P&F Projects

The abstracts for current P&F projects are listed below. This page will be updated as more information becomes available for each project.

Vinchi Abstract Collaborative P&F 2020

ABSTRACT: 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

ABSTRACT: 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 in situ 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

ABSTRACT: 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 Steap1, Steap2 or Steap4 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. We hypothesize that Lcytb and Steap3 are the reductases necessary for iron recycling in macrophages. 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 will utilize 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.