284 - Interleukin-33/ST2 Axis in Neonatal Hypoxic-Ischemic Encephalopathy: Clinical Findings and Early Experimental Approach toward Mechanistic Elucidation

Publication Number: 4280.284

Hiroki Hamano, Department of Pediatrics, Ube, Yamaguchi, Japan; Sasagu Kimura, Yamaguchi University, Ube, Yamaguchi, Japan; Yoshiko Nawata, Departnent of Pediatrics, Yamaguchi University Hospital, Ube, Yamaguchi, Japan; Hidenobu Kaneyasu, Yamaguchi University Hospital, Ube, Yamaguchi, Japan; Seigo Okada, Yamaguchi University, Ube, Yamaguchi, Japan; Kazumasa Takahashi, Yamaguchi University Graduate School of Medicine, Ube, Yamaguchi, Japan; Shunji Hasegawa, Yamaguchi University, Ube, Yamaguchi, Japan

Background: The interleukin-33 (IL-33)/ST2 axis regulates inflammatory and immune responses and plays a neuroprotective role in the central nervous system. In peripheral tissues, IL-33 acts as a pro-inflammatory cytokine released upon injury, whereas in the brain it may promote neuroprotection. In our previous study, serum soluble ST2 (sST2)-the decoy receptor of IL-33-was significantly elevated in infants with moderate-to-severe hypoxic-ischemic encephalopathy (HIE) compared with controls. Higher sST2 levels correlated with disease severity, magnetic resonance spectroscopy-derived lactate/N-acetylaspartate ratio, and adverse neurological outcomes. IL-33 was detected within 6 h after birth but declined below assay limits by day 2, possibly reflecting rapid degradation rather than true absence. We hypothesize that IL-33 exerts early neuroprotective effects, whereas sST2 counteracts them by sequestering IL-33. To explore this mechanism, we developed an in vitro brain-slice model of hypoxic-ischemic injury.

Objective: To establish an organotypic brain-slice in vitro HIE model under oxygen-glucose deprivation (OGD) to examine IL-33/ST2-mediated neuroprotection and injury response.

Design/Methods: Coronal brain slices from postnatal days 5-7 mice were cultured on semiporous membrane inserts at the air-medium interface using a neuron-specific medium. This system preserves the complex neuronal cytoarchitecture and cell interactions of the brain while allowing in vitro manipulation. Paraformaldehyde- or ethanol-fixed slices served as controls for propidium iodide (PI)-based cell-death validation. Cell death was assessed by PI staining under control and OGD conditions. The model is being optimized for reproducible injury quantification suitable for downstream cytokine assays.

Results: Clinically, sST2 remained persistently elevated in HIE and correlated with poor outcomes, while IL-33 levels declined rapidly after birth. Experimentally, slice cultures were maintained successfully, and preliminary PI staining showed fluorescence patterns consistent with previous reports. Quantitative analysis of OGD-induced injury and control validation are ongoing.

Conclusion(s): Our findings suggest that sST2 may attenuate IL-33-mediated neuroprotection in neonatal HIE. Although in vitro validation is in progress, this brain-slice OGD model provides a foundation for mechanistic exploration of the IL-33/ST2 axis and may ultimately guide therapeutic strategies targeting this pathway after hypoxic-ischemic injury.

Clinical changes in serum sST2 levels in control infants and those with mild-to-severe hypoxic-ischemic encephalopathy (HIE) at different time points.
sST2 levels were significantly higher and more persistent in moderate-to-severe HIE compared with controls. Comparisons were performed using the Kruskal-Wallis test followed by Dunn's post hoc test. *p < 0.05.

Proposed mechanism of the IL-33/ST2 axis in neonatal hypoxic-ischemic encephalopathy.
IL-33 released from damaged cells may promote neuroprotection through membrane-bound ST2L signaling, whereas soluble ST2 (sST2) acts as a decoy receptor that binds and neutralizes IL-33. This imbalance between IL-33 and sST2 may contribute to impaired neuroprotective signaling following hypoxic-ischemic injury.