158 - A Live Biotherapeutic Product Reduces Neutrophilic Inflammation in Bronchopulmonary Dysplasia through miR129-MMP9 Pathway

Disease progression in BPD is characterized by decreased lactobacillus, increased proteobacteria, and neutrophilic inflammation marked by matrix metalloproteinase 9 (MMP-9) pathway activity.

Objective:

We hypothesized that inhalation of live biotherapeutic AB1000 would attenuate neutrophilic inflammation in in vivo murine models of BPD lung injury.

Design/Methods:

Novel animal models: a) Gnotobiotic humanized mice model as published in AJRCMB (PMID:36287630) b) Hyperoxia/LPS double hit model as published in JCI Insight (PMID:29515035) c) miR129 KO mice by CRISPR/CAS.

Animal experiments: All three animal models were exposed to normoxia (21% FiO2) or hyperoxia (85% FiO2) from PN3-PN14. Mice were treated with either a vehicle control or AB1000 intranasally. AB1000 composes three live Lactobacillus strains in a dry powder commercially manufactured with a fine particle fraction that reaches deep lungs >73%. On PN14, pups were euthanized for BAL, lung tissue, blood, histology. Each group n=4-6. Neutrophil counts, MMP-9, CRP, NE, IL-6, MPO, pulmonary function (Flexivent), RAC and MLI were measured.

In vitro:

a) Human bronchial epithelial (HBE) and A549 alveolar basal epithelial cell lines were exposed to Escherichia coli, LPS, or otherwise indicated and cultured in cell culture chamber with 85% O2 or 21% O2.

b) p-CMV-miR-129 (ectopic overexpression) and miR-129 LNA inhibitor (knock down) were individually transfected at different concentrations into HBE cells by use of Lipofectamine 2000 reagent. miR-129-5p and MMP-9 mRNA were measured by RT-PCR.

Human samples: In a cohort study, human tracheal aspirate samples were collected from patients with established severe BPD and from gestation-matched full-term infants. MMP-9 and miR-129 expression in these samples were assayed by Duoset ELISA and qRT-PCR separately.

Results:

Markers of neutrophilic inflammation were significantly reduced, lung structure and function significantly improved in AB1000-treated as compared to controls in all models. Decreased miR-129 was associated with increased MMP-9 in BPD infants, in vitro cell lines, and BPD mouse model. miR-129 overexpression downregulated MMP-9 levels and miR-129 inhibition upregulated MMP-9 levels in a dose-dependent manner. Higher MMP-9 levels, worse lung function, and lower survival rates were observed in miR-129 KO mice compared to wild type.

Conclusion(s):

Administration of live biotherapeutic AB1000 to the lungs of mouse pups reduced the neutrophilic inflammatory response in BPD through the miR129-MMP9 pathway. This suggests a role for LBPs as interventions for preterm infants with chronic lung injury.