GSDMD Gene Knockout Prevents Hyperoxia-induced Lung and Retina Injury in Neonatal Mice

Background: Hyperoxia plays a key role in the pathogenesis of both bronchopulmonary dysplasia (BPD) and retinopathy of prematurity (ROP). Hyperoxia stimulates the inflammasome pathway which leads to activation of gasdermin D (GSDMD), a key executor of inflammatory cell death, also known as pyroptosis. Our research has shown that inhibition of GSDMD activation attenuates hyperoxia-induced lung injury in neonatal mice and recently obtained novel preliminary data shows that GSDMD is also activated in the retinas of neonatal mice. This suggests a critical role of GSDMD in the pathogenesis of BPD and ROP, however, the underlying mechanisms are unclear. Our objective was to determine whether GSDMD gene knockout (KO) affects hyperoxia-induced lung and retina injury in neonatal mice.

Methods: We utilized GSDMD KO mice and both hyperoxia-induced neonatal lung injury and ROP models. For the lung injury model, newborn GSDMD KO mice and their wildtype (WT) littermates were randomized to room air (RA, 21% O2) or hyperoxia (85% O2) from postnatal day (P) 1 to P14. Alveolarization was determined by mean linear intercept (MLI). Vascular development and vascular remodeling were assessed by vascular density (VD) and muscularized vessels on vWF and -SMA double immunofluorescence-staining. Lung inflammation was assessed by macrophage staining. Ki67 immunofluorescence and DAPI nuclear staining was used to determine cell proliferation index and total cell count. TUNEL assay was performed to quantify cell apoptotic index. For the retinopathy model, newborn GSDMD KO mice and their WT littermates were randomized to RA or hyperoxia (75% O2) from P7 to P12, then RA until P17. Vascularization was determined using isolectin immunostaining and quantification of avascular and neovascular area. Retinal thickness was determined by H&E staining and quantification of tissue thickness. The data are presented as Mean±SD and analyzed by ANOVA. A P-value of <0.05 was considered statistically significant.

Results: Hyperoxia-exposed WT mice (WT+O2) had decreased alveolarization, reduced vascular density and increased vascular remodeling, increased macrophage infiltration, decreased cell proliferation and total cell counts as well as increased apoptotic cells compared to RA-exposed WT mice (WT+RA). While RA-exposed KO mice (KO+RA) behaved similarly to WT+RA mice, hyperoxia-exposed KO mice (KO+O2) had improved alveolarization, vascular development and vascular remodeling, increased cell proliferation and total cell count as well as reduced apoptotic cells compared to WT+O2 mice. WT+O2 mice had increased vasoobliteration and neovascularization, and thinning of some retinal layers. Conversely, KO+O2 mice had improved retinal vascularization and decreased retinal thinning compared to WT+O2 mice.

Conclusion: GSDMD gene KO prevents hyperoxia-induced lung and retina injury in neonatal mouse models. This suggests that GSDMD plays a pathogenic role in both BPD and ROP and that targeting GSDMD may be beneficial in the prevention and treatment of BPD and ROP in preterm infants.