating early myeloid cell migration. Two CSF3 proteins have been identified in zebrafish, Csf3a and Csf3b. METHODS: This study investigated the roles of the Csf3a and Csf3b ligands as well as the downstream Janus kinase (JAK) and phosphatidylinositol 3-kinase (PI3K) pathways in mediating the effects of Csf3r in early myeloid cell development and function using gene knockdown and pharmacologic approaches. RESULTS: This study revealed that both Csf3a and Csf3b contribute to the developmental and emergency production of early myeloid cells, but Csf3a is responsible for the developmental migration of early neutrophils whereas Csf3b plays the major role in their wounding-induced migration, differentially participated in these responses, as did several downstream signaling pathways. Both JAK and PI3K signaling were required for developmental production and migration of early myeloid cells, but PI3K signaling was required for emergency production and initial migration in response to wounding, while JAK signaling mediated retention at the site of wounding. CONCLUSIONS: This study has revealed both distinct and overlapping functions for Csf3a and Csf3b and the downstream JAK and PI3K signaling pathways in early myeloid cell production and function.

Leukemia-associated truncation of granulocyte colony-stimulating factor receptor impacts granulopoiesis throughout the life-course.

Introduction: The granulocyte colony-stimulating factor receptor (G-CSFR), encoded by the CSF3R gene, is involved in the production and function of neutrophilic granulocytes. Somatic mutations in CSF3R leading to truncated G-CSFR forms are observed in acute myeloid leukemia (AML), particularly those subsequent to severe chronic neutropenia (SCN), as well as in a subset of patients with other leukemias. Methods: This investigation introduced equivalent mutations into the zebrafish csf3r gene via genome editing and used a range of molecular and cellular techniques to understand the impact of these mutations on immune cells across the lifespan. Results: Zebrafish harboring truncated G-CSFRs showed significantly enhanced neutrophil production throughout successive waves of embryonic hematopoiesis and a neutrophil maturation defect in adults, with the mutations acting in a partially dominant manner. Discussion: This study has elucidated new insights into the impact of G-CSFR truncations throughout the life-course and created a bone fide zebrafish model for further investigation.

G-CSF, the guardian of granulopoiesis.

A considerable amount of continuous proliferation and differentiation is required to produce daily a billion new neutrophils in an adult human. Of the few cytokines and factors known to control neutrophil production, G-CSF is the guardian of granulopoiesis. G-CSF/CSF3R signaling involves the recruitment of non-receptor protein tyrosine kinases and their dependent signaling pathways of serine/threonine kinases, tyrosine phosphatases, and lipid second messengers. These pathways converge to activate the families of STAT and C/EBP transcription factors. CSF3R mutations are associated with human disorders of neutrophil production, including severe congenital neutropenia, neutrophilia, and myeloid malignancies. More than three decades after their identification, cloning, and characterization of G-CSF and G-CSF receptor, fundamental questions remain about their physiology.

Endogenous Expression of G-CSF in Rat Dorsal Root Ganglion Neurons after Nerve Injury.

Granulocyte colony-stimulating factor (G-CSF) has been reported to modulate pain function following nerve injury. However, the expression of endogenous G-CSF in the dorsal root ganglion (DRG) and the response to nerve injury remain unclear. In the present study, we demonstrated that G-CSF and G-CSFR are mainly expressed in both small- and medium-diameter DRG neurons in rats and are responsible for transmitting pain responses. G-CSF and G-CSFR were co-expressed in certain nociceptive DRG neurons. In addition, G-CSF was expressed in satellite glial cells around large-diameter DRG neurons. After sciatic nerve injury, the number of G-CSF-positive DRG neurons was increased in both the ipsilateral and contralateral lesion sites in rats. However, G-CSF expression in satellite glial cells was not affected by nerve injury. To clarify the role of G-CSF in pain, exogenous G-CSF was administered to a rat model of neuropathic pain induced by partial sciatic nerve transaction (PST). Our results indicate that treatment with G-CSF did not attenuate but exacerbated neuropathic pain. In summary, G-CSF may directly activate sensory neurons and contribute to nociceptive signaling.

Application of the Moran Model in Estimating Selection Coefficient of Mutated CSF3R Clones in the Evolution of Severe Congenital Neutropenia to Myeloid Neoplasia.

Bone marrow failure (BMF) syndromes, such as severe congenital neutropenia (SCN) are leukemia predisposition syndromes. We focus here on the transition from SCN to pre-leukemic myelodysplastic syndrome (MDS). Stochastic mathematical models have been conceived that attempt to explain the transition of SCN to MDS, in the most parsimonious way, using extensions of standard processes of population genetics and population dynamics, such as the branching and the Moran processes. We previously presented a hypothesis of the SCN to MDS transition, which involves directional selection and recurrent mutation, to explain the distribution of ages at onset of MDS or AML. Based on experimental and clinical data and a model of human hematopoiesis, a range of probable values of the selection coefficient s and mutation rate µ have been determined. These estimates lead to predictions of the age at onset of MDS or AML, which are consistent with the clinical data. In the current paper, based on data extracted from published literature, we seek to provide an independent validation of these estimates. We proceed with two purposes in mind: (i) to determine the ballpark estimates of the selection coefficients and verify their consistency with those previously obtained and (ii) to provide possible insight into the role of recurrent mutations of the G-CSF receptor in the SCN to MDS transition.

G-CSF and G-CSFR Induce a Pro-Tumorigenic Macrophage Phenotype to Promote Colon and Pancreas Tumor Growth.

Tumor-associated macrophages (TAMs) in the gastrointestinal tumor microenvironment (TME) are known to polarize into populations exhibiting pro- or anti-tumoral activity in response to stimuli such as growth factors and cytokines. Our previous work has recognized granulocyte colony-stimulating factor (G-CSF) as a cytokine capable of influencing immune cells of the TME exhibiting pro-tumoral activity. Here, we aimed to focus on how G-CSF regulates TAM phenotype and function and the effects on gastrointestinal (GI) tumor progression. Thus, wildtype (WT) and G-CSFR-/- macrophages were examined for cytokine production, gene expression, and transcription factor activity. Adoptive transfer of WT or G-CSFR-/- macrophages into tumor-bearing mice was performed to study their influence in the progression of colon (MC38) and pancreatic (PK5L1940) tumor mouse models. Finally, the difference in cytotoxic potential between WT and G-CSFR-/- macrophages was examined both in vitro and in vivo. Our results indicate that G-CSF promotes increased IL-10 production and decreased IL-12 production, which was reversed in G-CSFR-/- macrophages for a pro-inflammatory phenotype. Furthermore, G-CSFR-/- macrophages were characterized by higher levels of NOS2 expression and NO production, which led to greater tumor related cytotoxicity both in vitro and in vivo. Our results suggest that in the absence of G-CSFR, macrophage-related tumor cytotoxicity was amplified. These findings, along with our previous reports, pinpoint G-CSF /G-CSFR as a prominent target for possible clinical applications that aim to control the TME and the GI tumor progression.

Cooperating, congenital neutropenia-associated Csf3r and Runx1 mutations activate pro-inflammatory signaling and inhibit myeloid differentiation of mouse HSPCs.

Patients with the pre-leukemia bone marrow failure syndrome called severe congenital neutropenia (CN) have an approximately 15% risk of developing acute myeloid leukemia (AML; called here CN/AML). Most CN/AML patients co-acquire CSF3R and RUNX1 mutations, which play cooperative roles in the development of AML. To establish an in vitro model of leukemogenesis, we utilized bone marrow lin- cells from transgenic C57BL/6-d715 Csf3r mice expressing a CN patient-mimicking truncated CSF3R mutation. We transduced these cells with vectors encoding RUNX1 wild type (WT) or RUNX1 mutant proteins carrying the R139G or R174L mutations. Cells transduced with these RUNX1 mutants showed diminished in vitro myeloid differentiation and elevated replating capacity, compared with those expressing WT RUNX1. mRNA expression analysis showed that cells transduced with the RUNX1 mutants exhibited hyperactivation of inflammatory signaling and innate immunity pathways, including IL-6, TLR, NF-kappaB, IFN, and TREM1 signaling. These data suggest that the expression of mutated RUNX1 in a CSF3R-mutated background may activate the pro-inflammatory cell state and inhibit myeloid differentiation.

SWATH-Proteomics of Ibrutinib's Action in Myeloid Leukemia Initiating Mutated G-CSFR Signaling.

PURPOSE: To evaluate cellular protein changes in response to treatment with an approved drug, ibrutinib, in cells expressing normal or mutated granulocyte-colony stimulating factor receptor (G-CSFR). G-CSFR mutations are associated with some hematological malignancies. Previous studies show the efficacy of ibrutinib (a Bruton's tyrosine kinase inhibitor) in mutated G-CSFR leukemia models but do not address broader signaling mechanisms. EXPERIMENTAL DESIGN: A label-free quantitative proteomics workflow to evaluate the cellular effects of ibrutinib treatment is established. This includes three biological replicates of normal and mutated G-CSFR expressed in a mouse progenitor cell (32D cell line) with and without ibrutinib treatment. RESULTS: The proteomics dataset shows about 1000 unique proteins quantified with nearly 400 significant changes (p value < 0.05), suggesting a highly dynamic network of cellular signaling in response to ibrutinib. Importantly, the dataset is very robust with coefficients of variation for quantitation at 13.0-20.4% resulting in dramatic patterns of protein differences among the groups. CONCLUSIONS AND CLINICAL RELEVANCE: This robust dataset is available for further mining, hypothesis generation, and testing. A detailed understanding of the restructuring of the proteomics signaling cascades by ibrutinib in leukemia biology will provide new avenues to explore its use for other related malignancies.

Ultra-Sensitive CSF3R Deep Sequencing in Patients With Severe Congenital Neutropenia.

High frequency of acquired CSF3R (colony stimulating factor 3 receptor, granulocyte) mutations has been described in patients with severe congenital neutropenia (CN) at pre-leukemia stage and overt acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS). Here, we report the establishment of an ultra-sensitive deep sequencing of a CSF3R segment encoding the intracellular "critical region" of the G-CSFR known to be mutated in CN-MDS/AML patients. Using this method, we achieved a mutant allele frequency (MAF) detection rate of 0.01%. We detected CSF3R mutations in CN patients with different genetic backgrounds, but not in patients with other types of bone marrow failure syndromes chronically treated with G-CSF (e.g., Shwachman-Diamond Syndrome). Comparison of CSF3R deep sequencing results of DNA and cDNA from the bone marrow and peripheral blood cells revealed the highest sensitivity of cDNA from the peripheral blood polymorphonuclear neutrophils. This approach enables the identification of low-frequency CSF3R mutant clones, increases sensitivity, and earlier detection of CSF3R mutations acquired during the course of leukemogenic evolution of pre-leukemia HSCs of CN patients. We suggest application of sequencing of the entire CSF3R gene at diagnosis to identify patients with inherited lost-of-function CSF3R mutations and annual ultra-deep sequencing of the critical region of CSF3R to monitor acquisition of CSF3R mutations.

Immunohistochemical Study on the Expression of G-CSF, G-CSFR, VEGF, VEGFR-1, Foxp3 in First Trimester Trophoblast of Recurrent Pregnancy Loss in Pregnancies Treated with G-CSF and Controls.

BACKGROUND: Recurrent Pregnancy Loss (RPL) is a syndrome recognizing several causes, and in some cases the treatment with Granulocyte Colony Stimulating Factor (G-CSF) may be successful, especially when karyotype of the previous miscarriage showed no embryo chromosomal abnormalities. In order to evaluate the effects of G-CSF treatment on the decidual and trophoblast expression of G-CSF and its receptor, VEGF and its receptor and Foxp3, specific marker of putative Tregs we conducted an immunohistochemical study. METHODS: This study was conducted on three groups of patients for a total of 38 women: in 8 cases decidual and trophoblast tissue were obtained from 8 women with unexplained RPL treated with G-CSF that miscarried despite treatment; in 15 cases the tissue were obtained from 15 women with unexplained RPL no treated; 15 cases of women who underwent voluntary pregnancy termination were used as controls. Tissue collected from these patients were used for immunohistochemistry studies testing the expression of G-CSF, G-CSFR, VEGF, VEGFR-1 and Foxp3. RESULTS: G-CSF treatment increased the concentration of cells expressing Foxp3, specific marker for Tregs, in the decidua, whereas in no treated RPL a reduction of these cells was found when compared to controls. Furthermore, G-CSF treatment increased the expression of G-CSF and VEGF in the trophoblast. CONCLUSIONS: Our study showed that G-CSF treatment increased the number of decidual Treg cells in RPL patients as well as the expression of G-CSF and VEGF in villus trophoblast. These finding may explain the effectiveness of this treatment in RPL, probably regulating the maternal immune response through Tregs recruitment in the decidua, as well as stimulating trophoblast growth.

Regulation of granulocyte colony-stimulating factor receptor-mediated granulocytic differentiation by C-mannosylation.

Granulocyte colony-stimulating factor (G-CSF) receptor (G-CSFR) is a type I cytokine receptor which is involved in hematopoietic cell maturation. G-CSFR has three putative C-mannosylation sites at W253, W318, and W446; however, it is not elucidated whether G-CSFR is C-mannosylated or not. In this study, we first demonstrated that G-CSFR was C-mannosylated at only W318. We also revealed that C-mannosylation of G-CSFR affects G-CSF-dependent downstream signaling through changing ligand binding capability but not cell surface localization. Moreover, C-mannosylation of G-CSFR was functional and regulated granulocytic differentiation in myeloid 32D cells. In conclusion, we found that G-CSFR is C-mannosylated at W318 and that this C-mannosylation has role(s) for myeloid cell differentiation through regulating downstream signaling.

Expression and role of granulocyte macrophage colony-stimulating factor receptor (GM-CSFR) and granulocyte colony-stimulating factor receptor (G-CSFR) on Ph-positive acute B lymphoblastic leukemia.

OBJECTIVE: We observed that ph + ALL patients administrated with recombinant human G-CSF (rhG-CSF) after intense chemotherapy have presented a trend of disease relapse. Thus, we aim to thoroughly investigate the expression and role of GM-CSFR and G-CSFR on ph + ALL patients. METHOD: SUP-B15, BALL-1 and primary leukemia cells were used in this study. Transcript levels were analyzed by quantitative PCR while cell viability was measured using a CCK-8 assay. Flow cytometry was used to assess the different stages of cell cycle. RESULTS: We found that the mRNA expression levels of GM-CSFR and G-CSFR were higher in patients with ph + ALL, as well as in SUP-B15 cells. rhG-CSF was also observed to promote the viability of SUP-B15 cells while inversely inhibiting BALL-1 cell viability. In addition, we also determined that rhG-CSF (100 ng/ml) decreased the sensitivity of SUP-B15 cells to imatinib and nilotinib, while the results were exactly the contrary for dasatinib. CONCLUSION: We demonstrated high expression levels of GM-CSFR and G-CSFR, as well as their promotable role for viability in ph + ALL cells. We further found that rhG-CSF influenced the sensitivity of SUP-B15 cells to TKIs.

E6AP inhibits G-CSFR turnover and functions by promoting its ubiquitin-dependent proteasome degradation.

Granulocyte colony-stimulating factor receptor (G-CSFR) plays a crucial role in regulating myeloid cell survival, proliferation, and neutrophilic granulocyte precursor cells maturation. Previously, we demonstrated that Fbw7α negatively regulates G-CSFR and its downstream signaling through ubiquitin-proteasome mediated degradation. However, whether additional ubiquitin ligases for G-CSFR exist is not known. Identifying multiple E3 ubiquitin ligases for G-CSFR shall improve our understanding of activation and subsequent attenuation of G-CSFR signaling required for differentiation and proliferation. Here, for the first time we demonstrate that E6 associated protein (E6AP), an E3 ubiquitin ligase physically associates with G-CSFR and targets it for ubiquitin-mediated proteasome degradation and thereby attenuates its functions. We further show that E6AP promoted G-CSFR degradation leads to reduced phosphorylation of signal transducer and activator of transcription 3 (STAT3) which is required for G-CSF dependent granulocytic differentiation. More importantly, our finding shows that E6AP also targets mutant form of G-SCFR (G-CSFR-T718), frequently observed in severe congenital neutropenia (SCN) patients that very often culminate to AML, however, at a quite slower rate than wild type G-CSFR. In addition, our data showed that knockdown of E6AP restores G-CSFR and its signaling thereby promoting granulocytic differentiation. Collectively, our data demonstrates that E6AP facilitates ubiquitination and subsequent degradation of G-CSFR leading to attenuation of its downstream signaling and inhibition of granulocytic differentiation.

The importance of the granulocyte-colony stimulating factor in oncology.

Granulocyte-colony stimulating factor (G-CSF) is a glycoprotein, the second CSF, sharing some common effects with granulocyte macrophage-colony stimulating factor (GM-CSF), interleukin-3 (IL-3) and interleukin-5 (IL-5). G-CSF is mainly produced by fibroblasts and endothelial cells from bone marrow stroma and by immunocompetent cells (monocytes, macrophages). The receptor for G-CSF (G-CSFR) is part of the cytokine and hematopoietin receptor superfamily and G-CSFR mutations cause severe congenital neutropenia. The main action of G-CSF - G-CSFR linkage is stimulation of the production, mobilization, survival and chemotaxis of neutrophils, but there are many other G-CSF effects: growth and migration of endothelial cells, decrease of norepinephrine reuptake, increase in osteoclastic activity and decrease in osteoblast activity. In oncology, G-CSF is utilized especially for the primary prophylaxis of chemotherapy-induced neutropenia, but it can be used for hematopoietic stem cell transplantation, it can produce monocytic differentiation of some myeloid leukemias and it can increase some drug resistance. The therapeutic indications of G-CSF are becoming more and more numerous: non neutropenic patients infections, reproductive medicine, neurological disturbances, regeneration therapy after acute myocardial infarction and of skeletal muscle, and hepatitis C therapy.

Granulocyte colony-stimulating factor receptor mutations in myeloid malignancy.

Granulocyte colony-stimulating factor is a cytokine able to stimulate both myelopoiesis and hematopoietic stem cell mobilization, which has seen it used extensively in the clinic to aid hematopoietic recovery. It acts specifically via the homodimeric granulocyte colony-stimulating factor receptor (G-CSFR), which is principally expressed on the surface of myeloid and hematopoietic progenitor cells. A number of pathogenic mutations have now been identified in CSF3R, the gene encoding G-CSFR. These fall into distinct classes, each of which is associated with a particular spectrum of myeloid disorders, including malignancy. This review details the various CSF3R mutations, their mechanisms of action, and contribution to disease, as well as discussing the clinical implications of such mutations.

E3 ubiquitin ligase Fbw7 negatively regulates granulocytic differentiation by targeting G-CSFR for degradation.

Tight control between activation and attenuation of granulocyte colony stimulating factor receptor (G-CSFR) signaling is essential to regulate survival, proliferation and differentiation of myeloid progenitor cells. Previous studies demonstrated negative regulation of G-CSFR through endosomal-lysosomal routing and ubiquitin-proteasome mediated degradation. However, very few E3 ubiquitin ligases are known to target G-CSFR for ubiquitin-proteasome pathway. Here we identified F-box and WD repeat domain-containing 7 (Fbw7), a substrate recognizing component of Skp-Cullin-F box (SCF) E3 ubiquitin Ligase physically associates with G-CSFR and promotes its ubiquitin-mediated proteasomal degradation. Our data shows that Fbw7 also interacts with and degrades G-CSFR-T718 (a truncated mutant of G-CSFR found in severe congenital neutropenia/acute myeloid leukemia (SCN/AML patients)) though at a quite slower rate compared to G-CSFR. We further show that glycogen synthase kinase 3 beta (GSK3ß), like Fbw7 also targets G-CSFR and G-CSFR-T718 for degradation; however, Fbw7 and GSK3ß are interdependent in targeting G-CSFR/G-CSFR-T718 for degradation because they are unable to degrade G-CSFR individually when either of them is knocked down. We further show that Fbw7 mediated downregulation of G-CSFR inhibits signal transducer and activator of transcription 3 (STAT3) phosphorylation which is required for G-CSF dependent granulocytic differentiation. In addition, our data also shows that inhibition of Fbw7 restores G-CSFR signaling leading to enhanced STAT3 activity resulting in massive granulocytic differentiation. These data indicate that Fbw7 together with GSK3ß negatively regulates G-CSFR expression and its downstream signaling.

Hematopoietic cell populations in dolphin bone marrow: Analysis of colony formation and differentiation.

Bone marrow biopsy is useful for diagnosis of hematopoietic diseases. We have recently reported that bone marrow biopsy from the flipper might be useful for diagnosis of hematopoietic diseases in dolphins. In this study, to demonstrate whether biopsy from the flipper is useful for clinical diagnosis, we investigated the gene expression profiles and proliferation and differentiation of bone marrow mononuclear cell (BMMC) isolated from the humeral bone marrow of bottlenose dolphins. BMMC exhibited gene expression profiles considered to be characteristic of hematopoietic cells. Similarly, a colony forming unit assay showed that dolphin BMMC possessed vigorous colony forming ability. The proliferation of hematopoietic progenitor cells resulted in the formation of three types of colonies, containing neutrophils, monocytes/macrophages and eosinophils with or without megakaryocytes, all of which could be identified based on the morphological characteristics and gene expression profiles typically associated with hematopoietic markers. Thus, dolphin BMMCs from humeral bone marrow contain many hematopoietic progenitor cells, and bone marrow biopsy from the flipper is suggested useful for clinical diagnosis for the dolphins.