P&F Projects 2017-2020

Vinchi Abstract Collaborative P&F 2020

Macrophages are key players in heme and iron metabolism as well as immune homeostasis. They exhibit remarkable phenotypic and functional plasticity, reflected by their capacity to integrate diverse signals from the microenvironment and acquire distinct phenotypes. According to a simple dichotomous nomenclature, macrophages are defined as pro-inflammatory ‘classically activated’ M1 or anti-inflammatory ‘alternatively activated’ M2. M1 macrophages produce pro-inflammatory cytokines, reactive oxygen species (ROS) and nitric oxide (NO), express markers such as MHCII, CD86 and iNOS, and show bactericidal activity. Conversely, M2 macrophages  are characterized by high expression of the mannose receptor CD206, produce anti-inflammatory cytokines, have immune-regulatory functions and are involved in cell growth control, matrix remodeling and tissue repair. Recently, we described the ability of heme and iron to induce an M1-like phenotypic switching of macrophages, which is prevented by heme and iron scavengers (e.g., hemopexin, transferrin, chelators). Heme-induced M1-like pro-inflammatory macrophages are of patho-physiologic relevance for hemolytic disorders and have been implicated in hepatic fibrosis in SCD (sickle cell disease). SCD is hallmarked by high circulating free heme and depletion of the heme scavenger Hemopexin as a result of intravascular hemolysis. Moreover, SCD is characterized by a chronic inflammatory state, which likely contributes to a number of complications associated with the disease. We suggest that M1 macrophage skewing triggered by free iron, heme and hemolysis is responsible for the chronic sterile inflammation in SCD and we believe that targeting the cellular and molecular mechanisms leading to macrophage phenotypic shift might be of therapeutic value in this disease. Although the underlying mechanisms have not been fully elucidated, our preliminary data indicate a role for TLR4 activation, ROS and NO production, and Arginase-1 suppression in heme/iron-driven M1 M polarization, suggesting a cell metabolic switching towards glycolysis. Emerging evidence on immunometabolism highlight the implication of metabolic intermediates in modulating and reprogramming macrophage immune functions. In this Pilot and Feasibility Program, we aim, in collaboration with the Metabolomics Core of the Center for Iron and Heme Disorders (CIHD) of the University of Utah, at exploring the link between metabolic skewing and immune reprogramming of macrophages by iron sources, with the hypothesis that cell inflammatory phenotypic switching is mediated by a specific metabolic response and adaptation to free heme and iron. We will test this concept by performing metabolic profiling of in vitro and ex vivo macrophages exposed to heme and iron. Finally, we will assess the metabolic profile of macrophages  isolated from SCD mice, with the hypothesis that heme-triggered metabolic skewing determines macrophage phenotypic switch and hence contributes to chronic inflammation in this hemolytic condition.
 

Medlock Abstract Collaborative P&F 2020

Heme is an essential cofactor for many cellular processes in mammalian cells including oxygen binding and delivery, redox reactions, detoxification, and regulation of transcription and translation. While all cells in mammals have the ability to synthesize heme de novo, the levels to which heme is required by different cell types vary greatly with developing erythrocytes synthesizing a large quantity (~109 molecules per cell) for hemoglobin production. Thus in the developing erythrocytes heme and globin synthesis must be coordinated in order to avoid pathologic conditions including thalassemias, porphyrias, and anemias. The regulation of heme synthesis is not well understood, with most studies focused at the transcriptional level of heme synthesis enzymes. Recent data has demonstrated that the mitochondrial enzymes of the pathway exist <em>in situ</em> as a complex, or metabolon, and that this metabolon is important in regulating porphyrin and heme synthesis. In addition to heme synthesis enzymes, other proteins involved in intermediary metabolism, mitochondrial structure and dynamics, and mitochondrial metabolite transport were also found to interact with the heme metabolon. While some of these proteins have known cellular functions, how they interact with the metabolon to regulate porphyrin and heme synthesis and homeostasis is unclear. We hypothesize that several of these interacting proteins serve crucial roles in the regulation of substrate synthesis and/or delivery, heme synthesis enzyme activity, and trafficking of completed heme. Herein we propose the creation of human erythroid cell lines in which metabolon components have been disrupted by CRISPR-Cas genome editing. These cells will be analyzed for differences in levels of various heme synthesis metabolites including heme, porphyrins IX, and other porphyrin synthesis intermediates. Cells will also be analyzed for alterations in peripheral metabolite pools such as amino acids, TCA cycle intermediates, and redox metabolites. Data resulting from these experiments will serve as preliminary data for further study of the metabolon in erythroid and non-erythroid cells through R01 funding from the NIDDK. Outcomes of these studies will further illuminate the processes by which heme synthesis and the numerous pathways linked thereto are regulated and coordinated. Importantly, this work could lend insight to potential treatments for conditions including anemias, porphyrias, and thalassemias.
 

Ward Abstract Collaborative P&F 2020

Macrophages play a critical role in mammalian iron metabolism as they are responsible for degrading senescent red blood cells and recycling iron back to plasma. They do this at a rate of 20-30 mg/day. Total body iron levels are approximately 3-4 g so that equates to about 1% of iron being recycled by macrophages/day, thus underscoring their important role in mammalian iron homeostasis. When iron is in excess macrophages store iron in cytosolic ferritin, which when iron is need can be broken down in the lysosome and iron released back to the cytosol for export into plasma. Iron in ferritin is stored in the Fe3+ state, but all iron transporters identified to date transport Fe2+, therefore, iron must be reduced to be exported from the lysosome. Reductases are found at the plasma membrane or in early endosomes (enterocyte Dcytb or Steap1-4) but the reductase involved in lysosomal iron reduction has not been identified. A candidate reductase for the lysosomal reductase,Cytb561a3 (Lcytb) was suggested years ago. While Lcytb is localized to the lysosome no evidence was provided that it is involved in lysosomal iron reduction. Using CrispR/Cas9 mutagenesis, we found that loss of Lcytb results in decreased iron export from lysosomes of RAW264.7 cells. We determined that RAW264.7 macrophages do not express <em>Steap1</em>, <em>Steap2</em> or <em>Steap4</em> mRNA. Again, using CrispR/Cas9, we determined that loss of the endosomal reductase Steap3 also decreases iron export from the lysosome and that loss of both is additive in limiting lysosomal iron export. This suggests that the mammalian lysosome can exist as an iron storage organelle similar to the vacuole in plants and yeast and that iron can be exported to the cytosol. <em>We hypothesize that Lcytb and Steap3 are the reductases necessary for iron recycling in macrophages.</em> Our preliminary results were done in an immortalized macrophage cell line RAW264.7 and we are currently confirming that Lcytb and Steap3 function in primary macrophages.

We utilized three cores sponsored by NIDDK (Utah – Metabolomics, Utah – Iron and Heme and Mutation Generation Detection Core) to determine the roles of these reductases in macrophage iron recycling and macrophage lysosome function.
 

Ramamoorthy Abstract

ATRX (α-thalassemia mental retardation, X-linked) is a member of the SWI/SNF family of chromatin remodelers. Mutations in ATRX lead to ATRX syndrome, a developmental disorder characterized by mental retardation and α-thalassemia. Exactly how these mutations lead to α-thalassemia, a loss of α-globin production due to repression of the HBA (hemoglobin α) gene, is yet unknown. We will investigate the sub-telomeric positioning of the HBA gene cluster (chr.16) and how telomeric cohesion impacts its expression in ATRX syndrome. We previously demonstrated that ATRX influences the cohesion at the telomeres through its interaction with a histone variant, macroH2A1.1. Interaction of ATRX with macroH2A1.1 sequesters the histone variant from localizing to the telomeres, allowing for cohesion resolution. Our preliminary data suggest that unlike WT ATRX, an ATRX patient mutation (ATRX L409S) does not interact with macroH2A1.1. We therefore hypothesize that ATRX L409S prevents α-globin gene expression by promoting persistent telomere cohesion. To test this hypothesis, we propose to create hiPSCs harboring the ATRX L409S mutations (Harvard NIDDK core) and differentiate them to erythroid lineage. Next, we will analyze the status of telomere cohesion and monitor the expression of the α-globin genes. These will be compared with WT hiPSCs and hiPSCs with ATRX knock downs (we have previously shown that reduction of ATRX leads to persistent cohesion). Finally, we will study the role of macroH2A1.1’s telomere localization on our phenotype. Successful remediation of the α-globin expression in the ATRX disease model by resolution of telomere cohesion will allow us to propose a treatment strategy to alleviate the α-globin deficiency. This will be through designing small molecules that that will mimic the ATRX domain, which can bind to and sequester macroH2A1.1 away from the telomeres, thereby potentially treat α-thalassemia in ATRX syndrome.
 

Scanlon Abstract

The long-term goal of my research is to elucidate the mechanisms that specify hematopoietic stem and progenitor cell fates. Such knowledge is critical for developing treatments for hematological diseases, as well as producing blood products for transfusion medicine. Currently there are many obstacles to overcome before we can manufacture a sufficient and safe supply of platelets and RBCs for the more than 10% of in-hospital patients receiving transfusions.

Both platelet-producing megakaryocytes (Mk) and RBCs can derive from the common bipotent progenitor called the Megakaryocytic-Erythroid Progenitor (MEP). Little is known regarding the mechanisms that control MEP fate specification, and a better understanding will inform in vitro derivation of platelets and RBCs.

Niche interactions, including communication between neighboring cells in the bone marrow, are key mediators of hematopoietic lineage commitment. Given the known role of bone marrow-derived macrophages (BMDM), bone marrow endothelial cells (BMEC) and mesenchymal stromal cells (MSC) in supporting hematopoietic stem cells, in conjunction with single cell RNAseq data from our lab showing MEP expression of cell surface proteins implicated in binding signals produced by these niche cells, I hypothesize that BMDM, BMEC and MSC regulate the fate of MEP. I propose to model the bone marrow niche in vitro and test the effects of BMDM, BMEC and MSC on MEP fate in collaboration with Dr. Beverly Torok-Storb. Her laboratory at the NIDDK-funded Fred Hutch Cooperative Center of Excellence in Hematology has successfully modeled a vascularized human bone marrow niche in vitro. With Dr. Torok-Storb’s support, I propose to establish an engineered, vascularized human bone marrow niche in vitro and grow MEP clonally to develop a model to elucidate the mechanism by which MEP self-renewal, or lineage commitment is regulated by BMDM, BMEC and MSC. Furthermore, I will explore the possibility of adapting this in vitro niche for live imaging to observe cell-cell interactions, as well as phenotypic/quantifiable features of MEP (cell division rate, motility, frequency of division types) that are influenced by these niche cells.

Successful completion of this project will reveal potential role(s) of macrophages, endothelial cells and marrow stromal cells in the bone marrow niche on bipotent MEP fate, which has implications for advancing our basic understanding of hematopoietic progenitor lineage commitment and deriving blood products to improve outcomes for patients receiving transfusions. Results of these pilot studies will be used as preliminary data in future grant proposals to the NIDDK and other relevant funding agencies.
 

Trowbridge Abstract

Hematopoietic stem cells (HSCs) are responsible for long-term maintenance and regeneration of the hematopoietic system. Loss of long-term (LT)-HSC function is a major contributor to decline in hematopoietic function with aging, leading to increased risk of infection, poor vaccination response, and increased susceptibility to hematologic malignancies. A number of LT-HSC-intrinsic alterations and LT-HSC-extrinsic changes in the bone marrow (BM) microenvironment have been associated with functional decline in aged LTHSCs, however, the initiating changes causing LT-HSC remain unclear. We took the novel approach of examining LT-HSC frequency and function in mice at a wide array of ages with the rationale that interventions to extend LT-HSC function will be most effective starting at or before the age of onset of functional hematopoietic decline. We found that canonical markers of LT-HSC aging significantly accumulate by middle age (9-12mo) in C57BL/6 mice, including increased phenotypic LT-HSC frequency, reduced regenerative capacity, myeloid lineage bias at both transcriptional and functional levels, increased gH2.AX staining, and loss of polarity of CDC42 and tubulin. Furthermore, we found by reciprocal transplantation studies of young LTHSCs into middle-aged recipient mice and middle-aged LT-HSCs into young recipient mice that LT-HSC extrinsic changes in the middle-aged BM microenvironment were necessary and sufficient to cause LT-HSC aging. By transcriptome analysis, we identified decreased IGF1 signaling in LT-HSCs as a candidate mechanism causing LT-HSC aging. We systematically identify mesenchymal stromal cells (MSCs) as the major local producer of IGF1 in the BM of young mice and determine that this production is diminished by middle age. To evaluate the specific effect of MSC-produced IGF1 on LT-HSCs, we co-cultured LT-HSCs with Igf1 conditional knockout MSCs, which was found to cause increased differentiation of LT-HSCs toward myeloid progenitor cells. In vivo, reduced IGF signaling in the BM microenvironment was modeled by transplantation of wild-type BM cells into Igf1 conditional knockout recipients, which phenocopied myeloid-biased hematopoiesis as observed in middle-aged mice. A similar myeloid-biased hematopoiesis phenotype was observed upon transplant of Igf1r conditional knockout LT-HSCs into wild-type recipients, supporting a model of direct communication between MSCs and LT-HSCs via IGF1 signaling. To determine whether restoration of IGF1 signaling had the capacity to rejuvenate middle-aged LT-HSCs, we applied short-term (18hr) in vitro treatment of recombinant IGF1. IGF1 treatment restored polarity of CDC42 and tubulin, decreased gH2.AX foci formation, and decreased myeloid-biased differentiation of middle-aged LT-HSCs both in vitro and in vivo. Transcriptional analysis identified an increase in mTOR signaling and decreased myeloid-biased LT-HSC and increased lymphoid-biased LT-HSC signatures. Other signatures upregulated upon IGF1 stimulation of middle-aged LT-HSCs included oxidative phosphorylation, cell cycle checkpoint and chromatin organization. We propose that restoration of local IGF1 signaling, or its downstream target pathways, represents an attractive prophylactic strategy for extending hematopoietic healthspan into older age. With the NIDDK-CCEH External Pilot Feasibility Grant, we propose to interrogate whether this same mechanism causes, and can be targeted to ameliorate, human HSC aging. This will provide critical preliminary data to enable translational and preclinical studies. We currently lack access to and direct funding for high-quality human samples to generate this preliminary data.
 

Yien Abstract

Antenatal anemia is a major public health issue, affecting approximately 40% of the world’s pregnant women, and is associated with adverse outcomes for the mother and child.  While the majority of these cases are attributable to nutritional iron deficiency and are currently treated by iron supplementation, the current regimen of iron supplementation itself has unpleasant side effects and decreases fractional iron uptake.  Further, iron supplementation is contraindicated in specific populations, such as patients who suffer form hemoglobinopathies.  The long-term goal of this project is to identify how pregnant mammals regulate erythropoiesis and iron metabolism to support the needs of the pregnant female, placenta and fetus for increased erythropoiesis and iron.  This project proposes to characterize erythroid development during the course of pregnancy.  Further, we propose to preparatively sort erythroid progenitors from pregnant females to enable analysis of gene expression changes in erythroid progenitor populations during pregnancy.  These studies will shed light on regulatory adaptations in transcriptional and metabolism networks that occur during pregnancy and will enable detailed, mechanistic studies that will facilitate the development of targeted therapies for pregnant women, who remain an underserved population.