Mechanisms of Pulmonary Microbiota-Induced Inflammation and Vascular Dysfunction in Neonatal Lung Injury

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PROJECT SUMMARY/ABSTRACT: This revised NIH K08 proposal describes a 5-year training and research plan for the candidate, a physician scientist with a long term goal of becoming an independent investigator in the field of neonatal pulmonary biology with expertise in understanding the role of the pulmonary microbiome in chronic lung disease of prematurity. To accomplish this goal, he and his mentoring committee put forth an integrated career development plan encompassing a novel research idea. Bronchopulmonary dysplasia (BPD), the most common pulmonary morbidity in extremely preterm infants is initiated by injury to the immature lung by early neutrophil influx, collagen degradation, remodeling, and arterial thickening. The candidate has discovered that the airways of newborn infants are not sterile but are occupied by a diverse microbiome even at birth, and that the microbiome is altered (dysbiosis) during the development of BPD. He has also recently discovered the role of exosomal microRNAs in BPD prediction and pathogenesis. His mentors have previously established that the tripeptide N-acetyl proline-glycine-proline (Ac-PGP) derived from the breakdown of the ECM plays a critical role in various chronic lung diseases by enhancing neutrophilic inflammation and endothelial permeability. The preliminary data in this proposal indicate that the airways of infants with BPD which have increased Gammaproteobacteria (¡-Proteobacteria) also have increased levels of Ac-PGP. Moreover, gain of Ac-PGP function creates the phenotype of BPD in murine models, whereas loss of Ac-PGP function reverses the BPD phenotype. Matrix metalloproteinase 9 (MMP9) and prolyl endopeptidase (PE) degrade collagen, leading to the release of Ac-PGP. MMP9 and PE are top predicted targets of exosomal miR 548m and miR 129-1-3 respectively and both these miRs are reduced in BPD. Collectively, these findings lead to the novel mechanistic hypothesis that ¡-Proteobacteria-induced reduction in exosomal miRs increase protease levels which in turn increase Ac-PGP release and cause chronic neutrophilic inflammation and vascular dysfunction in BPD. In addition to determining these mechanisms (Aim 1), the candidate will conduct a human study using independent ‘Discovery’ and ‘Validation’ cohorts of extremely preterm infants to test the hypothesis that decreased exosomal miRs 548m and miR 129- 1-3p and increased MMP9, PE and Ac-PGP, in relation to a dysbiotic airway microbota are early predictors of severe BPD (Aim 2). Thus the work proposed in this research proposal will generate novel information about the mechanisms of microbiota induced neutrophilic inflammation and vascular dysfunction in BPD, and will determine novel early biomarkers for BPD. The candidate has already assembled a research advisory committee with complementary intellect and content expertise that can guide him throughout the award period. In addition, the candidate will obtain didactic education to gain requisite knowledge culminating in the awarding of an MSPH degree. Through this integrated mentoring and didactic plan, the candidate will gain skill and expertise in pulmonary inflammation, microbial pathogenesis, microRNA and exosomal molecular biology, microbiome sequencing, bioinformatics, skills in manipulation of germ free mouse models and research ethics. This will enable the him to develop an independent research program and obtain NIH R01 funding.