174 - Anemia-Associated Endotoxemia Promotes Inflammatory Megakaryocytes and Reactive Platelet Formation in the Murine Liver

Publication Number: 2168.174

Arjun Surya Subrramanya, University of Texas Southwestern medical center, Dallas, TX, United States; Juanitaa George Raj, University of Texas Southwestern Medical Center, Dallas, TX, United States; Balamurugan Ramatchandirin, University of Texas Southwestern Medical School, Dallas, TX, United States; Marie Amalie Balamurugan, University if Texas Southwestern Medical Center, Dallas, TX, United States; Samantha Fine, University of Texas Southwestern Medical School, Dallas, TX, United States; Vishal Kapadia, University of Texas Southwestern Medical School, Dallas, TX, United States; Lisa M.. Scheid, University of Texas Southwestern Medical School, DALLAS, TX, United States; MohanKumar Krishnan, University of Texas Southwestern Medical School, Dallas, TX, United States

Background: Anemia is nearly universal in preterm infants and is associated with increased morbidity and mortality worldwide. We recently reported that anemia induces liver hematopoiesis, including myelopoiesis, stressed erythropoiesis with suppressed lymphopoiesis through tissue hypoxia and ‘leaky gut’-associated endotoxemia

Objective: To investigate the role of anemia-associated endotoxemia on liver immune megakaryocyte (MK) and platelet formation

Design/Methods: C57BL/6 mouse pups were randomly divided into two groups (n=6 per group): (1) naïve controls and (2) phlebotomy-induced anemia (PIA) on postnatal days P2 – P10 to maintain a hematocrit 20-24%. On P11, liver, bone marrow, and spleen tissues were collected, and flow cytometry was performed using megakaryocyte and its progenitor cell markers. Inflammatory response in megakaryocytes was analyzed to assess the endotoxin receptors and cytokine expression. To assess changes in ploidy, megakaryocytes were gated, and ploidy levels were analyzed using DNA stains. Platelet indices (P-LCR, MPV, and IPF) were monitored from P2 to P10. In a separate cohort, we used transgenic TLR4-/- mouse pups to test their impact on megakaryocyte’s inflammation and platelet release

Results: Flow analysis of anemic livers (P11) showed altered hematopoiesis with an increased megakaryocyte-erythroid progenitors (P < 0.001) and common myeloid progenitors (P < 0.033) than bone marrow and spleen compared to respective control organs. In anemic livers, we also found that the LSK population exhibited an increase in multipotent progenitors (P < 0.001) and decreased long-term hematopoietic stem cells (P < 0.002). Liver-derived megakaryocytes from anemic pups showed enhanced expression of endotoxin receptors (CD14, MD2, LBP, and specifically TLR4), pro-inflammatory cytokines and increased ploidy, which stimulated the production of reactive platelets (CD41+CD62P+) compared to bone marrow derived MKs. Interestingly, in anemic-TLR4-/- mice, the liver-derived megakaryocytes displayed reduced the expression of endotoxin receptors and as well as inflammatory response with decreased ploidy like bone marrow-derived cells. The release of reactive platelets (CD41+CD62P+) was drastically reduced in anemic-TLR4-/- mice, indicating TLR4-dependent mechanisms in anemia-associated MK inflammation and reactive platelet production thus increases thrombosis risk.

Conclusion(s): Phlebotomy-induced anemia in neonatal mice triggers hepatic TLR4-mediated inflammatory megakaryopoiesis and reactive platelet production driven by endotoxemia, providing a potential mechanistic link between neonatal anemia and thrombosis risk.