PHLDA3 deficiency attenuates neonatal sepsis-associated acute lung injury and is associated with reduced endoplasmic reticulum stress and NF-κB activation.

Abstract

Neonatal sepsis often causes acute lung injury (ALI), but the underlying molecular mechanisms of ALI have not been elucidated. Pleckstrin homology-like domain family A member 3 (PHLDA3) is a stress-responsive protein involved in inflammatory signaling, yet its role in neonatal sepsis-associated ALI has not been explored. Here, we established a neonatal polymicrobial sepsis model via intraperitoneal injection of cecal slurry (CS) and investigated the role of PHLDA3 in ALI using CRISPR/Cas9-generated constitutive PHLDA3 knockout (PHLDA3 KO) mice. CS induced time-dependent upregulation of PHLDA3 in neonatal lung tissues. CS triggered significant increases in the levels of IL-1β, IL-6, and TNF-α in lung tissues, impairing vascular integrity and causing severe alveolar damage. These harmful effects were alleviated by PHLDA3 KO. Transcriptomic analysis revealed enrichment of endoplasmic reticulum (ER) protein processing pathways. CS activated ER stress and unfolded protein response via PERK/eIF2α/ATF4 and ATF6 axes, which were attenuated by PHLDA3 deletion. Pharmacological induction of ER stress by thapsigargin (TG) reversed the protective effects of PHLDA3 KO, whereas ER stress inhibitor 4-phenylbutyric acid (4-PBA) mitigated inflammation and lung injury. In macrophage models, PHLDA3 knockdown was associated with reduced LPS-induced ER-stress-related signaling, NF-κB activation, and inflammatory responses, whereas TG attenuated these effects. Collectively, these findings indicate that constitutive PHLDA3 deficiency is associated with attenuated neonatal sepsis-associated lung injury, reduced ER-stress-related signaling, and decreased NF-κB activation, and support further investigation of PHLDA3 in neonatal sepsis-associated ALI.