Proteome of airway surface liquid and mucus in newborn wildtype and cystic fibrosis piglets

Respiratory Research · 6 authors, 2 centres

AI SUMMARY

FIDELITY 100%

POPULATIONNewborn wild-type (WT) and CFTR null (CF) piglets (Sus scrofa domesticus), less than 24 hours old, of both sexes

INTERVENTIONCarbachol (10 µM) stimulation of tracheobronchial explants; collection of five sequential fractions (four washable fractions and one Alcian blue-stained mucus fraction)

COMPARISONUnstimulated vs. carbachol-stimulated; WT vs. CF piglets; washable fractions (1–4) vs. mucus fraction (5)

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TL;DR

This study used mass spectrometry to analyze the proteome of airway surface liquid and mucus from newborn wild-type and cystic fibrosis (CF) piglets, finding a distinct separation between washable surface liquid proteins and mucus-associated proteins. Carbachol stimulation increased MUC5AC and MUC5B levels, and MUC5AC was more abundant than MUC5B in the mucus fraction. An additional cluster of acute phase proteins (fetuin-A, fetuin-B, apolipoprotein A1) was observed in CF piglet washable fractions, suggesting an early inflammatory response or altered lung development present at birth.

Full summary

4,686 CHARS

**Background:** The respiratory tract is protected by mucociliary clearance, mediated by mucus and cilia. Submucosal glands secrete linear MUC5B mucin polymers as bundled strands of 1000–5000 parallel molecules, while surface goblet cells secrete both MUC5AC and MUC5B. Understanding the protein composition of airway surface liquid versus mucus is important for understanding airway protection and diseases such as cystic fibrosis (CF).

**Methods:** The distal trachea and primary bronchi from newborn (<24 h old) WT and CFTR null (CF) piglets were excised, opened, and mounted mucosal side up on a tilted platform at 37°C. For unstimulated samples, 500 µl Krebs-Glucose buffer was added; for stimulated samples, 100 µl buffer containing 10 µM carbachol was added. Four sequential washable fractions (fraction 1–4) were collected at 10-min intervals by adding buffer to the cephalad end and collecting from the lowest point. After fraction 4, Alcian blue 8GX in buffer was added to stain mucus, and fraction 5 (mucus fraction) was collected by pipetting. A total of 80 samples were prepared from 10 WT piglets (4 non-stimulated, 6 stimulated) and 6 CF piglets (3 non-stimulated, 3 stimulated). Samples underwent filter-aided sample preparation (FASP) with LysC and trypsin digestion, followed by nanoLC-MS/MS on a Q-Exactive HF mass spectrometer. MaxQuant (version 1.5.7.4) was used against the UniProt pig proteome (20180301) with FDR at 1%. A total of 2328 proteins were identified. Hierarchical clustering used Euclidean distance with average linkage in Perseus software (version 1.5.6.0). Statistical analyses used two-tailed Mann–Whitney test or Kruskal–Wallis with Dunn’s multiple comparisons test; significance defined as P ≤ 0.05.

**Key Results:** Cluster analysis generated three distinct clusters for WT and CF piglets (fraction 1–4, fraction 2–4, and fraction 5) and an additional cluster for CF piglets in fractions 1–4. Fraction 5 (mucus fraction) contained MUC5AC and MUC5B, with higher MUC5AC than MUC5B in both unstimulated and stimulated WT and CF piglets. In unstimulated WT piglets, there was significantly more MUC5AC in fraction 5 than fraction 4 (P = 0.03), and similarly more MUC5B in fraction 5 than fraction 4 (P = 0.03). Carbachol stimulation increased MUC5AC and MUC5B. The mucus fraction also contained protein disulfide isomerases (AGR2, PDIA1, PDIA3) and lactoferrin. AGR2 was significantly higher in fraction 5 than fraction 4 in stimulated WT piglets (P = 0.04), PDIA1 was higher in fraction 5 than fraction 4 in stimulated WT piglets (P = 0.004), and PDIA3 was higher in fraction 5 than fraction 1 in stimulated WT piglets (P = 0.002). Lactoferrin was higher in fraction 5 than fraction 1 when all values were pooled (P = 0.03). The washable fractions (1–4) contained clusterin, alpha-1 antitrypsin, and orosomucoid 1. Clusterin was higher in fraction 4 than fraction 1 in pooled WT piglets (P = 0.0007) and higher in CF than WT piglets (P = 0.02). Alpha-1 antitrypsin was higher in CF than WT piglets (P = 0.009), and orosomucoid 1 was higher in CF than WT piglets (P = 0.02). The additional CF cluster included fetuin-A, fetuin-B, and apolipoprotein A1. Fetuin-A was higher in unstimulated CF than WT piglets (P = 0.002), higher in CF than WT in fraction 1 (P = 0.03), and higher in fraction 1 than fraction 5 (P = 0.0003). Fetuin-B was higher in unstimulated CF than WT piglets (P = 0.0004), higher in fraction 1–4 than fraction 5 in WT (P = 0.0005) and CF (P = 0.0005). Apolipoprotein A1 was higher in unstimulated CF than WT piglets (P = 0.002), higher in fraction 1–4 than fraction 5 in WT (P = 0.0008) and CF (P = 0.02). Immunostaining confirmed MUC5AC in surface goblet cells, MUC5B in submucosal glands, AGR2 and PDIA3 in submucosal glands, and orosomucoid 1 on the airway surface and in small capillaries.

**Clinical Implications:** The collection method successfully separates washable airway surface liquid proteins from mucus-associated proteins, providing a more refined approach than traditional bronchoalveolar lavage. The finding of an additional acute phase protein cluster (fetuin-A, fetuin-B, apolipoprotein A1) in CF piglet washable fractions, along with increased clusterin, alpha-1 antitrypsin, and orosomucoid 1, suggests that inflammation-promoting mechanisms are activated at birth in CF, even without overt signs of infection or inflammation. This supports the hypothesis that CF airway disease is present at birth and that treatment should commence directly after diagnosis. No differences in mucin levels were observed between WT and CF piglets, consistent with the absence of mucus overproduction in newborn CF piglets.

PICO

PPOPULATION

Newborn wild-type (WT) and CFTR null (CF) piglets (Sus scrofa domesticus), less than 24 hours old, of both sexes

IINTERVENTION

Carbachol (10 µM) stimulation of tracheobronchial explants; collection of five sequential fractions (four washable fractions and one Alcian blue-stained mucus fraction)

OOUTCOME

Protein composition and normalized intensities of identified proteins, including MUC5AC, MUC5B, protein disulfide isomerases (AGR2, PDIA1, PDIA3), lactoferrin, clusterin, alpha-1 antitrypsin, orosomucoid 1, fetuin-A, fetuin-B, and apolipoprotein A1

STUDY TYPE

other

SPECIALTY

pulmonology

SUMMARISED BY

AI pipeline

FIDELITY CHECK

100% · A

ORIGINAL PAPER

PMC10022022

Proteome of airway surface liquid and mucus in newborn wildtype and cystic fibrosis piglets

Respiratory Research

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ABSTRACT

Background The respiratory tract is protected from inhaled particles and microbes by mucociliary clearance, mediated by the mucus and the cilia creating a flow to move the mucus cephalad. Submucosal glands secrete linear MUC5B mucin polymers and because they pass through the gland duct before reaching the airway surface, bundled strands of 1000–5000 parallel molecules exit the glands. In contrast, the surface goblet cells secrete both MUC5AC and MUC5B. Methods We used mass-spectrometry based proteomic analysis of unstimulated and carbachol stimulated newborn wild-type (WT) and cystic fibrosis transmembrane conductance regulator (CFTR) null (CF) piglet airways to study proteins in the airway surface liquid and mucus, to investigate if levels of MUC5AC and MUC5B were affected by carbachol stimulation and whether the proteins clustered according to function. Results Proteins in the first four extracted fractions clustered together and the fifth fraction contained the mucus cluster, mucins and other proteins known to associate with mucins, whereas the traditional airway surface liquid proteins clustered to fraction 1–4 and were absent from the mucus fraction. Carbachol stimulation resulted in increased MUC5AC and MUC5B. Conclusions These results indicate a distinct separation between proteins in the washable surface liquid and the mucus fraction. In fractions 1–4 from newborn CF piglets an additional cluster containing acute phase proteins was observed, suggesting an early inflammatory response in CF piglets. Alternatively, increased levels of these proteins could indicate altered lung development in the CF piglets. This observation suggests that CF airway disease is present at birth and thus, treatment should commence directly after diagnosis. Supplementary Information The online version contains supplementary material available at 10.1186/s12931-023-02381-x.

CITATION EXPORT

Ana M. Rodriguez-Piñeiro, Florian Jaudas, Nikolai Klymiuk et al.. Proteome of airway surface liquid and mucus in newborn wildtype and cystic fibrosis piglets. Respiratory Research. (2023). https://doi.org/10.1186/s12931-023-02381-x

AUTHORS

6 · 2 CENTRES

Ana M. Rodriguez-Piñeiro, Florian Jaudas, Nikolai Klymiuk, Andrea Bähr, Gunnar C. Hansson, Anna Ermund

1grid.8761.80000 0000 9919 9582Department of Medical Biochemistry and Cell Biology, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden
2grid.5252.00000 0004 1936 973XCenter for Innovative Animal Models, Ludwig-Maximilians-University, Munich, Germany