Composition and origin of lung fluid proteome in premature infants and relationship to respiratory outcome

Abstract

Background: Infants born at extremely low gestational age are at high risk for bronchopulmonary dysplasia and continuing lung disease. There are no early clinical biomarkers for pulmonary outcome and limited therapeutic interventions.

Objectives: We performed global proteomics of premature infant tracheal aspirate (TA) and plasma to determine the composition and source of lung fluid proteins and to identify potential biomarkers of respiratory outcome.

Methods: TA samples were collected from intubated infants in the TOLSURF cohort before and after nitric oxide treatment, and plasma was collected from NO CLD infants. Protein abundance was assayed by HPLC/tandem mass spectrometry and Protein Prospector software. mRNA abundance in mid-gestation fetal lung was assessed by RNA sequencing. Pulmonary morbidity was defined as a need for ventilatory support at term and during the first year.

Results: Abundant TA proteins included albumin, hemoglobin, and actin-related proteins. 96 of 137 detected plasma proteins were present in TA (r = 0.69, p<0.00001). Based on lung RNAseq data, ~88% of detected TA proteins in injured infant lung are derived at least in part from lung epithelium with overrepresentation in categories of cell membrane/secretion and stress/inflammation. Comparing 37 infants at study enrollment (7-14 days) who did or did not develop persistent pulmonary morbidity, candidate biomarkers of both lung (eg., annexin A5) and plasma (eg., vitamin D-binding protein) origin were identified. Notably, levels of free hemoglobin were 2.9-fold (p = 0.03) higher in infants with pulmonary morbidity. In time course studies, hemoglobin decreased markedly in most infants after enrollment coincident with initiation of inhaled nitric oxide treatment.

Conclusions: We conclude that both lung epithelium and plasma contribute to the lung fluid proteome in premature infants with lung injury. Early postnatal elevation of free hemoglobin and heme, which are both pro-oxidants, may contribute to persistent lung disease by depleting nitric oxide and increasing oxidative/nitrative stress.

Fig 1. Comparison of abundance levels for proteins of TA and culture medium of human fetal lung explants.

By linear regression analysis of log transformed data there is a highly significant positive correlation (r = 0.56, p<10⁻³⁰).

Fig 2. Comparison of abundance levels for proteins present in both TA and plasma of premature infants.

By regression analysis r = 0.69 (p<0.00001) excluding albumin as an outlier, which had AI values of 179 and 315 for TA and plasma, respectively. Gene symbols are shown for some outlier proteins.

Fig 3. Comparison of abundance levels for total hemoglobin of TA by MS and ELISA assay.

Solid circles are infants with PM and open squares are No PM. n = 34, r = 34, p = 0.00001 by linear regression. Except for lower values, results by immunoassay correlate with MS results.

Fig 4. Time course for TA levels of total peptides, total hemoglobin, albumin and total IgG.

Example of one infant with TA samples collected between 10 and 18 d postnatal age, showing decreases over time in hemoglobin, but not other major plasma proteins. Values are AIx10⁸ except for total peptide.