CFTR-KO Mice Research Articles

Fecal dysbiosis and inflammation in intestinal-specific Cftr knockout mice on regimens preventing intestinal obstruction.

Chronic intestinal inflammation is a poorly understood manifestation of cystic fibrosis (CF), which may be refractory to ion channel CF transmembrane conductance regulator (CFTR) modulator therapy. People with CF exhibit intestinal dysbiosis, which has the potential for stimulating intestinal and systemic inflammation. CFTR is expressed in organ epithelia, leukocytes, and other tissues. Here, we investigate the contribution of intestinal epithelium-specific loss of Cftr [iCftr knockout (KO)] to dysbiosis and inflammation in mice treated with either of two antiobstructive dietary regimens necessary to maintain CF mouse models [polyethylene glycol (PEG) laxative or a liquid diet (LiqD)]. Feces collected from iCftr KO mice and their wild-type (WT) sex-matched littermates were used to measure fecal calprotectin to evaluate inflammation and to perform 16S rRNA sequencing to characterize the gut microbiome. Fecal calprotectin was elevated in iCftr KO relative to WT mice that consumed either PEG or LiqD. PEG iCftr KO mice did not show a change in α diversity versus WT mice but demonstrated a significant difference in microbial composition (β diversity) with included increases in the phylum Proteobacteria, the family Peptostreptococcaceae, four genera of Clostridia including C. innocuum, and the mucolytic genus Akkermansia. Fecal microbiome analysis of LiqD-fed iCftr KO mice showed both decreased α diversity and differences in microbial composition with increases in the Proteobacteria family Enterobacteriaceae, Firmicutes families Clostridiaceae and Peptostreptococcaceae, and enrichment of Clostridium perfringens, C. innocuum, C. difficile, mucolytic Ruminococcus gnavus, and reduction of Akkermansia. It was concluded that epithelium-specific loss of Cftr is a major driver of CF intestinal dysbiosis and inflammation with significant similarities to previous studies of pan Cftr KO mice.NEW & NOTEWORTHY Chronic intestinal inflammation is a manifestation of cystic fibrosis (CF), a disease caused by loss of the anion channel CF transmembrane conductance regulator (CFTR) that is expressed in many tissues. This study shows that intestinal epithelial cell-specific loss of CFTR [inducible Cftr knockout (KO)] in mice is sufficient to induce intestinal dysbiosis and inflammation. Experiments were performed on mice consuming two dietary regimens routinely used to prevent obstruction in CF mice.

EVALUATING THE ROLE OF GOBLET CELL ASSOCIATED ANTIGEN PASSAGES (GAPS) IN THE DEVELOPMENT OF MUCOSAL IMMUNE TOLERANCE IN THE CFTR KO INTESTINE

Greater than 70,000 individuals worldwide are living with the monogenetic disease cystic fibrosis (CF). The development of chronic intestinal inflammation, with clinical signs resembling inflammatory bowel disease-like conditions, is a common yet poorly understood occurrence in CF patients. This inflammation is typically neutrophilic in human and animal models with a heightened basal pro-inflammatory cytokine release. Prior research utilizing intestinal organoids (enteroids) cultured from Cftr knockout mice has shown that goblet cells in the CF mouse intestine demonstrate defective clearance of mucin granules and abnormal mucus retention. Goblet cell-associated antigen passages (GAPs), located in the small intestine and colon, deliver intraluminal antigens to antigen-presenting dendritic cells in the submucosa. This mechanism serves as an important step in the development and maintenance of tolerogenic dendritic cell populations expressing receptors to luminal antigens with involvement of regulatory T cell activation and release of IL-10. We hypothesized that mucus plugging of goblet cells in the CF intestine leads to defective GAP formation and a consequent decrease in the expansion of tolerogenic dendritic cells. To test this hypothesis, Cftrtm1Unc (Cftr KO) and wild type (WT) sex-matched littermate pairs (n=2) maintained on a commercially available liquid diet (Peptamen®) were anesthetized with ketamine/xylazine for a laparotomy to inject a luminal fluorescent 10kD dextran dye into the mid-jejunum. After 30 min, the mice were euthanized with CO2, and the intestine was collected for immunofluorescent staining to evaluate GAP formation. In the WT intestine, the dextran dye was observed within goblet cells outlined by CK18 immunofluorescence, a goblet cell marker. Punctate dextran dye was observed in the submucosa, suggestive of dendritic cell uptake. In contrast, the Cftr KO mice demonstrated defective GAP formation, i.e., without dye penetration of goblet cells, and the lack of punctate dextran fluorescence in the submucosa. To evaluate the population of tolerogenic dendritic cells, small intestinal segments from Cftr KO-WT sex-matched littermate pairs (3-female and 2-male pairs) were collected for FACS sorting of submucosal CD103+ (tolerogenic) and CD103- (pro-inflammatory) dendritic cells. The WT mice had a significantly higher population of CD103+ tolerogenic dendritic cells compared to the CF mice (WT: 20.5+/-2, CF: 9.2+/-3, P < 0.006). A trend towards an increase in CD103- dendritic cells was seen in the CF intestine. In summary, the CF mice were found to have defective intraluminal antigen transfer through the GAP pathway and a significant decrease in tolerogenic dendritic cells in the intestine.

EVALUATING THE ROLE OF GOBLET CELL ASSOCIATED ANTIGEN PASSAGES (GAPS) IN THE DEVELOPMENT OF MUCOSAL IMMUNE TOLERANCE IN THE CFTR KO INTESTINE

Abstract Greater than 70,000 individuals worldwide are living with the monogenetic disease cystic fibrosis (CF). The development of chronic intestinal inflammation, with clinical signs resembling inflammatory bowel disease-like conditions, is a common yet poorly understood occurrence in CF patients. This inflammation is typically neutrophilic in human and animal models with a heightened basal pro-inflammatory cytokine release. Prior research utilizing intestinal organoids (enteroids) cultured from Cftr knockout mice has shown that goblet cells in the CF mouse intestine demonstrate defective clearance of mucin granules and abnormal mucus retention. Goblet cell-associated antigen passages (GAPs), located in the small intestine and colon, deliver intraluminal antigens to antigen-presenting dendritic cells in the submucosa. This mechanism serves as an important step in the development and maintenance of tolerogenic dendritic cell populations expressing receptors to luminal antigens with involvement of regulatory T cell activation and release of IL-10. We hypothesized that mucus plugging of goblet cells in the CF intestine leads to defective GAP formation and a consequent decrease in the expansion of tolerogenic dendritic cells. To test this hypothesis, Cftrtm1Unc (Cftr KO) and wild type (WT) sex-matched littermate pairs (n=2) maintained on a commercially available liquid diet (Peptamen®) were anesthetized with ketamine/xylazine for a laparotomy to inject a luminal fluorescent 10kD dextran dye into the mid-jejunum. After 30 min, the mice were euthanized with CO2, and the intestine was collected for immunofluorescent staining to evaluate GAP formation. In the WT intestine, the dextran dye was observed within goblet cells outlined by CK18 immunofluorescence, a goblet cell marker. Punctate dextran dye was observed in the submucosa, suggestive of dendritic cell uptake. In contrast, the Cftr KO mice demonstrated defective GAP formation, i.e., without dye penetration of goblet cells, and the lack of punctate dextran fluorescence in the submucosa. To evaluate the population of tolerogenic dendritic cells, small intestinal segments from Cftr KO-WT sex-matched littermate pairs (3-female and 2-male pairs) were collected for FACS sorting of submucosal CD103+ (tolerogenic) and CD103- (pro-inflammatory) dendritic cells. The WT mice had a significantly higher population of CD103+ tolerogenic dendritic cells compared to the CF mice (WT: 20.5+/-2, CF: 9.2+/-3, P &amp;lt; 0.006). A trend towards an increase in CD103- dendritic cells was seen in the CF intestine. In summary, the CF mice were found to have defective intraluminal antigen transfer through the GAP pathway and a significant decrease in tolerogenic dendritic cells in the intestine.

Impaired renal HCO3- secretion in CFTR deficient mice causes metabolic alkalosis during chronic base-loading.

Cystic fibrosis patients have an increased risk of developing metabolic alkalosis presumably as a result of altered renal HCO3- handling. In this study, we directly assess the kidneys' ability to compensate for a chronic base-load in the absence of functional CFTR. Comprehensive urine and blood acid-base analyses were done in anaesthetized WT mice or mice lacking either CFTR or pendrin, with or without 7days of oral NaHCO3 loading. The in vivo experiments were complemented by a combination of immunoblotting and experiments with perfused isolated mouse cortical collecting ducts (CCD). Base-loaded WT mice maintained acid-base homeostasis by elevating urinary pH and HCO3- excretion and decreasing urinary net acid excretion. In contrast, pendrin KO mice and CFTR KO mice were unable to increase urinary pH and HCO3- excretion and unable to decrease urinary net acid excretion sufficiently and thus developed metabolic alkalosis in response to the same base-load. The expression of pendrin was increased in response to the base-load in WT mice with a paralleled increased pendrin function in the perfused CCD. In CFTR KO mice, 7days of base-loading did not upregulate pendrin expression and apical Cl- /HCO3- exchange function was strongly blunted in the CCD. CFTR KO mice develop metabolic alkalosis during a chronic base-load because they are unable to sufficiently elevate renal HCO3- excretion. This can be explained by markedly reduced pendrin function in the absence of CFTR.

Engineered mutant α-ENaC subunit mRNA delivered by lipid nanoparticles reduces amiloride currents in cystic fibrosis-based cell and mice models.

Cystic fibrosis (CF) results from mutations in the chloride-conducting CF transmembrane conductance regulator (CFTR) gene. Airway dehydration and impaired mucociliary clearance in CF is proposed to result in tonic epithelial sodium channel (ENaC) activity, which drives amiloride-sensitive electrogenic sodium absorption. Decreasing sodium absorption by inhibiting ENaC can reverse airway surface liquid dehydration. Here, we inhibit endogenous heterotrimeric ENaC channels by introducing inactivating mutant ENaC α mRNA (αmutENaC). Lipid nanoparticles carrying αmutENaC were transfected in CF-based airway cells in vitro and in vivo. We observed a significant decrease in macroscopic as well as amiloride-sensitive ENaC currents and an increase in airway surface liquid height in CF airway cells. Similarly, intranasal transfection of αmutENaC mRNA decreased amiloride-sensitive nasal potential difference in CFTRKO mice. These data suggest that mRNA-based ENaC inhibition is a powerful strategy for reducing mucus dehydration and has therapeutic potential for treating CF in all patients, independent of genotype.

A BAC Transgene Expressing Human CFTR under Control of Its Regulatory Elements Rescues Cftr Knockout Mice

Small-molecule modulators of cystic fibrosis transmembrane conductance regulator (CFTR) biology show promise in the treatment of cystic fibrosis (CF). A Cftr knockout (Cftr KO) mouse expressing mutants of human CFTR would advance in vivo testing of new modulators. A bacterial artificial chromosome (BAC) carrying the complete hCFTR gene including regulatory elements within 40.1 kb of DNA 5′ and 25 kb of DNA 3′ to the gene was used to generate founder mice expressing hCFTR. Whole genome sequencing indicated a single integration site on mouse chromosome 8 (8qB2) with ~6 gene copies. hCFTR+ offspring were bred to murine Cftr KO mice, producing hCFTR+/mCftr− (H+/m−) mice, which had normal survival, growth and goblet cell function as compared to wild-type (WT) mice. Expression studies showed hCFTR protein and transcripts in tissues typically expressing mCftr. Functionally, nasal potential difference and large intestinal short-circuit (Isc) responses to cAMP stimulation were similar in magnitude to WT mice, whereas small intestinal cAMP ΔIsc responses were reduced. A BAC transgenic mouse with functional hCFTR under control of its regulatory elements has been developed to enable the generation of mouse models of hCFTR mutations by gene editing for in vivo testing of new CF therapies.

Depletion of BAFF cytokine exacerbates infection in Pseudomonas aeruginosa infected mice

BackgroundCystic fibrosis (CF) is a genetic disease characterized by chronic inflammation of the lungs that is ineffective at clearing pathogens. B-cell activating factor (BAFF), a cytokine involved in the development of B-cells, is known to be elevated in CF patients with subclinical infections. We postulate that the elevated BAFF levels in CF patients might be triggered by Pseudomonas aeruginosa infection and it might play a protective role in the regulation of lung responses to infection. MethodsTo address this hypothesis, we used a well characterized model of CFTR.KO mice infected with a clinical strain of P. aeruginosa (PA508). We quantified cell types with flow cytometry, concentration of cytokines by ELISA tests, bacterial load by colony counting and lung physiology by metacholine-induced lung resistance. ResultsOur data demonstrates that BAFF is not elevated in uninfected CF mice, and infection with Pseudomonas leads to significant induction of this regulatory cytokine. We also demonstrate that the maintenance of BAFF levels and its induction during the infection is important for clearance of Pseudomonas infection as its depletion during the course of infection leads to decrease in the resolution of infection both in WT and CFTR-KO mice. Interestingly, the depletion of BAFF not only results in a depletion of B cells numbers but also to a significant decrease in the number of regulatory T cells in the non-infected lungs. ConclusionsOverall, our data demonstrate for the first time that BAFF is an important regulatory molecule helping to maintain the immunological response to infection and clearance of lung infection.

Anion and fluid secretory response of the murine jejunum to the heat‐stable Eschericia coli enterotoxin (STa) analogue linaclotide: Involvement of NHE3, Slc26a6, CFTR, proteinkinase GII (cGKII) and NHERF1‐3.

The different PDZ‐adaptors of the NHERF family differentially associate with intestinal electrolyte transporters and modulate their activity in an agonist‐specific fashion. STa induces secretory diarrhea via binding to the guanylate cyclase C, eliciting cGMP production, resulting both in a stimulation of CFTR‐mediated anion secretion and an inhibition of NHE3‐mediated fluid absorption, and these effects are modulated by different NHERF adaptor proteins in cell lines.AimWe studied the STa‐mediated short circuit current response in isolated jejunal mucosa as well as the STa action on in vivo fluid transport in anethstized mice, deficiet for NHE3, Slc26a6, CFTR and NHERF1‐3, to study how well in vitro and in vivo modulation of fluid transport by the above named transporters and NHERF proteins may correlate.Results10−7M of the STa analogue linacolide was applied to the luminal bath of chambered isolated jejunal mucosa of CFTR‐, NHE3‐, Slc26a6‐, cGKII, and NHERF1–3 deficient and WT mice, and short circuit current (Isc) response was measured. The same concentration of linaclotide was added to the jejunal perfusate during single pass perfusion of a jejunal segment in isoflurane‐anesthetized, acid/base‐ and blood pressure controlled KO and WT mice, and net fluid balance before and after linaclotide application assessed gravimetrically. The peak Isc response to linaclotide in chambered jejunal mucosa was absent in CFTR KO, reduced by 88% in cGKII KO, by 40% in NHERF1 KO, and not significantly in NHERF2, NHERF (PDZK1) and SLC26a6 KO isolated jejunal mucosa. In contrast, the magnitude of change from jejunal fluid absorption to secretion in vivo was reduced by 51% in cGKII KO, 49% in CFTR KO, 45% in NHERF1 KO, by 34% in NHERF2 KO, and not significantly in NHERF3 KO and Slc26a6 KO anesthetized mice. NHE3 deletion (48%), pharmacological NHE3 inhibition (50%) or Slc26a6 deletion (44%) also reduced the STa‐elicited change in fluid movement.ConclusionsThe results demonstrate that Isc measurements in isolated small intestinal mucosa are excellent models to selectively study agonist‐mediated CFTR activation, but do not accurately predict in vivo “secretory responses”, i.e. a relative decrease in the basal fluid absorptive flux. Activating the GCC receptor elicited a decrease in fluid absorption in CFTR KO mice, making this substance an interesting drug to improve gut fluidity in CF patients. Apart from direct interference with guanylate cyclase C activation, potential pharmacological strategies to prevent STa‐mediated fluid loss may be targeting both NHERF1 and NHERF2.Support or Funding InformationFunded by SFB621/C1, DFG grants SE460/13‐4, 19‐1 and 21‐1.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Novel role of cystic fibrosis transmembrane conductance regulator in maintaining adult mouse olfactory neuronal homeostasis.

The olfactory epithelium (OE) of mice deficient in cystic fibrosis transmembrane conductance regulator (CFTR) exhibits ion transport deficiencies reported in human CF airways, as well as progressive neuronal loss, suggesting defects in olfactory neuron homeostasis. Microvillar cells, a specialized OE cell-subtype, have been implicated in maintaining tissue homeostasis. These cells are endowed with a PLCβ2/IP3 R3/TRPC6 signal transduction pathway modulating release of neuropeptide Y (NPY), which stimulates OE stem cell activity. It is unknown, however, whether microvillar cells also mediate the deficits observed in CFTR-null mice. Here we show that Cftr mRNA in mouse OE is exclusively localized in microvillar cells and CFTR immunofluorescence is coassociated with the scaffolding protein NHERF-1 and PLCβ2 in microvilli. In CFTR-null mice, PLCβ2 was undetectable, NHERF-1 mislocalized, and IP3 R3 more intensely stained, along with increased levels of NPY, suggesting profound alteration of the PLCβ2/IP3 R3 signaling pathway. In addition, basal olfactory neuron homeostasis was altered, shown by increased progenitor cell proliferation, differentiation, and apoptosis and by reduced regenerative capacity following methimazole-induced neurodegeneration. The importance of CFTR in microvillar cells was further underscored by decreased thickness of the OE mucus layer and increased numbers of immune cells within this tissue in CFTR-KO mice. Finally, we observed enhanced immune responses to an acute viral-like infection, as well as hyper-responsiveness to chemical and physical stimuli applied intranasally. Taken together, these data strengthen the notion that microvillar cells in the OE play a key role in maintaining tissue homeostasis and identify several mechanisms underlying this regulation through the multiple functions of CFTR.

Understanding the role of host innate immune responses in viral vector-mediated gene delivery and strategies to achieve sustained transgene expression (P6357)

Abstract Stunning progress in genomic research has changed the way we address diseases. With the identification of disease causing genetic modifications, genomic research has helped us to decipher the patho-physiology of diseases and given us an extra tool to address the basic cause by correcting those genes. Only few drugs target the cause of disase, but for most of them we still need to deliver proper genes. Although many viral vectors are being used to deliver genes to target cells, most have problems related to safety and sustained expression. In our previous studies we have used HD-AD to successfully deliver cystic fibrosis correcting human-CFTR gene to mice achieving sustained expression and disease correction in CFTR KO mice. Where as in larger animals, there is an acute phase of reduction in transgene expression. Our preliminary studies suggest that, this difference may be due to difference in NK cell activity. To address this problem we have designed human cell based in-vitro evaluation assay to find out the role of NK cells and to find out effective small molecule to block NK cells during the window period of their activity. Our results show that NK cells are killing viral vector- transduced cells. We also demonstrate that this gene transduced cell killing can be inhibited by JAK and NF-kB inhibitors. These findings will help in designing better viral vector-based gene therapy to achieve sustained expression in large animals and in human studies.

Loss of CFTR Affects Biliary Epithelium Innate Immunity and Causes TLR4–NF-κB—Mediated Inflammatory Response in Mice

Loss of function of the cystic fibrosis transmembrane conductance regulator (CFTR) in the biliary epithelium reduces bile flow and alkalinization in patients with cystic fibrosis (CF). Liver damage is believed to result from ductal cholestasis, but only 30% of patients with CF develop liver defects, indicating that another factor is involved. We studied the effects of CFTR deficiency on Toll-like receptor 4 (TLR4)-mediated responses of the biliary epithelium to endotoxins. Dextran sodium sulfate (DSS) was used to induce colitis in C57BL/6J-Cftrtm1Unc (Cftr-KO) mice and their wild-type littermates. Ductular reaction and portal inflammation were quantified by keratin-19 and CD45 immunolabeling. Cholangiocytes isolated from wild-type and Cftr-KO mice were challenged with lipopolysaccharide (LPS); cytokine secretion was quantified. Activation of nuclear factor κB (NF-κB), phosphorylation of TLR4, and activity of Src were determined. HEK-293 that expressed the secreted alkaline phosphatase reporter and human TLR4 were transfected with CFTR complementary DNAs. DSS-induced colitis caused biliary damage and portal inflammation only in Cftr-KO mice. Biliary damage and inflammation were not attenuated by restoring biliary secretion with 24-nor-ursodeoxycholic acid but were significantly reduced by oral neomycin and polymyxin B, indicating a pathogenetic role of gut-derived bacterial products. Cftr-KO cholangiocytes incubated with LPS secreted significantly higher levels of cytokines regulated by TLR4 and NF-κB. LPS-mediated activation of NF-κB was blocked by the TLR4 inhibitor TAK-242. TLR4 phosphorylation by Src was significantly increased in Cftr-KO cholangiocytes. Expression of wild-type CFTR in the HEK293 cells stimulated with LPS reduced activation of NF-κB. CFTR deficiency alters the innate immunity of the biliary epithelium and reduces its tolerance to endotoxin, resulting in an Src-dependent inflammatory response mediated by TLR4 and NF-κB. These findings might be used to develop therapies for CF-associated cholangiopathy.

Evaluation of oxidant and antioxidant status in CFTR-KO mice: role of Peroxiredoxin 6

Evaluation of oxidant and antioxidant status in CFTR-KO mice: role of Peroxiredoxin 6

The adrenaline‐induced colonic K+ secretion is conducted by the ZERO splice variant of KCa1.1 (BK)

Colonic K+ secretion in mammals under go long term hormonal‐ and short term regulation by local agonists. The BK channel has previously been identified as the only luminal K+ channel in resting, Ca2+‐activated and aldosterone‐induced distal K+ secretion. Agonists elevating cAMP are potent activators of colonic K+ secretion. The K+ channel responsible for cAMP‐induced K+ secretion remains to be identified. In this study we used Ussing chambers to identify adrenalin‐induced electrogenic K+ secretion. In mouse colon we found that adrenaline‐induced ion secretion is a compound effect of a dominating anion secretion and a smaller electrically opposing K+ secretion. Using tissue from either BK or CFTR KO and WT mice we were able to isolate the adrenaline‐induced K+ secretion. We found that adrenaline‐induced K+ secretion: 1) is absent in BK−/− tissue, 2) is up‐regulated in mice on a high K+ diet and 3) is present as sustained positive current in colon from CFTR−/− mice. We also identified that colonic enterocytes express STREX and ZERO C‐terminal BK α‐subunit splice variants. Importantly, the ZERO variant known to be activated by cAMP is differentially up‐regulated in animals on high K+ diet. In summary, these results strongly indicate that the adrenalin‐induced distal colonic K+ secretion is mediated by the BK channel and likely involves aldosterone‐induced ZERO splice variant up‐regulation.

Fenretinide prevents the development of osteoporosis in Cftr-KO mice

Background The most recently described phenotype associated with Cystic Fibrosis (CF) is reduced bone mineral density which results in osteopenia and osteoporosis. The etiology of the early onset of osteoporosis in CF patients has remained to be established. It has been suggested that inadequate nutritional absorption of essential fatty acids may play a role in the altered bone metabolism. In this study, we characterized the protective effect of fenretinide [ N-(4-hydroxyphenyl) retinamide], a vitamin A derivative, on the early onset of osteoporosis in cystic fibrosis transmembrane conductance regulator knockout ( Cftr-KO) mice. Methods Using micro-computed-tomography we examined the effect of fenretinide on the bone composition and architecture in a Cftr-KO mouse model which was then confirmed with histological analyses. Plasma fatty acids were quantified using thin layer chromatography-ELISA method. Results Twice-weekly treatments with fenretinide, over four weeks dramatically increased trabecular bone volume compared to controls. This increase in bone volume was also related to an increased concentration of ceramide in the plasma resulting in the down regulation of phospholipid-bound AA in Cftr-KO mice. Conclusions To our knowledge, this is the first time that fenretinide's protective effect against osteoporosis has been demonstrated. The results of this study strongly suggest that fenretinide has potential to be used as a prophylaxis by preventing the early onset of osteoporosis.

Parthenolide Inhibits IκB Kinase, NF-κB Activation, and Inflammatory Response in Cystic Fibrosis Cells and Mice

Cystic fibrosis (CF) is characterized by prolonged and excessive inflammatory responses in the lung and increased activation of NF-kappaB. Parthenolide is a sesquiterpene lactone derived from the plant feverfew, which has been used in folk medicine for anti-inflammatory activity. Several studies suggest that this compound inhibits the NF-kappaB pathway, but the exact site is controversial. We hypothesized that parthenolide might ameliorate the excessive inflammatory response in CF models by inhibiting activation of NF-kappaB. This was tested in vitro, using two pairs of cell lines with defective versus normal CF transmembrane conductance regulator (CFTR) (antisense/sense transfected 16 HBE and IB-3/S9), and in vivo, using CFTR-knockout (KO) mice. All cell lines were pretreated with parthenolide and then stimulated with IL-1beta and/or TNF. Parthenolide significantly inhibited IL-8 secretion induced by these cytokines and prevented NF-kappaB activation, IkappaBalpha degradation, and IkappaB Kinase complex activity. CFTR-KO and wild-type mice were pretreated with parthenolide or vehicle alone then challenged intratracheally with LPS. Bronchoalveolar lavage was performed 3, 6, and 8 h later. Parthenolide pretreatment inhibited PMN influx as well as cytokine and chemokine production. This was also associated with inhibition of IkappaBalpha degradation and NF-kappaB activation. We thus conclude that parthenolide inhibits IkappaB kinase, resulting in stabilization of cytoplasmic IkappaBalpha, which in turn leads to inhibition of NF-kappaB translocation and attenuation of subsequent inflammatory responses. IkappaB kinase may be a good target, and parthenolide and/or feverfew might be promising treatments for the excessive inflammation in CF.

Elevated hepatic SULT1E1 activity in mouse models of cystic fibrosis alters the regulation of estrogen responsive proteins

Background Previous studies with cystic fibrosis transmembrane conductance regulator (CFTR) ΔF508 mice indicate that estrogen levels may play a role in the occurrence or severity of CF-associated liver disease. However, the underlying mechanisms of liver disease in CF are poorly understood. Methods The levels of SULT1E1 (estrogen sulfotransferase) were measured in livers of control and CFTR-knockout (KO) mice. The impact of increased SULT1E1 activity on hepatic protein expression was assessed by immunoblot and MALDI mass spectrometric analysis. Results SULT1E1 expression was significantly elevated in livers of several CFTR-KO mice. SULT1E1 and CFTR were specifically detected in hepatocytes and cholangiocytes, respectively. Elevated SULT1E1 activity may result in lower levels of free β-estradiol thereby altering estrogen-responsive hepatic protein expression. Estrogen receptors (ER)-α and β were differentially regulated in CFTR-KO and CFTR-ΔF508 mice. ERα expression was reduced in mice with high SULT1E1 activity. Glutathione S-transferase-P1 and carbonic anhydrase III were significantly decreased in CFTR (−/−) mice with high SULT1E1 activity. Furthermore, cytochrome P450 2B9, also estrogen regulated, was significantly induced in the livers of CFTR (−/−) mice with high SULT1E1 activity. Conclusions Elevated SULT1E1 levels and associated alterations in estrogen-regulated hepatic protein expression may play an important role in CF liver disease.

691. CFTR mRNA Is Expressed in Lungs of CFTR-KO Mice Following Administration of Adult Marrow Cells from Wild-Type Donor Mice

Top of pageAbstract A novel approach for treatment of cystic fibrosis and other lung diseases is suggested by recent studies demonstrating that airway and alveolar epithelium in adult mice can be repopulated with cells derived from bone marrow of adult donor mice. The mechanisms by which marrow-derived cells transform into epithelial cells are unknown. However pre-existing lung injury increases localization of bone marrow cells to lung epithelium suggesting that active repair or remodeling of lung epithelium may create a more conducive environment for bone marrow cells to lodge and transform into epithelial cells. We have previously demonstrated that donor-marrow-derived cells could localize to lungs of CFTR KO mice and that different experimentally-induced injury patterns could influence the distribution of the recruited marrow cells in the recipient lungs. We now demonstrate that wildtype CFTR mRNA is expressed in CFTR-KO lungs following administration of donor marrow cells from animals containing wtCFTR. Adult female CFTR-KO mice aged 6-12 weeks received intraperitoneal administration of naphthalene (275 mg/kg body weight of a stock solution of 10 ml/kg in sterile soybean oil) to induce airway lung epithelial injury. 3-4 days later, at the time of peak lung injury, treated and naive control CFTR KO mice received tail vein injection of 1 × 106 stromal marrow cells originally obtained from transgenic constitutive GFP-expressing donors (C57Bl/6 background) and cultured for 10 days prior to use. Mice were euthanized 1 week and 3 months after donor marrow cell administration and the lungs flash frozen in liquid N2, homogenized, and RNA extracted with Trizol. DNAse-treated purified RNA was reverse transcribed followed by PCR amplification using specific primers for murine CFTR (mCF11/mCF12). wtCFTR was observed in naphthalene-injured CFTR-KO mouse lungs collected 3 months but not 1 week after donor marrow administration. No wtCFTR expression was detected in un-injured transplanted CFTR-KO mice at either time point. Importantly, wtCFTR mRNA expression was not detected in donor marrow cells or in mature circulating leukocytes. These results suggest that donor marrow cells that traffic to lung subsequently express wtCFTR as they acquire an epithelial cell phenotype. Lung injury appears to increase this process. Importantly, these results suggest that marrow cells can directly traffic to lung and express a specific lung epithelial protein. Engraftment of the recipient marrow may not be necessary.