Absence of the cystic fibrosis transmembrane regulator (Cftr) from myeloid-derived cells slows resolution of inflammation and infection.

The absence or reduction of CFTR function causes CF and results in a pulmonary milieu characterized by bacterial colonization and unresolved inflammation. The ineffectiveness at controlling infection by species such as Pseudomonas aeruginosa suggests defects in innate immunity. Macrophages, neutrophils, and DCs have all been shown to express CFTR mRNA but at low levels, raising the question of whether CFTR has a functional role in these cells. Bone marrow transplants between CF and non-CF mice suggest that these cells are inherently different; we confirm this observation using conditional inactivation of Cftr in myeloid-derived cells. Mice lacking Cftr in myeloid cells overtly appear indistinguishable from non-CF mice until challenged with bacteria instilled into the lungs and airways, at which point, they display survival and inflammatory profiles intermediate in severity as compared with CF mice. These studies demonstrate that Cftr is involved directly in myeloid cell function and imply that these cells contribute to the pathophysiological phenotype of the CF lung.

Understanding the population structure of North American patients with cystic fibrosis.

It is generally presumed that the cystic fibrosis (CF) population is relatively homogeneous, and predominantly of European origin. The complex ethnic make-up observed in the CF patients collected by the North American CF Modifier Gene Consortium has brought this assumption into question, and suggested the potential for population substructure in the three CF study samples collected from North America. It is well appreciated that population substructure can result in spurious genetic associations. To understand the ethnic composition of the North American CF population, and to assess the need for population structure adjustment in genetic association studies with North American CF patients, genome-wide single-nucleotide polymorphisms on 3076 unrelated North American CF patients were used to perform population structure analyses. We compared self-reported ethnicity to genotype-inferred ancestry, and also examined whether geographic distribution and cystic fibrosis transmembrane regulator (CFTR) mutation type could explain the population structure observed. Although largely Caucasian, our analyses identified a considerable number of CF patients with admixed African-Caucasian, Mexican-Caucasian and Indian-Caucasian ancestries. Population substructure was present and comparable across the three studies of the consortium. Neither geographic distribution nor CFTR mutation type explained the population structure. Given the ethnic diversity of the North American CF population, it is essential to carefully detect, estimate and adjust for population substructure to guard against potential spurious findings in CF genetic association studies. Other Mendelian diseases that are presumed to predominantly affect single ethnic groups may also benefit from careful analysis of population structure.

A susceptibility gene for type 2 diabetes confers substantial risk for diabetes complicating cystic fibrosis.

AIMS/HYPOTHESIS: Insulin-requiring diabetes affects 25-50% of young adults with cystic fibrosis (CF). Although the cause of diabetes in CF is unknown, recent heritability studies in CF twins and siblings indicate that genetic modifiers play a substantial role. We sought to assess whether genes conferring risk for diabetes in the general population may play a risk modifying role in CF. METHODS: We tested whether a family history of type 2 diabetes affected diabetes risk in CF patients in 539 families in the CF Twin and Sibling family-based study. A type 2 diabetes susceptibility gene (transcription factor 7-like 2, or TCF7L2) was evaluated for association with diabetes in CF using 998 patients from the family-based study and 802 unrelated CF patients in an independent case-control study. RESULTS: Family history of type 2 diabetes increased the risk of diabetes in CF (OR 3.1; p = 0.0009). A variant in TCF7L2 associated with type 2 diabetes (the T allele at rs7903146) was associated with diabetes in CF in the family study (p = 0.004) and in the case-control study (p = 0.02; combined p = 0.0002). In the family-based study, variation in TCF7L2 increased the risk of diabetes about three-fold (HR 1.75 per allele, 95% CI 1.3-2.4; p = 0.0006), and decreased the mean age at diabetes diagnosis by 7 years. In CF patients not treated with systemic glucocorticoids, the effect of TCF7L2 was even greater (HR 2.9 per allele, 95% CI 1.7-4.9, p = 0.00011). CONCLUSIONS/INTERPRETATION: A genetic variant conferring risk for type 2 diabetes in the general population is a modifier of risk for diabetes in CF.

Modulation of cystic fibrosis lung disease by variants in interleukin-8.

Cystic fibrosis pulmonary disease is characterized by excessive and prolonged inflammation. CF Pulmonary disease severity exhibits considerable variation that, to some extent, appears to be due to the presence of modifier genes. Several components of the inflammatory response are known to have altered regulation in the CF lung. Genetic variants in 52 inflammatory genes were tested for associations with lung disease indices in a CF patient population (n=737) homozygous for the DeltaF508 cystic fibrosis transmembrane conductance regulator mutation. Variants in three inflammatory genes showed significant genotypic associations with CF lung disease severity, including IL8 and previously reported TGFbeta1 (P< or =0.05). When analyzed by gender, it was apparent that IL8 variant associations were predominantly due to males. The IL8 variants were tested in an additional CF population (n=385) and the association in males verified (P< or =0.01). The IL8 variants were in strong linkage disequilibrium with each other (R2> or =0.82), while variants in neighboring genes CXCL6, RASSF6 and PF4V1 did not associate (P> or =0.26) and were in weaker LD with each other and with the IL8 variants (0.01< or =R2< or =0.49). Studies revealed differential expression between the IL8 promoter variant alleles (P<0.001). These results suggest that IL8 variants modify CF lung disease severity and have functional consequences.

The cystic fibrosis transmembrane conductance regulator (Cftr) modulates the timing of puberty in mice.

BACKGROUND: Delayed puberty is common among individuals with cystic fibrosis (CF) and is usually attributed to chronic disease and/or poor nutrition. However, it has recently been recognised that pubertal delay can occur even in the setting of good nutritional and clinical status. This finding, along with evidence that Cftr is expressed in rat brain, human hypothalamus, and a gonadotropin releasing hormone secreting cell line, raises the possibility that some of the pubertal delay in CF could stem directly from alterations in Cftr function that affect the hypothalamic-pituitary-gonadal axis. METHODS: To examine this hypothesis, we investigated pubertal timing (as assessed by vaginal opening (VO)) in a mouse model of CF (Cftr(tm1Unc)) engineered to produce a truncated Cftr mRNA and referred to as S489X. Homozygous knockout, heterozygote, and wild type (WT) female mice were examined. RESULTS: As expected, the S489X-/S489X- knockout mice, which have chronic inflammation and gastrointestinal disease, grew more slowly and had later onset of puberty than WT animals. We anticipated that the S489X-/S489X+ heterozygotes, which have no clinical CF phenotype, might display an intermediate timing of puberty. Surprisingly, however, these mice had earlier VO than WT. These findings were confirmed in a second, independent model of CF engineered to generate the deltaF508 mutation in mice. Again, the homozygotes displayed later pubertal timing, while the heterozygotes displayed earlier VO than the WT animals. CONCLUSIONS: These data provide further evidence that Cftr can directly modulate the reproductive endocrine axis and raise the possibility that heterozygote mutation carriers may have a reproductive advantage.

Modifier genes and variation in cystic fibrosis.

The availability of molecular tools to carry out genotyping has led to a flurry of association studies between specific genes and clinical indices of disease or disease susceptibility. Human studies, for the most part, have a limited number of subjects available, precluding whole genome types of approaches. 'Candidate gene' strategies have consequently become widespread, probably in part due to the inherent similarity to clinical association studies. Such studies in cystic fibrosis have found tantalizing results in genes involved in infection and inflammation, but many other relevant pathways remain untapped. Genome scanning approaches may eventually uncover genes not currently recognized as important to cystic fibrosis. In the meantime, while thousands of polymorphisms are cataloged and other genomic resources become more available, the number of association studies with candidate genes will no doubt increase. To make sense of these studies, the choice of gene and phenotype must be carefully considered.

Examining basal chloride transport using the nasal potential difference response in a murine model.

Epithelia of humans and mice with cystic fibrosis are unable to secrete chloride in response to a chloride gradient or to cAMP-elevating agents. Bioelectrical properties measured using the nasal transepithelial potential difference (TEPD) assay are believed to reflect these cystic fibrosis transmembrane conductance regulator (CFTR)-dependent chloride transport defects. Although the response to forskolin is CFTR mediated, the mechanisms responsible for the response to a chloride gradient are unknown. TEPD measurements performed on inbred mice were used to compare the responses to low chloride and forskolin in vivo. Both responses show little correlation between or within inbred strains of mice, suggesting they are mediated through partially distinct mechanisms. In addition, these responses were assayed in the presence of several chloride channel inhibitors, including DIDS, diphenylamine-2-carboxylate, glibenclamide, and 5-nitro-2-(3-phenylpropylamino)-benzoic acid, and a protein kinase A inhibitor, the Rp diastereomer of adenosine 3',5'-cyclic monophosphothioate (Rp-cAMPS). The responses to low chloride and forskolin demonstrate significantly different pharmacological profiles to both DIDS and Rp-cAMPS, indicating that channels in addition to CFTR contribute to the low chloride response.

Construction of chromosome jumping and linking libraries in E. coli.

Chromosome jumping allows the use of one point on a chromosome as a starting point for exploring another potentially distant point on the same chromosome without cloning the intervening sequences as in chromosome walking. A linking library consists of genomic fragments that each contain a particular restriction site with a marker inserted into it. Because the sequences flanking the marker represent the termini of two contiguous genomic restriction fragments, the linking clones can be used, in conjunction with a specific jumping library made with the same restriction enzyme, to expedite jumping between consecutive restriction fragments. Separate protocols present strategies for constructing general and specific jumping libraries from total genomic DNA. An additional protocol details construction of a linking library from flow-sorted chromosomes. Support protocols provide instructions for preparing genomic insert DNA, supF gene fragments, and Chromosome jumping allows the use of one point on a chromosome as a starting point for exploring another potentially distant.

Stimulation of cystic fibrosis transmembrane conductance regulator-dependent short-circuit currents across DeltaF508 murine intestines.

BACKGROUND & AIMS: The cystic fibrosis transmembrane conductance regulator (CFTR) can be activated by pharmacological manipulation of the protein kinase A pathway in cell lines. Our goals were to stimulate wild-type CFTR in murine intestines via isoform-specific phosphodiesterase inhibition or protein kinase A activation and to apply the optimal stimulus to activate chloride secretion from homozygous DeltaF508 jejunum. METHODS: The response of T84 cells and sections of murine intestine to various inhibitors and activators was examined by Ussing chamber experiments. RESULTS: Maximal chloride secretion can be activated in T84 cells with application of class III phosphodiesterase inhibitors and in wild-type murine intestines with class I or III phosphodiesterase inhibitors or with activators of type II protein kinase A. Chloride secretion can be stimulated from homozygous DeltaF508 murine jejunum using a mixture of inhibitors and activators. CONCLUSIONS: DeltaF508 CFTR can be activated to levels 4% of wild-type when the combination of protein kinase A type II activators and phosphodiesterase class I and III inhibitors are used in murine jejunum. This result suggests that partial CFTR-mediated electrolyte transport can be restored in DeltaF508 murine jejunum by application of specific pharmacological agents.

Pitfall in the use of genotype analysis as the sole diagnostic criterion for cystic fibrosis.

In this report, we present an asymptomatic infant, seen for a second opinion, who was given the diagnosis of cystic fibrosis (CF) as a neonate based on the presence of two mutant alleles, DeltaF508 and R117H. The diagnosis of CF adversely affected the family's emotional, employment, and financial statuses. Our evaluation included sweat chloride, nasal transepithelial potential difference, and bronchoscopy with bronchoalveolar lavage measurements, all which were consistent with findings expected from an individual without CF. Genotype analysis for the sequence polymorphism in intron 8 of the cystic fibrosis transmembrane conductance regulator (CFTR) gene revealed the 7 thymidines and 9 thymidines alleles. We conclude that this patient probably expresses enough epithelial cell surface CFTR function such that she has a normal phenotype. Based on our evaluation, she does not meet the current diagnostic criteria for CF. Although genotype analysis can be an useful adjunct, it should not be the sole diagnostic criterion for CF.

Nitric oxide-mediated regulation of transepithelial sodium and chloride transport in murine nasal epithelium.

Transepithelial ion transport is regulated by a variety of cellular factors. In light of recent evidence that nitric oxide (NO) production is decreased in cystic fibrosis airways, we examined the role of NO in regulating sodium and chloride transport in murine nasal epithelium. Acute intervention with the inducible NO synthase (iNOS)-selective inhibitor S-methylisothiourea resulted in an increase of amiloride-sensitive sodium absorption observed as a hyperpolarization of nasal transepithelial potential difference. Inhibition of iNOS expression with dexamethasone also hyperpolarized transepithelial potential difference, but only a portion of this increase proved to be amiloride sensitive. Chloride secretion was significantly inhibited in C57BL/6J mice by the addition of both S-methylisothiourea and dexamethasone. Mice lacking iNOS expression [NOS2(-/-)] also had a decreased chloride-secretory response compared with control mice. These data suggest that constitutive NO production likely plays some role in the downregulation of sodium absorption and leads to an increase in transepithelial chloride secretion.

Inducible nitric oxide synthase expression is reduced in cystic fibrosis murine and human airway epithelial cells.

It has been reported that exhaled nitric oxide levels are reduced in cystic fibrosis (CF) patients. We have examined the inducible isoform of nitric oxide synthase (iNOS) in the airways by immunostaining and found that iNOS is constitutively expressed in the airway epithelia of non-CF mouse and human tissues but essentially absent in the epithelium of CF airways. We explored potential consequences of lost iNOS expression and found that iNOS inhibition significantly increases mouse nasal trans-epithelial potential difference, and hindered the ability of excised mouse lungs to prevent growth of Pseudomonas aeruginosa. The absence of continuous nitric oxide production in epithelial cells of CF airways may play a role in two CF-associated characteristics: hyperabsorption of sodium and susceptibility to bacterial infections.

Regulation of amiloride-sensitive sodium absorption in murine airway epithelium by C-type natriuretic peptide.

We have previously shown that C-type natriuretic peptide (CNP), a guanylate cyclase agonist, can stimulate cystic fibrosis transmembrane conductance regulator (CFTR)-mediated chloride secretion in murine airway epithelial cells via protein kinase (PK) A activation through the inhibition of cGMP-inhibited phosphodiesterases. In this paper, we show that CNP is also capable of reducing amiloride-sensitive sodium absorption in murine airway epithelium through a cGMP-dependent mechanism that is separate from the CFTR regulatory signaling pathway. Both murine tracheal and nasal tissues exhibit sensitivity to amiloride-sensitive sodium regulation by exogenously added CNP. CNP depolarized the nasal transepithelial potential difference by 6.3 +/- 0.5 mV, whereas the cGMP-inhibited phosphodiesterase inhibitor milrinone actually hyperpolarized the nasal transepithelial potential difference by 2.0 +/- 1.2 mV in mice homozygous for a CFTR stop mutation [CFTR(-/-)]. Inhibition of guanylate cyclase activity and PKG activity in normal mice resulted in an increase in amiloride-sensitive sodium absorption, suggesting that tonic regulation of amiloride-sensitive sodium absorption is in part due to basal cGMP levels and PKG activity.

Type II protein kinase A regulates CFTR in airway, pancreatic, and intestinal cells.

The type of protein kinase A (PKA) responsible for cystic fibrosis transmembrane conductance regulator (CFTR) activation was determined with adenosine 3', 5'-cyclic monophosphate analogs capable of selectively activating type I or type II PKA. The type II-selective pair stimulated chloride efflux in airway, pancreatic, and colonic epithelial cells; the type I-selective pair only stimulated a calcium-dependent efflux in airway cells. The type II-selective analogs activated larger increases in CFTR-mediated current than did the type I-selective analogs. Measurement of soluble PKA activity demonstrated similar levels stimulated by type I- and type II-selective analogs, creating an apparent paradox regarding PKA activity and current generated. Also, addition of forskolin after the type I-selective analogs resulted in an increase in current; little increase was seen after the type II-selective analogs. Measurement of insoluble PKA activity stimulated by the analogs resolved this paradox. Type II-selective analogs stimulated three times as much insoluble PKA activity as the type I-selective pair, indicating that differential activation of PKA in cellular compartments is important in CFTR regulation.

Cystic fibrosis transmembrane conductance regulator (CFTR) anion binding as a probe of the pore.

We compared the effects of mutations in transmembrane segments (TMs) TM1, TM5, and TM6 on the conduction and activation properties of the cystic fibrosis transmembrane conductance regulator (CFTR) to determine which functional property was most sensitive to mutations and, thereby, to develop a criterion for measuring the importance of a particular residue or TM for anion conduction or activation. Anion substitution studies provided strong evidence for the binding of permeant anions in the pore. Anion binding was highly sensitive to point mutations in TM5 and TM6. Permeability ratios, in contrast, were relatively unaffected by the same mutations, so that anion binding emerged as the conduction property most sensitive to structural changes in CFTR. The relative insensitivity of permeability ratios to CFTR mutations was in accord with the notion that anion-water interactions are important determinants of permeability selectivity. By the criterion of anion binding, TM5 and TM6 were judged to be likely to contribute to the structure of the anion-selective pore, whereas TM1 was judged to be less important. Mutations in TM5 and TM6 also dramatically reduced the sensitivity of CFTR to activation by 3-isobutyl 1-methyl xanthine (IBMX), as expected if these TMs are intimately involved in the physical process that opens and closes the channel.

In vivo activation of CFTR-dependent chloride transport in murine airway epithelium by CNP.

Inhibitors of guanosine 3',5'-cyclic monophosphate (cGMP)-inhibited phosphodiesterases stimulate Cl- transport across the nasal epithelia of cystic fibrosis mice carrying the delta F508 mutation [cystic fibrosis transmembrane conductance regulator (CFTR) (delta F/delta F)], suggesting a role for cGMP in regulation of epithelial ion transport. Here we show that activation of membrane-bound guanylate cyclases by C-type natriuretic peptide (CNP) stimulates hyperpolarization of nasal epithelium in both wild-type and delta F508 CFTR mice in vivo but not in nasal epithelium of mice lacking CFTR [CFTR(-/-)]. With the use of a nasal transepithelial potential difference (TEPD) assay, CNP was found to hyperpolarize lumen negative TEPD by 6.1 +/- 0.6 mV in mice carrying wild-type CFTR. This value is consistent with that obtained with 8-bromoguanosine 3',5'-cyclic monophosphate (6.2 +/- 0.9 mV). A combination of the adenylate cyclase agonist forskolin and CNP demonstrated a synergistic ability to induce Cl- secretion across the nasal epithelium of CFTR(delta F/delta F) mice. No effect on TEPD was seen with this combination when used on CFTR(-/-) mice, implying that the CNP-induced change in TEPD in CFTR(delta F/delta F) mice is CFTR-dependent.

Function of the R domain in the cystic fibrosis transmembrane conductance regulator chloride channel.

For a cystic fibrosis transmembrane conductance regulator (CFTR) channel to enter its open state, serine residues in the R domain must be phosphorylated by cAMP-dependent protein kinase, and intracellular ATP must bind to the nucleotide-binding folds and subsequently be hydrolyzed. CFTR with its R domain partially removed, DeltaR(708-835)-CFTR, forms a chloride channel that opens independently of protein kinase A phosphorylation, with open probability approximately one-third that of the wild type CFTR channel. Deletion of this portion of the R domain from CFTR alters the response of the channel to 5'-adenylylimidodiphosphate, pyrophosphate, and vanadate, compounds that prolong burst duration of the wild type CFTR channel but fail to do so in the DeltaR-CFTR. In addition, the addition of exogenous unphosphorylated R domain protein, which blocks the wild type CFTR channel, has no effect on the DeltaR-CFTR channel. However, when the exogenous R domain is phosphorylated, significant stimulation of the DeltaR-CFTR channel results; Po increases from 0.10 to 0.22. These data are consistent with a model for CFTR function in which the R domain in the unphosphorylated state interacts with the first nucleotide binding fold to inhibit either binding or hydrolysis of ATP or transduction of the effect to open the pore, but when the R domain is phosphorylated, it undergoes conformational change and interacts at a separate site in the first nucleotide binding fold to stimulate either binding or hydrolysis of ATP or transduction of the effect to open the pore.

C-type natriuretic peptide increases chloride permeability in normal and cystic fibrosis airway cells.

C-type natriuretic peptide (CNP), a hormone which stimulates particulate guanylate cyclase activity, was studied for its ability to stimulate chloride permeability through the cystic fibrosis transmembrane conductance regulator (CFTR) in airway epithelial cells. Two cell lines, Calu-3 and CF-T43, were used as models of normal and cystic fibrosis (CF) airway epithelial cells, respectively. Calu-3 cells, derived from a lung carcinoma, express relatively high levels of wild-type CFTR. CF-T43 is a transformed line derived from a nasal polyp and expresses the mutant CFTR, deltaF508. Calu-3 cells exposed to the nucleotide guanosine-3',5'-monophosphate (cGMP) analogue 8-Br-cGMP exhibit increased 36Cl- efflux, demonstrating that cGMP can mediate changes in chloride permeability. CNP induces a bumetanide-sensitive short circuit current across Calu-3 monolayers. Whole-cell currents stimulated by CNP display linear current-voltage relationships and have inhibitor pharmacology and ion selectivity consistent with CFTR channel activity. Sodium nitroprusside (SNP), an activator of soluble guanylate cyclase, and CNP both increase cGMP levels and short circuit current in Calu-3 cells. In contrast, exposure of CF-T43 cells to CNP resulted in an increased 36Cl- efflux rate only when combined with the adenylate cyclase agonist isoproterenol and the response was sensitive to kinase inhibitors. CF-T43 cells exposed to isoproterenol and SNP showed no increase in chloride efflux. Together, these data indicate that CNP can activate wild-type and mutant CFTR through a cAMP-dependent protein kinase pathway and that the sensitivity of Calu-3 cells for this stimulation is greater than that of the CF-T43 cells.

In vivo activation of the cystic fibrosis transmembrane conductance regulator mutant deltaF508 in murine nasal epithelium.

The gene causing cystic fibrosis (CF) encodes the CF transmembrane conductance regulator (CFTR), a cAMP-regulated chloride channel. Mutations in this gene result in reduced transepithelial chloride permeability across tissues affected in CF. Consequently, restoring chloride permeability to these tissues may prove therapeutic. Here we report that a combination of forskolin, an adenylate cyclase activator, and milrinone, an inhibitor of class III phosphodiesterases, increases the magnitude of the potential difference across nasal epithelium of mice homozygous for the most common CF mutation, delta F508, while neither drug alone has a significant effect on potential difference. Transgenic mice lacking CFTR do not respond to the milrinone/forskolin combination, indicating that the effect in delta F508 mice requires CFTR. These results suggest that, by pharmacological means, at least partial CFTR-mediated electrolyte transport can be restored in vivo to CF tissues expressing delta F508.

Human epithelial cystic fibrosis transmembrane conductance regulator without exon 5 maintains partial chloride channel function in intracellular membranes.

The cardiac isoform of the cystic fibrosis transmembrane conductance regulator (CFTR) is a splice variant of the epithelial CFTR, with lacks 30 amino acids encoded by exon 5 in the first intracellular loop. For examination of the role of exon 5 in CFTR channel function, a CFTR deletion mutant, in which exon 5 was removed from the human epithelial CFTR, was constructed. The wild type and delta exon5 CFTR were expressed in a human embryonic kidney cell line (293 HEK). Fully mature glycosylated CFTR (approximately 170 kDa) was immunoprecipitated from cells transfected with wild type CFTR cDNA, whereas cells transfected with delta exon5 CFTR express only a core-glycosylated from (approximately 140 kDa). The Western blot test performed on subcellular membrane fractions showed that delta exon5 CFTR was located in the intracellular membranes. Neither incubation at lower temperature (26 degrees C) nor stimulation of 293 HEK cells with forskolin or CPT-cAMP caused improvement in glycosylation and processing of delta exon5 CFTR proteins, indicating that the human epithelial CFTR lacking exon5 did not process properly in 293 HEK cells. On incorporation of intracellular membrane vesicles containing the delta exon5 CFTR proteins into the lipid bilayer membrane, functional phosphorylation- and ATP-dependent chloride channels were identified. CFTR channels with an 8-pS full-conductance state were observed in 14% of the experiments. The channel had an average open probability (Po) of 0.098 +/- 0.022, significantly less than that of the wild type CFTR (Po = 0.318 +/- 0.028). More frequently, the delta exon5 CFTR formed chloride channels with lower conductance states of approximately 2-3 and approximately 4-6 pS. These subconductance states were also observed with wild type CFTR but to a much lesser extent. Average Po for the 2-3-pS subconductance state, estimated from the area under the curve on an amplitude histogram, was 0.461 +/- 0.194 for delta exon5 CFTR and 0.332 +/- 0.142 for wild type (p = 0.073). The data obtained indicate that deleting 30 amino acids from the first intracellular loop of CFTR affects both processing and function of the CFTR chloride channel.