Regulation Of Myelopoiesis Research Articles

Abstract 2018: The Role Of Interleukin 27 Receptor Signaling In Regulation Of Intestinal Barrier, Gut Microbiota And Abdominal Aortic Aneurysm.

Abdominal aortic aneurysm (AAA) is a cardiovascular disease characterized by accumulation of immune cells, inflammation, and degradation of medial layer of the abdominal aorta. Angiotensin II (Ang II) plays a key role in controlling blood pressure but is also able to regulate various immune and non-immune cells. While some studies suggested the role of Ang II in dysbiosis, the link between alteration of intestinal microbiota and AAA is largely unexplored. Cytokines are established regulators of inflammatory milieu in the aortic wall in AAA, but their distant contribution, for example via control of intestinal microbial homeostasis and cooperation with Ang II, has never been explored. Interleukin (IL)27 is a member of the IL6/IL12 superfamily that regulates various immune and non-immune cells. We previously showed that IL27R signaling cooperates with Ang II in the regulation of myelopoiesis thereby promoting AAA. Myeloid progenitor expansion and myeloid cell production driven by AngII are blunted in Apoe -/- Il27ra -/- mice. Here we found that Apoe -/- Il27ra -/- mice were also protected from gut dysbiosis and contain less pro-inflammatory bacterial genera in their colons. Unexpectedly, microbiota depletion accelerated AAA development in Apoe -/- Il27ra -/- mice, while Apoe -/- controls were protected from the disease. RNA seq analysis revealed upregulation of inflammatory pathways and downregulation in tight junctions and collagen in colons of Apoe -/- Il27ra -/- mice with ablated microbiota. Transcriptomic analysis of Apoe -/- colonic organoids stimulated with recIL27 shows upregulation of pathways linked to cell proliferation, cell cycle, and junction genes, indicating direct role of IL27. The analysis of the bone marrow revealed elevation of early myeloid progenitors which correlated with heightened circulating monocytes and neutrophils in Apoe -/- Il27ra -/- mice with ablated microbiota. Non-targeted metabolomics revealed microbiota and IL27R signaling-dependent alteration of serum metabolites likely mediating the effect on myeloid cells and AAA. Overall, our work uncovers novel role of IL27R signaling in the regulation of AAA, mechanistically connecting intestinal barrier and microbiota to the pathogenesis of this CVD.

Human diseases and the mechanisms regulating myelopoiesis

Myelopoiesis is a process that produces myeloid cells including granulocytes and mononuclear phagocytes. The differentiation and proliferation of hematopoietic stem and progenitor cells are tightly regulated to meet demands for such myeloid cells both at steady state and under stressed conditions. CCAAT/enhancer-binding protein family transcription factors are involved not only in the appropriate regulation of myelopoiesis but also in dysregulated myelopoiesis. A recent study has revealed that inflammation, in addition to the established concepts or mechanisms of dysregulated myelopoiesis, triggers long-term epigenetic memory in hematopoietic stem/progenitor cells. Further, clonal hematopoiesis develops and impairs host health conditions via inflammatory conditions. Intensive studies covering both the basic and clinical aspects of myelopoiesis are required to establish therapeutic and even prophylactic approaches to different types of human diseases including hematopoietic and nonhematopoietic origins.

Age-related exacerbation of hematopoietic organ damage induced by systemic hyper-inflammation in senescence-accelerated mice

Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening systemic hyper-inflammatory disorder. The mortality of HLH is higher in the elderly than in young adults. Senescence-accelerated mice (SAMP1/TA-1) exhibit characteristic accelerated aging after 30 weeks of age, and HLH-like features, including hematopoietic organ damage, are seen after lipopolysaccharide (LPS) treatment. Thus, SAMP1/TA-1 is a useful model of hematological pathophysiology in the elderly with HLH. In this study, dosing of SAMP1/TA-1 mice with LPS revealed that the suppression of myelopoiesis and B-lymphopoiesis was more severe in aged mice than in young mice. The bone marrow (BM) expression of genes encoding positive regulators of myelopoiesis (G-CSF, GM-CSF, and IL-6) and of those encoding negative regulators of B cell lymphopoiesis (TNF-α) increased in both groups, while the expression of genes encoding positive-regulators of B cell lymphopoiesis (IL-7, SDF-1, and SCF) decreased. The expression of the GM-CSF-encoding transcript was lower in aged mice than in young animals. The production of GM-CSF by cultured stromal cells after LPS treatment was also lower in aged mice than in young mice. The accumulation of the TNF-α-encoding transcript and the depletion of the IL-7-encoding transcript were prolonged in aged mice compared to young animals. LPS dosing led to a prolonged increase in the proportion of BM M1 macrophages in aged mice compared to young animals. The expression of the gene encoding p16INK4a and the proportion of β-galactosidase- and phosphorylated ribosomal protein S6-positive cells were increased in cultured stromal cells from aged mice compared to those from young animals, while the proportion of Ki67-positive cells was decreased in stromal cells from aged mice. Thus, age-related deterioration of stromal cells probably causes the suppression of hematopoiesis in aged mice. This age-related latent organ dysfunction may be exacerbated in elderly people with HLH, resulting in poor prognosis.

Sclerostin Depletion Induces Inflammation in the Bone Marrow of Mice.

Romosozumab, a humanized monoclonal antibody specific for sclerostin (SOST), has been approved for treatment of postmenopausal women with osteoporosis at a high risk for fracture. Previous work in sclerostin global knockout (Sost−/−) mice indicated alterations in immune cell development in the bone marrow (BM), which could be a possible side effect in romosozumab-treated patients. Here, we examined the effects of short-term sclerostin depletion in the BM on hematopoiesis in young mice receiving sclerostin antibody (Scl-Ab) treatment for 6 weeks, and the effects of long-term Sost deficiency on wild-type (WT) long-term hematopoietic stem cells transplanted into older cohorts of Sost−/− mice. Our analyses revealed an increased frequency of granulocytes in the BM of Scl-Ab-treated mice and WT→Sost−/− chimeras, indicating myeloid-biased differentiation in Sost-deficient BM microenvironments. This myeloid bias extended to extramedullary hematopoiesis in the spleen and was correlated with an increase in inflammatory cytokines TNFα, IL-1α, and MCP-1 in Sost−/− BM serum. Additionally, we observed alterations in erythrocyte differentiation in the BM and spleen of Sost−/− mice. Taken together, our current study indicates novel roles for Sost in the regulation of myelopoiesis and control of inflammation in the BM.

Interleukin-33 regulates hematopoietic stem cell regeneration after radiation injury

BackgroundIL-33 is a pleiotropic cytokine of the IL-1 family, which has been reported to implicate in both innate and adaptive immune responses. Recent studies suggest IL-33 is crucial for regulation of myelopoiesis and myeloid cell activity. Here, we explore the potential effect of IL-33 against hematopoietic injury after total body irradiation (TBI).MethodsC57BL/6 mice were irradiated with a sublethal dose of radiation (600 cGy) and treated with IL-33 at a dose of 3 μg/dose i.p. once a day for seven consecutive days. H&E staining was used to determine the bone marrow cellularity. A flow cytometer was used to quantify the hematopoietic stem cell (HSC) population, cell proliferation, and apoptosis. The colony-forming assay was used to evaluate the clonogenic function of HSCs. RT-qPCR was used to determine the expression of apoptosis-associated genes.ResultsBone marrow HSCs from wild-type mice expressed functional IL-33 receptor (ST2), and treatment with IL-33 promoted the recovery of the HSC pool in vivo and improved the survival of mice after TBI. Conversely, mice with ST2 deficiency showed decreased HSC regeneration and mouse survival after TBI. Of note, IL-33 reduced radiation-induced apoptosis of HSCs and mediated this effect through repression of the p53-PUMA pathway.ConclusionsIL-33 regulates HSC regeneration after myelosuppressive injury through protecting HSCs from apoptosis and enhancing proliferation of the surviving HSCs.

Regulation of myelopoiesis by proinflammatory cytokines in infectious diseases.

Hematopoiesis is hierarchically orchestrated by a very small population of hematopoietic stem cells (HSCs) that reside in the bone-marrow niche and are tightly regulated to maintain homeostatic blood production. HSCs are predominantly quiescent, but they enter the cell cycle in response to inflammatory signals evoked by severe systemic infection or injury. Thus, hematopoietic stem and progenitor cells (HSPCs) can be activated by pathogen recognition receptors and proinflammatory cytokines to induce emergency myelopoiesis during infection. This emergency myelopoiesis counterbalances the loss of cells and generates lineage-restricted hematopoietic progenitors, eventually replenishing mature myeloid cells to control the infection. Controlled generation of such signals effectively augments host defense, but dysregulated stimulation by these signals is harmful to HSPCs. Such hematopoietic failure often results in blood disorders including chronic inflammatory diseases and hematological malignancies. Recently, we found that interleukin (IL)-27, one of the IL-6/IL-12 family cytokines, has a unique ability to directly act on HSCs and promote their expansion and differentiation into myeloid progenitors. This process resulted in enhanced production of neutrophils by emergency myelopoiesis during the blood-stage mouse malaria infection. In this review, we summarize recent advances in the regulation of myelopoiesis by proinflammatory cytokines including type I and II interferons, IL-6, IL-27, granulocyte colony-stimulating factor, macrophage colony-stimulating factor, and IL-1 in infectious diseases.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD)-mediated deregulation of myeloid and sebaceous gland stem/progenitor cell homeostasis.

Studies of TCDD toxicitystimulated identification of the responsible aryl hydrocarbon receptor (AHR), a multifunctional, ligand-activated transcription factor of the basic helix-loop-helix/Per-Arnt-Sim family. Accumulating evidence suggests a role of this receptor in homeostasis of stem/progenitor cells, in addition to its known role in xenobiotic metabolism. (1) Regulation of myelopoiesis is complex. As one example, AHR-mediated downregulation of human CD34+ progenitor differentiation to monocytes/macrophages is discussed. (2) Accumulation of TCDD in sebum leads to deregulation of sebocyte differentiation via Blimp1-mediated inhibition of c-Myc signaling and stimulation of Wnt-mediated proliferation of interfollicular epidermis. The resulting sebaceous gland atrophy and formation of dermal cysts may explain the pathogenesis of chloracne, the hallmark of TCDD toxicity. (3) TCDD treatment of confluent liver stem cell-like rat WB-F344 cells leads to release from cell-cell contact inhibition via AHR-mediated crosstalk with multiple signaling pathways. Further work is needed to delineate AHR function in crosstalk with other signaling pathways.

Apoptotic Molecules In Chronic Leukemia

Background: Chronic myeloid leukemia (CML) is characterized by the spread of malignant cells that exhibit resistance to caspase-mediated cell death (apoptosis) and this mechanism is proposed to play an important role in myeloid cell growth. However, the extent to which caspase-mediated cell death plays a crucial role in the regulation of myelopoiesis remains controversial.Objectives & Proceedure: The objectives of this study were to examine whether or not caspase-mediated cell death-related proteins take part in the development of CML and to also to detect the relationship between Fas, p53 and caspase-mediated cell death protease activating factor (Apaf-1) in 5 patients with CML using the real-time quantitative polymerase chain reaction. We demonstratedthatp53 and Apaf-1 messenger ribonucleic acid (mRNA) expression was moderately elevated (up to 5 fold, p<0.05) in 4 out of 5 CML patients. One patient with a p53 point mutation, exhibited a far greater elevation of p53 mRNA expression throughout their blast crisis, but in contrast, displayed a significant reduction in levels of Apaf-1 mRNA and Fas mRNA.Conclusion: Our results show in-vivo linkages between Fas, p53 and Apaf-1 transcription parameters suggesting that the key genes involved in the caspase-mediated cell death might contribute to CML disease development.Bangladesh Journal of Medical Science Vol.15(4) 2016 p.651-654

The Impact of NFAT Inhibition on Neutrophil Effector Functions in Patients after Allogeneic Hematopoietic Stem Cell Transplantation and on Neutrophil Antifungal Defense and Myelopoiesis in Cyclosporin Α Treated and NFATc1LysM Mice

Background and Aims: Immunosuppressive medication e.g. by calcineurin inhibitors substantially contributes to the risk for opportunistic fungal infections in patients after allogeneic transplantation (HSCT). It is well known that the nuclear factor of activated T cells (NFAT) is an important transcription factor downstream of calcineurin especially in T cells. Additionally, recent data in rodent models indicate that NFAT also seems to play a relevant role in innate antifungal immune responses by polymorphonuclear neutrophils (PMN), as well as in regulation of myelopoiesis and myeloid differentiation.Methods: Firstly, isolated PMN from healthy donors were analyzed in vitro in absence or presence of CsA regarding their effector functions and activation-induced release of inflammatory mediators. Consecutively, blood samples of CsA-treated patients after allogeneic HSCT (n=17) and healthy donors (n=8) were analyzed ex vivo at two different time points as described above. Secondly, we used a murine IPA model (C57BL/6) and treated mice with CsA (18 mg/kg/d) or vehicle and challenged them with Aspergillus fumigatus (A. f.) conidia intratracheally. PMN recruitment to the lungs and pulmonary fungal clearance were examined by analyzing bronchoalveolar lavages (BAL) and peripheral blood (PB) using flow cytometry and cytometric bead array and murine lungs by fungal culture assays and histopathologic examination. Furthermore, survival was studied with neutropenic animals serving as positive controls. Moreover, LysM-specific NFATc1 knockout (NFATc1LysM) mice were bred lacking NFATc1 expression solely in myelomonocytic cells. These animals were also infected with A. f. and analyzed as further mentioned. In addition, we investigated myelopoiesis and myeloid differentiation by quantifying bone marrow derived myeloid progenitor cells from CsA treated or NFATc1LysMmice using flow cytometry and simultaneously counting PMN in PB under steady state conditions.Results: CsA enhanced phagocytosis of PMN in vitro and ex vivo in patients’ blood samples (54.2 % +/- 4.1 (patients) vs. 43.8 +/- 1.5, LPS, p=0.006). Moreover, PMNs migratory capabilities were reduced in vitro, whereas other effector functions or release of IL-8 were rather unaffected. PMNs of CsA-treated patients showed increased activation, degranulation and production of inflammatory mediators, but production of ROS was slightly decreased. In our in vivo model, IPA was lethal in neutropenic mice whereas solely CsA or vehicle treated mice survived the infection. CsA treatment resulted in enhanced PMN recruitment in BAL by trend, while pulmonary inflammation and PMN counts in PB remained stable. Indeed, fungal clearance was clearly constrained in CsA treated animals (2.1 x 105 CFU/lung +/- 0.5 (CsA) vs. 1.7 x 105 +/- 0.2, p<0.005). In our murine knockout model, NFATc1LysM mice infected with A. f. showed unimpaired survival without displaying detectable differences in PMN recruitment or fungal clearance. However, pulmonary inflammation and PMN counts in PB seemed to be more pronounced in knockout mice. Interestingly, BALs of CsA treated mice showed increased levels of IL-6 by trend (4634 pg/mL +/- 1073 (CsA) vs. 3108 +/- 729, p=0.48) but decreased levels of MCP-1 and TNF-α. In contrast, MCP-1, RANTES and TNF-α were enhanced by trend in BALs of NFATc1LysM mice, while IL-6 was reduced compared to wild type controls (3762 pg/mL +/- 729 vs. 4770 +/- 1613, p=0.81). PMN counts in PB were unaffected in NFATc1LysM mice but distribution of bone marrow derived murine myeloid progenitor cells was clearly impaired especially in megakaryocyte-erythroid progenitor cells (1.2 x 105 cells +/- 0.2 (NFATc1LysM) vs. 2.7 +/- 0.6, p=0.015), whereas solely CsA treatment had no influence.Conclusion: Results from our in vitro and ex vivo studies on patients' blood samples as well as from our murine in vivo IPA model indicate that NFAT regulates not only myelopoiesis, but also PMN functionalities in mice and humans. Nevertheless, these interactions are obviously multidimensional and potentially derive from involvement of different pathways. The underlying molecular mechanisms and clinical relevance of our findings in HSCT remain to be determined. DisclosuresNo relevant conflicts of interest to declare.

The Impact of NFAT Inhibition on Neutrophil Antifungal Defense and Myelopoiesis in Cyclosporine A Treated and NFATc1LysM Mice

Background and Aims: Immunodeficient patients after allogeneic stem cell transplantation (HSCT) are heavily threatened by opportunistic fungal infections like invasive pulmonary aspergillosis (IPA), partly due to immunosuppressive medication e.g. by calcineurin inhibitors like cyclosporine A (CsA) or tacrolimus. It is well known that the nuclear factor of activated T cells (NFAT) is an important transcription factor downstream of calcineurin in the adaptive immune system especially in T cells. Additionally, there is a growing body of evidence that NFAT also plays a substantial role in innate immune response against invasive fungal diseases by polymorphonuclear neutrophils (PMN), as well as in regulation of myelopoiesis and myeloid differentiation, as indicated by recent data in rodent models.Methods: Firstly, we used a murine IPA model (C57BL/6) to clarify the role of NFAT in antifungal innate immune response in vivo. To do so, we treated mice intraperitoneally with CsA (18mg/kg/d) or vehicle for 2 weeks and challenged them with Aspergillus fumigatus (A. f.) conidia intratracheally. 24 hours later, some mice were sacrificed and PMN recruitment to the lungs and pulmonary fungal clearance were examined by analyzing bronchoalveolar lavages (BAL) and peripheral blood (PB) by flow cytometry and murine lungs by fungal culture assays and histopathologic examination. In addition, survival of remaining infected mice was studied with neutropenic animals (by depletion with anti-Gr1) serving as positive controls. Secondly, LysM-specific NFATc1 knockout (NFATc1LysM) mice were bred lacking NFATc1 expression solely in myelomonocytic cells (i.e. PMN and monocytes). Furthermore, these animals were infected with A. f. and analyzed as described above. Secondly, we investigated myelopoiesis and myeloid differentiation under steady state conditions by quantifying bone marrow derived myeloid progenitor cells from CsA treated or NFATc1LysM mice using flow cytometry and simultaneously counting PMN in PB.Results: While the infection was lethal in CsA or vehicle treated neutropenic mice, all CsA or vehicle treated mice survived the infection. CsA treated mice showed enhanced PMN recruitment in BAL by trend (55.2% +/- 12.0 (CsA) vs. 33.7% +/- 8.0 (control), mean +/- SEM, p=0.053), whereas pulmonary inflammation and PMN counts in PB were comparable to controls. In contrast, fungal clearance was clearly impaired in animals after CsA treatment (2.1 x 105 CFU/lung after 48 hours +/- 0.5 (CsA) vs. 1.7 x 105 +/- 0.2 (control), p<0.005). Along with that, NFATc1LysM mice infected with A. f. showed unimpaired survival. However, there were no detectable differences in PMN recruitment or fungal clearance, whereas pulmonary inflammation and PMN counts in PB seemed to be more pronounced in knockout mice (1.0 inflammation points/lung +/- 0.12 (NFATc1LysM) vs. 0.7 +/- 0.07 (control), p=0.057; 1.5 x 103 PMN/µl +/- 0.2 (NFATc1LysM) vs. 0.9 +/- 0.1 (control), p=0.036). Distribution of bone marrow derived murine myeloid progenitor cells was unaffected through NFAT inhibition by CsA but clearly impaired in NFATc1LysM mice especially in megakaryocyte-erythroid progenitor cells (1.2 x 105 cells +/- 0.2 (NFATc1LysM) vs. 2.7 +/- 0.6, p=0.015) whereas PMN blood counts in PB were unchanged.Conclusions: In a mouse model, NFAT inhibition via treatment with CsA does not influence survival after infection with A. f. in vivo but affects PMN recruitment and local fungal clearance. To some extent this may be due to impaired PMN phagocytic and migratory capabilities as indicated by our in vitro and ex vivo studies (data not shown). However, solely NFATc1 downregulation in PMN apparently results in slightly different effects given that infected NFATc1LysM mice displayed enhanced pulmonary inflammation and elevated PMN blood counts compared to controls. Additionally, NFATc1 inhibition in NFATc1LysM mice leads to constrained myelopoiesis under steady state conditions without affecting peripheral PMN blood counts compared to untreated wild type controls. Further studies are needed to clarify underlying mechanisms and clinical relevance in HSCT of our findings. DisclosuresNo relevant conflicts of interest to declare.

Abstract PR05: IRF8 regulates GM-CSF expression in T cells and tumor cells to mediate myeloid-derived suppressor cell differentiation

Abstract Myeloid cells are a heterogenous and abundant population of haematopoietic cells that are virtually present in all mammalian tissues, where they monitor local microenvironment to maintain homeostasis. All myeloid cells originate from the pluripotent hematopoietic stem cells that undergo progressive restriction in their lineage potential to give rise to mature granulocytes and macrophages. Lineage restriction and differentiation are regulated by timely activation of specific set of lineage-specific transcription factors in concert with down-regulation of other set(s) of transcription factors that are important for alternative cell lineage potential. Altered expression of these lineage-specific transcription factors often leads to deregulation of myelopoiesis and resultant hematopoietic disorders. Therefore, lineage-specific transcription factors are essential for myeloid cell lineage differentiation and maturation. Mice with a null mutation of irf8, the gene that encodes IFN regulatory factor 8 (IRF8), exhibit massive accumulation of CD11b+Gr1+ immature myeloid cells (IMCs). Therefore, IRF8 is a myeloid cell lineage-specific transcription factor that plays an essential function in the regulation of myelopoiesis. Particularly, IRF8 may determine differentiation, lineage commitment, and immune function of monocytes versus granulocytes under physiological conditions. A hallmark of cancer-bearing mice is the accumulation of CD11b+Gr1+ myeloid-derived suppressor cells (MDSCs). Interestingly, IRF8 is silenced in MDSCs from tumor-bearing mice. Therefore, IRF8 is apparently a key transcription factor that mediates MDSC differentiation. However, the molecular mechanism underlying IRF8 regulation of MDSCs is largely unknown. Because MDSCs is induced by inflammation, we therefore hypothesized that IRF8 may repress the expression of proinflammatory factors to mediate differentiation of MDSCs/IMCs under physiological and pathological conditions. To test this hypothesis, we made use of conventional IRF8 KO mice, mice with IRF8 deficiency only in myeloid cells, mice with IRF8 deficiency only in T cells, and tumor-bearing mouse models. Here we report an intriguing finding that although IRF8 conventional mice exhibit deregulated myeloid cell differentiation and resultant accumulation of CD11b+Gr1+ IMCs, surprisingly, mice with IRF8 deficiency only in myeloid cells exhibit normal myeloid cell lineage differentiation. Instead, mice with IRF8 deficiency only in T cells exhibited deregulated myeloid cell differentiation and IMC accumulation. We further demonstrated that IRF8-deficient T cells exhibit elevated GM-CSF expression and secretion. Treatment of mice with GM-CSF increased IMC accumulation, and adoptive transfer of IRF8-deficient T cells, but not GM-CSF-deficient T cells, increased IMC accumulation in the recipient chimera mice. Moreover, overexpression of IRF8 decreased GM-CSF expression in T cells. These data thus determine that IRF8 functions in T cells to repress GM-CSF expression to suppress IMCs. However, in tumor-bearing mice, IRF8 is silenced in MDSCs but not in T cells, suggesting a different mechanism of MDSC regulation by IRF8. We observed that silencing IRF8 using IRF8-specific siRNA dramatically increase GM-CSF expression in tumor cells. Therefore, IRF8 represses GM-CSF expression in tumor cells to mediate MDSC differentiation. In summary, we determine that IRF8 regulates GM-CSF expression in T cells and tumor cells, respectively, to mediate myelopoiesis under physiological and pathological conditions. This abstract is also presented as Poster A84. Citation Format: Amy Paschall, Ruihua Zhang, Kankana Bardhan, Chen-Feng Qi, Liang Peng, Geming Lu, Jianjun Yang, Miriam Merad, Mary Zimmerman, Tracy McGaha, Gang Zhou, Andrew Mellor, Scott I. Abrams, Herbert Morse, Keiko Ozato, Huabao Xiong, Kebin Liu. IRF8 regulates GM-CSF expression in T cells and tumor cells to mediate myeloid-derived suppressor cell differentiation. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr PR05.

Regulation of myelopoiesis by the transcription factor IRF8.

Interferon regulatory factor-8 (IRF8) is a transcription factor expressed in hematopoietic cells, particularly in mononuclear phagocytes [monocytes/macrophages and dendritic cells (DCs)] and their progenitors. Various studies have demonstrated that IRF8 is essential for the development of monocytes, DCs, eosinophils, and basophils. Conversely, IRF8 suppresses the generation of neutrophils. Accordingly, Irf8 (-/-) mice develop immunodeficiency and a chronic myeloid leukemia (CML)-like disease. Mutations and loss of expression of the human IRF8 gene are also associated with immunodeficiency and CML, respectively. Recent findings have begun to reveal the transcription factor network and epigenetic changes governed by IRF8. For example, in mononuclear phagocyte progenitors, IRF8 cooperates with PU.1 to promote the formation of promoter-distal enhancers to induce monocyte-related genes including the critical downstream transcription factor gene Klf4. On the other hand, IRF8 blocks C/EBPα activity to suppress the neutrophil differentiation program. Indeed, Irf8 (-/-) mononuclear phagocyte progenitors fail to efficiently generate monocytes and DCs and, instead, aberrantly give rise to neutrophils. This article provides an overview of recent advances in our understanding of the role of IRF8 in myelopoiesis and related diseases.

Regulation of Myelopoiesis by CD137L Signaling

CD137 ligand (CD137L) has emerged as a powerful regulator of myelopoiesis that links emergency situations, such as infections, to the generation of additional myeloid cells, and to their activation and maturation. CD137L is expressed on the cell surface of hematopoietic stem and progenitor cells (HSPC) and antigen presenting cells (APC) as a transmembrane protein. The signaling of CD137L into HSPC induces their proliferation and differentiation to monocytes and macrophages, and in monocytes CD137L signaling induces differentiation to potent dendritic cells (DC). CD137L signaling is initiated by CD137 which is expressed by T cells, once they become activated. Some of these activated, CD137-expressing T cells migrate from the site of infection to the bone marrow where they interact with HSPC to induce myelopoiesis, or they induce monocyte to DC differentiation locally at the site of infection. Therapeutically, induction of CD137L signaling can be utilized to reinitiate myeloid differentiation in acute myeloid leukemia cells, and to generate potent DC for immunotherapy.

CD137 ligand signaling enhances myelopoiesis during infections

CD137 and its ligand are expressed in the BM, and conflicting data exist on the regulation of myelopoiesis by the CD137 receptor-ligand system. CD137(-/-) mice have increased numbers of myeloid cells in the BM, indicating an inhibitory influence of CD137 on myelopoiesis. However, CD137 also induces proliferation of hematopoietic progenitor cells and their myeloid differentiation, arguing for an enhancing effect. Here we hypothesized that this latter case represents the situation during infections since expression of CD137 is activation dependent and strongly enhanced during inflammation. Indeed, infections with Influenza, Bordetella pertussis, Mycobacterium bovis, Bacille Calmette-Guérin or Escherichia coli or i.p. injection of LPS led to increased numbers of CD137-expressing cells, especially of CD4(+) T cells in the BM of mice. Coculture experiments confirmed that CD137 expression enables CD4(+) T cells to induce proliferation and myeloid differentiation of BM and hematopoietic progenitor cells. CD137 also enhances myelopoiesis in vivo since the infection-induced increase in myeloid cell proliferation and total myeloid cell numbers in the BM were significantly lower in CD137(-/-) mice. This study reconciles earlier conflicting data by demonstrating that while CD137-CD137L interactions inhibit myelopoiesis during steady-state conditions they increase myelopoiesis during infection.

Regulation of Myelopoiesis by Differentiation Stage-Specific Activities of HOXA9 and HOXA10

Abstract SCI-29HOXA9 and HOXA10 are homeodomain (HD) transcription factors that are implicated in control of myelopoiesis and contribute to myeloid leukemogenesis. These proteins are expressed coordinately during hematopoiesis, with maximal expression in granulocyte/monocyte progenitor (GMP) cells. Engineered overexpression of Hoxa9 or Hoxa10 in primary bone marrow cells expands the GMP population in vitro, and results in myeloproliferation in murine bone marrow transplant experiments. Mice transplanted with Hoxa9- or Hoxa10-overexpressing bone marrow develop acute myeloid leukemia (AML) over time. Consistent with this, increased and sustained expression of a set of HD proteins, including HOXA9 and HOXA10, is found in a subset of human AML, including AML with MLL gene translocations (11q23-AML). Since the DNA-binding HDs of HOXA9 and HOXA10 are highly conserved, we hypothesize that they recognize a common set of target genes. However, since HOXA9 and HOXA10 diverge outside the HD, we considered the unexplored possibility that they perform different functions in regulating such genes. To identify molecular mechanisms for HOX-induced GMP expansion and leukemogenesis, we performed a chromatin immunoprecipitation-based screen for HOXA10 target genes. Gene ontology studies determined that the identified set is enriched for genes encoding growth factors and receptors, including fibroblast growth factor 2 (FGF2). We found that production of FGF2 by Hoxa10-overexpressing GMP stabilizes β-catenin and induces proliferation in an autocrine manner. We also found that HOXA9 and HOXA10 activate common FGF2 cis elements. The Hoxa10-target-gene set is also enriched for HD-transcription factors, including CDX4. We determined that Cdx4 transcription is activated by HOXA10 in GMP, but repressed by HOXA9 in differentiating myeloid cells. CDX4 activates transcription of both Hoxa9 and Hoxa10, identifying a HOX-CDX cross-regulatory mechanism. This mechanism may be influenced by Fgf2, since Hoxa10 and Cdx4 are β-catenin target genes, but β-catenin activity decreases Hoxa9 expression. Gene expression profiling studies indicate that HOXA9, HOXA10, CDX4, and FGF2 are increased in 11q23-AML, suggesting clinical relevance. Arih2 (encoding the E3 ligase Triad1) is another common HOXA9 and HOXA10 target gene that may influence Fgf2 activity. We found that Arih2 transcription is repressed by HOXA9 in myeloid progenitors, but activated by HOXA10 in differentiating phagocytes. FGF receptors are destabilized by ubiquitination, and we found increased FGF-R ubiquitination in Hoxa10-overexpressing cells. Therefore, Triad1-dependent regulation of FGF-R stability is another mechanism for control of FGF2 activity and myeloproliferation by HOXA9 and HOXA10. Therefore, HOXA9 and HOXA10 regulate a common set of target genes that control GMP expansion in a manner that is antagonistic for some genes and cooperative for others. Clinical correlative studies suggest that coordinate control of these genes by HOXA9 and HOXA10 is dysregulated in HOX-overexpressing leukemia. Understanding HOX-regulated gene networks may identify therapeutic targets for HOX-overexpressing leukemias. For example, blocking FGF-related signaling pathways may ameliorate cytokine hypersensitivity in such leukemias, and would be a topic of interest for additional studies. Disclosures:No relevant conflicts of interest to declare.

Regulation of Granulocyte and Macrophage Populations of Murine Bone Marrow Cells by G-CSF and CD137 Protein

BackgroundGranulocytes and monocytes/macrophages differentiate from common myeloid progenitor cells. Granulocyte colony-stimulating factor (G-CSF) and CD137 (4-1BB, TNFRSF9) are growth and differentiation factors that induce granulocyte and macrophage survival and differentiation, respectively. This study describes the influence of G-CSF and recombinant CD137-Fc protein on myelopoiesis.Methodology/Principal FindingsBoth, G-CSF and CD137 protein support proliferation and survival of murine bone marrow cells. G-CSF enhances granulocyte numbers while CD137 protein enhances macrophage numbers. Both growth factors together give rise to more cells than each factor alone. Titration of G-CSF and CD137 protein dose-dependently changes the granulocyte/macrophage ratio in bone marrow cells. Both factors individually induce proliferation of hematopoietic progenitor cells (lin-, c-kit+) and differentiation to granulocytes and macrophages, respectively. The combination of G-CSF and CD137 protein further increases proliferation, and results in a higher number of macrophages than CD137 protein alone, and a lower number of granulocytes than G-CSF alone demonstrating that CD137 protein-induced monocytic differentiation is dominant over G-CSF-induced granulocytic differentiation. CD137 protein induces monocytic differentiation even in early hematopoietic progenitor cells, the common myeloid progenitors and the granulocyte macrophage progenitors.Conclusions/SignificanceThis study confirms earlier data on the regulation of myelopoiesis by CD137 receptor - ligand interaction, and extends them by demonstrating the restriction of this growth promoting influence to the monocytic lineage.

Implication of Genetic Variations of Ikzf1, ARIDB5, and CEBPE Genes In the Risk of Childhood Acute Lymphoblastic Leukemia In Korea

AbstractAbstract 3235 Background:Recent western studies have showed the implication of the germline genomic variations in IKZF1 gene at 7p12.2, ARIDB5 gene at 10q21.2, and CEBPE gene at 14q11.2 on the risk of childhood acute lymphoblastic leukemia (ALL); the most significant association was observed in the single nucleotide polymorphism (SNP) rs4132601 which located at 3' region of the IKZF1. IKZF1 plays important role in lymphocyte differentiation, proliferation and function, ARIDB5 in embryogenesis and growth regulation, and CEBPE in regulation of myelopoiesis. Genomic variants in these genes are therefore considered to be involved in transcriptional regulation and differentiation of B cell progenitors. However, there have been no reports on the role of germline variations in leukemogenesis of childhood ALL in Asian countries. The aim of this study is to show the impact of these genetic variants on childhood ALL in Korea. Patients and Methods:To examine the association between genetic variations (IKZF1 rs4132601, ARIDB5 rs7089424, and CEBPE rs2239633) and the risk of childhood ALL, we here analyzed 228 children with ALL and 508 healthy individuals in Korea. Results:In ARIDB5 rs7089424, TG and GG genotypes were significantly associated with a risk for ALL (odds ratio [OR], 1.63; 95% confidential interval [CI], 1.07–2.48; P=0.02 for TG genotype, OR, 2.69; 95% CI, 1.42–5.07; P=0.002 for GG genotype). The allele incidence of ARIDB5 rs7089424 was also significantly associated with a risk for ALL (OR, 1.66; 95% CI, 1.24–2.22; P=0.0006). CEBPE rs2239633 TT genotype showed a significant association with a decreased risk for ALL (OR, 0.54; 95% CI, 0.33–0.90; P=0.02 for TT genotype). The allele incidence of CEBPE rs2239633 was also associated with a decreased risk for ALL (OR, 0.77; 95% CI, 0.61–0.97; P=0.02). There was no significant association between IKZF1 rs4132601 polymorphism and a risk for ALL in this study. Conclusion:These results suggest that genomic variations of ARIDB5 and CEBPE may play an important role in the risk for childhood ALL in Korea, compared with findings from western countries showing a significant relation between IKZF1 and childhood ALL. Several factors should be considered to explain a discrepancy between our results and the previous studies, which include different genotype frequencies in polymorphisms and varied susceptibility to ALL in different ethnic groups. Further studies incorporating larger number of cases and analyzing other SNPs or other Asian countries are warranted in childhood ALL. Disclosures:No relevant conflicts of interest to declare.

Interferon regulatory factor-8−driven myeloid differentiation is regulated by 12/15-lipoxygenase−mediated redox signaling

Several transcription factors determine the cell fate decision between granulocytes and monocytes, but the upstream signal transduction pathways that govern myelopoiesis are largely unknown. Based on our observation of aberrant myeloid cell representation in hematopoietic tissues of 12/15-lipoxygenase (12/15-LOX)-deficient (Alox15) mice, we tested the hypothesis that polyunsaturated fatty acid metabolism regulates myelopoiesis. Multicolor flow cytometric analysis and methylcellulose assays were used to compare myelopoiesis and the differentiative capacity of progenitors from Alox15 and wild-type mice. Furthermore, we elucidated the mechanism by which 12/15-LOX is involved in regulation of myelopoiesis. Granulopoiesis in Alox15 mice is increased while monopoiesis is reduced. Moreover, there is an accumulation of granulocyte-macrophage progenitors that exhibit defective differentiation. Mechanistically, we demonstrate that transcriptional activity of interferon regulatory factor-8 (Irf8), which regulates myelopoiesis, is impaired in Alox15 progenitors and bone marrow-derived macrophages due to loss of 12/15-LOX-mediated redox regulation of Irf8 nuclear accumulation. Restoration of redox signaling in Alox15 bone marrow cells and granulocyte-macrophage progenitors reversed the defect in myeloid differentiation. These data establish 12/15-LOX-mediated redox signaling as a novel regulator of myelopoiesis and Irf8.

Regulation of myelopoiesis through syntenin-mediated modulation of IL-5 receptor output

AbstractThe granulocyte-macrophage colony-stimulating factor (GM-CSF)/interleukin (IL)–3/IL-5 receptor family regulates the production and function of myeloid cells. These cytokines signal through receptor complexes that consist of unique ligand-binding α-chains and common signaling β-chains. IL-5 is distinct from IL-3 and GM-CSF in its capacity to induce eosinophil development, however, the molecular mechanisms that generate functional diversity within this receptor family are mostly unknown. Here, we characterized the selective IL-5Rα–binding adapter protein syntenin in IL-5R function. Syntenin and IL-5Rα colocalize at the plasma membrane and in early endosomal compartments. Manipulation of syntenin expression by ectopic expression or knockdown selectively modulated IL-5R but not GM-CSF receptor signaling, and severely affected IL-5–induced eosinophil differentiation from primary human CD34+ hematopoietic progenitor cells. We found syntenin up-regulated during eosinophilopoiesis but down-regulated during neutropoiesis. Syntenin forms complexes with multiple IL-5Rα chains, suggesting that syntenin-enhanced IL-5R output may result from stabilization of an IL-5–induced oligomeric receptor complex. These data demonstrate that cytokine-specific functions can be transduced by unique receptor α-chain–associating adapter proteins.

P38 MAP Kinase Inhibits Neutrophil Development Through Phosphorylation of C/EBPα on Serine 21

Many extracellular stimuli regulate growth, survival, and differentiation responses through activation of the dual specificity mitogen activated protein kinase (MAPK) kinase three (MKK3) and its downstream effector p38 MAPK. Using CD34+ hematopoietic progenitor cells, here we describe a novel role for MKK3-p38MAPK in the regulation of myelopoiesis. Inhibition of p38MAPK utilizing the pharmacological inhibitor SB203580, enhanced neutrophil development ex vivo, but conversely reduced eosinophil differentiation. In contrast, constitutive activation of MKK3 dramatically inhibited neutrophil differentiation. Transplantation of beta2-microglobulin(-/-) nonobese diabetic/severe combined immune deficient (NOD/SCID) mice with CD34+ cells ectopically expressing constitutively active MKK3 resulted in reduced neutrophil differentiation in vivo, whereas eosinophil development was enhanced. Inhibitory phosphorylation of CCAAT/enhancer binding protein alpha (C/EBPalpha) on serine 21 was induced upon activation of p38MAPK. Moreover, ectopic expression of a non-phosphorylatable C/EBPalpha mutant was sufficient to abrogate MKK3-induced inhibition of neutrophil development. Furthermore, treatment of CD34+ progenitors from patients with severe congenital neutropenia with SB203580 restored neutrophil development. These results establish a novel role for MKK3-p38MAPK in the regulation of lineage choices during myelopoiesis through modulation of C/EBPalpha activity. This signaling module may thus provide an important therapeutic target in the treatment of bone marrow failure.