347 - Alveolar Type 1 Cell Dysfunction Drives Inflammation and Epithelial Barrier Breakdown in Neonatal ARDS

Publication Number: 1331.347

Atefeh Mohammadi, The Hospital for Sick Children, Toronto, ON, Canada; Julijana Ivanovska, The Hospital for Sick Children, Toronto, ON, Canada; Jeffrey Antwi, The Hospital for Sick Children, Toronto, ON, Canada; Estelle B.. Gauda, The Hospital for Sick Children, Toronto, ON, Canada

Background: Neonatal Acute Respiratory Distress Syndrome (NARDS) is a severe inflammatory lung injury affecting 34% of extremely preterm infants with a ~20% mortality rate. Respiratory failure and pulmonary edema are key features of NARDS, indicating injury to the alveolar Type 1 (AT1) cells that line 95% of the lungs. AT1 cells are crucial for gas exchange and fluid regulation, yet their role in NARDS pathogenesis remains unclear.

Objective: We hypothesize that AT1 cells contribute to NARDS pathogenesis by producing pro-inflammatory mediators in response to acute inflammatory stimuli that ultimately disrupt the alveolar epithelial barrier.

Design/Methods: Two in vitro models were used: primary AT1 cells isolated from neonatal rats on postnatal day 4 using fluorescence-activated cell sorting and the InSCREENex huArlo cell line. Primary AT1 cells were exposed to LPS (10µg/mL) and TNF-α (5ng/mL) for 18h. Conditioned media was collected for cytokine and chemokine analyses using multiplex ELISA assays. HuArlo cells were exposed to TNF-α (25ng/mL) and IFN- (30ng/mL) for 48h to assess membrane integrity using transepithelial electrical resistance (TEER), immunoblotting and immunostaining for tight junction proteins, and cell viability via MTT assay.

Results: Primary AT1 cells exposed to LPS and TNF-α had increased levels of IL-6, CCL2, GRO/KC, and CCL3 compared to control (p < 0.05, n=4). HuArlo cells exposed to TNF-α and IFN- had a significantly decreased membrane resistance (p < 0.0001, n=8) and decreased protein expression of e-cadherin (p < 0.01, n=5), occludin (p < 0.05, n=6), and claudin-4 (p < 0.01, n=5). Cell viability was not affected by exposure to inflammation (p>0.05, n=4).

Conclusion(s): We show for the first time that neonatal AT1 cells exposed to acute inflammatory stimuli produce significantly elevated levels of pro-inflammatory mediators, decreased membrane resistance, and reduced expression of key tight junction proteins. These results suggest a critical role for AT1 cells in NARDS pathogenesis and highlight the potential for therapies targeting the preservation of AT1 cell health and function.