MECHANISMS REGULATING THE RESPONSE TO ANEMIC STRESS

• Sponsoring Institution: State Agricultural Experiment Station
• Project Status: COMPLETE
• Funding Source: STATE
• Reporting Frequency: Annual
• Accession No.: 1005468
• Project Start Date: Jan 1, 2015
• Project End Date: Dec 31, 2019
• Program Code: [(N/A)]- (N/A)

Recipient Organization
PENNSYLVANIA STATE UNIVERSITY
208 MUELLER LABORATORY
UNIVERSITY PARK,PA 16802

Performing Department
Veterinary & Biomedical Sciences

Non Technical Summary
New erythrocytes must be made constantly during a person's life because erythrocytes have, on average, a lifespan of 120 days. Erythrocytes carry oxygen to the body's tissues. Any decrease in erythrocyte number or their ability to carry oxygen results in anemia, which is a highly prevalent condition with multiple etiologies. Anemia can cause significant morbidity and mortality, and it can have significant impact on quality of life. The goal of our work is to understand the mechanisms by which the body responds to anemic or hypoxic stress. Previous work from our group has identified a novel stress response pathway that is induced by anemic stress, which functions to rapidly produce new erythrocytes to relieve tissue hypoxia. Ultimately, we hope to apply our knowledge of this system to design therapeutic interventions to treat anemia.Anemia is a condition that results in the impaired delivery of oxygen, which leads to tissue hypoxia. Because oxygen is required for survival, a physiological response to tissue hypoxia has evolved. One aspect of this response is the generation of new erythrocytes to deliver oxygen to hypoxic tissues. We have discovered that the erythroid response to anemic stress relies on the expansion and differentiation of a specialized population of stress erythroid progenitors. These progenitors are distinct from steady state erythroid progenitors and they develop outside of the bone marrow in a process referred to as extra-medullary erythropoiesis. In mice, this process occurs primarily in the spleen, and the fetal and adult liver. The signals that regulate stress erythropoiesis are different as well. Our data show that the expansion of stress erythroid progenitors requires bone morphogenetic protein 4 (BMP4), Hedgehog (HH), and growth and differentiation factor 15 (GDF15). These factors act on a specific population of erythroid progenitors that express stem cell markers. During the expansion stage, these progenitor cells are unable to differentiate into new erythrocytes. Following the expansion of the cells, erythropoietin (Epo) and tissue hypoxia act on macrophages in the microenvironment of the spleen to promote the differentiation of the stress erythroid progenitors. Once they acquire the ability to differentiate, the progenitor cells respond to BMP4, stem cell factor (SCF), Epo and hypoxia which promotes the proliferation and terminal differentiation of new erythrocytes.GDF15 regulates the hypoxia response: Our initial experiments showed that mice mutant for GDF15 (GDF15-/-) have no defects in steady state erythropoiesis. However, if challenged with anemic stress, the mice are unable to respond and fail to survive. Using this phenotype, we discovered that GDF15 signaling plays an important role in the expansion of progenitor cells early during the response to anemia. Previously, we showed that tissue hypoxia results in the up-regulation of BMP4 expression in the spleen and liver. Hypoxia mediates this response through the hypoxia-inducible transcription factor Hif2a. Under hypoxic conditions, Hif2a protein is stable where it forms a dimer with the Hifb subunit and activates hypoxia-inducible gene expression. In normoxia, Hif2a is unstable and is degraded by the ubiquitin-mediated protein degradation system. The VHL protein that is part of ubiquitin ligase complex that promotes the degradation of Hif2a. In GDF15-/- mice, BMP4 expression is not maintained in the spleen, and the lack of BMP4 contributes to the inability of these mice to produce new erythrocytes in response to anemia. GDF15-dependent signaling maintains BMP4 expression by repressing the expression of VHL, which leads to stabilization of Hif2a. Although this aspect of GDF15 plays an essential role in stress erythropoiesis, we know GDF15-dependent signaling has other targets because we still observe defects in stress erythropoiesis when we supplement BMP4 in the absence of GDF15.Stress erythropoiesis relies on interactions between progenitor cells and the microenvironment: Much of our published work has focused on the responses of progenitor cells to signals that drive their expansion and differentiation into new erythrocytes. Recent data from us and others has shown that this process can occur only through the interaction between progenitor cells and their microenvironment and, in particular, red pulp macrophages in the spleen. Papers from the Rivella and Frenette labs showed that removing macrophages by genetic ablation or chemical treatment impaired the response to anemic stress by impeding the proliferation and differentiation of stress erythroid progenitors. In my laboratory, we have shown that removal of macrophages from cultures of stress erythroid progenitors blocks their development. Macrophages express BMP4 and HH ligands that are known to drive the expansion and differentiation of stress erythroid progenitors, but in cultures where we add exogenous HH and BMP4, we cannot rescue cultures lacking macrophages.We have established a developmental pathway for the expansion and differentiation of stress erythroid progenitors. In addition, we have identified a number signals that regulate this process. This research project will focus on three questions: (1) How do BMP4-, HH- and GDF15-dependent signaling combine to promote the expansion of stress erythroid progenitors with stem cell-like properties? (2) What are the signals expressed by macrophages that regulate stress erythropoiesis? (3) What are the molecular mechanisms that define the switch from progenitor cells that are stem cell-like and proliferating to progenitors that are actively differentiating?

Animal Health Component

0%

Research Effort Categories

Basic

100%

Applied

0%

Developmental

0%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
306	3840	1050	100%

Knowledge Area
306 - Environmental Stress in Animals;

Subject Of Investigation
3840 - Laboratory animals;

Field Of Science
1050 - Developmental biology;

Keywords

anemia

erythropoiesis

stem cells

Goals / Objectives
Our published and preliminary data support a model of stress erythropoiesis where a stem cell that originated in the bone marrow migrates into the spleen. Once in the spleen, these cells proceed through four stages of development (13): First, the multi-potential progenitor cells acquire the stress erythroid fate and become restricted to producing erythroid cells. Second, the cells now undergo rapid proliferation but maintain their stem cell characteristics. Third, Epo and hypoxia combine to switch the cells from proliferating stem cells to differentiating erythroid progenitors. Fourth, differentiating stress erythroid progenitors proliferate and undergo terminal differentiation.We will investigate the molecular mechanisms that affect this developmental pathway in three ways: First, we will determine the mechanism by which HH, BMP4, and signals derived from the macrophage microenvironment drive the stem cell-like expansion of early stress erythroid progenitors. Second, we will examine the signals generated by macrophages that regulate both the expansion and differentiation stress erythroid progenitors. Third, we will examine the changes in gene expression and chromatin structure when stress erythroid progenitors switch their development from stem cell-like proliferation to differentiation.

Project Methods
We have shown that HH and GDF15 signaling function to inhibit p53 activity. Primarily, p53 helps prevent cancer through its function in the response to genomic stress, but it also helps regulate stem cell self-renewal and promote differentiation of progenitor cells. In this situation, p53 acts to limit the expansion of stem cell-like stress erythroid progenitors. HH and GDF15 function to limit p53 expression and activity, and therefore, promote the expansion of these early stress erythroid progenitors. We hypothesize that decreased p53 activity results in increased Yap1 expression. Yap1 is a transcription factor that promotes the self-renewal of stem cells. The goal of this objective is to characterize the gene regulatory network that promotes the expansion of stem cell-like stress erythroid progenitors. To that end, we will utilize mice mutant in p53 or Yap1 to demonstrate the essential role of p53-mediated control of Yap1 expression in the regulation stress erythroid progenitor expansion. Bone marrow cells isolated from p53-/-, p53+/- and +/+ control mice will be tested in vitro by culturing cells in media that promote the development of stress erythroid progenitors. At regular time-points during the culture, cells will be removed and analyzed by flow cytometry for the development of stress erythroid progenitors, assayed for their function by colony assay; RNA samples will be isolated from specific populations of progenitor cells, and gene expression in control cells will be compared to p53+/- and p53-/- cells. We also will test the effects of p53 mutation in vivo by performing bone marrow transplants with donor cells isolated from p53-/-, p53+/-, and +/+ control mice. At regular time-points during the recovery from transplant, we will isolate spleen cells from the recipient mice. Donor-derived cells will be assayed by flow cytometry, and their ability to differentiate will be assayed by colony assay; specific populations will be isolated by flow cytometry, RNA isolated from them, and the expression of key genes in control cells will be compared to p53+/- and p53-/- donor cells.The role of Yap1 will be analyzed in a similar manner using both in vivo and in vitro approaches with Yap1+/-, Yap1-/-, and control +/+ cells. A key gene involved in promoting erythroid development is the transcription factor SCL, which acts as a heterodimer with the e-box protein E2A. Id2 is an inhibitor of the SCL-E47 complex because it can compete with E47 for SCL binding, but the SCL-Id2 complex cannot bind DNA. In neural stem cells, BMP4-dependent signaling activates the transcription factor Smad5. Smad5 forms a complex with Yap1 to up-regulate the expression of Id2, which is essential for the expansion of neural stem cells and inhibition of their differentiation. We will test whether BMP4 signaling in stress erythroid progenitors leads to formation of Yap1-Smad5 complexes, whether this complex up-regulates Id2 expression and whether Id2 complexes with SCL to block erythroid differentiation. Bone marrow cells from mice mutant in Yap1 or Smad5 or control cells will be cultured in vitro to generate stress erythroid progenitors. The expression of Id2 will be determined by q-RT-PCR. We predict that mutation of either Yap1 or Smad5 would block the expression of Id2. In control cells, the presence of Yap1-Smad5 complexes will be assayed by co-immunoprecipitation (Co-IP). We will examine the ability of Yap1-Smad5 complexes to bind to a previously identified site in the Id2 promoter by Chromatin-immunoprecipitation (ChIP). We will examine the ability of Id2 to bind to SCL by Co-IP.We will characterize signals derived from macrophages that regulate the expansion and differentiation of stress erythroid progenitors. We hypothesize that macrophages use Wnt family signals to accomplish this, and to test whether Wnt factors play a role, first we will analyze the expression of Wnt family factors by q-RT-PCR using RNA isolated from macrophages and sorted by flow cytometry from the spleens of recipient mice following bone marrow transplant. Specifically, recipient mice will be transplanted with donor cells at regular intervals during the recovery. Spleen cells will be isolated and F4/80+ macrophages will be isolated by flow cytometry. RNA will be isolated from the macrophages and the expression of Wnt 1, 2, 2b, 3, 3a, 4, 5a, 5b, 6, 7a, 7b, 8a, 8b, 9a, 9b, 10a, 10b, 11 and 16 will be tested by q-RT-PCR. Wnt signaling can be manipulated using chemical and biological inhibitors or activators. We will test the role of Wnt signaling in stress erythropoiesis using in vitro culture of stress erythroid progenitors. Bone marrow cells will be isolated and cultured under conditions that promote the expansion and differentiation of stress erythroid progenitors. In addition to control conditions, we also will culture cells in the presence of BIO, a chemical that constitutively activates Wnt signaling or Wif1, a protein that binds to and sequesters Wnt ligands from their receptors, thus inhibiting Wnt signaling. We will monitor the development of stress erythroid progenitors in the cultures by flow cytometry and colony assays. Once a role for Wnt signaling is established, we can test its role genetically by mutating beta-catenin, the effector protein of Wnt signaling in stress erythroid progenitors, and determining the effects of the mutation on the ability of mice to recovery from bone marrow transplant.Preliminary experiments show that proliferating stress erythroid progenitors can be separated from differentiating stress erythroid progenitors by flow cytometry. The proliferating cells express CD34 and CD133, while differentiating cells do not express either marker. We observed that proliferating cells do not express the erythropoietin receptor (EpoR), but differentiating cells do express the receptor. We hypothesize that the transition from proliferation to differentiation results in changes in gene expression, which are due to changes in chromatin structure. Our initial experiments will use the EpoR locus as a model gene to test this hypothesis. Bone marrow cells will be cultured under conditions that support the expansion of stress erythroid progenitors. At regular intervals, proliferating-progenitor (CD34+CD133+) and differentiating-progenitor (CD34-CD133-) cells will be isolated by flow cytometry. These two populations of cells will be tested for EpoR expression by q-RT-PCR. The presence of chromatin modifications associated with actively transcribed genes - H3K4 methylation (H3K4me), H3K27 acetylation (H3K27ac) - and DNAse I hypersensitivity, or genes having repressed expression - H3K27 tri methylation (H3K27me3) and DNASe I resistance - will be analyzed by ChIP assays using antibodies specific to the chromatin marks and primers specific for the promoter, the 1st intron enhancer, and a control region '3' to the EpoR locus. We will analyze whether levels of expression correlate with the active or repressed chromatin state. Once we have established this connection at the EpoR locus, we can expand this analysis genome wide. We will test gene expression by performing RNA sequencing of all mRNA transcripts isolated from proliferating and differentiating stress erythroid progenitors. We can then examine changes in chromatin structure by performing ChIP sequencing using the same set of chromatin marks we examined at the Epo locus. These experiments will give us a genome-wide analysis of the changes in chromatin structure and the accompanying changes in gene expression that occur during the development of stress erythroid progenitors.

Progress 01/01/15 to 12/31/19

Outputs
Target Audience:The target audience of our work includes scientists who are interested in the mechanisms that regulate the development of red blood cells, erythropoiesis and the mechanisms by which the body responds to anemia. Our research is also of interest to clinicians who study benign hematology. In addition, scientists from the pharmaceutical industry, who are developing new treatments for anemia would be targets of our work. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The work done on this project was performed by graduate students in the lab. During the project period, we held formal and informal professional development sessions. Students present their work in a student data club. These presentations are designed improve communication skills. Each student presented approximately twice per year. We have A Graduate student development group that meets monthly to career planning. My lab as weekly meetings to discuss research progress and I have an individual meeting with each student. How have the results been disseminated to communities of interest?As indicated in the list of products, during the project period, we have published peer reviewed papers in journals appropriate for our field. We have presented findings at national and international meetings. What do you plan to do during the next reporting period to accomplish the goals?As this report represents the final report for this project, the plan for future work will require the development of a new project

Impacts
What was accomplished under these goals? Overall the work completed on this proposal has advanced our knowledge of the mechanisms that regulate stress erythropoiesis. This response plays a key role in maintaining erythroid homeostasis during infection and inflammation and is the mechanism by which new erythrocytes are generated in response to anemia. During the project period, we developed a novel in vitro culture system that has allowed us to rapidly characterize the role of signaling pathways and specific gene function during stress erythropoiesis. Using this culture system, we refined our model for the development, proliferation and differentiation of stress erythroid progenitors (SEPs). Our studies showed that initially SEPs rapidly proliferate but do not differentiate. The increase in Erythropoietin (Epo) in blood stream drives the transition of these progenitors from proliferating cells that do not differentiate to erythroid progenitors committed to differentiation. Our analysis has defined clear signaling roles for Hedgehog, BMP4, SCF and GDF15 signals in the proliferation of SEPs. In addition, our work has characterized the components of the splenic niche where SEPs develop. It was well known that erythroid progenitors developed in close proximity with macrophages in structures called erythroblastic islands. We showed that during stress erythropoiesis, these erythroblastic islands are made from monocytes recruited into the spleen. Our data demonstrated that the niche develops in concert with the SEPs. These data resolved a long-standing question in the field. This analysis also led to the identification of a role for selenoproteins in the regulation of the niche and in the development of SEPs. Analysis of the niche macrophages also showed that another signaling pathway, canonical Wnt signaling, plays a key role in promoting the expansion of SEP populations in the spleen during the recovery from anemia. The expression of Wnt factors, however, must be turned off before the SEPs can commit to differentiation. Our analysis showed that Epo signaling in the macrophages induces the production of lipid messengers, prostaglandins, that inhibit the expression of Wnt factors and promote the differentiation of SEPs. The proliferation of SEPs is promoted by signals that regulate metabolism in SEPs. GDF15 signaling increases glycolysis and glutaminolysis. These two anabolic pathways generate building blocks for cell division and allow the for the rapid proliferation of immature cells. Yap1, a transcriptional activator regulated by the Hippo pathway also contributes to the maintenance of the proliferative metabolic state. Understanding the how the metabolism of SEPs changes during the transition from the expansion phase to the differentiation stage is a major focus of our current work. Previous work on stress erythropoiesis focused on the recovery from anemia. These analyses demonstrated that tissue hypoxia was a driver of the response. Although hypoxia is an important signal, we showed that inflammation is the primary signal that activates stress erythropoiesis. We used a model of sterile inflammation, that mimics the anemia of chronic inflammation, to demonstrate that pro-inflammatory signals promote the expansion of immature SEPs. In addition, inflammation increases erythrocyte turnover by red-pulp macrophages in the spleen. Although it is counter-intuitive, increased erythrocyte turnover initiates stress erythropoiesis. Hemoglobin from phagocytosed erythrocytes is broken down and the heme is transported into the nucleus where it inactivates the transcriptional repressor Bach1. Loss of this repressor increases the expression of SpiC, a transcription factor that induces the expression of GDF15 and BMP4, two key signals that initiate stress erythropoiesis. These data led to a new model that proposes that stress erythropoiesis is an integral part of the inflammatory response. Future work will investigate how manipulating stress erythropoiesis could be used to treat inflammatory anemia. In addition to our work on stress erythropoiesis, we have an ongoing collaboration with Dr. KS Prabhu at Penn State. We have identified novel prostaglandins that act as potent anti-leukemia drugs. We are investigating the mechanisms of action and demonstrating efficacy in animal models of acute myeloid leukemia.

Publications

• Type: Journal Articles Status: Under Review Year Published: 2019 Citation: Epo-receptor signaling in macrophages alters the splenic niche to promote erythroid differentiation. Y Chen, J Xiang, F Qian, BT Diwakar, B Ruan, S Hao, KS Prabhu and RF Paulson. Blood In revision
• Type: Journal Articles Status: Under Review Year Published: 2019 Citation: Yap promotes proliferation of transiently amplifying stress erythroid progenitors. S Hao, Y Matsui, Z-C Lai and RF Paulson. Experimental Hematology. In revision.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Crth2 receptor signaling down-regulates lipopolysaccharide-induced NF-?B activation in murine macrophages via changes in intracellular calcium. Diwakar BT, Yoast R, Nettleford S, Qian F, Lee TJ, Berry S, Huffnagle I, Rossi RM, Trebak M, Paulson RF, Prabhu KS. FASEB J. 2019 Sep 13:fj201802608R. doi: 10.1096/fj.201802608R. [Epub ahead of print]
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Downregulation of CD73 associates with T cell exhaustion in AML patients. Kong Y, Jia B, Zhao C, Claxton DF, Sharma A, Annageldiyev C, Fotos JS, Zeng H, Paulson RF, Prabhu KS, Zheng H. J Hematol Oncol. 2019 Apr 24;12(1):40. doi: 10.1186/s13045-019-0728-3.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Inflammation induces stress erythropoiesis through heme-dependent activation of SPI-C. Bennett LF, Liao C, Quickel MD, Yeoh BS, Vijay-Kumar M, Hankey-Giblin P, Prabhu KS, Paulson RF. Sci Signal. 2019 Sep 10;12(598). pii: eaap7336
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Gdf15 regulates murine stress erythroid progenitor proliferation and the development of the stress erythropoiesis niche. Hao S, Xiang J, Wu DC, Fraser JW, Ruan B, Cai J, Patterson AD, Lai ZC, Paulson RF. Blood Adv. 2019 Jul 23;3(14):2205-2217.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Dual Role of a C-Terminally Truncated Isoform of Large Tumor Suppressor Kinase 1 in the Regulation of Hippo Signaling and Tissue Growth. Matsui Y, Zhang Y, Paulson RF, Lai ZC. DNA Cell Biol. 2019 Jan;38(1):91-106. doi: 10.1089/dna.2018.4340.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Monocyte derived macrophages expand the murine stress erythropoietic niche during the recovery from anemia. Liao C, Prabhu KS, Paulson RF. Blood. 2018 Dec 13;132(24):2580-2593.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Mechanisms of erythrocyte development and regeneration: implications for regenerative medicine and beyond. Bresnick EH, Hewitt KJ, Mehta C, Keles S, Paulson RF, Johnson KD. Development. 2018 Jan 10;145(1). pii: dev151423. doi: 10.1242/dev.151423. Review.
• Type: Journal Articles Status: Accepted Year Published: 2017 Citation: Activation of PPARy by endogenous prostaglandin J2 mediates the antileukemic effect of selenium in murine leukemia. Finch ER, Tukaramrao DB, Goodfield LL, Quickel MD, Paulson RF, Prabhu KS. Blood. (2017) 129: 1802-1810
• Type: Journal Articles Status: Accepted Year Published: 2017 Citation: GATA Factor-Regulated Samd14 Enhancer Confers Red Blood Cell Regeneration and Survival in Severe Anemia. K. Hewitt, K. Katsumura, D. Matson, P. Devadas, N Tanimura, A. Hebert, J. Coon, JS Kim, C. Dewey, S Keles, S Hao, RF. Paulson and E. Bresnick. (2017) Developmental Cell 42: 213-225
• Type: Journal Articles Status: Accepted Year Published: 2017 Citation: SKI-178: A Multitargeted Inhibitor of Sphingosine Kinase and Microtubule Dynamics Demonstrating Therapeutic Efficacy in Acute Myeloid Leukemia Models. Hengst JA, Dick TE, Sharma A, Doi K, Hegde S, Tan SF, Geffert LM, Fox TE, Sharma AK, Desai D, Amin S, Kester M, Loughran TP, Paulson RF, Claxton DF, Wang HG, Yun JK. Cancer Transl Med. 2017 Jul-Aug;3(4):109-121
• Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: Epo-receptor signaling in macrophages alters the splenic niche to promote erythroid differentiation. Y Chen, J Xiang, F Qian, BT Diwakar, B Ruan, S Hao, KS Prabhu and RF Paulson. Red Cell Club, University of Rochester School of Medicine, Rochester, NY.
• Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: GDF15 signaling mediates the dynamic interplay between stress erythroid progenitors and the splenic niche that regulates stress erythropoiesis. Hao S, Xiang J, Wu DC, Fraser JW, Ruan B, Cai J, Patterson AD, Lai ZC, Paulson RF. Red Cells Gordon Research Conference, Salve Regina University, Newport, RI
• Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: Nrf2 regulates the transition of stress erythroid progenitors from expansion to differentiation during stress erythropoiesis. B Ruan and Paulson RF. Red Cells Gordon Research Conference, Salve Regina University, Newport, RI
• Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: Yap promotes proliferation of transiently amplifying stress erythroid progenitors. S Hao, Y Matsui, Z-C Lai and RF Paulson. Red Cells Gordon Research Conference, Salve Regina University, Newport, RI
• Type: Conference Papers and Presentations Status: Other Year Published: 2018 Citation: Epo dependent PGE2 signaling promotes the transition to differentiation of stress erythroid progenitors. Y Chen, B Ruan and RF Paulson. Red Cell Club, Yale University, New Haven CT.
• Type: Journal Articles Status: Accepted Year Published: 2015 Citation: In vitro culture of stress erythroid progenitors identifies distinct progenitor populations and analogous human progenitors. J. Xiang, D-C Wu, Y. Chen and RF Paulson. 2015 Blood 125:1803-1812.
• Type: Book Chapters Status: Published Year Published: 2018 Citation: Stress Erythropoiesis Model Systems. Bennett LF, Liao C, Paulson RF. Methods Mol Biol. 2018;1698:91-102. doi: 10.1007/978-1-4939-7428-3_5
• Type: Book Chapters Status: Published Year Published: 2017 Citation: The regulation of pathways of inflammation and resolution in immune cells and cancer stem cells by selenium. BT Diwakar, AM Korwar, RF Paulson and KS Prabhu. In Kenneth D. Tew and Francesco Galli, editors. Advances in Cancer Research Volume 136. Academic Press Burlington VT.
• Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: Stress erythropoiesis in the regulation of infection-induced inflammation  An emerging paradigm. #J Fraser, A Dey, S Nettleford, KS Prabhu, P Hankey Giblin and RF Paulson. 22nd Biennial meeting on Hemoglobin Switching.
• Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: Induction of stress erythropoiesis by inflammation (Invited Talk). Robert Paulson. 2017 Red Cell Gordon Conference. Newport, RI.
• Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: New roles of Epo induced PGE2-PERK signaling pathway in transitional and translational control during stress erythropoiesis. Yuanting Chen, Jie Xiang and Robert Paulson (poster) 2017 Red Cell Gordon Conference, Newport, RI.
• Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: Role of Yap1 in stress erythropoiesis. Siyang Hao and Robert Paulson (poster) 2017 Red Cell Gordon Conference, Newport, RI
• Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: Selenoproteins play a key role in stress erythropoiesis by regulating both the stress erythroid progenitors and the spleen microenvironment. Chang Liao, Ross C. Hardison, Mary J. Kennett, Bradley A. Carlson, Robert F. Paulson and K. Sandeep Prabhu (poster) 2017 Red Cell Gordon Conference, Newport, RI.
• Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: Inflammation induces stress erythropoiesis to maintain erythroid homeostasis in response to infection. Robert Paulson, Laura Bennett, Chang Liao and James Fraser. (Poster) International Society of Experimental Hematology, Frankfurt, Germany.
• Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: Translational regulation by PERK signaling pathway during stress erythropoiesis. Y Chen and RF Paulson. 22nd Biennial meeting on Hemoglobin Switching. Pacific Grove CA 2016.
• Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: Phenotypic dynamics of erythroblastic islands during stress erythropoiesis. #C. Liao and RF Paulson. 22nd Biennial meeting on Hemoglobin Switching. Pacific Grove CA 2016
• Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: Defect in Stress Erythropoiesis during Selenium Deficiency. Chang Liao, K. Sandeep Prabhu and Robert F. Paulson. Red Cells Gordon Conference, Hanover, NH.
• Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: Role of Glucocorticoid signaling in stress erythropoiesis. Siyang Hao and Robert Paulson. Red Cells Gordon Conference, Hanover NH.
• Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: Role of PGE2 dependent Perk kinase signaling in stress erythropoiesis. Yuanting Chen and Robert Paulson. Red Cells Gordon Conference, Hanover, NH.
• Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: Dynamic changes in the macrophage microenvironment during stress erythropoiesis. Robert Paulson (Invited Talk) 20th Biennial Hemoglobin Switching Conference. Asilomar Conference Center, Pacific Grove, CA.
• Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: Regulation of stress erythropoiesis by interactions between progenitor cells and the microenvironment. Robert F. Paulson, Jie Xiang, Laura Bennett, Sneha Hariharan, Yuanting Chen, Siyang Hao and Eun Hyeon Song. Red Cells Gordon Conference, Hanover, NH.
• Type: Theses/Dissertations Status: Accepted Year Published: 2019 Citation: Regulation of stress erythropoiesis via splenic erythroid microenvironment development and metabolic programming. Siyang Hao
• Type: Theses/Dissertations Status: Accepted Year Published: 2016 Citation: Study of growth of stress erythroid progenitors from peripheral blood mononuclear cells in murine and human anemic model systems by Sneha Hariharan
• Type: Theses/Dissertations Status: Accepted Year Published: 2015 Citation: Regulation of stress erythropoiesis: Interactions between microenvironment and stress erythroid progenitors. by Jie Xiang

Progress 10/01/17 to 09/30/18

Outputs
Target Audience:The target audience of our work includes scientists who are interested in the mechanisms that regulate the development of red blood cells, erythropoiesis and the mechanisms by which the body responds to anemia. Our research is also of interest to clinicians who study benign hematology. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The work done in the lab was performed by graduate students. Throughout the year there are formal and informal programs for professional development. Students give presentations to the faculty on a regular basis. Each student will present approximately twice a year. We have A Graduate student development group that meets monthly to career planning. My lab as weekly meetings to discuss research progress and I have an individual meeting with each student. How have the results been disseminated to communities of interest?We have reported our data at two international meetings, the American Association of Immunology Annual Meeting and the 21st Conference on Hemoglobin Switching. In addition, our work is reported in peer reviewed publications and book chapters. What do you plan to do during the next reporting period to accomplish the goals?The focus of the laboratory will continue to be the regulation stress erythropoiesis. We are working on two main areas. First, we are investigating the mechanisms that regulate the proliferation and differentiation of stress erythroid progenitors. In the past year we have made considerable progress analyzing the role of metabolism is regulating differentiation. We are working to finish these studies so that they can be submitted for publication. Second, we are expanding our analysis of stress erythropoiesis in the context of infection and in the development of the chronic anemia of inflammatory disease. On this project we have identified a number of novel mechanisms, which we are working to analyze and submit for publication. In addition we have started a collaboration with a lab studying malaria and have exciting preliminary data concerning the role of stress erythropoiesis in the progression of malaria.

Impacts
What was accomplished under these goals? One graduate student project focused on two areas of stress erythropoiesis. One part of her thesis focused on the role of the micronutrient selenium in the regulation of stress erythropoiesis. She showed that dietary deficiency of selenium leads to exacerbated anemia in our models of anemic stress. Selenium is incorporated into proteins as a novel amino acid, selenocysteine. Selenoproteins play a key role in regulating the proliferation and differentiation of stress erythroid progenitors. Her work identified Selenoprotein Was a key selenoprotein in this process. She also showed that mice that are selenium deficient have defects in microenvironment that surrounds and regulates stress erythroid progenitors. This work was published in Blood, the journal of the American Society of Hematology. The paper was well received and was one of three papers that was highlighted with a news and views on the paper. The same student also completed a comprehensive analysis of the development of stress erythropoietic niche in the spleen. These data addressed a longstanding question in the field of the origin of macrophages in this niche. Our data show that macrophages that interact with developing stress erythroid progenitors in structures referred to as erythroblastic islands are not tissue resident macrophages, but rather are derived from monocytes. This analysis showed that the development of the niche parallels the development of stress erythroid progenitors. This student was co-advised by myself and Dr. Sandeep Prabhu in the department. My lab continues to focus on the interactions between stress erythropoiesis and the immune response. Another graduate student in my laboratory demonstrated that infection induces stress erythropoiesis to maintain erythroid development during the response to infection. It is well known that infection blocks bone marrow steady state erythropoiesis and additionally leads to sequestration of the body's iron stores, which further exacerbates the anemia. We showed that stress erythropoiesis is induced during infection to maintain erythrocyte production. This switch in the sites of erythropoiesis is possible because the signals that inhibit bone marrow erythropoiesis stimulate stress erythropoiesis in the spleen. The surprising result, however came when we analyzed the response of mice with mutations that block stress erythropoiesis to infection. We expected that the mice would develop anemia, but instead they developed a lethal inflammatory disease. These data suggest that inflammation and infection activate stress erythropoiesis, but stress erythropoiesis inhibits inflammation and is needed to resolve infection. These data were presented at the American Association of Immunology Annual Meeting and the student presenting won a graduate student travel award. We are continuing our work with Dr. Prabhu on the development prostaglandin J3 based drugs for the treatment of leukemia.

Publications

• Type: Journal Articles Status: Published Year Published: 2018 Citation: Monocyte derived macrophages expand the murine stress erythropoietic niche during the recovery from anemia. Liao C, Prabhu KS, Paulson RF. Blood. 2018 Oct 15. pii: blood-2018-06-856831. doi: 10.1182/blood-2018-06-856831. [Epub ahead of print
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Mtf2-PRC2 control of canonical Wnt signaling is required for definitive erythropoiesis. Rothberg JLM, Maganti HB, Jrade H, Porter CJ, Palidwor GA, Cafariello C, Battaion HL, Khan ST, Perkins TJ, Paulson RF, Ito CY, Stanford WL. Cell Discov. 2018 May 1;4:21. doi: 10.1038/s41421-018-0022-5. eCollection 2018
• Type: Journal Articles Status: Published Year Published: 2018 Citation: The intricate role of selenium and selenoproteins in erythropoiesis. Liao C, Carlson BA, Paulson RF, Prabhu KS. Free Radic Biol Med. 2018 Nov 1;127:165-171. doi: 10.1016/j.freeradbiomed.2018.04.578. Epub 2018 Apr 30. Review
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Neuroprotective Role of the Ron Receptor Tyrosine Kinase Underlying Central Nervous System Inflammation in Health and Disease. Dey A, Allen JN, Fraser JW, Snyder LM, Tian Y, Zhang L, Paulson RF, Patterson A, Cantorna MT, Hankey-Giblin PA. Front Immunol. 2018 Mar 19;9:513. doi: 10.3389/fimmu.2018.00513. eCollection 2018
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Selenoproteins regulate stress erythroid progenitors and spleen microenvironment during stress erythropoiesis. Liao C, Hardison RC, Kennett MJ, Carlson BA, Paulson RF, Prabhu KS. Blood. 2018 Jun 7;131(23):2568-2580. doi: 10.1182/blood-2017-08-800607. Epub 2018 Apr 3
• Type: Conference Papers and Presentations Status: Other Year Published: 2018 Citation: EPO Dependent PGE2 Signaling Promotes the Transition to Differentiation of Stress Erythroid Progenitors. Y Chen, B Ruan and RF Paulson. 21st Hemoglobin Switching Conference. Pembroke College, Oxford, UK
• Type: Conference Papers and Presentations Status: Other Year Published: 2018 Citation: GDF15 and BMP4 co-regulate stress erythropoiesis and Treg development to maintain erythroid homeostasis and resolve inflammation during infection. Fraser, JW., Dey, A., Nettleford, S., Zhao, L., Giblin, P., Prabhu, KS., Xiong, N. & Paulson, RF. American Association of Immunology Annual Meeting Austin, TX.
• Type: Theses/Dissertations Status: Accepted Year Published: 2018 Citation: Regulation of stress erythropoiesis and the niche: a novel role for selenoproteins. Chang Liao

Progress 10/01/16 to 09/30/17

Outputs
Target Audience:The target audience of our project is the community of scientists who are interested in the mechanisms that regulate red blood cell development and the causes of anemia. This group would include clinicians who practice Hematology and Oncology as well as basic scientists who are interested in erythroid development and disease. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Work on this project is done by graduate students. We have several programs to enhance the training of graduate students. We have a regular biweekly research meeting where students present their work. In addition, a Graduate student development group meets monthly to discuss career issues. Finally my own lab has regular meetings to discuss training and research results. How have the results been disseminated to communities of interest?Our experimental data was reported at two international meetings. Several of my students presented posters at the Red Cell Gordon Conference and I was invited to speak. I also presented a poster at the International Society of Experimental Hematology Annual meeting in Frankfurt Germany. We also published several manuscripts. What do you plan to do during the next reporting period to accomplish the goals?We will continue our work on stress erythropoiesis. The lab works on two aspects of this process. One concerns the molecular characterization of the signals that regulate stress erythropoiesis. The second project concerns the role of stress erythropoiesis in the response to inflammation and infection. This work is generating new hypotheses to explain the anemia of chronic disease. We are working to understand how inflammation induces stress erythropoiesis.

Impacts
What was accomplished under these goals? We have made considerable progress towards our goals. One graduate student finished her thesis work. This project changed the focus of our research. Our original work examined stress erythropoiesis in the context of anemia, but recent work has shown that this pathway is induced by inflammation. One common form of anemia is the anemia of chronic disease, where the anemia is secondary to other infection, inflammation or trauma. The anemia of chronic disease results in significant morbidity and decreased quality of life. It is a major human health problem. Our previous work showed that low tissue oxygen levels (hypoxia) leads to the activation of stress erythropoiesis. This pathway is designed to rapidly produce a bolus of new erythrocytes that maintain homeostasis until the bone marrow can resume producing erythrocytes. In these new studies we show that stress erythropoiesis is induced by inflammatory signals. These signals lead to the expression of key factors, GDF15 and BMP4, that regulate stress erythropoiesis. However, hypoxia dependent signals are not involved. Instead inflammation induces the turnover of erythrocytes by macrophages in the spleen. Breakdown of erythrocyte hemoglobin in the macrophages releases heme, which in turn acts a signaling molecule. Heme signaling leads to the induction of stress erythropoiesis. Preliminary data suggest that this mechanism functions in other types of anemia making it a new paradigm for the field, which we will continue to pursue. In addition to these findings we have worked in collaboration with other labs to address other questions in the regulation of stress erythropoiesis. We worked with Dr. Emery Bresnick at the University of Wisconsin to demonstrate a role for Samd14 in the regulation of Stem Cell Factor (SCF) signaling during the recovery from acute anemia. SCF dependent signaling promotes the expansion of early stress erythroid progenitors. Samd14 potentiates that signal. Working Dr. William Stanford from the Ottawa Hospital Research Institute, we helped to show that Mtf2, a protein that regulates chromatin stricture as part of the polycomb complex plays a key role in stress erythropoiesis. Mtf2 regulates signaling by two pathways, the Wnt pathway and Hippo pathway. We also collaborated with Dr. Sandeep Prabhu in our Department to demonstrate a role for selenoproteins in stress erythropoiesis. This work showed that dietary selenium deficiency leads to defects in the response acute anemic stress. In addition the work on stress erythropoiesis, my lab also works on developing treatments for leukemia. We published studies showing that prostaglandin J2 activates Peroxisome proliferator activating receptor gamma (PPARg), which decreases leukemia stem cells. Working with investigators at the Hershey Medical School we showed that Sphingosine Kinase inhibitors can target acute myeloid leukemia cells.

Publications

• Type: Theses/Dissertations Status: Accepted Year Published: 2017 Citation: Analysis of stress erythropoiesis during inflammation: stimulation of TLRs induces erythrophagocytosis and activates stress erythropoiesis. By Laura Bennett
• Type: Journal Articles Status: Accepted Year Published: 2017 Citation: Activation of PPAR? by endogenous prostaglandin J2 mediates the antileukemic effect of selenium in murine leukemia. Finch ER, Tukaramrao DB, Goodfield LL, Quickel MD, Paulson RF, Prabhu KS. Blood. (2017) 129: 1802-1810
• Type: Journal Articles Status: Accepted Year Published: 2017 Citation: GATA Factor-Regulated Samd14 Enhancer Confers Red Blood Cell Regeneration and Survival in Severe Anemia. K. Hewitt, K. Katsumura, D. Matson, P. Devadas, N Tanimura, A. Hebert, J. Coon, JS Kim, C. Dewey, S Keles, S Hao, RF. Paulson and E. Bresnick. (2017) Developmental Cell 42: 213-225
• Type: Journal Articles Status: Accepted Year Published: 2017 Citation: SKI-178: A Multitargeted Inhibitor of Sphingosine Kinase and Microtubule Dynamics Demonstrating Therapeutic Efficacy in Acute Myeloid Leukemia Models. Hengst JA, Dick TE, Sharma A, Doi K, Hegde S, Tan SF, Geffert LM, Fox TE, Sharma AK, Desai D, Amin S, Kester M, Loughran TP, Paulson RF, Claxton DF, Wang HG, Yun JK. Cancer Transl Med. 2017 Jul-Aug;3(4):109-121
• Type: Journal Articles Status: Under Review Year Published: 2017 Citation: Selenoproteins regulate stress erythroid progenitors and the spleen microenvironment during stress erythropoiesis. Chang Liao, Ross C. Hardison, Mary J. Kennett, Bradley A. Carlson, Robert F. Paulson, and K. Sandeep Prabhu. Submitted to Blood
• Type: Journal Articles Status: Under Review Year Published: 2017 Citation: Mtf2 dynamically regulates Wnt and Hippo signaling to control hematopoiesis. Janet L. Manias Rothberg, Harinad B. Maganti, Hani Jrade, Christopher J. Porter, Gareth A. Palidwor, Christopher Cafariello, Theodore J. Perkins, Robert F. Paulson, Caryn Ito, William L. Stanford. Submitted to Cell Discovery.
• Type: Journal Articles Status: Under Review Year Published: 2017 Citation: Mechanisms of erythrocyte development and regeneration: implications for regenerative medicine and beyond. Emery H. Bresnick, Kyle J. Hewitt, Charu Mehta, Robert F. Paulson and Kirby D. Johnson. In Revision at Development.
• Type: Book Chapters Status: Published Year Published: 2017 Citation: Stress erythropoiesis model systems. Laura Bennett, Chang Liao and Robert Paulson. In Methods in Molecular Biology: Erythropoiesis-Methods and Protocols. Ed. Joyce Lloyd. Springer New York, NY.
• Type: Book Chapters Status: Published Year Published: 2017 Citation: The regulation of pathways of inflammation and resolution in immune cells and cancer stem cells by selenium. BT Diwakar, AM Korwar, RF Paulson and KS Prabhu. In Kenneth D. Tew and Francesco Galli, editors. Advances in Cancer Research Volume 136. Academic Press Burlington VT.
• Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: Translational regulation by PERK signaling pathway during stress erythropoiesis. Y Chen and RF Paulson. 22nd Biennial meeting on Hemoglobin Switching. Pacific Grove CA 2016.
• Type: Journal Articles Status: Under Review Year Published: 2017 Citation: Inflammation induces stress erythropoiesis through heme dependent activation of Spi-C. Laura F. Bennett, Michael D. Quickel, Beng San Yeoh, Matam Vijay-Kumar, Pamela Hankey-Giblin, Robert F. Paulson. Science Signaling under review.
• Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: Induction of stress erythropoiesis by inflammation (Invited Talk). Robert Paulson. 2017 Red Cell Gordon Conference. Newport, RI.
• Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: New roles of Epo induced PGE2-PERK signaling pathway in transitional and translational control during stress erythropoiesis. Yuanting Chen, Jie Xiang and Robert Paulson (poster) 2017 Red Cell Gordon Conference, Newport, RI.
• Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: Role of Yap1 in stress erythropoiesis. Siyang Hao and Robert Paulson (poster) 2017 Red Cell Gordon Conference, Newport, RI.
• Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: Selenoproteins play a key role in stress erythropoiesis by regulating both the stress erythroid progenitors and the spleen microenvironment. Chang Liao, Ross C. Hardison, Mary J. Kennett, Bradley A. Carlson, Robert F. Paulson and K. Sandeep Prabhu (poster) 2017 Red Cell Gordon Conference, Newport, RI.
• Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: Inflammation induces stress erythropoiesis to maintain erythroid homeostasis in response to infection. Robert Paulson, Laura Bennett, Chang Liao and James Fraser. (Poster) International Society of Experimental Hematology, Frankfurt, Germany

Progress 10/01/15 to 09/30/16

Outputs
Target Audience:The target audience of our project is clinicians who practice Hematology and Oncology as well as basic scientists who are interested in the mechanisms that regulate red blood cell production at normal times and during disease states. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This work is being done by graduate students in the laboratory. Professional development activities for these students include a regular lab meeting where students discuss their findings. In addition I meet weekly with all students to discuss their progress. Students also present their data in a biweekly meeting called Data Club where students from all the labs in the Center for Molecular Immunology and Infectious Disease present their work. Finally the individual graduate programs have professional development activities for their students. How have the results been disseminated to communities of interest?Our experimental data was reported at an international meeting. Several of my students presented posters and I was invited to speak at the 22nd Biennial meeting on Hemoglobin Switching in Pacific Grove CA. In addition we are working on revised and new manuscripts for publication. What do you plan to do during the next reporting period to accomplish the goals?We will continue our work analyzing the molecular mechanisms that regulate the expansion and differentiation of red blood cell precursors in the spleen in response to anemic stress. We are presently finishing some work concerning the role of this pathway in the response to infection and inflammation. In addition to that work, we are also examining the mechanisms that regulate the production of hemoglobin during erythrocyte development. We have identified a series of signaling pathways that enable differentiating red blood cell precursors to massively up-regulate their ability to translate proteins so that large quantities of globin protein can be made and incorporated into hemoglobin.

Impacts
What was accomplished under these goals? Over the past year we have made several advances towards achieving our goals. Furthermore new discoveries have moved our research into new areas. One major goal of our work is to understand the molecular mechanisms that regulate the expansion of red cell progenitors in response to anemic stress. We have discovered that a change in the signals produced by support cells in the spleen microenvironment coordinate the initial expansion of precursor cells and their transition to differentiation into erythrocytes. The expansion phase is driven by the expression Wnt family factors by macrophages in the spleen. While the differentiation of the progenitor cells is driven by repression of Wnt expression and the production of PGE2 by the same macrophages. We identified the systemic signal that promotes this transition as erythropoietin. These data identify a novel mechanism by which a systemic signal, erythropoietin, alters the microenvironment to induce the production of new erythrocytes during times of anemic stress. These data will be reported in a manuscript in preparation.

Publications

• Type: Theses/Dissertations Status: Accepted Year Published: 2016 Citation: Study of growth of stress erythroid progenitors from peripheral blood mononuclear cells in murine and human anemic model systems by Sneha Hariharan.
• Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: Translational regulation by PERK signaling pathway during stress erythropoiesis. Y Chen and RF Paulson. 22nd Biennial meeting on Hemoglobin Switching. Pacific Grove CA 2016.
• Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: Phenotypic dynamics of erythroblastic islands during stress erythropoiesis. C. Liao and RF Paulson. 22nd Biennial meeting on Hemoglobin Switching. Pacific Grove CA 2016
• Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: Dynamic changes in the macrophage microenvironment during stress erythropoiesis. Robert Paulson (Invited Talk) 20th Biennial Hemoglobin Switching Conference. Asilomar Conference Center, Pacific Grove, CA.
• Type: Journal Articles Status: Under Review Year Published: 2016 Citation: GDF15 represses VHL to regulate hypoxia dependent transcription during the recovery from anemia. J Xiang, DC Wu, E Ellsworth, S Hegde, JL Miller and RF Paulson. Stem Cell Reports in revision.
• Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: Stress erythropoiesis in the regulation of infection-induced inflammation  An emerging paradigm. #J Fraser, A Dey, S Nettleford, KS Prabhu, P Hankey Giblin and RF Paulson. 22nd Biennial meeting on Hemoglobin Switching. Pacific Grove CA 2016

Progress 01/01/15 to 09/30/15

Outputs
Target Audience:Our work is targeted to other members of the field who study the development of red blood cells and disorders of red cell production. This audience includes other scientists as well as clinicians. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Training and professional development activities included educating and mentoring graduate (PhD) students who are working on the project. How have the results been disseminated to communities of interest?The results have been disseminated at national meetings. This past year we attended the Red Cells Gordon Conference, which is an international meeting attended by leading experts in the field of erythropoiesis. In addition, we have published our findings in peer-reviewed journal articles, as indicated in the "Products" section. What do you plan to do during the next reporting period to accomplish the goals?Over the next year we will continue to address the goals set for this project. In particular, our recent work has focused on characterizing the microenvironment and how it changes during recovery from anemia. We have new data showing that complexes known as erythroblastic islands are generated during the recovery from anemia, and these structures allow macrophages to pass signals and other nutrients to the developing stress erythroid progenitors. We are working to understand the development of the erythroblastic islands and understand how they signal to the stress erythroid progenitors. In addition, our observation that stress erythroid progenitors transition from stem cell-like progenitors that are unable to differentiate to stress erythroid progenitors that are capable of differentiation suggests that there is a fundamental molecular change in the cells. We are working in collaboration with Ross Hardison in the Biochemistry and Molecular Biology Department at Penn State to understand changes in chromatin structure that underlie this transition.

Impacts
What was accomplished under these goals? During the past year, we made major advances towards achieving our goals. First of all, we developed an in vitro culture system that allows us to generate mouse and human stress erythroid progenitors by culturing unfractionated bone marrow in defined media conditions. This work, which was published in Blood, allowed us to identify key signals that are involved in the expansion of early stem cell-like stress erythroid progenitors, the transition of progenitors to differentiating stress erythroid progenitors, and the terminal differentiation of stress erythroid progenitors. This culture system is now being used in other labs to analyze stress erythroid progenitors. Second, we have identified GDF15 as a key factor in regulating the expansion of stress erythroid progenitors. Our data show that GDF15, produced by the stress erythroid progenitors themselves, is able to induce the expression of another key factor, BMP4, by macrophages in the microenvironment. GDF15 promotes the expression of BMP4 by inducing the hypoxia response. despite the fact that tissue hypoxia is not present. These data fit with other observations that GDF15 is induced when tissue damage occurs and aids in the repair of tissues by promoting expansion of cells. These findings are reported in our manuscript that is under revision at Stem Cell Reports.

Publications

• Type: Theses/Dissertations Status: Other Year Published: 2015 Citation: Regulation of stress erythropoiesis: Interactions between microenvironment and stress erythroid progenitors. The Penn State University. Jie Xiang.
• Type: Journal Articles Status: Under Review Year Published: 2015 Citation: Xiang, J., Wu, D. C., Ellsworth, E., Hegde, S., Miller, J. L., and Paulson, R. F. GDF15 represses VHL to regulate hypoxia dependent transcription during the recovery from anemia. Stem Cell Reports (in revision).
• Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: Paulson, R. F., Xiang, J., Bennett, L., Hariharan, S., Chen,Y., Hao, S., and Song, E. H. Regulation of stress erythropoiesis by interactions between progenitor cells and the microenvironment. Red Cells Gordon Conference, Hanover, NH.
• Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: Liao, C., Prabhu, K. S., and Paulson, R. F. Defect in stress erythropoiesis during selenium deficiency. Red Cells Gordon Conference, Hanover, NH.
• Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: Hao, S. and Paulson, R. Role of Glucocorticoid signaling in stress erythropoiesis. Red Cells Gordon Conference, Hanover NH.
• Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: Chen, Y. and Paulson, R. Role of PGE2 dependent Perk kinase signaling in stress erythropoiesis. Red Cells Gordon Conference, Hanover, NH.
• Type: Journal Articles Status: Accepted Year Published: 2015 Citation: Xiang,J., Wu, D. C., Chen, Y., and Paulson, R. F. In vitro culture of stress erythroid progenitors identifies distinct progenitor populations and analogous human progenitors. Blood 2015 Mar 12;125(11):1803-1812.