Pancreatic damage in fetal and newborn cystic fibrosis pigs involves the activation of inflammatory and remodeling pathways

Abstract

Pancreatic disease has onset in utero in humans with cystic fibrosis (CF), and progresses over time to complete destruction of the organ. The exact mechanisms leading to pancreatic damage in CF are incompletely understood. Inflammatory cells are present in the pancreas of newborn pigs with CF (CF pigs) and humans, which suggests that inflammation may have a role in the destructive process. We wondered whether tissue inflammation and genes associated with inflammatory pathways were increased in the pancreas of fetal CF pigs [83 to 90 days gestation (normal pig gestation is ~114 days)] and newborn pigs. Compared with fetal pigs without CF (non-CF pigs), in fetal CF pigs, the pancreas exhibited patchy inflammation and acinar atrophy, with progression in distribution and severity in neonatal CF pigs. Large-scale transcript profiling revealed that the pancreas in fetal and newborn CF pigs exhibited significantly increased expression of proinflammatory, complement cascade, and profibrotic genes when compared with fetal and newborn non-CF pigs. Acinar cells exhibited increased apoptosis in the pancreas of fetal and newborn CF pigs. α-Smooth muscle actin and transforming growth factor β1 were increased in both fetal and newborn CF pig pancreas, suggesting activation of profibrotic pathways. Cell proliferation and mucous cell metaplasia were detected in newborn, but not fetal, CF pigs, indicating that they were not an initiator of pathogenesis but a response. Proinflammatory, complement cascade, proapoptotic, and profibrotic pathways are activated in CF pig pancreas, and likely contribute to the destructive process.

Figure 1

CF and non-CF pancreata at 83 to 90 days gestation and at birth. Eosinophilic zymogen material is abundant in all pancreatic acini in non-CF animals (eosinophilic staining). At 83 to 90 days gestation, CF pancreata exhibit reduced zymogen-filled mature acini (black arrows), which was associated with increased acini plugging and degeneration by luminal eosinophilic zymogen secretions (red arrow). This eventually results in further loss of normal zymogen-filled acini by birth (arrow). While less extensive in fetal versus neonatal CF pancreas, discrete patchy aggregates of lymphoid inflammation (arrowheads) expand the interstitium, with scattered neutrophils and macrophages occasionally seen in the lumen of adjacent ducts and acini, similar to the localization pattern of inflammation previously described in CF pig pancreas. H&E. Scale bar = 100 μm.

Figure 2

Loss of CFTR function alters the transcriptome in the CF pancreas. A: Unsupervised hierarchical clustering in 8 fetal and 12 newborn CF samples reveals significant differences in the transcriptome profile of CF and non-CF samples at fetal and newborn stages. B: One-way ANOVA reveals differentially expressed genes in the fetal and newborn CF samples (P < 0.01). C and D: Quantitative RT-PCR of the fetal and newborn pancreas samples shows the relative abundance of genes. Each bar represents data from eight non-CF and seven CF samples tested in quadruplicate. Error bars indicate mean (SD). *P < 0.01, CF versus non-CF.

Figure 3

Apoptosis is increased in both fetal and newborn CF pig pancreas. Activated caspase-3 staining (arrows) of non-CF and CF pig pancreas at 83 days gestation and in newborn pigs (IHC). Caspase-3 index was measured in 6 non-CF and 3 CF fetuses (left graph) and 10 non-CF and 18 CF newborn pigs (right graph), which show increased immunostaining in the CF pig pancreas at both time points. *P < 0.05. Scale bar = 35 μm.

Figure 4

Top panels: Fetal and newborn Non-CF pig pancreata showing a normal duct that is empty and one filled by normal secretions, respectively. Mucous cell metaplasia is a late event in pancreatic disease in CF. Left panels: Mucous cell change in the duct epithelium is lacking in non-CF and CF pancreata at 90-days gestation (arrows). Bottom right panel: In the newborn pigs with CF, obstructed ducts were often distended with mucus (magenta area), and the mucous cells were prominent in the epithelium (arrows). Mucous cell metaplasia was not detected in non-CF neonatal pig pancreas. dPAS stain. Scale bar = 45 μm.

Figure 5

Increased cell proliferation in the newborn CF pig pancreas. Ki-67 immunostaining is increased in newborn CF pig pancreas (n = 20) compared with non-CF pancreas (n = 10) (*P < 0.001) but is similar between fetal pig groups (CF, n = 4; non-CF, n = 6). Ki-67-positive cells are often associated with proliferative ductuloacinar units. Scale bar = 100 μm.

Figure 6

CF pig pancreas exhibit increased α-SMA immunostaining in fetal pancreas at 90 days gestation and in newborn pigs. Top panels: In non-CF pig pancreas, α-SMA staining is detected, as normally expected, surrounding the ducts (arrows) and vessels (arrowheads). Bottom panels: At 90-days gestation, CF pancreas exhibits patchy mild to moderate staining adjacent to relatively unaffected exocrine tissue, whereas neonatal CF pancreas demonstrate widespread α-SMA staining surrounding proliferative and dilated ducts. Scale bar = 350 μm.