Christina Caputo

Macrophages Directly Contribute to the Exaggerated Inflammatory Response in Cystic Fibrosis Transmembrane Conductance Regulator−/− Mice

Pulmonary infection with an exaggerated inflammatory response is the major cause of morbidity and mortality in cystic fibrosis (CF). The objective of this study was to determine whether differences in the innate immune system underlie the exaggerated immune response in CF. We established a model that recapitulates the exaggerated immune response in a CF mouse model by exposure to Pseudomonas aeruginosa LPS and assessed the pulmonary cellular and cytokine responses of wild-type (WT) and CF mice. Compared with WT mice, CF mice had increased numbers of neutrophils and increased proinflammatory cytokines in their bronchoalveolar lavage fluid after LPS exposure. Based on the increased levels of IL-1alpha, IL-6, granulocyte colony-stimulating factor (G-CSF), and keratinocyte chemoattractant, all of which are known to be produced by macrophages, we tested whether two populations of macrophages, bone marrow-derived macrophages and alveolar macrophages, directly contribute to the elevated cytokine response of CF mice to LPS. After in vitro stimulation of bone marrow-derived macrophages and alveolar macrophages with LPS, IL-1alpha, IL-6, G-CSF, and monocyte chemoattractant protein-1 were higher in CF compared with WT cell supernatants. Quantitative analyses for IL-6 and keratinocyte chemoattractant revealed that LPS-stimulated CF macrophages have higher mRNA and intracellular protein levels compared with WT macrophages. Our data support the hypothesis that macrophages play a role in the exuberant cytokine production and secretion that characterizes CF, suggesting that the macrophage response may be an important therapeutic target for decreasing the morbidity of CF lung disease.

Partial correction of cystic fibrosis defects with PLGA nanoparticles encapsulating curcumin.

Cystic fibrosis (CF) is a common life threatening genetic disease (incidence: approximately 1 in 2500 live births). CF is caused by mutations in CFTR, a chloride channel involved in epithelial secretion of fluid and electrolytes. The most common mutation entails the deletion of a phenylalanine in position 508 that causes protein misfolding and abnormal CFTR processing. The DeltaF508 mutation accounts for approximately 70% of all CF alleles, and about 90% of CF patients carry at least one copy of DeltaF508 CFTR. Curcumin, a natural constituent of Curcuma longa (turmeric spice), is a nontoxic low-affinity SERCA (sarco (endo)plasmic reticulum calcium ATPase) pump inhibitor thought to permit DeltaF508 CFTR escape from the ER. The compound has been shown to be capable of correcting the defect in cell lines and mice expressing DeltaF508 CFTR. In this work, poly lactic-co-glycolic acid (PLGA) nanoparticles encapsulating curcumin were synthesized and used to treat two different CF mouse strains in an effort to correct the defects associated with CF by improving bioavailability of the compound, which has previously been a challenge in treatment with curcumin. Our results suggest that oral administration of PLGA nanoparticles encapsulating curcumin enhances the effects of curcumin therapy in CF mice, as compared to delivery of nonencapsulated curcumin.

Abnormal Trafficking and Degradation of TLR4 Underlie the Elevated Inflammatory Response in Cystic Fibrosis

Morbidity and mortality in cystic fibrosis (CF) are due not only to abnormal epithelial cell function, but also to an abnormal immune response. We have shown previously that macrophages lacking CF transmembrane conductance regulator (CFTR), the gene mutated in CF, contribute significantly to the hyperinflammatory response observed in CF. In this study, we show that lack of functional CFTR in murine macrophages causes abnormal TLR4 subcellular localization. Upon LPS stimulation, CFTR macrophages have prolonged TLR4 retention in the early endosome and reduced translocation into the lysosomal compartment. This abnormal TLR4 trafficking leads to increased LPS-induced activation of the NF-κB, MAPK, and IFN regulatory factor-3 pathways and decreased TLR4 degradation, which affects downregulation of the proinflammatory state. In addition to primary murine cells, mononuclear cells isolated from CF patients demonstrate similar defects in response to LPS. Moreover, specific inhibition of CFTR function induces abnormal TLR4 trafficking and enhances the inflammatory response of wild-type murine cells to LPS. Thus, functional CFTR in macrophages influences TLR4 spatial and temporal localization and perturbs LPS-mediated signaling in both murine CF models and patients with CF.

Nanoparticles that deliver triplex-forming peptide nucleic acid molecules correct F508del CFTR in airway epithelium

Cystic fibrosis (CF) is a lethal genetic disorder most commonly caused by the F508del mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. It is not readily amenable to gene therapy because of its systemic nature and challenges including in vivo gene delivery and transient gene expression. Here, we use triplex-forming PNA molecules and donor DNA in biodegradable polymer nanoparticles to correct F508del. We confirm modification with sequencing and a functional chloride efflux assay. In vitro correction of chloride efflux occurs in up to 25% of human cells. Deep sequencing reveals negligible off-target effects in partially homologous sites. Intranasal application of nanoparticles in CF mice produces changes in nasal epithelium potential differences consistent with corrected CFTR, with gene correction also detected in lung tissue. This work represents facile genome engineering in vivo with oligonucleotides using a nanoparticle system to achieve clinically relevant levels of gene editing without off-target effects.Cystic fibrosis (CF) is a lethal genetic disorder most commonly caused by the F508del mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. It is not readily amenable to gene therapy because of its systemic nature and challenges including in vivo gene delivery and transient gene expression. Here we use triplex-forming peptide nucleic acids and donor DNA in biodegradable polymer nanoparticles to correct F508del. We confirm modification with sequencing and a functional chloride efflux assay. In vitro correction of chloride efflux occurs in up to 25% of human cells. Deep-sequencing reveals negligible off-target effects in partially homologous sites. Intranasal delivery of nanoparticles in CF mice produces changes in the nasal epithelium potential difference assay, consistent with corrected CFTR function. Also, gene correction is detected in the nasal and lung tissue. This work represents facile genome engineering in vivo with oligonucleotides using a nanoparticle system to achieve clinically relevant levels of gene editing without off-target effects.

ΔF508 mutation results in impaired gastric acid secretion

The cystic fibrosis transmembrane conductance regulator (CFTR) is recognized as a multifunctional protein that is involved in Cl– secretion, as well as acting as a regulatory protein. In order for acid secretion to take place a complex interaction of transport proteins and channels must occur at the apical pole of the parietal cell. Included in this process is at least one K+ and Cl– channel, allowing for both recycling of K+ for the H,K-ATPase, and Cl– secretion, necessary for the generation of concentrated HCl in the gastric gland lumen. We have previously shown that an ATP-sensitive potassium channel (KATP) is expressed in parietal cells. In the present study we measured secretagogue-induced acid secretion from wild-type and ΔF508-deficient mice in isolated gastric glands and whole stomach preparations. Secretagogue-induced acid secretion in wild-type mouse gastric glands could be significantly reduced with either glibenclamide or the specific inhibitor CFTR-inh172. In ΔF508-deficient mice, however, histamine-induced acid secretion was significantly less than in wild-type mice. Furthermore, immunofluorescent localization of sulfonylurea 1 and 2 failed to show expression of a sulfonylurea receptor in the parietal cell, thus further implicating CFTR as the ATP-binding cassette transporter associated with the KATP channels. These results demonstrate a regulatory role for the CFTR protein in normal gastric acid secretion.

Myosin Ia is Required for CFTR Brush Border Membrane Trafficking and Ion Transport in the Mouse Small Intestine

In enterocytes of the small intestine, endocytic trafficking of CFTR channels from the brush border membrane (BBM) to the subapical endosomes requires the minus‐end motor, myosin VI (Myo6). The subapical localization of Myo6 is dependent on myosin Ia (Myo1a) the major plus‐end motor associated with the BBM, suggestive of functional synergy between these two motors. In villus enterocytes of the Myo1a KO mouse small intestine, CFTR accumulated in syntaxin‐3 positive subapical endosomes, redistributed to the basolateral domain and was absent from the BBM. In colon, where villi are absent and Myo1a expression is low, CFTR exhibited normal localization to the BBM in the Myo1a KO similar to WT. cAMP‐stimulated CFTR anion transport in the small intestine was reduced by 58% in the KO, while anion transport in the colon was comparable to WT. Co‐immunoprecipitation confirmed the association of CFTR with Myo1a. These data indicate that Myo1a is an important regulator of CFTR traffic and anion transport in the BBM of villus enterocytes and suggest that Myo1a may power apical CFTR movement into the BBM from subapical endosomes. Alternatively, it may anchor CFTR channels in the BBM of villus enterocytes as was proposed for Myo1as role in BBM localization of sucrase‐isomaltase.

Modified Poly(lactic-co-glycolic Acid) Nanoparticles for Enhanced Cellular Uptake and Gene Editing in the Lung

Surface-modified poly(lactic-co-glycolic acid) (PLGA)/poly(β-aminoester)(PBAE)nanoparticles (NPs) have shown great promise in gene delivery. In this work, the pulmonary cellular uptake of these NPs is evaluated and surface-modified PLGA/PBAE NPs are shown to achieve higher cellular association and gene editing than traditional NPs composed of PLGA or PLGA/PBAE blends alone.

Calcium-Modulated Chloride Pathways Contribute to Chloride Flux in Murine Cystic Fibrosis-Affected Macrophages

Cystic fibrosis (CF), a common lethal inherited disorder defined by ion transport abnormalities, chronic infection, and robust inflammation, is the result of mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) protein, a cAMP-activated chloride (Cl−) channel. Macrophages are reported to have impaired activity in CF. Previous studies suggest that Cl− transport is important for macrophage function; therefore, impaired Cl− secretion may underlie CF macrophage dysfunction. To determine whether alterations in Cl− transport exist in CF macrophages, Cl− efflux was measured using N-[ethoxycarbonylmethyl]-6-methoxy-quinolinium bromide (MQAE), a fluorescent indicator dye. The contribution of CFTR was assessed by calculating Cl− flux in the presence and absence of cftrinh-172. The contribution of calcium (Ca2+)-modulated Cl− pathways was assessed by examining Cl− flux with varied extracellular Ca2+ concentrations or after treatment with carbachol or thapsigargin, agents that increase intracellular Ca2+ levels. Our data demonstrate that CFTR contributed to Cl− efflux only in WT macrophages, while Ca2+-mediated pathways contributed to Cl− transport in CF and WT macrophages. Furthermore, CF macrophages demonstrated augmented Cl− efflux with increases in extracellular Ca2+. Taken together, this suggests that Ca2+-mediated Cl− pathways are enhanced in CF macrophages compared with WT macrophages.

Rectal potential difference and the functional expression of CFTR in the gastrointestinal epithelia in cystic fibrosis mouse models.

Cystic fibrosis (CF) is an autosomal recessive disease that results from mutations in the CF transmembrane conductance regulator (CFTR) gene. The effect of interventions aimed at correcting the CF electrophysiologic phenotype has been primarily measured using in vitro methods in gastrointestinal and respiratory epithelia. A reliable in vivo assay of CFTR function would be of great value in the investigation of pharmacologic interventions for CF mouse models. We performed the in vivo rectal potential difference (RPD) assay on three different mouse models. We then compared the in vivo data with the results obtained using the in vitro Ussing chamber method. The results from the in vitro method correlated closely with the results acquired using the in vivo method and were reproducible. The data suggest that the in vivo RPD assay is a reliable assay of functional CFTR expression in CF mouse models.

Restoration of cytoskeletal and membrane tethering defects but not defects in membrane trafficking in the intestinal brush border of mice lacking both myosin Ia and myosin VI.

Myosin Ia (Myo1a), the most prominent plus‐end directed motor and myosin VI (Myo6) the sole minus‐end directed motor, together exert opposing tension between the microvillar (MV) actin core and the apical brush border (BB) membrane of the intestinal epithelial cell (IEC). Mice lacking Myo1a or Myo6 each exhibit a variety of defects in the tethering of the BB membrane to the actin cytoskeleton. Double mutant (DM) mice lacking both myosins revealed that all the defects observed in either the Myo1a KO or Snells waltzer (sv/sv) Myo6 mutant mouse are absent. In isolated DM BBs, Myo1a crosslinks between MV membrane and MV actin core are absent but the gap (which is lost in Myo1a KO) between the MV core and membrane is maintained. Several myosins including Myo1c, d, and e and Myo5a are ectopically recruited to the BB. Consistent with the restoration of membrane tethering defects by one or more of these myosins, upward ATP‐driven shedding of the BB membrane, which is blocked in the Myo1a KO, is restored in the DM BB. However, Myo1a or Myo6 dependent defects in expression of membrane proteins that traffic between the BB membrane and endosome (NaPi2b, NHE3, CFTR) are not restored. Compared to controls, Myo1a KO, sv/sv mice exhibit moderate and DM high levels of hypersensitivity to dextran sulfate sodium‐induced colitis. Consistent with Myo1a and Myo6 playing critical roles in maintaining IEC integrity and response to injury, DM IECs exhibit increased numbers of apoptotic nuclei, above that reported for Myo1a KO.