Spontaneous rescue from cystic fibrosis in a mouse model

Abstract

Background: From the original CftrTgH(neoim)Hgu mutant mouse model with a divergent genetic background (129P2, C57BL/6, MF1) we have generated two inbred CftrTgH(neoim)Hgu mutant strains named CF/1-CftrTgH(neoim)Hgu and CF/3-CftrTgH(neoim)Hgu, which are fertile and show normal growth and lifespan. Initial genome wide scan analysis with microsatellite markers indicated that the two inbred strains differed on the genetic level. In order to further investigate whether these genetic differences have an impact on the disease phenotype of cystic fibrosis we characterised the phenotype of the two inbred strains.

Results: Reduced amounts, compared to wild type control animals, of correctly spliced Cftr mRNA were detected in the nasal epithelia, lungs and the intestine of both inbred CftrTgH(neoim)Hgu strains, with higher residual amount observed for CF/1-CftrTgH(neoim)Hgu than CF/3-CftrTgH(neoim)Hgu for every investigated tissue. Accordingly the amounts of wild type Cftr protein in the intestine were 9% for CF/1-CftrTgH(neoim)Hgu and 4% for CF/3-CftrTgH(neoim)Hgu. Unlike the apparent strain and/or tissue specific regulation of Cftr mRNA splicing, short circuit current measurements in the respiratory and intestinal epithelium revealed that both strains have ameliorated the basic defect of cystic fibrosis with a presentation of a normal electrophysiology in both tissues.

Conclusion: Unlike the outbred CftrTgH(neoim)Hgu insertional mouse model, which displayed the electrophysiological defect in the gastrointestinal and respiratory tracts characteristic of cystic fibrosis, both inbred CftrTgH(neoim)Hgu strains have ameliorated the electrophysiological defect. On the basis of these findings both CF/1-CftrTgH(neoim)Hgu and CF/3-CftrTgH(neoim)Hgu offer an excellent model whereby determination of the minimal levels of protein required for the restoration of the basic defect of cystic fibrosis can be studied, along with the modulating factors which may affect this outcome.

Figure 1

Quantification of wild type Cftr protein in apical membranes (only band C present) of enterocytes of F₂₈ CF/1-Cftr^{TgH(neoim)Hgu} (n = 4) and F₂₇ CF/3-Cftr^{TgH(neoim)Hgu} (n = 4) mutant mice. Blots were labelled using the Cftr specific R3195 antibody.

Figure 2

Alcian blue staining of deparaffinised sections of jejunum (A, B, C, magnification ×200) and ileum (D, E, F, magnification ×400) from wild type MF1 (A, D) and mutant F₂₈ CF/1-Cftr^{TgH(neoim)Hgu} (B, E) and F₂₇ CF/3-Cftr^{TgH(neoim)Hgu} (C, F) animals showed focal hypertrophy of goblet cells in the ileum of the two inbred Cftr^{TgH(neoim)Hgu} mutant mice. Goblet cell counts are given as percentage of all epithelial cells. From each strain two mice and three sections/mouse were counted. Goblet cell count: CF/1 = 22.7% ± 2.3 (p = 0.112, p-value CF/1 compared to MF1), CF/3 = 23% ± 2.8 (p = 0.172, p-value CF/3 compared to MF1), MF1 = 16.8% ± 1.2. The jejunal sections of the same mice did not show any signs of goblet cell hypertrophy and were similar to wild type MF1 (Goblet cell count: CF/1 = 7.4% ± 0.4 (p = 0.18, p-value CF/1 compared to MF1), CF/3 = 8.5% ± 1.2 (p = 0.77, p-value CF/3 compared to MF1), MF1 = 8.8% ± 0.7.

Figure 3

Immunohistological analysis of Cftr^{TgH(neoim)Hgu} Cftr expression. Immunocytochemical staining of Cftr in the duodenum, jejunum, ileum of wild type mice MF1 (A, B, C), duodenum (E, magnification ×200), jejunum (F, magnification ×200) and ileum (G, magnification ×400) of homozygous F₂₈ CF/1-Cftr^{TgH(neoim)Hgu} and duodenum (H, magnification ×200), jejunum (I, magnification ×200) and ileum (J, magnification ×400) of F₂₇ CF/3-Cftr^{TgH(neoim)Hgu} mice was performed with the R3195 anti-Cftr antibody as described in Methods. Wild type mice showed intense Cftr staining of the apical border of epithelial cells. In CF/1-Cftr^{TgH(neoim)Hgu} and CF/3-Cftr^{TgH(neoim)Hgu} mutant mice Cftr staining was weaker and more diffuse mainly in the upper villus region. Ileum of a CF-Knock out mouse (D) was used as negative control.

Figure 4

Cftr immunostaining in the nasal epithelium of wild type MF1 (A, magnification ×400), C57BL/6J (B, ×400), F₃₀ CF/1-Cftr^{TgH(neoim)Hgu} (C ×400), F₂₉ CF/3-Cftr^{TgH(neoim)Hgu} (D, ×400).

Figure 5

Short-circuit current (Isc) measurements. delta Isc represents the change in short-circuit current from baseline after addition of either forskolin, genistein, carbachol, or amiloride. Upper panel. Bioelectric characteristics of the F₂₈ CF/1-Cftr^{TgH(neoim)Hgu} (n = 4) and F₂₇ CF/3-Cftr^{TgH(neoim)Hgu} (n = 4) in the ileum compared with those of wild type MF1 (n = 5) and C57BL/6J (n = 4) mice. *, significant p-values compared to MF-1. Lower panel. Bioelectric characteristics of the F₂₈ CF/1-Cftr^{TgH(neoim)Hgu} and F₂₇ CF/3-Cftr^{TgH(neoim)Hgu} in the nasal epithelium compared with those of wild type MF1 and C57BL/6J. The amiloride response is significantly increased in both CF/1-Cftr^{TgH(neoim)Hgu} and CF/3-Cftr^{TgH(neoim)Hgu} mice compared to MF1 wild type outbred mice. C57BL/6J inbred wild type mice exhibited less chloride secretion than MF1. The forskolin evoked chloride secretion in both CF/1-Cftr^{TgH(neoim)Hgu} Cftr^{TgH(neoim)Hgu} and CF/3-Cftr^{TgH(neoim)Hgu} was reduced compared to wild type MF1 but elevated compared to wild type C57BL/6J inbred mice.