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Iron plays a critical role in the immune response to inflammation and infection due to its role in the catalysis of reactive oxygen species (ROS) through the Haber-Weiss and Fenton reactions. However, ROS overproduction can be harmful and damage healthy cells. Therefore, iron chelation represents an innovative pharmacological approach to limit excess ROS formation and the related pro-inflammatory mediator cascades. The present study was designed to investigate the impact of the iron chelator, DIBI, in an experimental model of LPS-induced acute lung injury (ALI). DIBI was administered intraperitoneally in the early and later stages of lung inflammation as determined by histopathological evaluation. We found that lung tissues showed significant injury, as well as increased NF-κB p65 activation and significantly elevated levels of various inflammatory mediators (LIX, CXCL2, CCL5, CXCL10, IL-1𝛽, IL-6) 4 h post ALI induction by LPS. Mice treated with DIBI (80 mg/kg) in the early stages (0 to 2 h) after LPS administration demonstrated a significant reduction of the histopathological damage score, reduced levels of NF-κB p65 activation, and reduced levels of inflammatory mediators. Intravital microscopy of the pulmonary microcirculation also showed a reduced number of adhering leukocytes and improved capillary perfusion with DIBI administration. Our findings support the conclusion that the iron chelator, DIBI, has beneficial anti-inflammatory effects in experimental ALI.

Background and Objective: Lung-protective mechanical ventilation is known to attenuate ventilator-associated lung injury (VALI), but often at the expense of hypoventilation and hypercapnia. It remains unclear whether the main mechanism by which VALI is attenuated is a product of limiting mechanical forces to the lung during ventilation, or a direct biological effect of hypercapnia. Methods: Acute lung injury (ALI) was induced in 60 anesthetized rats by the instillation of 1.25 M HCl into the lungs via tracheostomy. Ten rats each were randomly assigned to one of six experimental groups and ventilated for 4 h with: 1) Conventional HighV E Normocapnia (high V T , high minute ventilation, normocapnia), 2) Conventional Normocapnia (high V T , normocapnia), 3) Protective Normocapnia (V T 8 ml/kg, high RR), 4) Conventional iCO 2 Hypercapnia (high V T , low RR, inhaled CO 2 ), 5) Protective iCO 2 Hypercapnia (V T 8 ml/kg, high RR, added CO 2 ), 6) Protective endogenous Hypercapnia (V T 8 ml/kg, low RR). Blood gasses, broncho-alveolar lavage fluid (BALF), and tissue specimens were collected and analyzed for histologic and biologic lung injury assessment. Results: Mild ALI was achieved in all groups characterized by a decreased mean PaO 2 /FiO 2 ratio from 428 to 242 mmHg ( p < 0.05), and an increased mean elastance from 2.46 to 4.32 cmH 2 O/L ( p < 0.0001). There were no differences in gas exchange among groups. Wet-to-dry ratios and formation of hyaline membranes were significantly lower in low V T groups compared to conventional tidal volumes. Hypercapnia reduced diffuse alveolar damage and IL-6 levels in the BALF, which was also true when CO 2 was added to conventional V T . In low V T groups, hypercapnia did not induce any further protective effect except increasing pulmonary IL-10 in the BALF. No differences in lung injury were observed when hypercapnia was induced by adding CO 2 or decreasing minute ventilation, although permissive hypercapnia decreased the pH significantly and decreased liver histologic injury. Conclusion: Our findings suggest that low tidal volume ventilation likely attenuates VALI by limiting mechanical damage to the lung, while hypercapnia attenuates VALI by limiting pro-inflammatory and biochemical mechanisms of injury. When combined, both lung-protective ventilation and hypercapnia have the potential to exert an synergistic effect for the prevention of VALI., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Ismaiel, Whynot, Geldenhuys, Xu, Slutsky, Chappe and Henzler.)

Vasoactive intestinal peptide (VIP), a 28-amino acid neuropeptide with potent anti-inflammatory, bronchodilatory and immunomodulatory functions, is secreted by intrinsic neurons innervating all exocrine glands, including the pancreas, in which it exerts a regulatory function in the secretion of insulin and glucagon. Cystic fibrosis-related diabetes (CFRD) is the most common co-morbidity associated with cystic fibrosis (CF), impacting approximately 50% of adult patients. We recently demonstrated a 50% reduction of VIP abundance in the lungs, duodenum and sweat glands of C57Bl/6 CF mice homozygous for the F508del-CFTR disease-causing mutation. VIP deficiency resulted from a reduction in VIPergic and cholinergic innervation, starting before signs of CF disease were observed. As VIP functions as a neuromodulator with insulinotropic effect on pancreatic beta cells, we sought to study changes in VIP in the pancreas of CF mice. Our goal was to examine VIP content and VIPergic innervation in the pancreas of 8- and 17-week-old F508del-CFTR homozygous mice and to determine whether changes in VIP levels would contribute to CFRD development. Our data showed that a decreased amount of VIP and reduced innervation are found in CF mice pancreas, and that these mice also exhibited reduced insulin secretion, up-regulation of glucagon production and high random blood glucose levels compared to same-age wild-type mice. We propose that low level of VIP, due to reduced innervation of the CF pancreas and starting at an early disease stage, contributes to changes in insulin and glucagon secretion that can lead to CFRD development., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Vasoactive Intestinal Peptide (VIP) is the major physiological agonist of the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) chloride channel activity. VIP functions as a neuromodulator and neurotransmitter secreted by neurons innervating all exocrine glands. VIP is also a potent vasodilator and bronchodilator that regulates exocrine gland secretions, contributing to local innate defense by stimulating the movement of water and chloride transport across intestinal and tracheobronchial epithelia. Previous human studies have shown that the rich intrinsic neuronal networks for VIP secretion around exocrine glands could be lost in tissues from patients with cystic fibrosis. Our research has since confirmed, in vitro and in vivo, the need for chronic VIP exposure to maintain functional CFTR chloride channels at the cell surface of airways and intestinal epithelium, as well as normal exocrine tissues morphology [1]. The goal of the present study was to examine changes in VIP in the lung, duodenum and sweat glands of 8- and 17-weeks old F508del/F508del mice and to investigate VIPergic innervation in the small intestine of CF mice, before important signs of the disease development. Our data show that a low amount of VIP is found in CF tissues prior to tissue damage. Moreover, we found a specific reduction in VIPergic and cholinergic innervation of the small intestine. The general innervation of the primary and secondary myenteric plexus was lost in CF tissues, with the presence of enlarged ganglionic cells in the tertiary layer. We propose that low amount of VIP in CF tissues is due to a reduction in VIPergic and cholinergic innervation and represents an early defect that constitutes an aggravating factor for CF disease progression., Competing Interests: Conflict of Interest Statement There are no conflicts of interest to declare., (Copyright © 2020. Published by Elsevier B.V.)

The CFTR chloride channel is regulated by phosphorylation at PKA and PKC consensus sites within its regulatory region (R-region) through a mechanism, which is still not completely understood. We used a split-CFTR construct expressing the N-term-TMD1-NBD1 (Front Half; FH), TMD2-NBD2-C-Term (Back Half; BH), and the R-region as separate polypeptides (Split-R) in BHK cells, to investigate in situ how different phosphorylation conditions affect the R-region interactions with other parts of the protein. In proximity ligation assays, we studied the formation of complexes between the R-region and each half of the Split-CFTR. We found that at basal conditions, the density of complexes formed between the R-region and both halves of the split channel were equal. PKC stimulation alone had no effect, whereas PKA stimulation induced the formation of more complexes between the R-region and both halves compared to basal conditions. Moreover, PKC + PKA stimulation further enhanced the formation of FH-R complexes by 40% from PKA level. In cells expressing the Split-R with the two inhibitory PKC sites on the R-region inactivated (SR-S641A/T682A), density of FH-R complexes was much higher than in Split-R WT expressing cells after PKC or PKC + PKA stimulation. No differences were observed for BH-R complexes measured at all phosphorylation conditions. Since full-length CFTR channels display large functional responses to PKC + PKA in WT and S641A/T682A mutant, we conclude that FH-R interactions are important for CFTR function. Inactivation of consensus PKC site serine 686 (S686A) significantly reduced the basal BH-R interaction and prevented the PKC enhancing effect on CFTR function and FH-R interaction. The phospho-mimetic mutation (S686D) restored basal BH-R interaction and the PKC enhancing effect on CFTR function with enhanced FH-R interaction. As the channel function is mainly stimulated by PKA phosphorylation of the R-region, and this response is known to be enhanced by PKC phosphorylation, our data support a model in which the regulation of CFTR activation results from increased interactions of the R-region with the N-term-TMD1-NBD1. Also, serine S686 was found to be critical for the PKC enhancing effect which requires a permissive BH-R interaction at basal level and increased FH-R interaction after PKC + PKA phosphorylation., Competing Interests: There are no conflict of interest to declare., (© 2019 The Authors.)

Background: Adults with cystic fibrosis (CF) must continuously manage their condition, while working for a living, and want a normal life. Adherence rates to treatments/medications are less than optimal. Existing theory offers little to explain adherence rates. The purpose of this study was to develop a theory to further the understanding of how people with CF manage their condition in an adherence-driven health care system., Methods: Constructivist Grounded Theory methodology was used to conduct 27 semistructured interviews with adults with CF, family members, and health care providers. Data collection and analysis were simultaneous, using constant comparative methods, initial and focused coding, and category identification and reduction to develop a theory., Results: Doing what works to balance life and CF is the theory generated from this study. The main concern of participants was to be seen as normal. The theory depicts what participants with CF and their family members do about their concerns and involves 4 interrelated processes: working overtime, receiving support, passing as normal, and facing disease progression., Conclusion: Participants did not relate to the term nonadherent; rather they described working overtime to manage CF, to work, and to have a normal life. Health care provider and researcher perspectives on adherence differ from those of people with CF. Engaging adults with CF and health care providers in a dialogue in which expectations are shared may lead to individualized treatment regimens that work, because adults with CF will do what works., (Copyright © 2019 by Daedalus Enterprises.)

Our understanding of the multiorgan pathology of cystic fibrosis (CF) has improved impressively during the last decades, but we still lack a full comprehension of the disease progression. Animal models have greatly contributed to the elucidation of specific mechanisms involved in CF pathophysiology and the development of new therapies. Soon after the cloning of the CF transmembrane conductance regulator ( CFTR ) gene in 1989, the first mouse model was generated and this model has dominated in vivo CF research ever since. Nonetheless, the failure of murine models to mirror human disease severity in the pancreas and lung has led to the generation of larger animal models such as pigs and ferrets. The following review presents and discusses data from the current animal models used in CF research.

Cystic fibrosis (CF) is an autosomal recessive genetic disorder that results in defective cystic fibrosis transmembrane conductance regulator (CFTR) protein expression and function in various tissues. The leading cause of CF mortality and morbidity is the progressive destruction of the lungs due to recurrent infections and chronic inflammation. CFTR defect also affects immune cells, including neutrophils, resulting in ineffective, severe and persistent inflammatory response. Since unopposed recruitment of neutrophils significantly contributes to lung tissue damage through the generation of reactive oxygen species (ROS), we hypothesize that the administration of iron chelators could serve as a novel treatment to attenuate chronic inflammation in CF lungs since iron is significantly involved in ROS production by neutrophils. Ideally, the iron chelator should sequester host iron effectively, prevent bacterial access to chelator-bound iron and penetrates lung tissues efficiently, e.g. by inhalational route of administration., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Background: The progression of cystic fibrosis (CF) in patients with the rare mutation P67L was examined to determine if it induced a milder form of CF compared to the common severe ΔF508 mutation., Methods: Parameters of lung function, level of bacterial infection, nutritional status and hospitalization were used to represent CF progression. Age at diagnosis and pancreatic status were used to assess CF presentation. Analysis of data from the CF Canada Registry collected over a 15-year period included 266 ΔF508/ΔF508 homozygote patients from CF clinics in Atlantic Canada and 26 compound heterozygote patients with the rare P67L mutation from clinics across Canada., Results: Late age at diagnosis, high incidence of pancreatic sufficiency, maintained Body Mass Index (BMI) with age, delayed life-threatening bacterial infection, and fewer days in hospital were observed for P67L heterozygote patients included in this study. Although the decline of lung function did not differ from ΔF508 homozygotes, the fact that a greater proportion of P67L heterozygotes live to an older age suggests that lung function is not the primary factor determining CF progression for P67L heterozygote patients., Conclusion: The P67L mutation is associated with a mild disease, even when combined with the severe ΔF508 mutation., (Copyright © 2017 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.)

Ghrelin is a 28-amino acid (aa) stomach-derived peptide discovered in 1999 as the endogenous ligand for growth hormone secretagogue-receptor (GHS-R). Ghrelin-producing cells constitute a distinct group of endocrine cells dispersed throughout the gastric mucosa and to a lesser extent in the small intestine and the endocrine pancreas. Ghrelin plasma levels rise during fasting and chronic caloric restriction to stimulate food intake and fat storage and to prevent life-threatening falls in blood glucose. Plasma ghrelin levels decrease after a meal is consumed and in conditions of energy surplus (such as obesity). Ghrelin has emerged as a key player in the regulation of appetite and energy homeostasis. Ghrelin achieves these functions through binding the ghrelin receptor GHS-R in appetite-regulating neurons and in peripheral metabolic organs including the endocrine pancreas. Ghrelin levels are negatively correlated with body mass index (BMI) and insulin resistance. In addition, ghrelin secretion is impaired in obesity and insulin resistance. Several studies highlight an important role for ghrelin in glucose homeostasis. Genetic, immunological, and pharmacological blockade of ghrelin signaling resulted in improved glucose tolerance and insulin sensitivity. Furthermore, exogenous ghrelin administration was shown to decrease glucose-induced insulin release and increase glucose level in both humans and rodents. GHS-R was shown to be expressed in pancreatic β-cells and ghrelin suppressed insulin release via a Ca2+-mediated pathway. In this review, we provide a detailed summary of recent advances in the field that focuses on the role of insulin and insulin resistance in the regulation of ghrelin secretion and on the role of ghrelin in glucose-stimulated insulin secretion (GSIS).

Vasoactive intestinal peptide (VIP), a neuropeptide, controls multiple functions in exocrine tissues, including inflammation, and relaxation of airway and vascular smooth muscles, and regulates CFTR-dependent secretion, which contributes to mucus hydration and local innate defense of the lung. We had previously reported that VIP stimulates the VPAC1 receptor, PKCϵ signaling cascade, and increases CFTR stability and function at the apical membrane of airway epithelial cells by reducing its internalization rate. Moreover, prolonged VIP stimulation corrects the molecular defects associated with F508del, the most common CFTR mutation responsible for the genetic disease cystic fibrosis. In the present study, we have examined the impact of the absence of VIP on CFTR maturation, cellular localization, and function in vivo using VIP knockout mice. We have conducted pathological assessments and detected signs of lung and intestinal disease. Immunodetection methods have shown that the absence of VIP results in CFTR intracellular retention despite normal expression and maturation levels. A subsequent loss of CFTR-dependent chloride current was measured in functional assays with Ussing chamber analysis of the small intestine ex vivo, creating a cystic fibrosis-like condition. Interestingly, intraperitoneal administration of VIP corrected tissue abnormalities, close to the wild-type phenotype, as well as associated defects in the vital CFTR protein. The results show in vivo a primary role for VIP chronic exposure in CFTR membrane stability and function and confirm in vitro data., (Copyright © 2014 the American Physiological Society.)

Vasoactive intestinal peptide (VIP) is a topical airway gland secretagogue regulating fluid secretions, primarily by stimulating cystic fibrosis transmembrane conductance regulator (CFTR)-dependent chloride secretion that contributes to the airways innate defense mechanism. We previously reported that prolonged VIP stimulation of pituitary adenylate cyclase-activating peptide receptors (VPAC1) in airway cells enhances CFTR function by increasing its membrane stability. In the present study, we identified the key effectors in the VIP signaling cascade in the human bronchial serous cell line Calu-3. Using immunocytochemistry and in situ proximity ligation assays, we found that VIP stimulation increased CFTR membrane localization by promoting its colocalization and interaction with the scaffolding protein Na(+)/H(+) exchange factor 1 (NHERF1), a PDZ protein known as a positive regulator for CFTR membrane localization. VIP stimulation also increased phosphorylation, by protein kinase Cε of the actin-binding protein complex ezrin/radixin/moesin (ERM) and its interaction with NHERF1 and CFTR complex. On the other hand, it reduced intracellular CFTR colocalization and interaction with CFTR associated ligand, another PDZ protein known to compete with NHERF1 for CFTR interaction, inducing cytoplasmic retention and lysosomal degradation. Reducing NHERF1 or ERM expression levels by specific siRNAs prevented the VIP effect on CFTR membrane stability. Furthermore, iodide efflux assays confirmed that NHERF1 and P-ERM are necessary for VIP regulation of the stability and sustained activity of membrane CFTR. This study shows the cellular mechanism by which prolonged VIP stimulation of airway epithelial cells regulates CFTR-dependent secretions., (Copyright © 2014 the American Physiological Society.)

F508del is the most common cystic fibrosis-causing mutation that induces early degradation and poor trafficking of cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels to the apical membrane of epithelial cells. Our previous work in bronchial serous cells showed that vasoactive intestinal peptide (VIP) stimulation of the VPAC(1) receptor enhances CFTR-dependent chloride secretion by increasing its membrane insertion by a protein kinase C (PKC)-dependent pathway. In the present study, we investigated the effect of VIP on F508del-CFTR activity and membrane insertion in the human nasal epithelial cell line JME/CF15, which also expresses the VPAC(1) receptor. At reduced temperature (27 degrees C), which rescues F508del-CFTR trafficking, acute stimulation by VIP of rescued F508del-CFTR channels was protein kinase A (PKA)- and PKC-dependent. One hour of treatment with VIP strongly increased F508del-CFTR activity, with iodide efflux peaks three times higher than with untreated cells. At 37 degrees C, VIP-treated cells, but not untreated controls, showed significant iodide efflux peaks that were sensitive to the CFTR inhibitor 3-[(3-trifluoromethyl)phenyl]-5-[(4-carboxyphenyl)methylene]-2-thioxo-4-thiazolidinone (CFTR(inh)-172). Immunostaining, biotinylation assays, and Western blots confirmed a VIP-induced maturation and membrane insertion of F508del-CFTR at 37 degrees C. The corrector effect of VIP was abolished by the PKA inhibitor N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamidedihydrochloride (H89), whereas Galpha(s) stimulation by cholera toxin significantly increased F508del-CFTR trafficking. On the other hand, membrane localization, but not maturation, of F508del-CFTR was significantly reduced by the PKC inhibitor bisindolylmaleimide X and the G(i/o) protein inhibitor pertussis toxin. VIP treatment had no effect on intracellular calcium or proteasome activity. These results indicate that, in human nasal cells, VIP rescues trafficking and membrane insertion of functional F508del-CFTR channels at physiological temperature by stimulating both PKA- and PKC-dependent pathways.

The unphosphorylated regulatory (R) domain of the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) is often viewed as an inhibitor that is released by phosphorylation. To test this notion, we studied domain interactions using CFTR channels assembled from three polypeptides. Nucleotides encoding the R domain (aa 635-836) were replaced with an internal ribosome entry sequence so that amino- and carboxyl-terminal half-molecules would be translated from the same mRNA transcript. Although only core glycosylation was detected on SplitDeltaR, biotinylation, immunostaining, and functional studies clearly demonstrated its trafficking to the plasma membrane. SplitDeltaR generated a constitutive halide permeability, which became responsive to cAMP when the missing R domain was coexpressed. Each half-molecule was co-precipitated by antibody against the other half. Contrary to expectations, GST-R domain was pulled down only if prephosphorylated by protein kinase A, and coexpressed R domain was precipitated with SplitDeltaR much more efficiently when cells were stimulated with cAMP. These results indicate that phosphorylation regulates CFTR by promoting association of the R domain with other domains rather than by causing its dissociation from an inhibitory site.

Protein kinase C (PKC) phosphorylation stimulates the cystic fibrosis transmembrane conductance regulator (CFTR) channel and enhances its activation by protein kinase A (PKA) through mechanisms that remain poorly understood. We have examined the effects of mutating consensus sequences for PKC phosphorylation and report here evidence for both stimulatory and inhibitory sites. Sequences were mutated in subsets and the mutants characterized by patch clamping. Activation of a 4CA mutant (S707A/S790A/T791A/S809A) by PKA was similar to that of wild-type CFTR and was enhanced by PKC, whereas responses of 3CA (T582A/T604A/S641A) and 2CA (T682A/S686A) channels to PKA were both drastically reduced (>90%). When each mutation in the 3CA and 2CA constructs was studied individually in a wild-type background, T582, T604, and S686 were found to be essential for PKA activation. Responses were restored when these three residues were reintroduced simultaneously into a 9CA mutant lacking all nine PKC consensus sequences (R6CA revertant); however, PKC phosphorylation was not required for this rescue. Nevertheless, two of the sites (T604 and S686) were phosphorylated in vitro, and PKC alone partially activated wild-type CFTR, the 4CA mutant, and the point mutants T582A and T604A, but not S686A channels, indicating that PKC does act at S686. The region encompassing S641 and T682 is inhibitory, because S641A enhanced activation by PKA, and T682A channels had 4-fold larger responses to PKC compared to wild-type channels. These results identify functionally important PKC consensus sequences on CFTR and will facilitate studies of its convergent regulation by PKC and PKA.