ABCMdb

PMID: 11978765

Oceandy D, McMorran BJ, Smith SN, Schreiber R, Kunzelmann K, Alton EW, Hume DA, Wainwright BJ
Gene complementation of airway epithelium in the cystic fibrosis mouse is necessary and sufficient to correct the pathogen clearance and inflammatory abnormalities.
Hum Mol Genet. 2002 May 1;11(9):1059-67., 2002-05-01 [PubMed]

Sentences

No. Mutations Sentence Comment

2 ABCC7 p.Gly551Asp We generated G551D CF mice transgenically expressing the human CFTR gene in two tissue compartments previously demonstrated to mediate a CFTR-dependent inflammatory response: lung epithelium and alveolar macrophages. Login to comment 24 ABCC7 p.Ser489* CF mice with the null S489X mutation (Cftrm1UNC ) when infected with P. aeruginosa embedded in agar beads produce more proinflammatory cytokines such as tumor necrosis factor a (TNF-a), macrophage inflammatory protein 2 (mip-2) and KC compared with wild-type animals (23). Login to comment 25 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp Another CF mouse model, the G551D mouse (CftrG551D/G551D ), exhibits abnormal lung inflammation in response to bacterial lipopolysaccharide (LPS), and the bone-marrow-derived macrophages (BMM) from these animals demonstrate hypersensitivity to LPS-induced cytokine production (24). Login to comment 28 ABCC7 p.Gly551Asp We generated G551D CF mice expressing human CFTR cDNA in airway epithelia, or in macrophages using lineage-specific promoters. Login to comment 29 ABCC7 p.Gly551Asp The G551D homozygous mice have a reduced capacity to clear bacteria compared with normal mice after pulmonary infection with P. aeruginosa embedded in beads (27). Login to comment 32 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp RESULTS Establishment of the G551D-K18 and G551D-fms bitransgenic mice We used the human cytokeratin 18 (K18) promoter to direct CFTR cDNA expression selectively to respiratory epithelium. Login to comment 45 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp From three transgenic founders bearing the D6.7fms-CFTR transgene, two transmitted the transgene to their offspring. We then mated K18-CFTR mice and D6.7fms-CFTR mice with G551D CF mice (CftrG551D/G551D ). Login to comment 46 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp Then, by intercross breeding of F1 mice, we generated bitransgenic CftrG551D/ G551D K18-CFTR þ / 7 (G551D-K18), which are CF mice expressing human CFTR in the respiratory epithelial cells, and CftrG551D/G551D D6.7fms-CFTR þ / 7 (G551D-fms) which are CF mice expressing human CFTR in macrophages. Login to comment 47 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp Subsequent studies were focused on those mice together with G551D CF mice (CftrG551D/G551D ) and normal mice (CftrG551D/ þ and Cftr þ / þ ) that are the littermates of both G551D-K18 and G551D-fms as controls. Login to comment 62 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp The electrophysiological responses to IBMX and forskolin were examined in excised tracheal and caecal tissues from G551D, G551D-K18, G551D-fms and normal mice. Login to comment 63 ABCC7 p.Gly551Asp In trachea, the change in short-circuit current (DISC) after IBMX/forskolin challenge was significantly reduced in G551D mice compared with normal animals (P < 0.05) (Fig. 3A). Login to comment 64 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp In the G551D-K18 mice, the DISC was significantly higher (P ¼ 0.01) and the average was increased by approximately 2.5 fold compared with G551D animals, indicating that the human transgene had restored function to the murine CF trachea. Login to comment 65 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp By contrast, the IBMX/ forskolin response in the trachea of G551D-fms animals was not different from that in G551D mice, suggesting that expression of the transgene in the macrophages did not restore the chloride channel activity in the trachea. Login to comment 67 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp The forskolin response in the cecum was markedly reduced in G551D compared with normal mice (P < 0.001), and in G551D-K18 mice the DISC was increased by approximately 40% of the value of normal mice and significantly higher (P < 0.05) than G551D animals (Fig. 3B). Login to comment 68 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp As in the trachea, the forskolin response in the cecum of G551D-fms mice was comparable to that in G551D mice. Login to comment 70 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp Expression of human CFTR in airway epithelial cells corrects P. aeruginosa clearance in the lung To test whether complementation of human CFTR in airway epithelial cells or in alveolar macrophages corrected the pathogen clearance defect in CF mice, we studied the effects of chronic lung infection in G551D-K18 and G551D-fms mice. Login to comment 72 ABCC7 p.Gly551Asp After 3 days of infection, G551D mice had a significantly higher (P < 0.05) number of P. aeruginosa in the lung than normal mice (Fig. 4A and Table 1), indicating an impaired clearance capacity. Login to comment 74 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp The bacterial burden in the lung of G551D-K18 mice was comparable to that in normal mice and significantly (P ¼ 0.01) lower than that in G551D mice. However, there was no significant difference between G551D and G551D-fms animals. Login to comment 77 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp One out of 14 (7.1%) G551D mice and 2 out of 11 (18.2%) G551D-fms mice died by 3 days after infection, whereas none of the G551D-K18 and normal mice died (Table 1). Login to comment 78 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp Although it did not reach statistical significance, this result supports the quantitative bacterial clearance assay that G551D and G551D-fms mice are more susceptible to P. aeruginosa lung infection. Login to comment 80 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp Two days after infection, weight loss stabilized in normal and G551D-K18 mice, whereas it continued in G551D and G551D-fms mice (Fig. 4B). Login to comment 81 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp Thus, by day 3 after infection, G551D and G551D-fms mice lost more weight than G551D-K18 and normal mice, although this did not reach statistical significance (P < 0.2). Login to comment 84 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp (A) The response to IBMX and forskolin on in vitro short-circuit current change (DISC) in the trachea of normal (n ¼ 8), G551D (n ¼ 8), G551D-K18 (n ¼ 6) and G551D-fms (n ¼ 4) mice. Login to comment 85 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp (B) Change in ISC in response to forskolin in the cecum of normal (n ¼ 18), G551D (n ¼ 7), G551D-K18 (n ¼ 4) and G551D-fms (n ¼ 4) mice. Login to comment 87 ABCC7 p.Gly551Asp (P < 0.05) elevated in the G551D mice compared with normal animals after 3 days of infection with P. aeruginosa agar beads (Fig. 5). Login to comment 88 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp In the G551D-K18 mice, the concentrations of these molecules were comparable to those in normal mice, and significantly lower (P < 0.01) than there in G551D mice. Login to comment 90 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp Bacterial burden in the lung and mortality after 3 days of P. aeruginosa beads infection Bacterial burden Mortality Mean Median Normal 6.23 Â 106 6.78 Â 103 0/11 (0%) G551D 7.74 Â 107 5.22 Â 105 1/14 (7.1%) G551D-K18 8.17 Â 104 1.56 Â 104 0/13 (0%) G551D-fms 2.88 Â 107 2.88 Â 104 2/11 (18.2%) Values for bacterial burden are expressed in CFU/ml lung homogenates. Login to comment 94 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp (A) Bacterial burden in lung homogenates after 3 days of P. aeruginosa infection in normal (squares, n ¼ 11), G551D (circles, n ¼ 13), G551D-K18 (triangles, n ¼ 13) and G551D-fms (crosses, n ¼ 10) mice. Login to comment 98 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp (B) Change in body weight during P. aeruginosa infection: squares normal mice; circles, G551D; triangles, G551D-K18; crosses, G551D-fms. Login to comment 105 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp Sample size are as follows: normal, n ¼ 11; G551D, n ¼ 13; G551D-K18, n ¼ 12; G551D-fms, n ¼ 9. Login to comment 107 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp If upregulation of the inflammatory response in CF mice is caused by altered CFTR function in macrophages, we anticipated correction in the G551D-fms mice. However, levels of these cytokines were not significantly different in these mice compared to G551D animals. Login to comment 110 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp Thus, the higher concentration of cytokine/chemokines in the BAL fluid of G551D and G551D-fms mice was most likely a consequence of ineffective bacterial clearance by the pulmonary epithelium, resulting in the accumulation of bacterial products and stimulation of macrophages. Login to comment 115 ABCC7 p.Gly551Asp In keeping with these findings, G551D CF mice that carry a type III mutation in the Cftr gene also show reduced bacterial clearance after infection with P. aeruginosa-laden agar beads (27). Login to comment 118 ABCC7 p.Gly551Asp On average, the G551D mice retained 12-fold more bacteria in the lung than normal mice after 3 days of P. aeruginosa infection (Table 1). Login to comment 119 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp In contrast, the bacterial clearance capacity of G551D-K18 mice was comparable to that of normal animals and significantly better than that of G551D mice. However, the bacterial clearance defect was not corrected in the G551D-fms mice - suggesting that expression of exogenous CFTR in macrophages had no effect in eliminating bacterial infection. Login to comment 121 ABCC7 p.Gly551Asp Thomas and co-workers (24) reported that G551D CF mice have an abnormal inflammatory response in the lung as well as in isolated macrophages after bacterial LPS induction. Login to comment 125 ABCC7 p.Gly551Asp However, G551D-fms mice exhibited an excessive inflammatory response after P. aeruginosa infection, as indicated by higher concentrations of TNF-a, mip-2 and KC in BAL fluid. Login to comment 126 ABCC7 p.Gly551Asp In contrast, G551D-K18 mice showed reduced cytokine/chemokine production, comparable to normal levels. Login to comment 133 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp Each symbol represents an individual mouse: squares, normal mice; circles, G551D; triangles, G551D-K18; crosses, G551D-fms. Login to comment 145 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp Female transgenic offspring were mated with CftrG551D/G551D males (129Sv/CD1), which are homozygous for the G551D mutation in the murine Cftr gene (44). Login to comment 148 ABCC7 p.Gly551Asp PCR followed by restriction enzyme digestion was used to identify the G551D mutation as described (44). Login to comment 157 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp Electrophysiological measurements Electrophysiological studies were performed in excised trachea and cecum from G551D, bitransgenic G551D-K18, G551D-fms and normal mice. Login to comment