681 - Severe Anemia in Murine Neonates Alters Liver Hematopoiesis with Inflammatory Megakaryocytes

Publication Number: 681.6946

Arjun S. Subrramanya, University of Nebraska Medical Center, Omaha, NE, United States; Juanitaa George Raj, University of Nebraska College of Medicine, Omaha, NE, United States; Balamurugan Ramatchandirin, University of Nebraska Medical Center, Omaha, NE, United States; Marie Amalie Balamurugan, University of Nebraska Medical Center, Omaha, NE, United States; Pranavi Prakash, University of Nebraska Medical Center, Omaha, NE, United States; Zainab D. Lawal, University of Nebraska College of Medicine, OMAHA, NE, United States; Megan M. Ferris, University of Nebraska College of Medicine, Omaha, NE, United States; Mohan Krishnan, University of Nebraska Medical Center, Omaha, NE, United States

Background: Severe anemia is common among preterm infants and frequently leads to heightened gut permeability, which permits endotoxins (LPS) to enter the bloodstream, sensitizing immune cells and causing systemic inflammation. Nevertheless, the comprehensive effects of anemia-associated endotoxin on hematopoiesis, particularly its influence on megakaryopoiesis, and the production of reactive platelets, have not been thoroughly investigated.

Objective: To study the effect of severe anemia-induced endotoxin release on liver hematopoiesis, megakaryocyte inflammation, and reactive platelet release.

Design/Methods: C57BL/6 mouse pups were randomly divided into two groups (n=6 per group): naïve controls and severely anemic pups, with anemia induced by facial vein phlebotomy on postnatal days P2 – P10 to maintain a hematocrit < 20%. On P11, liver, bone marrow (BM), and spleen tissues were collected for single-cell suspensions, and flow cytometry was performed using hematopoietic and progenitor cell markers. Inflammatory markers in megakaryocytes were analyzed to assess LPS receptor and cytokine expression. To assess changes in ploidy, megakaryocytes were gated, and ploidy levels were analyzed using DNA stain. Platelet indices (P-LCR, MPV, and IPF) were monitored from P2 to P10. In a separate cohort, an intestinal permeability blocker, let-7e-5p inhibitor was administered i.p during each phlebotomy from P6 onwards and evaluate its impact on platelet indices.

Results: Flow cytometry analysis revealed altered hematopoiesis in the liver than other hematopoietic organs such as BM and spleen.  A notable decrease in lineage-negative cells in anemic pups, accompanied by an increase in megakaryocyte-erythroid progenitors (**p < 0.002) and a reduction in granulocyte-monocyte progenitors ((**p < 0.002) and common myeloid progenitors (*p < 0.033).  Megakaryocytes showed enhanced expression of LPS receptors (TLR2; ***p < 0.001 and TLR9; **p < 0.002), pro-inflammatory cytokines (IL-6; **p < 0.002, IL-1β; ***p < 0.001, TNF-∝; **p < 0.002, IL-12a; **p < 0.002, and IFN-Ɣ; ***p < 0.001), and increased ploidy, which stimulated the production of reactive platelets. By day 8, platelet indices had significantly risen and when treated with let-7e-5p inhibitor the platelet indices returned to normal levels.

Conclusion(s): Severe anemia-induced endotoxin release causes altered liver hematopoiesis with higher ploidy inflamed megakaryocytes, resulting in the production of reactive platelets, and these MK- and platelet-associated responses were reduced on let-7e-5p treatment.