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Hepatobiliary disease causes significant morbidity in people with cystic fibrosis (CF), yet this problem remains understudied. We previously found that newborn CF pigs have microgallbladders with significant luminal obstruction in the absence of infection and consistent inflammation. In this study, we sought to better understand the early pathogenesis of CF pig gallbladder disease. We hypothesized that loss of CFTR would impair gallbladder epithelium anion/liquid secretion and increase mucin production. CFTR was expressed apically in non-CF pig gallbladder epithelium but was absent in CF. CF pig gallbladders lacked cAMP-stimulated anion transport. Using a novel gallbladder epithelial organoid model, we found that Cl−or HCO3−was sufficient for non-CF organoid swelling. This response was absent for non-CF organoids in Cl−/HCO3−-free conditions and in CF. Single-cell RNA-sequencing revealed a single epithelial cell type in non-CF gallbladders that coexpressed CFTR, MUC5AC, and MUC5B. Despite CF gallbladders having increased luminal MUC5AC and MUC5B accumulation, there was no significant difference in the epithelial expression of gel-forming mucins between non-CF and CF pig gallbladders. In conclusion, these data suggest that loss of CFTR-mediated anion transport and fluid secretion contribute to microgallbladder development and luminal mucus accumulation in CF.

Loss of cystic fibrosis transmembrane conductance regulator (CFTR) function causes cystic fibrosis (CF), predisposing the lungs to chronic infection and inflammation. In young infants with CF, structural airway defects are increasingly recognized before the onset of significant lung disease, which suggests a developmental origin and a possible role in lung disease pathogenesis. The role(s) of CFTR in lung development is unclear and developmental studies in humans with CF are not feasible. Young CF pigs have structural airway changes and develop spontaneous postnatal lung disease similar to humans; therefore, we studied lung development in the pig model (non-CF and CF). CF trachea and proximal airways had structural lesions detectable as early as pseudoglandular development. At this early developmental stage, budding CF airways had smaller, hypo-distended lumens compared to non-CF airways. Non-CF lung explants exhibited airway lumen distension in response to forskolin/IBMX as well as to fibroblast growth factor (FGF)-10, consistent with CFTR-dependent anion transport/secretion, but this was lacking in CF airways. We studied primary pig airway epithelial cell cultures and found that FGF10 increased cellular proliferation (non-CF and CF) and CFTR expression/function (in non-CF only). In pseudoglandular stage lung tissue, CFTR protein was exclusively localized to the leading edges of budding airways in non-CF (but not CF) lungs. This discreet microanatomic localization of CFTR is consistent with the site, during branching morphogenesis, where airway epithelia are responsive to FGF10 regulation. In summary, our results suggest that the CF proximal airway defects originate during branching morphogenesis and that the lack of CFTR-dependent anion transport/liquid secretion likely contributes to these hypo-distended airways.

BackgroundCystic fibrosis (CF) lung disease is defined by large numbers of neutrophils and associated damaging products in the airway. Delayed neutrophil apoptosis is described in CF although it is unclear whether this is a primary neutrophil defect or a response to chronic inflammation. Increased levels of neutrophil extracellular traps (NETs) have been measured in CF and we aimed to investigate the causal relationship between these phenomena and their potential to serve as a driver of inflammation. We hypothesised that the delay in apoptosis in CF is a primary defect and preferentially allows CF neutrophils to form NETs, contributing to inflammation.MethodsBlood neutrophils were isolated from patients with CF, CF pigs and appropriate controls. Neutrophils were also obtained from patients with CF before and after commencing ivacaftor. Apoptosis was assessed by morphology and flow cytometry. NET formation was determined by fluorescent microscopy and DNA release assays. NET interaction with macrophages was examined by measuring cytokine generation with ELISA and qRT-PCR.ResultsCF neutrophils live longer due to decreased apoptosis. This was observed in both cystic fibrosis transmembrane conductance regulator (CFTR) null piglets and patients with CF, and furthermore was reversed by ivacaftor (CFTR potentiator) in patients with gating (G551D) mutations. CF neutrophils formed more NETs and this was reversed by cyclin-dependent kinase inhibitor exposure. NETs provided a proinflammatory stimulus to macrophages, which was enhanced in CF.ConclusionsCF neutrophils have a prosurvival phenotype that is associated with an absence of CFTR function and allows increased NET production, which can in turn induce inflammation. Augmenting neutrophil apoptosis in CF may allow more appropriate neutrophil disposal, decreasing NET formation and thus inflammation.

Pancreatic disease has onset in uteroin humans with cystic fibrosis (CF), and progresses over time to complete destruction of the organ. The exact mechanisms leading to pancreatic damage in CF are incompletely understood. Inflammatory cells are present in the pancreas of newborn pigs with CF (CF pigs) and humans, which suggests that inflammation may have a role in the destructive process. We wondered whether tissue inflammation and genes associated with inflammatory pathways were increased in the pancreas of fetal CF pigs [83 to 90 days gestation (normal pig gestation is ∼114 days)] and newborn pigs. Compared with fetal pigs without CF (non-CF pigs), in fetal CF pigs, the pancreas exhibited patchy inflammation and acinar atrophy, with progression in distribution and severity in neonatal CF pigs. Large-scale transcript profiling revealed that the pancreas in fetal and newborn CF pigs exhibited significantly increased expression of proinflammatory, complement cascade, and profibrotic genes when compared with fetal and newborn non-CF pigs. Acinar cells exhibited increased apoptosis in the pancreas of fetal and newborn CF pigs. α-Smooth muscle actin and transforming growth factor β1 were increased in both fetal and newborn CF pig pancreas, suggesting activation of profibrotic pathways. Cell proliferation and mucous cell metaplasia were detected in newborn, but not fetal, CF pigs, indicating that they were not an initiator of pathogenesis but a response. Proinflammatory, complement cascade, proapoptotic, and profibrotic pathways are activated in CF pig pancreas, and likely contribute to the destructive process.

Recent advances in basic science have greatly expanded our understanding of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR), the chloride and bicarbonate channel that is encoded by the gene, which is mutated in patients with CF. We review the structure, function, biosynthetic processing, and intracellular trafficking of CFTR and discuss the five classes of mutations and their impact on the CF phenotype. The therapeutic discussion is focused on the significant progress toward CFTR mutation-specific therapies. We review the results of encouraging clinical trials examining orally administered therapeutics, including agents that promote read-through of class I mutations (premature termination codons); correctors, which overcome the CFTR misfolding that characterizes the common class II mutation F508del; and potentiators, which enhance the function of class III or IV mutated CFTR at the plasma membrane. Long-term outcomes from successful mutation-specific treatments could finally answer the question that has been lingering since and even before the CFTRgene discovery: Will therapies that specifically restore CFTR-mediated chloride secretion slow or arrest the deleterious cascade of events leading to chronic infection, bronchiectasis, and end-stage lung disease?

Cystic fibrosis (CF), which is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR), is characterized by multiorgan pathology that begins early in life. To better understand the initial stages of disease, we studied the gastrointestinal pathology of CFTR−/−pigs. By studying newborns, we avoided secondary changes attributable to environmental interactions, infection, or disease progression. Lesions resembling those in humans with CF were detected in intestine, pancreas, liver, gallbladder, and cystic duct. These organs had four common features. First, disease was accelerated compared with that in humans, which could provide a strategy to discover modifying factors. Second, affected organs showed variable hyperplastic, metaplastic, and connective tissue changes, indicating that remodeling was a dynamic component of fetal life. Third, cellular inflammation was often mild to moderate and not always present, which raises new questions as to the role of cellular inflammation in early disease pathogenesis. Fourth, epithelial mucus-producing cells were often increased, producing a striking accumulation of mucus with a layered appearance and resilient structure. Thus, mucus cell hyperplasia and mucus accumulation play prominent roles in early disease. Our findings also have implications for CF lung disease, and they lay the foundation for a better understanding of CF pathogenesis.

Background: Alcohol abuse and infection with HIV individually compromise immune function, but the consequence of both conditions together is poorly understood owing to the difficulties of performing appropriate studies in human subjects. Simian immunodeficiency virus (SIV) infection of rhesus macaques is considered to closely model HIV disease in that the virus infects CD4+ cells and this infection leads to a similar AIDS state. This study was initiated to study the combined effects of chronic binge alcohol consumption on the primary stage of SIV infection.

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Background: Tumor necrosis factor‐α (TNF‐α), a product of monocytes and macrophages, functions as an important proinflammatory cytokine in the host's response to invading pathogens.

Background: Ethanol (EtOH) is known to alter host immune responses and cytokine production. Acute EtOH exposure can suppress tumor necrosis factor (TNF)‐α production, which attenuates pulmonary defense against infection. Previous studies in our laboratory show that acute EtOH inhibited TNF‐α production by a posttranscriptional process, namely suppression of TNF‐α‐converting, enzyme‐mediated, ectodomain shedding. However, chronic EtOH has been shown to augment TNF‐α production, and this has been associated with EtOH‐induced liver injury. To further characterize this paradoxical effect of EtOH on TNF‐α production, we developed an in vitro model by using Mono Mac 6 cells, a human monocytic cell line.

Neutropenia and impairment of neutrophil function are commonly observed in patients with human immunodeficiency virus (HIV) infections. The HIV regulatory protein Tat is known to exert immunosuppressive effects. Alcohol is known also to be an immunosuppressive factor, and as alcohol abuse is common among HIV infected hosts, both factors may interact in an additive or synergistic fashion to further impair the host defenses of these patients. In order to test this possibility, endotoxin‐induced neutrophil β2‐integrins CD11b and CD18 expression, phagocytosis, and hydrogen, peroxide generation were examined in normal and HIV‐1 Tat‐transgenic mice in the absence and presence of ethanol intoxication. Acute ethanol intoxication was induced in mice (body weight of 25 ± 1 g) by an intraperitoneal Op) injection of ethanol (3 g/kg, 20% in normal saline). Thirty min later, the animals were given an ip injection of endotoxin (20 tig in 0.2 ml of saline/mouse). Vehicle‐treated controls received an ip injection of saline without ethanol or endotoxin. Two hr after endotoxin administration, the animals were killed to determine neutrophil functions with flow cytometry. The baseline expression of CD11b and CD18 was similar in normal nontransgenic and Tat‐transgenic mice. Endotoxin administration significantly up‐regulated CD11b and CD18 expression in normal mice. This up‐regulation was suppressed in Tat‐transgenic mice. Ethanol intoxication inhibited endotoxin‐induced CD11b and CD18 expression in normal mice and totally abolished endotoxin‐induced C011b and CD18 expression in Tat‐transgenic mice. Neutrophil phagocytic activity in normal and Tat‐transgenic mice was similar. Ethanol intoxication produced a similar inhibition of phagocytosis in both study groups. Endotoxin suppressed phagocytosis in normal mice, and this suppression was more pronounced in Tat‐transgenic mice. Spontaneous and phorbol myristate acetate (PMA)‐stimulated hydrogen peroxide generation by circulating neutrophils (PMNs) were similar in normal and Tat‐transgenic mice. Neither ethanol nor endotoxin affected hydrogen peroxide generation by PMNs. These data show that both Tat and alcohol significantly impair PMN function, and this may be a mechanism leading to the increased susceptibility of the HIV‐infected host to bacterial infection.

The effects of granulocyte colony-stimulating factor (G-CSF) on the functional activities of circulating and lung-recruited neutrophils (PMNs) and alveolar macrophages (AMs) were studied in rats to further elucidate the mechanisms underlying G-CSF-enhanced pulmonary host defense. Animals received G-CSF or vehicle twice a day for 2 days, followed by an intratracheal challenge with endotoxin or saline. G-CSF up-regulated CD11b/c expression and mean channel fluorescence intensity of phagocytosis in circulating PMNs. G-CSF also enhanced phagocytic activities, reflected by both the percentage of phagocytosis and mean channel fluorescence intensity in lung-recruited PMNs and AMs in intratracheal endotoxin-challenged rats. The endotoxin-induced increase in pulmonary production of tumor necrosis factor-α and cytokine-induced neutrophil chemoattractant was not affected by G-CSF pretreatment. These data demonstrate that G-CSF-enhanced pulmonary recruitment of PMNs is primarily based on the effects of G-CSF on the PMNs themselves, rather than the generation of certain chemotactic stimuli, i.e., cytokine-induced neutrophil chemoattractant and tumor necrosis factor-α. The enhanced phagocytic activities of lung-recruited PMNs and AMs also augment pulmonary host defenses in G-CSF-pretreated animals.

Alcohol has long been recognized as an immunosuppressive drug and a risk factor for a spectrum of infectious diseases. Among these infections, bacterial pneumonias are most closely correlated with alcohol abuse. One potential mechanism of ethanol‐induced immunosuppression is through its ability to suppress alveolar macrophage production of tumor necrosis factor (TNF‐α). This defect can be reversed by priming macrophages with interferon‐γ (IFN‐γ). We hypothesized that macrophage priming in vivo in a model of acute ethanol intoxication could augment pulmonary host defenses. To test this hypothesis, we used adenoviral‐mediated gene transfer of the IFN‐γ gene. This strategy resulted in prolonged expression of IFN‐γ in vivo. Moreover, in a model of acute ethanol intoxication, this vector significantly enhanced lipopolysaccharide‐induced TNF‐α responses and lung polymorphonuclear leukocyte recruitment. Furthermore, pulmonary host defenses against Klebsiella pneumoniaewere were significantly augmented. These enhanced host defenses were not reversed with pretreatment with a polyclonal anti‐TNF‐α antibody, suggesting that IFN‐γ's effect was through a non‐TNF‐α‐dependent mechanism. These data demonstrate that ethanol‐induced suppression of pulmonary host defenses can be reversed with IFN‐γ gene therapy.

The effects of acute ethanol intoxication on neutrophil [polymorphonuclear leukocyte (PMN)] adhesion molecule expression and certain other functional properties during endotoxemia were studied in rats to elucidate the mechanisms underlying the immunosuppressive effects of ethanol. Acute ethanol intoxication was induced by an intraperitoneal injection of 20% ethanol at a dose of 5.5 g of ethanol/kg. Control animals received an intraperitoneal injection of saline. Thirty minutes after intraperitoneal injection, animals were given a 90‐min intravenous infusion of Escherichia coliendotoxin (total dose of 112.5 μg/rat in 2.5 ml of saline) or saline. Certain rats received granulocyte colony‐stimulating factor (G‐CSF; 50 μg/kg in 5% dextrose, subcutaneous injection twice daily) or vehicle pretreatment for 2 days before intravenous endotoxin infusion. Endotoxemia significantly upregulated CD11b/c and CD18 expression on PMNs when compared with those of saline‐infused rats. Acute ethanol intoxication inhibited this endotoxin‐induced upregulation of CD11b/c and CD18 expression on PMNs. Ethanol intoxication also suppressed the phagocytic activities of PMNs in saline‐infused rats, but this suppression failed to reach statistical significance in endotoxin‐infused rats. Hydrogen peroxide generation by PMNs in saline‐ or endotoxin‐infused rats was not affected by ethanol intoxication. Histological examination showed extensive PMN sequestration in the liver after endotoxin infusion, and ethanol intoxication significantly attenuated this hepatic sequestration of PMNs. G‐CSF pretreatment enhanced neutrophil phagocytosis, CD11b/c and CD18 expression in endotoxin‐infused rats, and prevented the ethanol‐induced inhibition of neutrophil CD18 expression and phagocytosis. The impairment of β2‐integrin expression on PMNs may be one mechanism underlying ethanol‐induced defects of neutrophil delivery into tissue sites of infection. G‐CSF may be of benefit to the infected alcoholic host by enhancing leukocyte defense functions.

Alcohol's suppressive effects on polymorphonuclear leukocyte (PMN) production and function increases host susceptibility to a wide variety of infections and impairs the ability of these effector cells to seek and destroy invading pathogens. Granulocyte colony‐stimulating factor (G‐CSF), an important regulator of PMN production and function, is known to be increased in the plasma during infectious episodes. In previous studies we found acute alcohol intoxication to suppress the tumor necrosis factor‐α (TNFα) response to in vivo challenges with bacteria or lipopolysaccharide. The present study was initiated to determine the impact of alcohol intoxication on the plasma G‐CSF response to Gram‐negative infection. For this purpose, rats received an intravenous challenge of Escherichia coli(106CFU) 30 min after an intraperitoneal injection of ethanol (5.5 g/kg) or an equivalent volume of saline (control). Ethanol‐intoxicated rats had a greater 48 hr mortality to live E. coliinjection than did unintoxicated animals (45% vs. 8%). Despite an increased bacterial burden in both the lung and liver at 24 hr after initiating E. coliinfection in alcohol‐intoxicated animals, PMN tissue recruitment, indexed as myeloperoxidase activity, did not differ between control and alcohol‐treated rats. Moreover, alcohol suppressed blood PMN phagocytic capacity to a greater extent in animals given alcohol than controls at 5 and 24 hr after initiating infection. In control animals after intravenous E. coliinjection, bioactive G‐CSF increased in plasma and peaked near 300 ng/ml at 8 hr. In rats pretreated with alcohol, the plasma G‐CSF response was markedly suppressed in response to intravenous E. coli (p0.05). In a second experiment, neutralization of the E. coli‐induced plasma TNFα response by pretreatment with anti‐TNFα antibody similarly inhibited the plasma G‐CSF response. These results support the postulate that alcohol‐induced inhibition of TNFα directly contributes to the adverse effects of alcohol on PMN function by suppressing the normal autocrine amplification pathway responsible for G‐CSF production.

Infectious diseases continue to be a major cause of morbidity and mortality in the United States. Novel strategies directed at amplifying critical components of the host's immune system may be one potential adjuvant therapy. To date, studies with granulocyte colonystimulating factor GCSF treatment have demonstrated favorable results, decreasing both morbidity and mortality in a variety of infectious disease models in nonneutropenic hosts. The recent completion of two clinical trials has also provided supportive data that GCSF is effective in patients with serious infections. Most important, GCSF does not produce any adverse consequences, which were of concern to some because of the capacity of GCSF to enhance neutrophil production and function. This review focuses on current data suggesting that augmentation of the immune system with GCSF is a promising new approach to adjuvant therapy in infectious diseases.

Objectives: In cystic fibrosis (CF), loss of CFTR-mediated bicarbonate secretion reduces the airway surface liquid (ASL) pH causing airway host defense defects. Aerosolized sodium bicarbonate can reverse these defects, but its effects are short-lived. Aerosolized tromethamine (THAM) also raises the ASL pH but its effects are much longer lasting. In this pilot study, we tested the hypothesis that nasally administered THAM would alter the nasal bacterial composition in adults with and without CF.Methods: Subjects (n = 32 total) received intranasally administered normal saline or THAM followed by a wash out period prior to receiving the other treatment. Nasal bacterial cultures were obtained prior to and after each treatment period.Results: At baseline, nasal swab bacterial counts were similar between non-CF and CF subjects, but CF subjects had reduced microbial diversity. Both nasal saline and THAM were well-tolerated. In non-CF subjects, nasal airway alkalinization decreased both the total bacterial density and the gram-positive bacterial species recovered. In both non-CF and CF subjects, THAM decreased the amount of Corynebacterium accolensdetected, but increased the amount of Corynebacterium pseudodiphtheriticumrecovered on nasal swabs. A reduction in Staphylococcus aureusnasal colonization was also found in subjects who grew C. pseudodiphtheriticum.Conclusions: This study shows that aerosolized THAM is safe and well-tolerated and that nasal airway alkalinization alters the composition of mucosal bacterial communities.Clinical Trial Registration: NCT00928135 (https://clinicaltrials.gov/ct2/show/NCT00928135).

A common mutation in human cystic fibrosis, CFTR-ΔF508, results in misprocessed CFTR and a cystic fibrosis–like clinical phenotype in pigs.

The lungs of just-born piglets with cystic fibrosis fail to efficiently eliminate bacteria, suggesting that lung problems in cystic fibrosis patients may be secondary to impaired antibacterial defense mechanisms.