Cystic Fibrosis Transmembrane Conductance Regulator Knockout Mice Research Articles

Synthesis of ionizable lipopolymers using split-Ugi reaction for pulmonary delivery of various size RNAs and gene editing.

We present an efficient approach for synthesizing cationic poly(ethylene imine) derivatives using the multicomponent split-Ugi reaction to rapidly create a library of complex functional ionizable lipopolymers. We synthesized a diverse library of 155 polymers, formulated them into polyplexes to establish structure-activity relationships crucial for endosomal escape and efficient transfection. After discovering a lead structure, lipopolymer-lipid hybrid nanoparticles are introduced to preferentially deliver to and elicit effective mRNA transfection in lung endothelium and immune cells, including T cells with low in vivo toxicity. The lipopolymer-lipid hybrid nanoparticles showed 300-fold improvement in systemic mRNA delivery to the lung compared to in vivo -JetPEI ® . Lipopolymer-lipid hybrid nanoparticles demonstrated efficient delivery of mRNA-based therapeutics for treatment of two different disease models. Lewis Lung cancer progression was significantly delayed after treatment with loaded IL-12 mRNA in U155@lipids after repeated i.v. administration. Systemic delivery of human CFTR (hCFTR) mRNA resulted in production of functional form of CFTR protein in the lungs. The functionality of hCFTR protein was confirmed by restoration of CFTR- mediated chloride secretion in conductive airway epithelia in CFTR knockout mice after nasal instillation of hCFTR mRNA loaded U155@lipids. We further showed that, U155@lipids nanoparticles can deliver complex CRISPR-Cas9 based RNA cargo to the lung, achieving 5.6 ± 2.4 % gene editing in lung tissue. Moreover, we demonstrated successful PD-1 gene knockout of T cells in vivo . Our results highlight a versatile delivery platform for systemic delivering of mRNA of various sizes for gene therapy for a variety of therapeutics.

DRA involvement in linaclotide-stimulated bicarbonate secretion during loss of CFTR function.

Duodenal bicarbonate secretion is critical to epithelial protection, as well as nutrient digestion and absorption, and is impaired in cystic fibrosis (CF). We examined if linaclotide, typically used to treat constipation, may also stimulate duodenal bicarbonate secretion. Bicarbonate secretion was measured in vivo and in vitro using mouse and human duodenum (biopsies and enteroids). Ion transporter localization was identified with confocal microscopy, and de novo analysis of human duodenal single-cell RNA sequencing (scRNA-Seq) data sets was performed. Linaclotide increased bicarbonate secretion in mouse and human duodenum in the absence of cystic fibrosis transmembrane conductance regulator (CFTR) expression (Cftr-knockout mice) or function (CFTRinh-172). Na+/H+ exchanger 3 inhibition contributed to a portion of this response. Linaclotide-stimulated bicarbonate secretion was eliminated by down-regulated in adenoma (DRA, SLC26A3) inhibition during loss of CFTR activity. ScRNA-Seq identified that 70% of villus cells expressed SLC26A3, but not CFTR, mRNA. Loss of CFTR activity and linaclotide increased apical brush border expression of DRA in non-CF and CF differentiated enteroids. These data provide further insights into the action of linaclotide and how DRA may compensate for loss of CFTR in regulating luminal pH. Linaclotide may be a useful therapy for CF individuals with impaired bicarbonate secretion.

Prominent role of gut dysbiosis in the pathogenesis of cystic fibrosis-related liver disease in mice

Cystic fibrosis-related liver disease (CFLD) is a chronic cholangiopathy that increases morbidity and mortality in patients with CF. Current treatments are unsatisfactory, and incomplete understanding of CFLD pathogenesis hampers therapeutic development. We have previously shown that mouse CF cholangiocytes respond to lipopolysaccharide with excessive inflammation. Thus, we investigated the role of the gut-liver axis in the pathogenesis of CFLD. Wild-type (WT), whole-body Cftr knockout (CFTR-KO) and gut-corrected (CFTR-KO-GC) mice were studied. Liver changes were assessed by immunohistochemistry and single-cell transcriptomics (single-cell RNA sequencing), inflammatory mediators were analysed by proteome array, faecal microbiota by 16S ribosomal RNA sequencing and gut permeability by FITC-dextran assay. The livers of CFTR-KO mice showed ductular proliferation and periportal inflammation, whereas livers of CFTR-KO-GC mice had no evident pathology. Single-cell RNA sequencing analysis of periportal cells showed increased presence of neutrophils, macrophages and T cells, and activation of pro-inflammatory and pathogen-mediated immune pathways in CFTR-KO livers, consistent with a response to gut-derived stimuli. CFTR-KO mice exhibited gut dysbiosis with enrichment of Enterobacteriaceae and Enterococcus spp., which was associated with increased intestinal permeability and mucosal inflammation, whereas gut dysbiosis and inflammation were absent in CFTR-KO-GC mice. Treatment with nonabsorbable antibiotics ameliorated intestinal permeability and liver inflammation in CFTR-KO mice. Faecal microbiota transfer from CFTR-KO to germ-free WT mice did not result in dysbiosis nor liver pathology, indicating that defective intestinal CFTR is required to maintain dysbiosis. Defective CFTR in the gut sustains a pathogenic microbiota, creates an inflammatory milieu, and alters intestinal permeability. These changes are necessary for the development of cholangiopathy. Restoring CFTR in the intestine or modulating the microbiota could be a promising strategy to prevent or attenuate liver disease. Severe cystic fibrosis-related liver disease (CFLD) affects 10% of patients with cystic fibrosis (CF) and contributes to increased morbidity and mortality. Treatment options remain limited due to a lack of understanding of disease pathophysiology. The cystic fibrosis transmembrane conductance regulator (CFTR) mediates Cl- and HCO3- secretion in the biliary epithelium and its defective function is thought to cause cholestasis and excessive inflammatory responses in CF. However, our study in Cftr-knockout mice demonstrates that microbial dysbiosis, combined with increased intestinal permeability caused by defective CFTR in the intestinal mucosa, acts as a necessary co-factor for the development of CFLD-like liver pathology in mice. These findings uncover a major role for the gut microbiota in CFLD pathogenesis and call for further investigation and clinical validation to develop targeted therapeutic strategies acting on the gut-liver axis in CF.

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.

The Anion Channel TMEM16a/Ano1 Modulates CFTR Activity, but Does Not Function as an Apical Anion Channel in Colonic Epithelium from Cystic Fibrosis Patients and Healthy Individuals.

Studies in human colonic cell lines and murine intestine suggest the presence of a Ca2+-activated anion channel, presumably TMEM16a. Is there a potential for fluid secretion in patients with severe cystic fibrosis transmembrane conductance regulator (CFTR) mutations by activating this alternative pathway? Two-dimensional nondifferentiated colonoid-myofibroblast cocultures resembling transit amplifying/progenitor (TA/PE) cells, as well as differentiated monolayer (DM) cultures resembling near-surface cells, were established from both healthy controls (HLs) and patients with severe functional defects in the CFTR gene (PwCF). F508del mutant and CFTR knockout (null) mice ileal and colonic mucosa was also studied. HL TA/PE monolayers displayed a robust short-circuit current response (ΔIeq) to UTP (100 µM), forskolin (Fsk, 10 µM) and carbachol (CCH, 100 µM), while ΔIeq was much smaller in differentiated monolayers. The selective TMEM16a inhibitor Ani9 (up to 30 µM) did not alter the response to luminal UTP, significantly decreased Fsk-induced ΔIeq, and significantly increased CCH-induced ΔIeq in HL TA/PE colonoid monolayers. The PwCF TA/PE and the PwCF differentiated monolayers displayed negligible agonist-induced ΔIeq, without a significant effect of Ani9. When TMEM16a was localized in intracellular structures, a staining in the apical membrane was not detected. TMEM16a is highly expressed in human colonoid monolayers resembling transit amplifying cells of the colonic cryptal neck zone, from both HL and PwCF. While it may play a role in modulating agonist-induced CFTR-mediated anion currents, it is not localized in the apical membrane, and it has no function as an apical anion channel in cystic fibrosis (CF) and healthy human colonic epithelium.

In Vivo Inflammation Caused by Achromobacter spp. Cystic Fibrosis Clinical Isolates Exhibiting Different Pathogenic Characteristics.

Achromobacter spp. lung infection in cystic fibrosis has been associated with inflammation, increased frequency of exacerbations, and decline of respiratory function. We aimed to evaluate in vivo the inflammatory effects of clinical isolates exhibiting different pathogenic characteristics. Eight clinical isolates were selected based on different pathogenic characteristics previously assessed: virulence in Galleria mellonella larvae, cytotoxicity in human bronchial epithelial cells, and biofilm formation. Acute lung infection was established by intratracheal instillation with 10.5 × 108 bacterial cells in wild-type and CFTR-knockout (KO) mice expressing a luciferase gene under control of interleukin-8 promoter. Lung inflammation was monitored by in vivo bioluminescence imaging up to 48 h after infection, and mortality was recorded up to 96 h. Lung bacterial load was evaluated by CFU count. Virulent isolates caused higher lung inflammation and mice mortality, especially in KO animals. Isolates both virulent and cytotoxic showed higher persistence in mice lungs, while biofilm formation was not associated with lung inflammation, mice mortality, or bacterial persistence. A positive correlation between virulence and lung inflammation was observed. These results indicate that Achromobacter spp. pathogenic characteristics such as virulence and cytotoxicity may be associated with clinically relevant effects and highlight the importance of elucidating their mechanisms.

Cftr deletion in mouse epithelial and immune cells differentially influence the intestinal microbiota

Cystic fibrosis (CF) is a life-threatening genetic disorder, caused by mutations in the CF transmembrane-conductance regulator gene (cftr) that encodes CFTR, a cAMP-activated chloride and bicarbonate channel. Clinically, CF lung disease dominates the adult patient population. However, its gastrointestinal illness claims the early morbidity and mortality, manifesting as intestinal dysbiosis, inflammation and obstruction. As CF is widely accepted as a disease of epithelial dysfunction, it is unknown whether CFTR loss-of-function in immune cells contributes to these clinical outcomes. Using cftr genetic knockout and bone marrow transplantation mouse models, we performed 16S rRNA gene sequencing of the intestinal microbes. Here we show that cftr deletion in both epithelial and immune cells collectively influence the intestinal microbiota. However, the immune defect is a major factor determining the dysbiosis in the small intestine, while the epithelial defect largely influences that in the large intestine. This finding revises the current concept by suggesting that CF epithelial defect and immune defect play differential roles in CF intestinal disease.

Platelet CFTR inhibition enhances arterial thrombosis via increasing intracellular Cl- concentration and activation of SGK1 signaling pathway.

Platelet hyperactivity is essential for thrombus formation in coronary artery diseases (CAD). Dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) in patients with cystic fibrosis elevates intracellular Cl- levels ([Cl-]i) and enhanced platelet hyperactivity. In this study, we explored whether alteration of [Cl-]i has a pathological role in regulating platelet hyperactivity and arterial thrombosis formation. CFTR expression was significantly decreased, while [Cl-]i was increased in platelets from CAD patients. In a FeCl3-induced mouse mesenteric arteriole thrombosis model, platelet-specific Cftr-knockout and/or pre-administration of ion channel inhibitor CFTRinh-172 increased platelet [Cl-]i, which accelerated thrombus formation, enhanced platelet aggregation and ATP release, and increased P2Y12 and PAR4 expression in platelets. Conversely, Cftr-overexpressing platelets resulted in subnormal [Cl-]i, thereby decreasing thrombosis formation. Our results showed that clamping [Cl-]i at high levels or Cftr deficiency-induced [Cl-]i increasement dramatically augmented phosphorylation (Ser422) of serum and glucocorticoid-regulated kinase (SGK1), subsequently upregulated P2Y12 and PAR4 expression via NF-κB signaling. Constitutively active mutant S422D SGK1 markedly increased P2Y12 and PAR4 expression. The specific SGK1 inhibitor GSK-650394 decreased platelet aggregation in wildtype and platelet-specific Cftr knockout mice, and platelet SGK1 phosphorylation was observed in line with increased [Cl-]i and decreased CFTR expression in CAD patients. Co-transfection of S422D SGK1 and adenovirus-induced CFTR overexpression in MEG-01 cells restored platelet activation signaling cascade. Our results suggest that [Cl-]i is a novel positive regulator of platelet activation and arterial thrombus formation via the activation of a [Cl-]i-sensitive SGK1 signaling pathway. Therefore, [Cl-]i in platelets is a novel potential biomarker for platelet hyperactivity, and CFTR may be a potential therapeutic target for platelet activation in CAD.

Alkalosis-induced hypoventilation in cystic fibrosis: The importance of efficient renal adaptation

The lungs and kidneys are pivotal organs in the regulation of body acid-base homeostasis. In cystic fibrosis (CF), the impaired renal ability to excrete an excess amount of HCO3- into the urine leads to metabolic alkalosis [P. Berg etal., J. Am. Soc. Nephrol. 31, 1711-1727 (2020); F. Al-Ghimlas, M. E. Faughnan, E. Tullis, Open Respir. Med. J. 6, 59-62 (2012)]. This is caused by defective HCO3- secretion in the β-intercalated cells of the collecting duct that requires both the cystic fibrosis transmembrane conductance regulator (CFTR) and pendrin for normal function [P. Berg etal., J. Am. Soc. Nephrol. 31, 1711-1727 (2020)]. We studied the ventilatory consequences of acute oral base loading in normal, pendrin knockout (KO), and CFTR KO mice. In wild-type mice, oral base loading induced a dose-dependent metabolic alkalosis, fast urinary removal of base, and a moderate base load did not perturb ventilation. In contrast, CFTR and pendrin KO mice, which are unable to rapidly excrete excess base into the urine, developed a marked and transient depression of ventilation when subjected to the same base load. Therefore, swift renal base elimination in response to an acute oral base load is a necessary physiological function to avoid ventilatory depression. The transient urinary alkalization in the postprandial state is suggested to have evolved for proactive avoidance of hypoventilation. In CF, metabolic alkalosis may contribute to the commonly reduced lung function via a suppression of ventilatory drive.

INTESTINAL EPITHELIAL CELL-SPECIFIC CFTR KNOCKOUT MICE EXHIBIT FECAL MICROBIAL DYSBIOSIS

Abstract BACKGROUND Chronic intestinal inflammation is a poorly understood manifestation of Cystic Fibrosis (CF) disease. Fecal microbial dysbiosis has the potential of inducing intestinal inflammation and has been reported in people with CF as well as CF mouse models. CF mice consuming an osmotic polyethylene glycol (PEG) laxative in drinking water to prevent intestinal obstruction have low levels of intestinal inflammation and low intestinal bacterial loads. In contrast, CF mice consuming a nutritionally complete liquid diet to prevent intestinal impaction exhibit intestinal inflammation and high intestinal bacterial loads (R. C. De Lisle, et al., Am J Physiol 293, G577-G584, 2007) To investigate the development of dysbiosis in a CFTR-deficient intestine, we transitioned adult intestinal epithelial cell-specific knockout and wild-type (WT) mice from life-long consumption of PEG laxative to a nutritionally-complete liquid diet for a two week period before collection of fecal samples. METHODS Fecal samples collected from B6.Cg-Tg(Vil1-cre)-Cftrf10/f10 intestinal-specific Cftr knockout mice (iCftr KO) and their WT sex-matched littermates were DNA extracted and prepared for 16S rRNA sequencing and evaluation of the microbiome. Microbial community composition, richness and diversity indices, and relative abundance of bacterial species of interest were evaluated. RESULTS 16S rRNA sequencing and metagenomic analysis revealed significant differences in microbial composition with intestinal-specific loss of CFTR as compared to WT, as well as a significant decrease in Shannon diversity and Chao-1 richness indices. A significant deficit of taxa was seen in the intestinal-specific CF mice, as compared to WT, which corresponded with a significant increase in bacterial species that potentially serve as provocateurs of intestinal inflammation including Helicobacter typhlonius, Clostridium perfringens, Ruminococcus gnavus, and Tyzerella spp. CONCLUSIONS As compared to WT, the transition from PEG laxative to a nutritionally-complete liquid diet resulted in overt differences in the fecal microbiota composition in iCftr KO mice which included enrichment of potential bacterial pathobionts capable of stimulating intestinal inflammation. These studies suggest that the CFTR-deficient intestinal epithelium provides an environment selective for potential bacterial pathobionts. The paradigm of the study may prove useful for investigating the development of dysbiosis and corresponding changes in intestinal mucosal immunity.

679: Host defense defects and inflammation within the nasal airways of CFTR knockout mice

679: Host defense defects and inflammation within the nasal airways of CFTR knockout mice

Abstract LB230: Inactivation of the CFTR gene in duodena of mice exposed to hexavalent chromium (Cr(VI)) in drinking water supports its tumor-suppressor status and implies its role in Cr(VI)-induced carcinogenesis of the small intestines

Abstract Hexavalent chromium [Cr (VI)] compounds are used in metallurgical, chemical and refractory industries. Their carcinogenicity has been supported by a number of epidemiological and animal studies; however, their carcinogenic mode of action is still incompletely understood. To identify mechanisms involved in cancer development from toxicologically relevant exposures to Cr(VI), we analyzed gene expression data from duodena of mice exposed for 7 or 90 days to Cr(VI) in drinking water at concentrations 0.3-520 mg/L. This analysis included identification of upstream regulatory molecules that are likely responsible for the observed gene expression changes, and identification of annotated gene expression data from public repositories that correlate with gene expression changes in duodena of Cr(VI)-exposed mice. We identified the CFTR gene among top scoring upstream regulators, including at low exposure levels that, for the given timepoints, did not induce observable tissue damage. Furthermore, the expression data from duodena of Cr(VI)-exposed mice displayed a positive correlation with other datasets in public repositories associated with the inactive CFTR gene, of which the top scoring were datasets for ilea and duodena of Cftr-knockout mice. Our results support the tumor-suppressor role of the CFTR gene, which was previously suggested for intestinal carcinogenesis in humans and mice. Furthermore, our findings indicate, for the first time, the role of CFTR inactivation in chemical carcinogenesis and expand the range of plausible mechanisms that may be operative in Cr(VI)-mediated carcinogenesis of intestinal and possibly other tissues. Disclaimer: The views expressed are those of the authors and do not necessarily represent the views or policies of the US EPA. Citation Format: Roman Mezencev, Scott S. Auerbach. Inactivation of the CFTR gene in duodena of mice exposed to hexavalent chromium (Cr(VI)) in drinking water supports its tumor-suppressor status and implies its role in Cr(VI)-induced carcinogenesis of the small intestines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB230.

CFTR Deficiency Affects Glucose Homeostasis via Regulating GLUT4 Plasma Membrane Transportation.

Cystic Fibrosis (CF) is an autosomal recessive disorder caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene. CF-related diabetes (CFRD) is one of the most prevalent comorbidities of CF. Altered glucose homeostasis has been reported in CF patients. The mechanism has not been fully elucidated. Besides the consequence of pancreatic endocrine dysfunction, we focus on insulin-responsive tissues and glucose transportation to explain glucose homeostasis alteration in CFRD. Herein, we found that CFTR knockout mice exhibited insulin resistance and glucose tolerance. Furthermore, we demonstrated insulin-induced glucose transporter 4 (GLUT4) translocation to the cell membrane was abnormal in the CFTR knockout mice muscle fibers, suggesting that defective intracellular GLUT4 transportation may be the cause of impaired insulin responses and glucose homeostasis. We further demonstrated that PI(4,5)P2 could rescue CFTR related defective intracellular GLUT4 transportation, and CFTR could regulate PI(4,5)P2 cellular level through PIP5KA, suggesting PI(4,5)P2 is a down-stream signal of CFTR. Our results revealed a new signal mechanism of CFTR in GLUT4 translocation regulation, which helps explain glucose homeostasis alteration in CF patients.

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.

CFTR plays an important role in the regulation of vascular resistance and high-fructose/salt-diet induced hypertension in mice

BackgroundThe pathophysiological roles of cystic fibrosis transmembrane-conductance regulator (CFTR) Cl− channels in the regulation of blood pressure (BP) remain controversial. Here we studied the function of CFTR Cl− channels in regulation of BP and in the high-fructose-salt-diet (HFSD) induced hypertension in mice. MethodsThe systolic, diastolic and mean BP (SBP, DBP and MBP, respectively) were continuously monitored from unrestricted conscious wild-type (cftr+/+) FVB and CFTR-knockout (cftr−/−) mice (8-week old, male). HFSD (64.7% fructose, 2% NaCl water) or control normal starch diet (CNSD, 58.9% corn starch, 0 NaCl water) was given for 8 weeks and vascular Doppler were performed. Real-time PCR and Western blot were used to examine mRNA and protein expression, respectively. ResultsThe aortic stiffness, daytime and nighttime SBP, DBP, and MBP of the cftr−/− mice were significantly higher than those in the age- and gender-matched cftr+/+ mice, which is consistent with the findings of increased vascular resistance in cystic fibrosis patients. The aortic stiffness, daytime and nighttime SBP, DBP, and MBP of cftr+/+ mice fed with HFSD were all significantly higher than those fed with CNSD. Importantly, HFSD caused a significant decrease in mRNA and protein expression of WINK1, WINK4 and CFTR in aorta and mesenteric arteries, but not in the kidney, corroborating that HSFD-induced downregulation of WINKs and loss of CFTR function specifically in the arteries may mediate the increased BP. ConclusionsCFTR regulates peripheral arterial resistance and BP in vivo. HFSD-induced CFTR downregulation specifically in the arteries may be a novel mechanism for hypertension.

Transcriptomic analysis of CFTR-impaired endothelial cells reveals a pro-inflammatory phenotype.

Cystic fibrosis (CF) is a life-threatening disorder characterised by decreased pulmonary mucociliary and pathogen clearance, and an exaggerated inflammatory response leading to progressive lung damage. CF is caused by bi-allelic pathogenic variants of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes a chloride channel. CFTR is expressed in endothelial cells (ECs) and EC dysfunction has been reported in CF patients, but a role for this ion channel in ECs regarding CF disease progression is poorly described.We used an unbiased RNA sequencing approach in complementary models of CFTR silencing and blockade (by the CFTR inhibitor CFTRinh-172) in human ECs to characterise the changes upon CFTR impairment. Key findings were further validated in vitro and in vivo in CFTR-knockout mice and ex vivo in CF patient-derived ECs.Both models of CFTR impairment revealed that EC proliferation, migration and autophagy were downregulated. Remarkably though, defective CFTR function led to EC activation and a persisting pro-inflammatory state of the endothelium with increased leukocyte adhesion. Further validation in CFTR-knockout mice revealed enhanced leukocyte extravasation in lung and liver parenchyma associated with increased levels of EC activation markers. In addition, CF patient-derived ECs displayed increased EC activation markers and leukocyte adhesion, which was partially rescued by the CFTR modulators VX-770 and VX-809.Our integrated analysis thus suggests that ECs are no innocent bystanders in CF pathology, but rather may contribute to the exaggerated inflammatory phenotype, raising the question of whether normalisation of vascular inflammation might be a novel therapeutic strategy to ameliorate the disease severity of CF.

Protease inhibitors elicit anti-inflammatory effects in CF mice with Pseudomonas aeruginosa acute lung infection.

Pseudomonas aeruginosa is the major respiratory pathogen in patients with cystic fibrosis (CF). P. aeruginosa-secreted proteases, in addition to host proteases, degrade lung tissue and interfere with immune processes. In this study, we aimed at evaluating the possible anti-inflammatory effects of protease inhibitors Marimastat and Ilomastat in the treatment of P. aeruginosa infection. Lung infection with the P. aeruginosa PAO1 strain was established in wild-type and cystic fibrosis transmembrane conductance regulator (CFTR) knock-out C57BL/6 mice expressing a luciferase gene under control of bovine interleukin (IL)-8 promoter. After intratracheal instillation with 150µM Marimastat and Ilomastat, lung inflammation was monitored by in-vivo bioluminescence imaging and bacterial load in the lungs was assessed. In vitro, the effects of protease inhibitors on PAO1 growth and viability were evaluated. Acute lung infection was established in both wild-type and CFTR knock-out mice. After 24h, the infection induced IL-8-dependent bioluminescence emission, indicating lung inflammation. In infected mice with ongoing inflammation, intratracheal treatment with 150µM Marimastat and Ilomastat reduced the bioluminescence signal in comparison to untreated, infected animals. Bacterial load in the lungs was not affected by the treatment, and in vitro the same dose of Marimastat and Ilomastat did not affect PAO1 growth and viability, confirming that these molecules have no additional anti-bacterial activity. Our results show that inhibition of protease activity elicits anti-inflammatory effects in cystic fibrosis (CF) mice with acute P. aeruginosa lung infection. Thus, Marimastat and Ilomastat represent candidate molecules for the treatment of CF patients, encouraging further studies on protease inhibitors and their application in inflammatory diseases.

Impaired Renal HCO3- Excretion in Cystic Fibrosis.

Patients with cystic fibrosis (CF) do not respond with increased urinary HCO3- excretion after stimulation with secretin and often present with metabolic alkalosis. By combining RT-PCR, immunohistochemistry, isolated tubule perfusion, in vitro cell studies, and in vivo studies in different mouse models, we elucidated the mechanism of secretin-induced urinary HCO3- excretion. For CF patients and CF mice, we developed a HCO3- drinking test to assess the role of the cystic fibrosis transmembrane conductance regulator (CFTR) in urinary HCO3-excretion and applied it in the patients before and after treatment with the novel CFTR modulator drug, lumacaftor-ivacaftor. β-Intercalated cells express basolateral secretin receptors and apical CFTR and pendrin. In vivo application of secretin induced a marked urinary alkalization, an effect absent in mice lacking pendrin or CFTR. In perfused cortical collecting ducts, secretin stimulated pendrin-dependent Cl-/HCO3- exchange. In collecting ducts in CFTR knockout mice, baseline pendrin activity was significantly lower and not responsive to secretin. Notably, patients with CF (F508del/F508del) and CF mice showed a greatly attenuated or absent urinary HCO3--excreting ability. In patients, treatment with the CFTR modulator drug lumacaftor-ivacaftor increased the renal ability to excrete HCO3-. These results define the mechanism of secretin-induced urinary HCO3- excretion, explain metabolic alkalosis in patients with CF, and suggest feasibility of an in vivo human CF urine test to validate drug efficacy.

Myeloid CFTR loss-of-function causes persistent neutrophilic inflammation in cystic fibrosis.

Persistent neutrophilic inflammation is a hallmark of cystic fibrosis (CF). However, the mechanisms underlying this outstanding pathology remain incompletely understood. Here, we report that CFTR in myeloid immune cells plays a pivotal role in control of neutrophilic inflammation. Myeloid CFTR-Knockout (Mye-Cftr-/-) mice and congenic wild-type (WT) mice were challenged peritoneally with zymosan particles at different doses, creating aseptic peritonitis with varied severity. A high-dose challenge resulted in significantly higher mortality in Mye-Cftr-/- mice, indicating an intrinsic defect in host control of inflammation in mice whose myeloid cells lack CF. The low-dose challenge demonstrated an impaired resolution of inflammation in Mye-Cftr-/- mice, reflected by a significant overproduction of proinflammatory cytokines, including neutrophil chemokines MIP-2 and KC, and sustained accumulation of neutrophils. Tracing neutrophil mobilization in vivo demonstrated that myeloid CF mice recruited significantly more neutrophils than did WT mice. Pulmonary challenge with zymosan elicited exuberant inflammation in the lung and recapitulated the findings from peritoneal challenge. To determine the major type of cell that was primarily responsible for the over-recruitment of neutrophils, we purified and cultured ex vivo zymosan-elicited peritoneal neutrophils and macrophages. The CF neutrophils produced significantly more MIP-2 than did the WT counterparts, and peripheral blood neutrophils isolated from myeloid CF mice also produced significantly more MIP-2 after zymosan stimulation in vitro. These data altogether suggest that CFTR dysfunction in myeloid immune cells, especially neutrophils, leads to hyperinflammation and excessive neutrophil mobilization in the absence of infection. Thus, dysregulated inflammation secondary to abnormal or absent CFTR in myeloid cells may underlie the clinically observed neutrophilic inflammation in CF.