025 - Targeting TREM1 Attenuates Anemia-Associated Microglial Activation and Neuroinflammation in the Neonatal Mouse Brain

Publication Number: 25.6953

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

Background: Anemia is a nearly universal diagnosis in preterm infants, caused primarily by phlebotomy essential for medical
care, though anemia may contribute to neuroinflammation. Microglial cells, the brain’s primary immune cells, may respond to systemic stress like anemia and cause. The triggered receptor expressed on myeloid cells-1 (TREM1) amplifies immune responses, but its role in anemia-induced microglial activation and neuroinflammation is not well understood, highlighting its potential as a therapeutic target.

Objective: To investigate the role of trem1 in anemia-induced neuroinflammation and assess the therapeutic potential of trem1 inhibition in anemia-associated inflammatory responses in the neonatal murine brain.

Design/Methods: C57BL/6 mouse pups were studied in 2 groups (n=6 each): (1) naïve controls; and (2) anemic (hematocrit 20 24%). Severe anemia was induced by facial vein phlebotomy on postnatal day (P) 2, 4, 6, 8, and 10. After 24 hours, whole brain tissue was subjected to qRT-PCR and Luminex Multiplex Array for quantifying inflammatory cytokine expression. Flow cytometry was performed to identify the microglial activation with trem1 expression. The murine BV2 microglial cells were treated with lipopolysaccharide (LPS) to explore the mechanistic role of trem1 and evaluate the potential benefits of LQVTDSGLYRCVIYHPP (LP17) peptide (10µM for 2hrs) treatment by in vitro. The LP17 was also intraperitoneally administered to experimental mouse pups during each phlebotomy from P6 onwards and the inflammatory response, microglial activation, and trem1 expression were measured by using the above methods.

Results: Severe anemia contributes to hyper-inflammatory activation in the brain, evidenced by significantly elevated mRNA levels of IL1β, IFN-γ, and IL6, (***p≤ 0.001) and their protein expression (pg/50µg of brain tissue) by mouse 32-plex Luminex arrays (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001). Microglial cells in the anemic brain exhibited heightened immunoreactivity to P2RY12, indicating strong microglial activation with higher trem1 expression than control resting microglia. The in vitro study showed that LPS (500ng/ml for 4hrs) treatment increased trem1 expression, and inflammatory response, whereas LP17 treatment significantly reduced trem1 expression and microglial activation thus reducing inflammatory response. The in vivo treatment with LP17 alleviates anemia-associated inflammatory responses by inhibiting trem1-associated microglial activation.

Conclusion(s): Inhibiting trem1 effectively reduces anemia-associated microglial activation and its inflammatory response in neonatal murine brains.