Long-term dietary (n-3) polyunsaturated fatty acids show benefits to the lungs of Cftr F508del mice

Abstract

Introduction: The pro-inflammatory status of cystic fibrosis (CF) patients promotes pulmonary colonization with opportunist and pathogenic bacteria, which is favored by a sticky mucus. Oral supplementation with (n-3) long chain polyunsaturated fatty acids (LC-PUFA) has shown anti-inflammatory effects. The aim of this study was to demonstrate the positive effects of a long-term diet enriched in (n-3) LC-PUFA on the lungs of Cftr F508del mice.

Materials and methods: Breeding CftrΔF508del/+ mice received a control diet or a diet enriched in (n-3) LC-PUFA for 5 weeks before mating, gestation and lactation. After weaning, the offspring were given the same diet as their mother until post-natal day 60. The effects of (n-3) LC-PUFA supplementation on the lungs were evaluated in homozygous Cftr F508del mice and their wild-type littermates after acute lung inflammation induced by Pseudomonas aeruginosa lipopolysaccharide (LPS) inhalation.

Results: (n-3) LC-PUFA enrichment of mothers contributes to enrichment of mammary milk and cell membrane of suckling pups. Cftr F508del mice exhibited growth retardation and lung damage with collapsed alveoli, hyperplasia of bronchial epithelial cells and inflammatory cell infiltration. The (n-3) LC-PUFA diet corrected the growth delay of Cftr F508del mice and decreased hyperplasia of bronchial epithelial cells. Besides decreasing metaplasia of Club cells after LPS inhalation, (n-3) LC-PUFA modulated lung inflammation and restricted lung damage.

Conclusion: Long-term (n-3) LC-PUFA supplementation shows moderate benefits to the lungs of Cftr F508del mice.

Fig 1. Early (n-3) LC-PUFA consumption improves fatty acid incorporation in tissues of CftrΔF508 mice.

(n-3) LC-PUFA incorporation was quantified in pup liver and lungs at post-natal (PN) day 1–3 and in adult liver on PN60 (5–9 mice/group). CTRL: control diet group; n-3: (n-3) LC-PUFA diet group; WT: wild-type mice; ΔF508: CftrΔF508 mice.

Fig 2. Early (n-3) LC-PUFA consumption corrects growth retardation in male CftrΔF508 mice.

Body mass of CftrΔF508 (ΔF) and brother-sister wild-type (WT) mice at weaning (A) and adulthood (B) (4–10 pup mice and 6–17 adult mice/group). NS: not significant; CTRL: control diet group; n-3: (n-3) LC-PUFA diet group; WT: wild-type mice; ΔF508: CftrΔF508 mice.

Fig 3. Histologic score for lung damage.

(A) Total histologic score is the sum of the score for each criterion (lung injury areas, collapsed alveoli, hyperplasia and metaplasia of bronchial epithelial cells, mucus plugs defined as mucus material in bronchi and total inflammatory cells). The total inflammatory cell parameter was established by scoring infiltration of inflammatory cells into lung tissue and specific infiltration of peribronchial and perivascular inflammatory cells separately. Total histologic score results are mean ± SEM. The higher the histologic score, the more impaired the lung (8–16 mice/group, 3–14 lung sections/mouse scored). CTRL: control diet group; n-3: (n-3) LC-PUFA diet group; WT: wild-type mice; ΔF508: CftrΔF508 mice. (B) Representative lung sections from CftrΔF508 mice fed with control (a) or (n-3) LC-PUFA (b) diets after LPS challenge showing PAS positive mucus plug (P, zoom) with inflammatory cells. (C) Representative lung sections stained with HE 24 h after PBS (control) or LPS challenge. Black arrows outline collapsed alveoli; asterisks show inflammatory cells; red arrow heads indicate metaplasia; blue arrow heads indicate hyperplasia. Lu: lumen of bronchi.

Fig 4. Detection of pro-inflammatory cytokines in lung homogenates by ELISA.

(A) KC, (B) TNF-α and (C) IFNγ. Pro-inflammatory cytokine levels in wild-type (WT) and CftrΔF508 (ΔF508) mice were normalized to the concentration of total proteins (9–18 mice/group). CTRL: control diet group; n-3: (n-3) LC-PUFA diet group; WT: wild-type mice; ΔF508: CftrΔF508 mice.

Fig 5. PPAR expression in lung homogenates by RT-qPCR.

(A) PPARα and (B) PPARγ. Transcripts were normalized to 18S. Data shown are mean ± standard error (5–20 mice/group). CTRL: control diet group; n-3: (n-3) LC-PUFA diet group; WT: wild-type mice; ΔF508: CftrΔF508 mice.