Volume 133, Issue 6, Pages 1928-1937.e3 (December 2007) Amelioration of Cystic Fibrosis Intestinal Mucous Disease in Mice by Restoration of mCLCA3  Fiona D. Young, Susan Newbigging, Caroline Choi, Mary Keet, Geraldine Kent, Richard F. Rozmahel  Gastroenterology  Volume 133, Issue 6, Pages 1928-1937.e3 (December 2007) DOI: 10.1053/j.gastro.2007.10.007 Copyright © 2007 AGA Institute Terms and Conditions

Figure 1 TgrIFABP-mCLCA3 transgene and expression. (A) The TgrIFABP-mCLCA3 transgene was constructed by fusing the rat intestinal fatty acid binding protein (rIFABP) promoter, a synthetic intron from the pS1 mammalian (IS) expression plasmid, the complete coding region of mCLCA3, and an SV40 Late poly(A) addition sequence. The priming sites for detection of transgene-specific mCLCA3 mRNA expression are indicated by P1 (FABP-1162) and P2 (Gob5-785R). (B) Results of RT-PCR with (+RT) and without (−RT) reverse transcriptase enzyme are shown using transgene specific primers P1 and P2. The transgene-specific mCLCA3 mRNA product with intron spliced out is present in duodenum (D), ileum (I) and colon (C) but absent from lung (L), kidney (K), and brain (B). RT-PCR of β-actin is shown as an RNA control. (C) Real-time PCR analysis of ileal mCLCA3 expression levels normalized to β-actin. Level of mCLCA3 expression normalized to β-actin in wild-type (WT) is designated a value of 1 (n = 3 mice, 3 runs, 3 replications each). Results of CF (n = 3 mice, 3 runs for each) and TgrIFABP-mCLCA3/CF (n = 3 mice, 3 replications each) mice are shown as mean ± SEM. *Indicates significant difference (P ≤ .05) from wild-type. (D) Cell-specific expression of the TgrIFABP-mCLCA3 transgene by RNA in situ hybridization. Composite bright- and dark-field photomicrographs are shown for the ilea of 3-week-old wild-type (WT), CF, and TgrIFABP-mCLCA3/CF (CF/Tg+) mice hybridized with mCLCA3 antisense (AS) and sense (S) probes to adjacent sections. mCLCA3 appears to be expressed exclusively in goblet cells in WT and to a lower level in CF ileum. Expression is considerably more robust than WT in the TgrIFABP-mCLCA3/CF ileum and appears in both goblet cells and a subset of enterocytes of the villus, as well as crypt cells. Sections are counterstained with H&E, and photomicrographs were taken at ×40 magnification. Gastroenterology 2007 133, 1928-1937.e3DOI: (10.1053/j.gastro.2007.10.007) Copyright © 2007 AGA Institute Terms and Conditions

Figure 2 Protein analyses. Incubation of a Western blot of in vitro translated mCLCA3 with the AbMCLCA3(252–267) antibody shows the appropriate 100-kilodalton band (A). Incubation of a Western blot of ileal protein lysates (B) shows the posttranslationally cleaved 75-kilodalton mCLCA3 band in WT, which is severalfold more abundant in the TgrIFABP-mCLCA3 (WT/Tg+)ileum. The CF ilea appear to have higher levels of mCLCA3 compared with wild-type; however, this is explained by its luminal accumulation in inspissated mucous content. In contrast, TgrIFABP-mCLCA3/CF (CF/Tg+) ilea show robust mCLCA3 protein. Blocking of specific binding by preincubation with the mCLCA3 peptide (C) shows subtraction of the 75-kilodalton band. Hybridization with a β-actin antibody (43-kilodalton band) shows relative protein levels among the samples. (D) Immunohistochemistry of ileal villi shows specific mCLCA3 staining to goblet cells and the perivillar mucous layer in WT, and to goblet cells, enterocytes, and the perivillar mucous layer in WT/Tg+. (E) Photomicrographs of WT, CF, and CF/Tg+ ileal villi stained with the mCLCA3 antibody show specific staining to goblet cells and the perivillar mucous layer in WT, and the CF mice also shows robust staining throughout the lumen corresponding to inspissated mucous content. CF/Tg+ ileal villi show robust signal in enlarged goblet cells, moderate staining of enterocytes and the mucous layer, and no staining in the lumen. Photomicrographs of WT, CF, and CF/Tg+ ileal crypts stained with the mCLCA3 antibody shows minimal staining in WT, whereas the CF crypt has robust staining throughout, corresponding to goblet cells and mucous concretions. Crypts of CF/Tg+ ilea show markedly less staining, which appears primarily in undifferentiated enterocytes, goblet cells, with only minimal mucous content. Photomicrographs in D and crypts in E are ×100 magnification, whereas villi in E are ×40 magnification. Gastroenterology 2007 133, 1928-1937.e3DOI: (10.1053/j.gastro.2007.10.007) Copyright © 2007 AGA Institute Terms and Conditions

Figure 3 Histopathology. The jejunum and ileum of WT (A and D, respectively), CF (B and E, respectively), and TgrIFABP-mCLCA3/CF (CF/Tg+) (C and F, respectively) stained with H&E and periodic acid schiff shows characteristic intestinal disease in the CF, including goblet cell hyperplasia, copious amounts of luminal mucous, extensive villi damage, epithelial cell sloughing and villi fusion, mucous occluded and engorged crypts, along with severely enlarged lymphatic vessels. In contrast, the jejunum and ileum of CF/Tg+ mice appear relatively normal with only minor indications of mucous inspissation and no pronounced villar atrophy or epithelial cell sloughing, although continuing to show goblet cell hyperplasia. The intestinal crypts of CF/Tg+ mice appear normal at the gross level, in contrast to CF. Photomicrographs are ×40 magnification. Gastroenterology 2007 133, 1928-1937.e3DOI: (10.1053/j.gastro.2007.10.007) Copyright © 2007 AGA Institute Terms and Conditions

Figure 4 Measure of CF intestinal phenotypes. Differences in CF-relevant intestinal phenotypes among WT, TgrIFABP-mCLCA3 (WT/TG+), CF, and TgrIFABP-mCLCA3/CF (CF/Tg+) mice (n = 6 each genotype, 10 sections for each) were assessed. (A) Goblet cell numbers per villus show that WT/Tg+ are not different from WT, whereas CF mice show a roughly 70% increase, and CF/Tg+ have an approximate 150% elevation, suggestive of goblet cell hyperplasia in CF, which is more pronounced in CF/Tg+. *Indicates P ≤ .05 difference from wild-type; **Indicates P ≤ .05 significant difference from both wild-type and CF. (B) Morphometric analysis of goblet cell size shows no difference among the WT, WT/Tg+, and CF intestines; however, CF/Tg+ have a significant ∼25% increase, suggestive of goblet cell hypertrophy (WT: 1366 goblet cells; WT/Tg+: 1003 goblet cells; CF: 817 goblet cells; CF/Tg+: 981 goblet cells). *Indicates P ≤ .05 difference from wild-type. (C) Morphometric analysis of crypt luminal area (mucous engorgement) between CF and CF/Tg+ jejunum and ileum (CF: 239 crypts; CF/Tg+: 242 crypts) shows significant amelioration of this phenotype in both intestinal sections of CF/Tg+ animals. *Indicates P ≤ .0001 difference from CF. Gastroenterology 2007 133, 1928-1937.e3DOI: (10.1053/j.gastro.2007.10.007) Copyright © 2007 AGA Institute Terms and Conditions

Figure 5 Gross phenotype analysis. (A) Measurement of weights at weaning (21 days of age) shows that the CF mice (n = 14) are significantly (P ≤ .05) smaller than wild-type (WT) (n = 24), whereas the TgrIFABP-mCLCA3/CF (CF/Tg+) animals (n = 19) show a marginal (not significant) improvement (n = 20). (B) At 5 weeks of age, CF mice are considerably smaller than WT sibs, and, whereas CF/Tg+ mice appear somewhat larger than age-matched CF, they continue to be smaller than age-matched WT mice. (C) Survival at weaning shows a significant deficiency (12% of genotyped animals compared with the expected 25% from Mendelian ratio) of Cftr−/− (CF) mice without the TgrIFABP-mCLCA3 transgene (left), whereas CF mice with the TgrIFABP-mCLCA3 transgene (right) are near the expected frequency (22% of genotyped animals compared with the expected 25% from Mendelian segregation). (D) Survival of CF mice without the TgrIFABP-mCLCA3 transgene is severely compromised, with less than 70% survival by 4-weeks of age (▲; n = 21). In contrast, CF mice with the TgrIFABP-mCLCA3 transgene (CF/Tg+) display 80% survival at 4 weeks of age (■; n = 34) and over 60% at 8 weeks, indicating a significant (P ≤ .0001) effect of the transgene on survival. Gastroenterology 2007 133, 1928-1937.e3DOI: (10.1053/j.gastro.2007.10.007) Copyright © 2007 AGA Institute Terms and Conditions

Figure 6 Intestinal electrophysiology. Ussing chamber measurements of (A) basal potential difference (PD), (B) A23187-activated (10 μmol/L) response, and (C) forskolin-activated (10 μmol/L) response in ilea of 4-week-old wild-type (WT) (n = 6), wild-type with the TgrIFABP-mCLCA3 transgene (WT/Tg+) (n = 6), CF (n = 6), and CF with the TgrIFABP-mCLCA3 transgene (CF/Tg+) (n = 8). (A) Measure of basal PD of CF mice shows a significant decrease from WT (P < .001), whereas CF/Tg+ ilea show a slight (although not significant, P = .10) improvement. No difference is observed between WT and WT/Tg+ ilea. (B) No A23187-activated response is detected in both CF and CF/Tg+ ilea, although a minor (not significantly different from WT, P = .18) response was observed in WT/Tg+. (C) No forskolin-activated response is detected in CF and CF/Tg+ ilea, and no significant difference (P = .22) is evident between WT and WT/Tg+ ilea. Gastroenterology 2007 133, 1928-1937.e3DOI: (10.1053/j.gastro.2007.10.007) Copyright © 2007 AGA Institute Terms and Conditions