406 - Systemic Inflammation in Neonatal Mice Downregulates Microglial Inflammatory Gene Expression in Response to LPS Stimulation

Poster Number: 406
Publication Number: 406.236

Garima Singh, University of Minnesota Medical School, Minneapolis, MN, United States; Wen Sheng, University of Minnesota, Minneapolis, MN, United States; Shuxian hu, University of Minnesota Medical School, Minneapolis, MN, United States; James R. Lokensgard, University of Minnesota, Minneapolis, MN, United States; Tate A. Gisslen, University of Minnesota, Edina, MN, United States

Background: Preterm infants commonly experience multiple systemic inflammatory illnesses such as early-onset sepsis, late-onset sepsis, or necrotizing enterocolitis. Long-term neurodevelopmental outcomes for infants that experience postnatal inflammatory events are worse than for infants that do not. In preclinical models, neonatal sepsis results in activation of microglia, the tissues specific macrophage of the brain. Microglial activation likely contributes to poor neurodevelopmental outcomes, but the mechanisms involved are not well understood.

Objective: To determine how microglia respond to ex vivo LPS stimulation when previously activated by in vivo systemic inflammatory challenge.

Design/Methods: Neonatal mice were injected i.p. with LPS (0.1 mg/kg in 30 uL) to model sepsis or equivalent volume of saline at postnatal day (P) 5. At postnatal day (P) 7, whole brains were digested to obtain single cell preparations. Microglia were obtained by positive selection using CD11b beads and magnetic separating column and then 500,000 cells were cultured per well of a 24-well plate. Cells were stimulated with LPS (100 ng/uL) for 4 or 20 hours to assess microglial gene responses. Cells were then collected and lysed to prepare RNA and cDNA. Gene expression of microglial inflammatory markers was determined by PCR (n=3-4/group) assayed in duplicate and normalized against the reference gene S18.

Results: Peak expression of most microglial inflammatory genes occurred 4 hours post-LPS stimulation. Microglial gene expressions of CCL2, CXCL10, CD86, and NFkB1 at 4 hours post-LPS stimulation were downregulated after i.p. LPS compared to i.p. saline controls. Gene expression of IL-6 at 4 hours post LPS stimulation demonstrated a trend downregulation. MHCII expression peaked 20 hours post-LPS stimulation and was downregulated by i.p. LPS compared to controls. Genes associated with anti-inflammatory responses, IL-10 and Arg1, had a peak expression 20 hours post-LPS stimulation and showed a trend upregulation after i.p. LPS.Conclusion(s): Systemic neonatal inflammation altered the function of microglia such that they had a diminished inflammatory gene expression response to LPS stimulation. This phenotypic change of microglia may indicate their impaired developmental function in the developing brain that contributes to poor neurodevelopmental outcomes.