Anemia in Lymphoma: The Role of Cytokines

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 5103-5103
Author(s):  
Zonghong Shao ◽  
Ting Wang ◽  
Meifeng Tu ◽  
Jun Zhu ◽  
Wen Zheng
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Inverse Correlation ◽  
Mild Anemia ◽  
Tnf Α ◽  
Moderate Anemia ◽  
High Bone ◽  
Ifn Γ

Abstract Abstract 5103 Purpose This study was to investigate the role that cytokines play in the pathogenesis of lymphoma associated anemia. Methods IFN-γ, IL-6, TNF-α, IL-1β and EPO in plasma samples from 45 lymphoma patients and 12 controls were analyzed using enzyme-linked immunosorbent assays and EPOR on their bone marrow cells were detected by flow cytometry. Their bone marrow CFU-E were cultured in vitro also. Results Of 45 initial lymphoma patients, 25 (55. 6%) had anemia before diagnosis, 13(28. 9%) had anemia during therapy, 7(15. 5%)never had anemia. Plasma IFN-γ and TNF-α levels were significantly higher in severe and moderate anemia patients than those in mild anemia patients, no anemia patients and controls. Patient's plasma EPO, IL-6 and IFN-γlevels showed an inverse correlation to their Hb. Lymphoma without anemia patients had significantly high bone marrow EPOR level than that of lymphoma associated anemia patients and controls. Patients bone marrow CFU-E showed positive correlation to their Hb and EPOR. Conclusion Increased plasma IFN-γ, TNF-α and IL-6 might contribute to anemia in lymphoma, EPO and EPOR levels were elevated to balance such negetive effects and maintain hemotopoiesis. Disclosures: No relevant conflicts of interest to declare.

CD74 Expression by AML Cell Lines and Bone Marrow Specimens, and Augmented In Vitro Cytotoxicity of Anti-CD74 Antibody After Interferon-Gamma (IFN-γ) Treatment

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2888-2888
Author(s):  
Abhinav B. Chandra ◽  
Jack Burton ◽  
Rhona Stein ◽  
Susan Chen ◽  
Nidhi Mishra ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Cellular Signaling ◽  
In Vitro Cytotoxicity ◽  
Cancer Type ◽  
Phosphorylated Akt ◽  
Wide Range ◽  
Ifn Γ

Abstract Abstract 2888 Background: CD74 (HLA-DR-associated invariant chain) is expressed alone or along with DR in a wide range of hematologic cancers and solid tumors. Humanized anti-CD74 mAb, milatuzumab (Immunomedics, Morris Plains, NJ), exhibits direct cytotoxicity for NHL, CLL and MM cell lines, and is undergoing clinical evaluation for treatment of these malignancies. CD74 is upregulated by interferons in hematologic and epithelial cancer cell lines. Here we present the results of our analysis of CD74 expression and function in AML, and the effect of CD74 upregulation by treatment with IFN-γ on the cytotoxicity of milatuzumab for AML cell lines. Methods: CD74 expression in bone marrow biopsy (BMB) specimens from non-M3 AML patients was evaluated by immunohistochemistry and, for the 3 human AML cell lines, by flow cytometry, with/without permeabilization and with/without IFN-γ (40 and 200 U/mL). These cell lines were also tested in proliferation assays for responses to milatuzumab, with/without IFN-γ. Also, assessment of apoptosis and cellular signaling was performed. Results: In the initial group of AML cases, 13/14 BMB specimens showed moderate to strong CD74 expression by leukemic blasts, which was mostly intracellular, usually with a perinuclear distribution. Three AML cell lines also showed moderate to strong expression of CD74, which was mostly intracellular. Without IFN-γ, surface expression of CD74 was present, but IFN-γ treatment of these 3 lines resulted in upregulation of surface CD74 by 69–117%. Much higher levels of intracellular CD74 were observed in all 3 lines (with and without IFN-γ), with IFN-γ-induced upregulation of intracellular CD74 in all 3 lines (from 85%-868%; P<0.001). In 2/3 lines, IFN-γ increased milatuzumab-mediated growth inhibition (23.7 to 44.8% and -3.9 to 30.9%, P=0.01 and P<0.05, respectively). Cytotoxicity was in part due to apoptosis, as significant increases in Annexin V binding (P=0.01) were observed after treatment with IFN-γ plus milatuzumab. Initial experiments addressing cellular signaling suggest a role for AKT, because phosphorylated AKT levels increased (P=0.06) in response to IFN-γ + milatuzumab. Conclusions: CD74 is expressed in AML patient specimens and in AML cell lines, with the majority of CD74 expression found intracellularly. Cell surface and cytoplasmic expression of CD74 were upregulated in AML lines after IFN-γ exposure. This increased expression resulted in increased cytotoxicity of the anti-CD74 mAb, milatuzumab, in 2/3 AML lines. This effect was through apoptosis and involved the AKT pathway. Thus, AML is another cancer type where combined IFN-γ and milatuzumab treatment may be useful. Supported in part by NIH grant PO1-CA103985 (DMG). Disclosures: No relevant conflicts of interest to declare.

JAK2 Inhibition with TG101348 Inhibits Monosomy 7 Myelodysplastic Syndromes (MDS) Bone Marrow Cells In Vitro: a Potential Targeted Therapy for Monosomy 7 MDS

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 973-973 ◽  
Author(s):  
Matthew J. Olnes ◽  
Andrea Poon ◽  
Zachary Tucker ◽  
Neal S. Young ◽  
Elaine M Sloand
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Myelodysplastic Syndromes ◽  
Mononuclear Cells ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Myeloid Cells ◽  
Dependent Manner ◽  
Monosomy 7

Abstract Abstract 973 The myelodysplastic syndromes (MDS) are bone marrow disorders characterized by cytopenias and a variable risk of progression to acute myeloid leukemia (AML). Monosomy 7 is the second most common cytogenetic abnormality in MDS, and the most frequent karyotypic aberration occurring in aplastic anemia patients following immunosuppressive therapy. Monosomy 7 MDS carries a particularly poor prognosis, with patients manifesting severe cytopenias and a high propensity to develop treatment-refractory AML. There are currently no targeted therapies for this disorder. We previously reported that monosomy 7 bone marrow mononuclear cells (BMMNCs) express high levels of a differentiation-defective granulocyte colony stimulating factor (G-CSF) receptor isoform (IV), an alternative splice variant that exhibits constitutive signaling through the JAK-2 and STAT-1 pathway, while levels of STAT-3 and -5 are unchanged (Sloand et al, PNAS, 2006, 103:14483). As a result, the cell's ability to differentiate is limited, while its ability to proliferate remains intact. Here we examine the effects of the highly selective JAK2 inhibitor TG101348 on monosomy 7 aneuploidy in BMMNCs, as well as the activity of this compound on CD34+ stem cells and CD13+ myeloid cells in culture, and on the JAK-2 signaling apparatus. Incubation of BMMNCs with TG101348 for 5 days significantly decreased absolute numbers of monosomy 7 aneuploid cells in a concentration dependent manner versus vehicle- treated controls (0.187 × 106 vs 1.08 × 106, P=0.007), while diploid cell numbers remained stable (0.338 × 106 vs 0.213 × 106, P=0.50). Flow cytometry experiments demonstrated that incubation with increasing concentrations of TG101348 decreased the absolute number of CD34+CD13- stem cells, and increased numbers of more differentiated CD34-CD13+ myeloid cells, with median CD34+/CD13+ ratios of 6.547 and 2.216 for cells treated with vehicle and 100 nM TG101348, respectively. By immunoblot, STAT-1 protein expression in monosomy 7 BMMNCs treated with 1uM TG101348 was decreased relative to vehicle- treated controls, while there was no difference in STAT-3 and STAT-5 levels. Thus TG101348 decreases monosomy 7 MDS blasts in vitro through inhibition of JAK-2/STAT-1 signaling, a finding that warrants further study of this agent in clinical trials for patients with monosomy 7 MDS and AML. Disclosures: No relevant conflicts of interest to declare.

Autophagy Regulation and Dysfunction in Bone Marrow Malignancies

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4623-4623
Author(s):  
Fernando V Pericole ◽  
Mariana Lazarini ◽  
Adriana S. S. Duarte ◽  
João Machado-Neto ◽  
Sara T. Olalla Saad
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Low Risk ◽  
Control Group ◽  
Hematopoietic Stem ◽  
Combination Treatments ◽  
Molecular Machinery ◽  
Total Bone Marrow

Abstract Abstract 4623 Introduction: Autophagy is a catabolic pathway by which cytoplasmic materials are degraded into the lysosome and it is also a quality control system for proteins and organelles. Autophagy plays an important role in cell adaptation to starvation, hypoxia, cell survival and cancer. Its core molecular machinery is tightly linked to metabolic pathways, such as LKB1/AMPK and mTORC1. Autophagy has been shown to play several important roles in cancer. Indeed, multiple autophagy genes have been characterized as tumor suppressor genes. In hematopoietic system, autophagy is required during myeloid and lymphoid differentiation, terminal erythroid mitochondrial clearance, production of proplatelets and also differentiation of monocytes into macrophages. Interestingly, autophagy seems disturbed in most bone marrow malignancies. Evidence in mice suggests that autophagy suppression (ATG7 or ATG5 knockdown models) in hematopoietic stem cells may be implicated in Acute Myeloid Leukemia (AML) pathogenesis. In Multiple Myeloma (MM), in vitro studies using cell lines showed autophagy activation and lysosome inhibitors (such as chloroquine) are currently been used in various combination treatments in clinical trials. Aim: The aim was to characterize the expression of autophagy machinery key genes (BECN1, MAP1LC3A, SQSTM1), as well as hypoxia master regulator (HIF1A) in total bone marrow cells from bone marrow malignancies: myelodysplasia (MDS), MM and AML patients, excluding acute promyelocytic leukemia. Methods: BECN1, MAP1LC3A, SQSTM1 and HIF1A levels were verified, by q-PCR, in diagnostic (or without any treatment) BM aspirates from 22 normal donors, 30 MDS (17 low-risk and 13 high-risk, according 2008 WHO classification), 43 AML and 11 MM patients. Results: BECN1 gene expression was increased in MM, compared with control group. All other groups did not differ from the control group. Comparing diseases amongst each other, AML had a lower BECN1 expression, compared with low-risk MDS and with MM (Figure 1A). Disclosures: No relevant conflicts of interest to declare.

An intrinsic thyrotropin-mediated pathway of TNF-α production by bone marrow cells

Blood ◽  
2003 ◽  
Vol 101 (1) ◽  
pp. 119-123 ◽  
Author(s):  
Heuy-Ching Wang ◽  
Jolene Dragoo ◽  
Qin Zhou ◽  
John R. Klein
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Hematopoietic Cells ◽  
Thyroid Stimulating Hormone ◽  
Appreciable Change ◽  
Tnf Α ◽  
Factor Α ◽  
Inductive Signal ◽  
Necrosis Factor

Abstract Recent studies have identified a role for thyroid-stimulating hormone (TSH; ie, thyrotropin) as an inductive signal for tumor necrosis factor-α (TNF-α) secretion by bone marrow (BM) cells, although the features of that activation pathway have not been defined. Using intracellular TSH staining and enzyme-linked immunoassay for detection of secreted TSH, we demonstrate that TSH synthesis in BM cells occurs within CD45+ (leukocyte common antigen) hematopoietic cells and that the majority of that activity resides in a component of CD11b+ BM cells that are not mature T cells, B cells, or Thy-1+ cells in the BM. Conversely, TSH-responsive BM cells defined by expression of TSH receptor (TSHR) using flow cytometry were selectively associated with a nonerythroid CD11b− lymphocyte precursor population. In vitro culture of magnetic-activated cell sorted CD11b− and CD11b+ cells with titrated amounts of purified TSH resulted in significantly higher levels of TNF-α secretion from CD11b− BM cells compared to non-TSH–treated cells, with no appreciable change in TNF-α production from CD11b+cells. These findings are the first to demonstrate TSH production by BM hematopoietic cells, and they demonstrate that TSH may be involved in the regulation of TNF-α by CD11b− BM cells. They also indicate that TSH-mediated regulation of TNF-α secretion within the BM most likely operates through an intrinsic network of TSH production and use between different types of BM cells, and they suggest that local TSH may be an important homeostatic regulator of hematopoiesis mediated by TNF-α.

scholarly journals Overexpression of tumor necrosis factor (TNF)-α and interferon (IFN)-γ by bone marrow cells from patients with myelodysplastic syndromes

Leukemia ◽  
1997 ◽  
Vol 11 (12) ◽  
pp. 2049-2054 ◽  
Author(s):  
M Kitagawa ◽  
I Saito ◽  
T Kuwata ◽  
S Yoshida ◽  
S Yamaguchi ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Tumor Necrosis Factor ◽  
Myelodysplastic Syndromes ◽  
Tumor Necrosis ◽  
Bone Marrow Cells ◽  
Tnf Α ◽  
Ifn Γ ◽  
Necrosis Factor ◽  
Marrow Cells

Decitabine Increase Platelet Count by Two Mechanisms.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3507-3507
Author(s):  
Jianhui Wang ◽  
Zongdong Li
Keyword(s):  
Bone Marrow ◽  
Platelet Count ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Current Data ◽  
Dna Demethylation ◽  
Mouse Bone Marrow ◽  
Platelet Release ◽  
Marrow Cells

Abstract Abstract 3507 Poster Board III-444 Thrombocytopenia is frequently associated with the myelodysplastic syndromes (MDS). 5-aza-2'-deoxycytidine (Decitabine) has been used to treat MDS with an approximately 20% response rate in thrombocytopenia. In this study, we have investigated the effect of Decitabine on platelet count in mouse. We report here that enhanced platelet release and maturation of megakaryocyte are two mechanisms involved in Decitabine induced elevation of platelet count. We first noted that a 30% of platelet count increase was found in the Balb/c mice 12 hours after the injection of Dectiabine at a clinically relevant dose (15 mg/m2) suggesting an instant platelet release from spleen or from megkaryocyte of bone marrow. The effect of Decitabine on megakaryocyte maturation was studied in in vitro differentiation of mouse bone marrow cells and megakaryoblastic cell line L8057. Decitabine (2.5 mm) is able to induce L8057 cells to differentiate into a megakaryocyte like polyploidy cells with positive marker of acetylcholinesterase and αIIb integrin. High expression of αIIb integrin was also found in the primary bone marrow cells cultured with both thrombopoietin and Decitabine as compared to thrombopoietin alone. The demethylation-induced transcription of GP6 has been reported in thrombopoietin induced megakaryocyte differentiation. Since Decitabine is a DNA demethylation reagent, we have investigated the GP6 expression in Decitabine treated L8057 cells and have found upregualtion of GP6 expression. Although the role of DNA demethylation in megkaryoctye differentiation still needs to be verified, our current data support that Decitabine is able to drive magakaryocyte maturation. Disclosures: No relevant conflicts of interest to declare.

Potential Role Of RUNX1 In The Pathogenesis Of Juvenile Myelomonocytic Leukemia (JMML)

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 45-45 ◽  
Author(s):  
Hui Huang ◽  
Daniel E. Bauer ◽  
Mignon L. Loh ◽  
Govind Bhagat ◽  
Alan B. Cantor ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Tyrosine Phosphorylation ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasm ◽  
Brdu Incorporation ◽  
Juvenile Myelomonocytic Leukemia ◽  
Ras Signaling ◽  
Myelomonocytic Leukemia

Juvenile myelomonocytic leukemia (JMML) is an aggressive myeloproliferative neoplasm of young children. The only current curative treatment is bone marrow transplantation. Yet even with this aggressive therapy, ∼50% of children still die from their disease. Somatic mutations leading to constitutive activation of the tyrosine phosphatase Shp2 (also called PTPN11) or of RAS signaling occur in ∼90% cases of JMML. However, the transcription factors that act downstream of these aberrant signaling events have not been identified. We recently showed that RUNX1 is a direct interacting partner of Shp2 in megakaryocytic cells (Huang et al. 2012. Genes Dev 26: 1587-1601). Moreover, we showed that RUNX1 is normally negatively regulated by src-family kinase (SFK) mediated tyrosine phosphorylation in megakaryocytes and T-lymphocytes, and that Shp2 contributes to RUNX1 tyrosine dephosphorylation. We now show that overexpression of a mutant RUNX1 (RUNX1Y260F, Y375F, Y378F, Y379F, Y386F, “RUNX1-5F”), which is expected to mimic constitutive dephosphorylation by Shp2 in murine Lin- Sca-1+ c-kit+ (LSK) bone marrow cells is resistant to SFK-mediated tyrosine phosphorylation and leads to a dramatic expansion of CFU-M/CFU-GM and Gr1+Mac1+ cells in vitro and in vivo. In contrast, these effects are not seen when wild type RUNX1 or RUNX1Y260D, Y375D, Y378D, Y379D, Y386D (“RUNX1-5D”; mimicking constitutive RUNX1 tyrosine phosphorylation) are overexpressed. The RUNX1-5F expressing cells also have increased replating activity in serial colony forming assays, increased proliferation (BrdU incorporation), decreased apoptosis, and reduced cytokine dependence. This partially phenocopies conditional knock-in mice that express JMML associated activating Shp2 mutations. Flow sorted Gr1+Mac1+ cells from the RUNX1-5F transduced cultures expressed higher levels of the direct RUNX1 target gene PU.1, which plays a role in myelomonocytic growth, and Cyclin D1. To test whether RUNX1 is required for the myelomonocytic hyperproliferation in JMML, CD34+ peripheral blood cells from a patient with JMML and known activating Shp2 mutation (Shp2E76G) were lentivirally transduced with doxycycline-inducible RUNX1-5D or RUNX1-5F expression constructs and cultured under myeloid growth conditions. Upon doxycycline induction, the RUNX1-5D overexpressing cells (resistant to Shp2) exhibited at 32% reduction in BrdU incorporation. In contrast, the control RUNX1-5F expressing cells had no significant reduction in proliferation. These results are consistent with RUNX1 acting as an essential downstream target of activated Shp2 in JMML. As ERK mediated phosphorylation (downstream of RAS/MEK) is also known to increase RUNX1 activity, we propose that RUNX1 may be a common downstream transcriptional target of both activated Shp2 and RAS signaling in the pathogenesis of JMML. Disclosures: No relevant conflicts of interest to declare.

Effective Targeting Of Chronic Myeloid Leukemia Initiating Activity With The Combination Of Arsenic Trioxide and Interferon Alpha

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 5171-5171
Author(s):  
Rabab El Eit ◽  
Iskandarani Ahmad ◽  
Jessica Saliba ◽  
Mark Jabbour ◽  
Rami Mahfouz ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Chronic Myeloid Leukemia ◽  
Arsenic Trioxide ◽  
Interferon Alpha ◽  
Myeloid Leukemia ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Clinical Activity ◽  
Retroviral Transduction

Abstract Imatinib is the standard of care in the treatment of Chronic Myeloid Leukemia (CML) patients. However, imatinib is not curative since most patients who discontinue therapy will relapse, stressing the need for novel therapies that target leukemia initiating cells (LIC). Arsenic trioxide induces complete remission in patients with acute promyelocytic leukemia (APL). We previously demonstrated that, through full degradation of PML-RARA, the combination of arsenic and retinoic acid eradicates APL LICs and cures the disease. Interferon alpha (IFN) induces hematologic and cytogenetic remissions and interestingly, improved outcome was reported with the combination of interferon and imatinib. We investigated the effects of arsenic and IFN on human CML cell lines or primary cells and the bone marrow retroviral transduction/transplantation murine CML model. In vitro, the combination of arsenic and IFN inhibited proliferation and activated apoptosis. Importantly, arsenic and IFN synergistically reduced the clonogenic activity of primary bone marrow cells derived from CML patients. Moreover, in a murine transplantation model of CML, we show that arsenic/IFN prolonged survival of primary leukemic mice and sharply diminished transplantation of CML cells in secondary recipients, pointing to exhaustion of CML LICs. Our results also suggest that IFN/arsenic may clear LICs independently from oncoprotein catabolism. Interestingly, arsenic/IFN effect on CML LIC activity was significantly superior to that of imatinib.These studies plea for a clinical exploration of this combination, knowing that IFN and arsenic have both shown clinical activity in CML, alone or in combination with imatinib. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Fractionated Irradiation of Right Thorax Induces Abscopal Damage on Bone Marrow Cells via TNF-α and SAA

2021 ◽  
Vol 22 (18) ◽  
pp. 9964
Author(s):  
Yimeng Song ◽  
Songling Hu ◽  
Junling Zhang ◽  
Lin Zhu ◽  
Xinrui Zhao ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Thoracic Irradiation ◽  
Amyloid A ◽  
Tnf Α ◽  
Polychromatic Erythrocytes ◽  
Underlying Mechanisms ◽  
The Right ◽  
Marrow Cells

Radiation-induced abscopal effect (RIAE) outside of radiation field is becoming more attractive. However, the underlying mechanisms are still obscure. This work investigated the deleterious effect of thoracic irradiation (Th-IR) on distant bone marrow and associated signaling factors by irradiating the right thorax of mice with fractionated doses (8 Gy × 3). It was found that this localized Th-IR increased apoptosis of bone marrow cells and micronucleus formation of bone marrow polychromatic erythrocytes after irradiation. Tandem mass tagging (TMT) analysis and ELISA assay showed that the concentrations of TNF-α and serum amyloid A (SAA) in the mice were significantly increased after Th-IR. An immunohistochemistry assay revealed a robust increase in SAA expression in the liver rather than in the lungs after Th-IR. In vitro experiments demonstrated that TNF-α induced SAA expression in mouse hepatoma Hepa1–6 cells, and these two signaling factors induced DNA damage in bone marrow mesenchymal stem cells (BMSCs) by increasing reactive oxygen species (ROS). On the other hand, injection with TNF-α inhibitor before Th-IR reduced the secretion of SAA and attenuated the abscopal damage in bone marrow. ROS scavenger NAC could also mitigated Th-IR/SAA-induced bone marrow damage in mice. Our findings indicated that Th-IR triggered TNF-α release from lung, which further promoted SAA secretion from liver in a manner of cascade reaction. Consequently, these signaling factors resulted in induction of abscopal damage on bone marrow of mice.

The Transcription Factor IRF8 is a Key Transcription Factor for Basophil Development

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1197-1197
Author(s):  
Daisuke Kurotaki ◽  
Haruka Sasaki ◽  
Naoki Osato ◽  
Izumi Sasaki ◽  
Chika Kaneda ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Transcription Factor ◽  
Peripheral Blood ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Promoter Regions ◽  
Retroviral Transduction ◽  
Pass Through ◽  
Myeloid Progenitors

Abstract Basophils are the rarest granulocytes circulating in the peripheral blood. They play critical roles in anti-parasite Th2-type immune responses and chronic allergic disorders. The developmental pathway for basophils has been recently demonstrated; myeloid progenitors pass through common myeloid progenitors, granulocyte-monocyte progenitors, granulocyte-committed progenitors (GPs), and basophil-committed progenitors (BaPs) in the bone marrow. BaPs then give rise to mature basophils. However, our understanding of how this pathway is regulated remains still elusive. Interferon Regulatory Factor-8 (IRF8), a hematopoietic cell-specific IRF transcription factor, is essential for the development of monocytes, dendritic cells, and eosinophils, while it inhibits neutrophil differentiation. Its role in the development of basophils has yet to be analyzed. In this study, we investigated whether IRF8 has any role in the development of the basophil lineage. We found that Irf8–/– mice displayed a severe reduction of basophil counts in the bone marrow, peripheral blood and spleen compared to wild-type (WT) mice. Irf8–/– mice retained GPs but lacked BaPs. Cell transfer experiments revealed that the defect of basophil development in Irf8–/– mice resides in bone marrow cells. We utilized IRF8-GFP chimera knock-in mice to examine IRF8 protein expression in the basophil lineage at a single cell level. We found that GPs, but not BaPs and mature basophils, expressed IRF8. Furthermore, purified Irf8–/– GPs failed to efficiently give rise to basophils in vitro. These results indicate that IRF8 acts at the stage of GPs in a cell-intrinsic manner. To understand the mechanism by which IRF8 promotes basophil development, we performed transcriptome analysis of purified GPs from WT and Irf8–/– mice by microarray. Because IRF8 is no more expressed in BaPs, we envisaged that IRF8 acts by inducing downstream transcription factors in GPs. The expression of several transcription factor genes such as Gata2 and Spib was reduced in Irf8–/– GPs compared to WT GPs. Analysis of DNA motifs in the promoter regions of genes downregulated in Irf8–/– GPs predicted that GATA transcription factor(s) may act downstream of IRF8. Indeed, retroviral transduction of GATA2, known to be essential for basophil development, into Irf8–/– hematopoietic progenitor cells rescued basophil differentiation in vitro. On the other hand, Spib–/– mice showed no obvious defects in basophil development. Taken together, these results suggest that the IRF8-GATA2 axis in GPs critically regulates basophil development. Disclosures: No relevant conflicts of interest to declare.

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