Infection with Anaplasma phagocytophilum Inhibits Proliferation and Differentiation of Myeloid Progenitors: New Insight into Infection-Related Pancytopenia.
Authors: Gaines Peter , Thomas Venetta , Fikrig Erol , Berliner Nancy 
Published: April 2020

Abstract
Human granulocytic anaplasmosis (HGA) is a potentially fatal tick-borne disease caused by the obligate intracellular bacterium Anaplasma phagocytophilum, which has a tropism for neutrophils. Clinical symptoms of HGA include fever, headache, myalgia, neutropenia and thrombocytopenia. Our group and others have identified several mechanisms utilized by A. phagocytophilum to establish a safe environment within the normally hostile mature neutrophil to facilitate its survival and propagation, including inhibition of the phagocyte respiratory burst, inhibition of apoptosis, and induction of cytokine secretion to recruit fresh neutrophils. However, the occurrence of thrombocytopenia as the most prominent hematologic abnormality in infected individuals suggests that neutrophils are not the only reservoir of infection. We postulated that A. phagocytophilum infects bone marrow progenitors and inhibits their proliferation and maturation to cause more global marrow suppression and induce pancytopenia. To test this hypothesis, we infected several hematopoietic cell models and determined whether infection inhibits the proliferation and differentiation of myeloid progenitors at several distinct stages of development. Using three distinct in vitro myeloid models, we have shows that A. phagocytophilum can efficiently infect promyelocytes and late myeloblasts, and that infection inhibits their responses to both proliferative and differentiation-inducing signals. The first model is the EPRO cell line, which shows dramatic decreases in colony formation upon GM-CSF-induced growth when infected with A. phagocytophilum. For our second model we used a myeloblast cell line generated by transducing bone marrow progenitors with an empty pBabepuro vector with subsequent selection in puromycin and growth in stem cell factor (SCF). These cells rapidly undergo neutrophil differentiation upon induction with G-CSF in the absence of SCF. Infection with A. phagocytophilum causes a pronounced decrease in G-CSF induced differentiation, and inhibits the initial stages of proliferation prior to differentiation. Using a third cell line model, we show that A. phagocytophilum can also infect the EML multipotent progenitor cell line. Furthermore, we demonstrate that A. phagocytophilum infection causes decreased proliferation in response to SCF. Parallel studies in primary progenitor cells are ongoing. Together these data suggest that A. phagocytophilum can infect progenitors at multiple stages of blood cell development, and that infection inhibits both the proliferation of myeloid progenitors and their ability to differentiate towards mature neutrophils. Whether infection also inhibits thrombopoiesis is currently under investigation. Our studies provide a new paradigm of susceptibility to A. phagocytophilum, where both peripheral blood neutrophils and bone marrow cells become infected upon exposure to the bacterium. These data also suggest that the pancytopenia observed in A. phagocytophilum-infected patients may be due to inhibitory effects on the growth and development of bone marrow progenitors.