Sinus hypoplasia precedes sinus infection in a porcine model of cystic fibrosis

Abstract

Objectives/hypothesis: Chronic sinusitis is nearly universal in humans with cystic fibrosis (CF) and is accompanied by sinus hypoplasia (small sinuses). However, whether impaired sinus development is a primary feature of loss of the cystic fibrosis transmembrane conductance regulator (CFTR) or a secondary consequence of chronic infection remains unknown. Our objective was to study the early pathogenesis of sinus disease in CF.

Study design: Animal/basic science research.

Methods: Sinus development was studied in a porcine CF model.

Results: Porcine sinus epithelia expressed CFTR and exhibited transepithelial anion transport. Disruption of the CFTR gene eliminated both. Sinuses of newborn CF pigs were not infected and showed no evidence of inflammation, yet were hypoplastic at birth. Older CF pigs spontaneously developed sinus disease similar to that seen in humans with CF.

Conclusions: These results define a role for CFTR in sinus development and suggest the potential of the CF pig as a genetic model of CF-sinus disease in which to test therapeutic strategies to minimize sinus-related CF morbidity.

Fig. 1

Histopathology of newborn CFTR^+/+ and CFTR^−/− pig sinus epithelia are similar. Hematoxylin and eosin stains of CFTR^+/+ respiratory (A) and olfactory (B) epithelia show no differences to CFTR^−/− respiratory and olfactory epithelia in regard to structure, inflammation, or remodeling. Bars: 29 μm.

Fig. 2

CFTR is expressed in pig sinus epithelia. (A) Reverse-transcriptase polymerase chain reaction of sinus epithelia with primers for CFTR (155 bp) and β-actin control. (B) Immunohistochemistry of sinus epithelia staining for CFTR protein in airway epithelia of CFTR^+/+ pigs. (C) Absence of staining in CFTR^−/− pig airway epithelia. Bars: 8 μm.

Fig. 3

CFTR^−/− pig sinus epithelia lack Cl⁻ conductance, and have decreased Na+ conductance. Data are presented as mean ± standard error from sinus epithelia cultures of CFTR^+/+ (open symbols and bars) and CFTR^−/− (closed symbols and bars) pigs. Studies performed on three cultures from four different pigs (n = 4) *P < .05. (A, D) Change in short-circuit current (Isc) and transepithelial electrical conductance (Gt) after adding amiloride (Amil) (100 μM) apically. (B, E) Change in Isc and Gt induced by adding 10 mM forskolin and 100 mM 3-isobutyl-2-methylxanthine (F&I). (C, F) Change in Isc and Gt following addition of GlyH-101 (GlyH) (100 μM).

Fig. 4

Cystic fibrosis (CF) piglets have ethmoid hypoplasia at birth. (A) Three-dimensional reconstruction view of skull (transparent overlay) and sinus of newborn pig. (B) Non-CF and (C) CFTR^−/− newborn piglet three-dimensional sinus scan with ethmoid (red) and maxillary (green) sinus. Dotted line dividing anterior and posterior ethmoid sinus. Volumetric analysis performed on three-dimensional reconstructions of segmented sinus computed tomography scans. Data points represent mean ± standard error of non-CF (n = 7; open symbols) and CFTR^−/− (n = 8; closed symbols) littermate pigs. *P < .05. (C) Volume of ethmoid and maxillary sinuses. (D) Volume of skull. (E) Weight of newborn pigs.

Fig. 5

Cystic fibrosis (CF) pigs have sinus hypoplasia and delayed frontal sinus development. Volumetric analysis performed on three-dimensional reconstructions of segmented sinus computed tomography scans. Data points represent mean volumes of one to four non-CF (open symbols) and CF (closed symbols) pigs. Ethmoid (A), maxillary (B), and frontal volumes (C).

Fig. 6

Cystic fibrosis (CF) pigs spontaneously develop sinus disease. Comparison of sinus computed tomography (CT) findings, gross necropsy at death, and microbiology culture results of CF pigs with sinus disease. Dotted white lines on CT highlight the area viewed on gross necropsy. Green arrows highlight thick viscous mucus in the sinuses. Microbiology cultures taken from infected sinus mucus.

Fig. 7

Cystic fibrosis (CF) pig sinus disease shows mucus accumulation and epithelial remodeling on histology. (A, B) Moderate sinus disease in CF pigs showed goblet cell hyperplasia with mucus accumulation and submucosal gland hypertrophy, periodic acid-Schiff (PAS) stain, scale: 50 μm. (C, D) Severe sinus disease with mucus from goblet cells and submucosal glands, PAS stain, bar: 55 μm.

Fig. 8

Mucopurulent obstruction of affected sinuses. (A) Lamellar mucus accumulating in the sinus from goblet cells, periodic acid-Schiff stain, bar: 95 μm. (B) Neutrophilic inflammation with bacteria, bar: 40 μm.