Overview of Session 3 Mechanisms of Decreased Erythropoiesis and Erythroid Cell Injury

Abstract

This session, held during the 41st Annual STP Symposium, focused on mechanisms of decreased erythropoiesis and erythroid cell injury. The speakers provided comprehensive overviews of physiologic and pathologic erythropoiesis, reviewed various mechanisms of erythroid cell injury, and shared innovative investigative research with the audience.

The 41st Annual STP Symposium, titled “Toxicologic Pathology of the Hematopoietic System,” was held in Austin, Texas, June 19-23, 2022, at the Marriott Downtown Hotel. Session 3 occurred on Tuesday, June 21, 2022; was planned and facilitated by Drs AE Schultze and L Ramaiah; and focused on “Mechanisms of Decreased Erythropoiesis and Erythroid Cell Injury.” Four subject matter experts, including a physician scientist, a toxicologist, and two veterinary clinical pathologists, provided comprehensive overviews of physiologic and pathologic erythropoiesis, reviewed various mechanisms of erythroid cell injury, and shared innovative investigative research with the audience. The speakers were selected to give oral presentations based upon their publication records and unique training and research interests in hematology.

The first speaker was Leif Ludwig (MD, PhD). Dr Ludwig is an Emmy Noether Research Group Leader at the Berlin Institute of Health (BIH) at Charité Universitätsmedizin Berlin, Germany. His presentation was titled “Mechanisms of Physiologic and Pathologic Erythropoiesis and Functional Studies of GATA1.”

Dr Ludwig reviewed in detail the process of human hematopoiesis with emphasis on erythropoiesis. He described the value of incorporating human genetics and genomic approaches as research tools in hematology. He provided an overview about insights that can be gained from epigenomic and transcriptional time course data across the process of human erythropoiesis, including the role of several regulatory genes for human red blood cell production including GATA1 (also known as GATA-binding factor or erythroid transcription factor), and he described rare variants of GATA1 that disrupt erythropoiesis. Much information was provided that correlated stage-specific natural variation of erythropoiesis cell cycle progression (due to genetic modulation of cyclin D3 expression) with resulting variation in erythrocyte numbers and red blood cell size.

Several GATA1 mutations are associated with anemia in people. Diamond-Blackfan anemia, a hypoplastic anemia in children, is characterized by macrocytic anemia, reticulocytopenia, and relatively normal bone marrow cellularity with decreased numbers of erythroid precursors. Dr Ludwig used Diamond-Blackfan anemia to illustrate how specific defects caused by mutations in ribosomal proteins such as RPS19 may cause alterations in translation of GATA mRNA that then result in impaired erythropoiesis. In contrast, R307R/C mutations in GATA1 may affect distinct functions of GATA1 and result in hemolytic anemia in people that is characterized by decreased red blood cell mass, reticulocytosis, normal osmotic fragility, and erythroid hyperplasia without dysplasia.

Deidre Dalmas, PhD, presented the “Effects of p38 MAP Kinase Inhibitors on the Differentiation and Maturation of Erythroid Progenitors.” Dr Dalmas is a Director of US Global Investigative Safety and a Fellow at GlaxoSmithKline with more than 25 years of experience. She reviewed the process of erythropoiesis; Mitogen Activated Protein (MAP) Kinase-induced hematosuppression in mice, rats, and nonhuman primates (NHPs); and use of unique endpoints and tools to evaluate the effects of drugs that inhibit erythropoiesis.

MAP Kinases are central elements in cellular signal transduction. They are large and highly conserved protein kinases that are associated with early embryonic development, regulation of apoptosis and cellular proliferation, cytokine production in the immune response, and regulation of secretion, depolarization, and certain metabolic responses. In health, p38 MAP Kinases are involved in erythroid proliferation and differentiation.

Inhibition of p38 MAP Kinase results in transiently decreased reticulocyte counts, decreased red blood cell mass, and bone marrow hypocellularity in the mouse, rat, and NHP. These effects may be detected within 24 hours of administration. Dr Dalmas reviewed several cases of p38 MAP Kinase hematosuppression in laboratory animals and provided information on numerous in vitro tests that can be used to monitor decreased cellular production including colony forming unit (CFU) assays and liquid culture systems. Using a novel liquid culture system, Dr Dalmas and colleagues investigated the effects of MAP Kinases on erythroid (burst-forming unit-erythroid [BFU-E]) generation, erythroid lineage-specific gene expression, protein expression using flow cytometric analysis, and erythroid cell morphology at multiple timepoints throughout the experiment.

The third presentation was given by Erica L. Behling-Kelly, DVM, PhD, Diplomate ACVP, who is an Associate Professor in the Department of Population Medicine and Diagnostic Sciences in the College of Veterinary Medicine at Cornell University and is the Director of the Clinical Pathology Laboratory. Dr Behling-Kelly presented remotely and spoke on “Erythroid Injury: Mechanisms, Detection, and Adversity.” She emphasized two broad mechanisms of erythroid cell injury: hemolysis and decreased production of erythroid cells.

Cases of hemolysis were subdivided into intravascular and extravascular causes. Characteristic morphologic alterations in erythrocytes were detailed, and case examples of naturally occurring and xenobiotic-induced hemolysis were provided. Hematologic and biochemical methods to detect and monitor hemolytic events were reviewed. Light microscopy, electron microscopy, atomic force microscopy, and flow cytometric evaluation were discussed. Challenges of using newer instruments incorporating digital morphometry such as the CellaVision were demonstrated. Infrequently used techniques to evaluate erythrocyte membrane integrity such as osmotic fragility were presented.

Mechanisms of decreased erythrocyte production including dyserythropoiesis and erythropoietic arrest were reviewed. Naturally occurring and mouse models with receptor defects were used to highlight different mechanisms for decreased erythrocyte production. The importance of a complete evaluation of erythroid, leukocyte, and megakaryocyte precursors; bone marrow stroma; and iron stores was emphasized.

Assessing adversity in cases of hemolysis and decreased erythrocyte production can be challenging. Concurrent evaluation of the anemia, indicators of bone marrow regeneration, extramedullary hematopoiesis, vascular inflammation, and alterations in tests of hemostasis and thrombosis were discussed as critical considerations in the final assessment.

The last presentation of the session was delivered by Michelle Cora, DVM, Diplomate ACVP. Dr Cora is currently a Staff Clinical Pathologist at Antech Diagnostics. Previously, she was a Veterinary Medical Officer/Principal Clinical Pathologist at the Cellular and Molecular Pathology Branch of the Division of National Toxicology Program (NTP) and National Institute of Environmental Health Sciences (NIEHS) in Research Triangle Park, NC. The work she presented, titled “Black Cohosh Herbal Extract and Hematologic Alterations in B6C3F1/N Mice,” was completed while employed at NTP.

Black Cohosh (Actaea racemose) is a perennial plant found in many woodlands in North America. The roots and rhizomes of this plant have been used by herbalists as treatments for painful menstruation and labor, various neuralgias, and rheumatism. Extracts of this plant are marketed commercially as Remifemin and Estroven for treatment of menopausal symptoms. Black Cohosh was studied at NTP to investigate the potential for reproductive and developmental toxicities in humans.

In a subchronic toxicity study, B6C3F1 weanling female mice were administered Black Cohosh (0, 62.5, 125, 250, 500, or 1000 mg/kg) by oral gavage daily for 90 days. Mice developed mild to moderate decreases in red blood cell mass that were accompanied by erythrocyte morphologic changes, basophilic stippling, and increased red blood cell micronucleus (Howell-Jolly bodies) counts. The findings were consistent with megaloblastic anemia—a dyshematopoietic condition that results from deranged DNA synthesis and affects all hematopoietic cell lines.

Dr Cora discussed causes for megaloblastic anemias and provided overviews of folate and cobalamin metabolism. A follow-up, mechanistic study in female mice given 1000 mg/kg Black Cohosh by oral gavage daily for 90 days confirmed characteristic changes in red blood cell mass and morphology and increased concentrations of homocysteine and methylmalonic acid that were consistent with megaloblastic anemia. Additional assays used in the diagnosis of megaloblastic anemias such as bone marrow cytology and mouse liver gene microarray were presented and discussed in detail. Cytologic examination of erythroid progenitor cells in the bone marrow identified dysplastic features such as multilobulated nuclei, micronuclei, nuclear to cytoplasmic asynchrony, gigantism, and nuclear fragmentation. The gene microarray results revealed that genes associated with drug metabolizing enzymes were upregulated. The pathways related to hypoxia/decreased erythron as it relates to hypoxia-inducible factor 1-alpha (HIF-1α) were highly affected.

All speakers in this session were invited to submit articles that described their research interests. More details on the topics listed above may be found in the articles that follow.