Innate lung defenses and compromised Pseudomonas aeruginosa clearance in the malnourished mouse model of respiratory infections in cystic fibrosis

Abstract

Cystic fibrosis (CF) is characterized by dysfunction of the digestive and respiratory tracts resulting in generalized malnutrition and chronic respiratory infections. Chronic lung infections with Pseudomonas aeruginosa, intense neutrophil-dominated airway inflammation, and progressive lung disease are the major cause of high morbidity and mortality in CF. Here we investigated the effects of malnutrition in CF on innate lung defenses, susceptibility to P. aeruginosa colonization, and associated inflammation, using aerosol models of acute and chronic infections in normal, malnourished, and transgenic mice. CFTR(m1Unc-/-) knockout mice displayed body weight variations and showed variable pulmonary clearance of P. aeruginosa. This variability was not detected in bitransgenic CFTR(m1Unc-/-)(FABP-hCFTR) mice in which the intestinal defect had been corrected. Diet-induced protein calorie malnutrition in C57BL/6J mice resulted in impaired pulmonary clearance of P. aeruginosa. Tumor necrosis factor alpha (TNF-alpha) and nitrite levels detected upon exposure to P. aeruginosa aerosols were lower in the lungs of the malnourished C57BL/6J mice relative than in lungs of mice fed a normal diet. The role of TNF-alpha and reactive nitrogen intermediates in P. aeruginosa clearance was tested in TNF-alpha and inducible nitric oxide synthase (iNOS) knockout mice. P. aeruginosa clearance was diminished in transgenic TNF-alpha- and iNOS-deficient mice. In contrast to the effects of TNF-alpha and iNOS, gamma interferon knockout mice retained a full capacity to eliminate P. aeruginosa from the lung. Malnutrition also contributed to excessive inflammation in C57BL/6J mice upon chronic challenge with P. aeruginosa. The repeatedly infected malnourished host did not produce interleukin-10, a major anti-inflammatory cytokine absent or diminished in the bronchoalveolar fluids of CF patients. These results are consistent with a model in which defective CFTR in the intestinal tract leads to nutritional deficiency which in turn contributes to compromised innate lung defenses, bacterial colonization, and excessive inflammation in the CF respiratory tract.

FIG. 1

(A) Pseudomonas clearance from the lungs of control C57BL/6J mice (normal-protein diet), PEM mice, and PEM-R mice (as defined in Materials and Methods). P. aeruginosa was delivered as aerosol (initial deposition was 2 × 10⁶ to 8 × 10⁶ CFU/g of lung tissue). Bacterial survival was expressed as the fraction of the initially deposited CFU (determined by sacrificing a group of animals immediately following exposure) remaining in the lung 18 h following infection (relative bacterial survival). (B) Pulmonary clearance in transgenic iNOS, TNF-α, and IFN-γ knockout mice. ∗∗, P < 0.01 (ANOVA post hoc t test; relative to control in panel A or relative to C57BL/6J in panel B).

FIG. 2

TNF-α (A) and total nitrite (B) in the lungs of control C57BL/6J and PEM mice 18 h following exposure to P. aeruginosa aerosols; levels of MIP-2 (C), KC (C), and MPO (D) in the lungs of control (fed normal-protein diet) and PEM mice 18 h following exposure to P. aeruginosa aerosols. ∗∗, P < 0.01; ∗, P < 0.05 (ANOVA post hoc t test).

FIG. 3

Increased neutrophil and inflammatory cell infiltration in PEM mice exposed to P. aeruginosa. (A) Control C57BL/6J mice fed normal-protein diet; (B) malnourished mice (PEM) fed low-protein diet. Both groups of mice were exposed as in Fig. 1.

FIG. 4

(A) Pulmonary clearance in chronically infected PEM mice. Mice were repeatedly exposed (eight times, every 72 h) to P. aeruginosa as previously described (39). (B and C) IL-10 (B), MIP-2 (C), KC (C), and MPO (C) in chronically infected mice. ∗∗, P < 0.01; ∗, P < 0.05 (ANOVA post hoc t test).