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BackgroundThe extension of sepsis encompassing the preterm newborn’s brain is often overlooked due to technical challenges in this highly vulnerable population, yet it leads to substantial long-term neurodevelopmental disabilities. In this study, we demonstrate how neonatal neuroinflammation following postnatal E. coli lipopolysaccharide (LPS) exposure in rat pups results in persistent reduction in sialylation of cerebral glycoproteins.MethodsMale Sprague-Dawley rat pups at postnatal day 3 (P3) were injected in the corpus callosum with saline or LPS. Twenty-four hours (P4) or 21 days (P24) following injection, brains were extracted and analyzed for neuraminidase activity and expression as well as for sialylation of cerebral glycoproteins and glycolipids.ResultsAt both P4 and P24, we detected a significant increase of the acidic neuraminidase activity in LPS-exposed rats. It correlated with significantly increased neuraminidase 1 (Neu1) mRNA in LPS-treated brains at P4 and with neuraminidases 1 and 4 at P24 suggesting that these enzymes were responsible for the rise of neuraminidase activity. At both P4 and P24, sialylation of N-glycans on brain glycoproteins decreased according to both mass-spectrometry analysis and lectin blotting, but the ganglioside composition remained intact. Finally, at P24, analysis of brain tissues by immunohistochemistry showed that neurons in the upper layers (II–III) of somatosensory cortex had a reduced surface content of polysialic acid.ConclusionsTogether, our data demonstrate that neonatal LPS exposure results in specific and sustained induction of Neu1 and Neu4, causing long-lasting negative changes in sialylation of glycoproteins on brain cells. Considering the important roles played by sialoglycoproteins in CNS function, we speculate that observed re-programming of the brain sialome constitutes an important part of pathophysiological consequences in perinatal infectious exposure.

This is an open access article under the terms of the Creat ive Commo ns Attri butio n-NonCo mmercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes. contributed equally to this study.Abbreviations: ASD, autism spectrum disorder; CpG, cytosine followed by guanine; DMR, differentially methylated region; DOHaD, developmental origins of health and disease; E. coli, Escherichia coli; GO, gene ontology; LPS, lipopolysaccharide; P3, postnatal day 3; P4, postnatal day 4; P24, postnatal day 24; PBS, phosphate-buffered saline; PFA, paraformaldehyde; RRBS, reduced representation bisulfite sequencing; WMI, white matter injury. AbstractPreterm infants are vulnerable to inflammation-induced white matter injury (WMI), which is associated with neurocognitive impairment and increased risk of neuropsychiatric diseases in adulthood. Epigenetic mechanisms, particularly DNA methylation, play a role in normal development and modulate the response to pathological challenges. Our aims were to determine how WMI triggered DNA methylation alterations in brains of neonatal rats and if such changes persisted over time. We used a robust model of WMI by injecting lipopolysaccharide (LPS) or sterile saline in the corpus callosum of 3-day-old (P3) rat pups. Brains were collected 24 hours (P4) and 21 days post-injection (P24). We extracted genomic DNA from the brain to establish genome-wide quantitative DNA methylation profiles using reduced representation bisulfite sequencing. Neonatal LPS exposure induced a persistent increased methylation of genes related to nervous system development and a reduced methylation of genes associated with inflammatory pathways. These findings suggest that early-life neuroinflammatory exposure impacts the cerebral methylation landscape with determining widespread epigenetic modifications especially in genes related to neurodevelopment. K E Y W O R D SDNA methylation, epigenetics, lipopolysaccharide, periventricular leukomalacia

Targeting b-adrenergic receptor signaling with propranolol has emerged as a potential candidate to counteract choroidal neovascularization (CNV). Little is known of its effect on macrophages, which play a critical role in CNV. We investigated the effect of propranolol on angiogenic response of mononuclear phagocytes (MPs). METHODS. The angiogenic effect of propranolol was evaluated in laser-induced CNV model. Mice received intraperitoneal injections of propranolol (6 mg/kg/d) or vehicle. CNV area and inflammatory cells were determined respectively by using lectin staining and an anti-IBA-1 antibody on RPE/choroid flat mounts. Inflammatory gene expression was evaluated by quantitative (q) PCR analysis. Mechanisms of propranolol was studied in MP cell lines J774 and RAW264.7 and in primary peritoneal macrophages. Expression of pro-and antiangiogenic mediators was studied. In addition, effects of propranolol treatment of MPs was assessed on choroidal explant. RESULTS. CNV was attenuated by propranolol and concomitantly associated with decreased inflammatory mediators IL-6 and TNFa, albeit with accumulation of (b-adrenoceptor harboring) MPs in the CNV area. Conditioned media from MPs preincubated with propranolol exerted antiangiogenic effects. Treatment of J774 confirmed the attenuation of inflammatory response to propranolol and increased cleaved caspase-3 on choroidal explant. We found that propranolol increased pigment epithelium-derived factor (PEDF) expression in MPs. Trapping of PEDF with an antibody abrogated antiangiogenic effects of propranolol. PEDF was also detected in CNV-associated MPs. CONCLUSIONS. We hereby show that propranolol confers on MPs antiangiogenic properties by increasing PEDF expression, which complements its effects on vascular tissue resulting in inhibition of choroidal vasoproliferation in inflammatory conditions. The study supports possible use of propranolol as a therapeutic modality for CNV.