Oxysterol-Mediated Modulation of NFï•«B -Linked Systemic Inflammatory Response in a Neonatal Mouse Model of Intestinal Perforation 2259826

Abstract

Neonatal intestinal perforations are a major complication of preterm birth, strongly associated with mortality and neurodevelopmental impairment (NDI). Our prior work links intestinal perforation to NFκB-mediated inflammatory injury in the subventricular zone (SVZ), a neural stem cell niche critical for brain development. We hypothesize that the choroid plexus (ChP) mediates SVZ injury through NFκB-activated monocyte/macrophage trafficking after modeled intestinal perforation (MIP), and that modulation of this inflammatory cascade by 20α-hydroxycholesterol (20HC), a breast milk—derived oxysterol with anti-inflammatory activity, protects the SVZ. Using single-nucleus RNA sequencing (snRNA-seq) and spatial transcriptomics, we observed peripheral monocyte/macrophage transit through the ChP and colonization of the SVZ after MIP, resulting in cytokine exposure, ependymal activation, astroglial scarring, and reduced progenitor production. For transcriptomic analyses, ChP samples were obtained from control, MIP-induced, and 20HC+MIP-treated mice. Neonatal mice received daily subcutaneous 20HC (100 mg/kg/day in MCT oil) from postnatal day 3 (P3); MIP was induced on P5, and ChP harvested 24 h later for scRNA-seq to characterize immune activation. Complementary in vitro studies used 20HC-pretreated macrophage and microglia stimulated with lipopolysaccharide (LPS) to assess NFκB-linked cytokine expression by qPCR. MIP induction in P5 mice caused bacterial seeding in brain, lungs, and spleen within 48 hours and elevated NFκB-linked cytokines (TNFα, IL-1β, IL-6) across organs. 20HC treatment significantly reduced cytokine expression, preserved SVZ integrity, and decreased inflammatory pathway enrichment in ChP macrophage clusters. In vitro, 20HC suppressed IL-1β and IL-6 via NFκB inhibition. These findings identify the ChP as a mediator of inflammation-induced SVZ injury and highlight 20HC as a promising therapy to protect the developing brain after neonatal intestinal perforation. Pathogenic Mechanisms of Inflammatory Subventricular Zone Injury in Preterm Infants-National Institutes of Health (5R01-NS140419-02) Neuroimmunology (NEUR)