A pathogenic role for cystic fibrosis transmembrane conductance regulator in celiac disease.

Intestinal handling of dietary proteins usually prevents local inflammatory and immune responses and promotes oral tolerance. However, in ~ 1% of the world population, gluten proteins from wheat and related cereals trigger an HLA DQ2/8-restricted TH1 immune and antibody response leading to celiac disease. Prior epithelial stress and innate immune activation are essential for breaking oral tolerance to the gluten component gliadin. How gliadin subverts host intestinal mucosal defenses remains elusive. Here, we show that the α-gliadin-derived LGQQQPFPPQQPY peptide (P31-43) inhibits the function of cystic fibrosis transmembrane conductance regulator (CFTR), an anion channel pivotal for epithelial adaptation to cell-autonomous or environmental stress. P31-43 binds to, and reduces ATPase activity of, the nucleotide-binding domain-1 (NBD1) of CFTR, thus impairing CFTR function. This generates epithelial stress, tissue transglutaminase and inflammasome activation, NF-κB nuclear translocation and IL-15 production, that all can be prevented by potentiators of CFTR channel gating. The CFTR potentiator VX-770 attenuates gliadin-induced inflammation and promotes a tolerogenic response in gluten-sensitive mice and cells from celiac patients. Our results unveil a primordial role for CFTR as a central hub orchestrating gliadin activities and identify a novel therapeutic option for celiac disease.

Mutation-specific therapies and drug repositioning in cystic fibrosis.

Cystic fibrosis (CF) is an inherited, prematurely lethal rare disease affecting more than 85,000 people worldwide. CF is caused by more than 2000 loss-of-function mutations in the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR). This review summarizes recent advances in the etiological therapies of CF that aim at repairing the functional defect of CFTR by means of CFTR modulators. We will discuss the state of art of the mutation-specific treatments that are designed to target different steps of the CFTR biogenesis perturbed by mutations in CFTR gene. Moreover, we will discuss how drug repositioning, namely the use of drugs already approved for the treatment of other human diseases, may be repurposed in CF patients to circumvent CFTR dysfunction. Finally, we highlight how the combined use of two or more compounds acting on different disease mechanisms is required to achieve clinical benefit in CF population.

Inhaled medications in cystic fibrosis beyond antibiotics.

Structural lung disease begins very early in children with cystic fibrosis (CF), often in the first three months of life. Inhaled medications represent an attractive therapeutic approach in CF that are routinely used as early intervention strategies. Two aerosolized solutions, hypertonic saline and dornase alfa, have significant potential benefits by improving mucociliary clearance, with minimal associated side-effects. In particular, they favor rehydration of airway surface liquid and cleavage of extracellular DNA in the airways, respectively, consequently reducing rate of pulmonary disease exacerbations. Indirect anti-inflammatory effects have been documented for both drugs, addressing each of the three interrelated elements in the vicious cycle of lung disease in CF: airway obstruction, inflammation and infection. This short review aimed to summarize the main papers that support potential clinical impact of inhaled solutions on pulmonary disease in CF.

Personalization of therapies in rare diseases: a translational approach for the treatment of cystic fibrosis.

High variability in the response rates to treatments can make the interpretation of data from clinical trials very difficult, particularly in rare genetic diseases in which the enrolment of thousands of patients is problematic. Personalized medicine largely depends on the establishment of appropriate early detectors of drug efficacy that may guide the administration (or discontinuation) of specific treatments. Such biomarkers should be capable of predicting the therapeutic response of individual patients and of monitoring early benefits of candidate drugs before late clinical benefits become evident. The identification of these biomarkers implies a rigorous stepwise process of translation from preclinical evaluation in cultured cells, suitable animal models or patient-derived freshly isolated cells to clinical application. In this review, we will discuss how a process of research translation can lead to the implementation of functional and mechanistic disease-relevant biomarkers. Moreover, we will address how preclinical data can be translated into the clinic in a personalized medical approach that can provide the right drug to the right patient within the right timeframe.

Reduced caveolin-1 promotes hyperinflammation due to abnormal heme oxygenase-1 localization in lipopolysaccharide-challenged macrophages with dysfunctional cystic fibrosis transmembrane conductance regulator.

We have previously reported that TLR4 signaling is increased in LPS-stimulated cystic fibrosis (CF) macrophages (MΦs), contributing to the robust production of proinflammatory cytokines. The heme oxygenase-1 (HO-1)/CO pathway modulates cellular redox status, inflammatory responses, and cell survival. The HO-1 enzyme, together with the scaffold protein caveolin 1 (CAV-1), also acts as a negative regulator of TLR4 signaling in MΦs. In this study, we demonstrate that in LPS-challenged CF MΦs, HO-1 does not compartmentalize normally to the cell surface and instead accumulates intracellularly. The abnormal HO-1 localization in CF MΦs in response to LPS is due to decreased CAV-1 expression, which is controlled by the cellular oxidative state, and is required for HO-1 delivery to the cell surface. Overexpression of HO-1 or stimulating the pathway with CO-releasing molecules enhances CAV-1 expression in CF MΦs, suggesting a positive-feed forward loop between HO-1/CO induction and CAV-1 expression. These manipulations re-established HO-1 and CAV-1 cell surface localization in CF MΦs. Consistent with restoration of HO-1/CAV-1-negative regulation of TLR4 signaling, genetic or pharmacological (CO-releasing molecule 2) induced enhancement of this pathway decreased the inflammatory response of CF MΦs and CF mice treated with LPS. In conclusion, our results demonstrate that the counterregulatory HO-1/CO pathway, which is critical in balancing and limiting the inflammatory response, is defective in CF MΦs through a CAV-1-dependent mechanism, exacerbating the CF MΦ response to LPS. This pathway could be a potential target for therapeutic intervention for CF lung disease.

Anakinra restores cellular proteostasis by coupling mitochondrial redox balance to autophagy.

Autophagy selectively degrades aggregation-prone misfolded proteins caused by defective cellular proteostasis. However, the complexity of autophagy may prevent the full appreciation of how its modulation could be used as a therapeutic strategy in disease management. Here, we define a molecular pathway through which recombinant IL-1 receptor antagonist (IL-1Ra, anakinra) affects cellular proteostasis independently from the IL-1 receptor (IL-1R1). Anakinra promoted H2O2-driven autophagy through a xenobiotic sensing pathway involving the aryl hydrocarbon receptor that, activated through the indoleamine 2,3-dioxygenase 1-kynurenine pathway, transcriptionally activated NADPH oxidase 4 independent of the IL-1R1. By coupling the mitochondrial redox balance to autophagy, anakinra improved the dysregulated proteostasis network in murine and human cystic fibrosis. We anticipate that anakinra may represent a therapeutic option in addition to its IL-1R1-dependent antiinflammatory properties by acting at the intersection of mitochondrial oxidative stress and autophagy with the capacity to restore conditions in which defective proteostasis leads to human disease.

The gliadin-CFTR connection: new perspectives for the treatment of celiac disease.

Familial loss-of-function mutations of the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR) channel protein cause cystic fibrosis (CF), the most frequent inherited life-threatening disease in the Caucasian population. A recent study indicates that the gluten/gliadin-derived peptide (P31-43) can cause CFTR inhibition in intestinal epithelial cells, thus causing a local stress response that contributes to the immunopathology of celiac disease (CD). Accordingly, an increased prevalence of CD has been observed in several cohorts of CF patients. CD is characterized by a permanent intolerance to gluten/gliadin proteins occurring in a proportion of susceptible individuals who bear the human leukocyte antigen (HLA) DQ2/DQ8. In CD, perturbations of the intestinal environment, together with the activation of the innate immune system by P31-43, are essential for rendering other immunodominant gliadin peptide fully antigenic, thus triggering an adaptive immune response with an autoimmune component. P31-43-induced CFTR inhibition elicits the danger signals that ignite the epithelial stress response and perturb epithelial proteostasis. Importantly, potentiators of CFTR channel gating, such as the FDA-approved drug Ivacaftor, prevent P31-43 driven CFTR inhibition and suppress the gliadin-induced stress response in cells from celiac patients, as well as the immunopathology developing in gliadin-sensitive mice. Thus, CFTR potentiators may represent a novel therapeutic option for celiac patients.

Defective proteostasis in celiac disease as a new therapeutic target.

Cystic fibrosis (CF) is a disease caused by loss-of-function mutations affecting the CF transmembrane conductance regulator (CFTR), a chloride channel. Recent evidence indicates that CFTR is inhibited by a gluten/gliadin-derived peptide (P31-43), causing an acquired state of CFTR inhibition within the gut that contributes to the pathogenesis of celiac disease (CD). Of note, CFTR inhibition does not only cause intra- and extracellular ion imbalances but also affects proteostasis by activating transglutaminase-2 (TGM2) and by disabling autophagy. These three phenomena (CFTR inhibition, TGM2 activation, and autophagy impairment) engage in multiple self-amplifying circuitries, thus forming an "infernal trio". The trio hinders enterocytes from returning to homeostasis and instead locks them in an irreversible pro-inflammatory state that ultimately facilitates T lymphocyte-mediated immune responses against another gluten/gliadin-derived peptide (P57-68), which,upon deamidation by activated TGM2, becomes fully antigenic. Hence, the pathogenic protein gliadin exemplifies a food constituent the exceptional immunogenicity of which arises from a combination of antigenicity (conferred by deaminated P57-68) and adjuvanticity (conferred by P31-43). CF can be treated by agents targeting the "infernal trio" including CFTR potentiators, TGM2 inhibitors, and autophagy enhancers. We speculate that such agents may also be used for CD therapy and indeed could constitute close-to-etiological treatments of this enteropathy.

To Be or Not to Be a Pathogen: Candida albicans and Celiac Disease.

Celiac disease (CD) is an immune-mediated disorder triggered by the ingestion of gluten and characterized by reversible small-bowel mucosal atrophy in genetically predisposed subjects. Although the prevalence of CD has increased, many aspects of this pathology are still unrecognized. Candida albicans, a commensal of the human gastrointestinal tract, has been linked to CD for a long time based, among others, upon the observation of similarity between the fungal wall component, hyphal wall protein 1, and CD-related gliadin T-cell epitopes. We have recently demonstrated that Candida may switch from commensal to pathogen contingent upon several players, including mast cells, key sentinels of the immune system at the interface between the environment and the host, and the pleiotropic cytokine IL-9. However, other factors are likely to play a role by altering the balance between inflammation and tolerance. In this regard, tryptophan and its metabolites are increasingly being recognized in promoting mucosal homeostasis by balancing the immune response to external cues. Based on these premises, we will discuss how the output of Candida colonization in the gut is highly contextual, being determined at the intersection of many immunological (IL-9/mast cells) and metabolic (tryptophan) pathways that ultimately dictate the Candida commensalism vs. pathogenicity in CD, thus paving the way for novel therapeutic opportunities in CD.

Autophagy suppresses the pathogenic immune response to dietary antigens in cystic fibrosis.

Under physiological conditions, a finely tuned system of cellular adaptation allows the intestinal mucosa to maintain the gut barrier function while avoiding excessive immune responses to non-self-antigens from dietary origin or from commensal microbes. This homeostatic function is compromised in cystic fibrosis (CF) due to loss-of-function mutations in the CF transmembrane conductance regulator (CFTR). Recently, we reported that mice bearing defective CFTR are abnormally susceptible to a celiac disease-like enteropathy, in thus far that oral challenge with the gluten derivative gliadin elicits an inflammatory response. However, the mechanisms through which CFTR malfunction drives such an exaggerated response to dietary protein remains elusive. Here we demonstrate that the proteostasis regulator/transglutaminase 2 (TGM2) inhibitor cysteamine restores reduced Beclin 1 (BECN1) protein levels in mice bearing cysteamine-rescuable F508del-CFTR mutant, either in homozygosis or in compound heterozygosis with a null allele, but not in knock-out CFTR mice. When cysteamine restored BECN1 expression, autophagy was increased and gliadin-induced inflammation was reduced. The beneficial effects of cysteamine on F508del-CFTR mice were lost when these mice were backcrossed into a Becn1 haploinsufficient/autophagy-deficient background. Conversely, the transfection-enforced expression of BECN1 in human intestinal epithelial Caco-2 cells mitigated the pro-inflammatory cellular stress response elicited by the gliadin-derived P31-43 peptide. In conclusion, our data provide the proof-of-concept that autophagy stimulation may mitigate the intestinal malfunction of CF patients.

Author Correction: Thymosin α1 represents a potential potent single-molecule-based therapy for cystic fibrosis.

In the version of this article originally published, some labels in Fig. 1f are incorrect. The "ß-actin" labels on the second and fourth rows of blots should instead be "ß-tubulin". The error has been corrected in the HTML and PDF versions of this article.

Publisher Correction: Thymosin α1 represents a potential potent single-molecule-based therapy for cystic fibrosis.

In the version of this article originally published, the amino acid sequence for Tα1 described in the Online Methods is incorrect. The sequence is described as "Ac-SDAAVDTSSEITTJDLKEKKEVVEEAEN-OH". It should be "Ac-SDAAVDTSSEITTKDLKEKKEVVEEAEN-OH". The error has been corrected in the HTML and PDF versions of this article.

Cysteamine re-establishes the clearance of Pseudomonas aeruginosa by macrophages bearing the cystic fibrosis-relevant F508del-CFTR mutation.

Cystic fibrosis (CF), the most common lethal monogenic disease in Caucasians, is characterized by recurrent bacterial infections and colonization, mainly by Pseudomonas aeruginosa, resulting in unresolved airway inflammation. CF is caused by mutations in the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR) protein, which functions as a chloride channel in epithelial cells, macrophages, and other cell types. Impaired bacterial handling by macrophages is a feature of CF airways, although it is still debated how defective CFTR impairs bacterial killing. Recent evidence indicates that a defective autophagy in CF macrophages leads to alterations of bacterial clearance upon infection. Here we use bone marrow-derived macrophages from transgenic mice to provide the genetic proof that defective CFTR compromises both uptake and clearance of internalized Pseudomonas aeruginosa. We demonstrate that the proteostasis regulator cysteamine, which rescues the function of the most common F508del-CFTR mutant and hence reduces lung inflammation in CF patients, can also repair the defects of CF macrophages, thus restoring both bacterial internalization and clearance through a process that involves upregulation of the pro-autophagic protein Beclin 1 and re-establishment of the autophagic pathway. Altogether these results indicate that cysteamine restores the function of several distinct cell types, including that of macrophages, which might contribute to its beneficial effects on CF.

Thymosin α1 represents a potential potent single-molecule-based therapy for cystic fibrosis.

Cystic fibrosis (CF) is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) that compromise its chloride channel activity. The most common mutation, p.Phe508del, results in the production of a misfolded CFTR protein, which has residual channel activity but is prematurely degraded. Because of the inherent complexity of the pathogenetic mechanisms involved in CF, which include impaired chloride permeability and persistent lung inflammation, a multidrug approach is required for efficacious CF therapy. To date, no individual drug with pleiotropic beneficial effects is available for CF. Here we report on the ability of thymosin alpha 1 (Tα1)-a naturally occurring polypeptide with an excellent safety profile in the clinic when used as an adjuvant or an immunotherapeutic agent-to rectify the multiple tissue defects in mice with CF as well as in cells from subjects with the p.Phe508del mutation. Tα1 displayed two combined properties that favorably opposed CF symptomatology: it reduced inflammation and increased CFTR maturation, stability and activity. By virtue of this two-pronged action, Tα1 has strong potential to be an efficacious single-molecule-based therapeutic agent for CF.

Manipulating proteostasis to repair the F508del-CFTR defect in cystic fibrosis.

Cystic fibrosis (CF) is a lethal monogenic disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that entails the (diagnostic) increase in sweat electrolyte concentrations, progressive lung disease with chronic inflammation and recurrent bacterial infections, pancreatic insufficiency, and male infertility. Therapies aimed at restoring the CFTR defect have emerged. Thus, a small molecule which facilitates chloride channel opening, the potentiator Ivacaftor, has been approved for the treatment of CF patients bearing a particular class of rare CFTR mutations. However, small molecules that directly target the most common misfolded CFTR mutant, F508del, and improve its intracellular trafficking in vitro, have been less effective than expected when tested in CF patients, even in combination with Ivacaftor. Thus, new strategies are required to circumvent the F508del-CFTR defect. Airway and intestinal epithelial cells from CF patients bearing the F508del-CFTR mutation exhibit an impressive derangement of cellular proteostasis, with oxidative stress, overactivation of the tissue transglutaminase (TG2), and disabled autophagy. Proteostasis regulators such as cysteamine can rescue and stabilize a functional F508del-CFTR protein through suppressing TG2 activation and restoring autophagy in vivo in F508del-CFTR homozygous mice, in vitro in CF patient-derived cell lines, ex vivo in freshly collected primary patient's nasal cells, as well as in a pilot clinical trial involving homozygous F508del-CFTR patients. Here, we discuss how the therapeutic normalization of defective proteostasis can be harnessed for the treatment of CF patients with the F508del-CFTR mutation.

Restoration of CFTR function in patients with cystic fibrosis carrying the F508del-CFTR mutation.

Restoration of BECN1/Beclin 1-dependent autophagy and depletion of SQSTM1/p62 by genetic manipulation or autophagy-stimulatory proteostasis regulators, such as cystamine, have positive effects on mouse models of human cystic fibrosis (CF). These measures rescue the functional expression of the most frequent pathogenic CFTR mutant, F508del, at the respiratory epithelial surface and reduce lung inflammation in Cftr(F508del) homozygous mice. Cysteamine, the reduced form of cystamine, is an FDA-approved drug. Here, we report that oral treatment with cysteamine greatly reduces the mortality rate and improves the phenotype of newborn mice bearing the F508del-CFTR mutation. Cysteamine was also able to increase the plasma membrane expression of the F508del-CFTR protein in nasal epithelial cells from F508del homozygous CF patients, and these effects persisted for 24 h after cysteamine withdrawal. Importantly, this cysteamine effect after washout was further sustained by the sequential administration of epigallocatechin gallate (EGCG), a green tea flavonoid, both in vivo, in mice, and in vitro, in primary epithelial cells from CF patients. In a pilot clinical trial involving 10 F508del-CFTR homozygous CF patients, the combination of cysteamine and EGCG restored BECN1, reduced SQSTM1 levels and improved CFTR function from nasal epithelial cells in vivo, correlating with a decrease of chloride concentrations in sweat, as well as with a reduction of the abundance of TNF/TNF-alpha (tumor necrosis factor) and CXCL8 (chemokine [C-X-C motif] ligand 8) transcripts in nasal brushing and TNF and CXCL8 protein levels in the sputum. Altogether, these results suggest that optimal schedules of cysteamine plus EGCG might be used for the treatment of CF caused by the F508del-CFTR mutation.

Towards a rational combination therapy of cystic fibrosis: How cystamine restores the stability of mutant CFTR.

Cystic fibrosis (CF) is most frequently due to homozygous ΔF508-CFTR mutation. The ΔF508-CFTR protein is unstable in the plasma membrane (PM), even if it is rescued by pharmacological agents that prevent its intracellular retention and degradation. Restoring defective autophagy in CF airways by proteostasis regulators (such as cystamine and its reduced form, cysteamine) can rescue and stabilize ΔF508-CFTR at the PM, thus enabling the action of CFTR potentiators, which are pharmacological agents that stimulate the function of CFTR as an ion channel. The effects of cystamine extend for days (in vitro) and weeks (in vivo) beyond washout, suggesting that once peripheral proteostasis has been re-established, PM-resident ΔF508-CFTR sustains its own stability. We demonstrated that the pharmacological inhibition of wild-type CFTR [cystic fibrosis transmembrane conductance regulator (ATP-binding cassette subfamily C, member 7)], in bronchial epithelial cells decreases the stability of the CFTR protein by inhibiting autophagy, elevating the abundance of SQSTM1/p62 and its interaction with CFTR at the PM, increasing the ubiqutination of CFTR, stimulating the lysosomal degradation of CFTR and avoiding its recycling. All these effects could be inhibited by cystamine. Moreover, CFTR-sufficient epithelia generate permissive conditions for incorporating ΔF508-CFTR into the PM and stabilizing it at this location. These results provide the rationale for a combination therapy of CF in which pretreatment with cystamine or cysteamine enables the later action of CFTR potentiators.

Nebulized hyaluronan ameliorates lung inflammation in cystic fibrosis mice.

RATIONALE: Chronic lung inflammation with increased susceptibility to bacterial infections cause much of the morbidity and mortality in patients with cystic fibrosis (CF), the most common severe, autosomal recessively inherited disease in the Caucasian population. Exogenous inhaled hyaluronan (HA) can exert a protective effect against injury and beneficial effects of HA have been shown in experimental models of chronic respiratory diseases. Our objective was to examine whether exogenous administration of nebulized HA might interfere with lung inflammation in CF. STUDY DESIGN/METHODS: F508del homozygous mice (Cftr(F508del) ) and transgenic mice overexpressing the ENaC channel ß-subunit (Scnn1b-Tg) were treated with nebulized HA (0.5 mg/mouse/day for 7 days). Tumor necrosis factor-alpha (TNFα), macrophage inflammatory protein-2 (MIP-2), myeloperoxidase (MPO) levels, and macrophage infiltration were assessed on lung tissues. IB3-1 and CFBE41o-epithelial cell lines were cultured with HA (24 hr, 100 µg/ml) and Reactive Oxygen Species (ROS), Tissue Transglutaminase (TG2) SUMOylation and Peroxisome Proliferator Activated Receptor gamma (PPARγ) and phospho-p42/p44 levels were measured by dichlorodihydrofluorescein assay, or fluorescence resonance energy transfer (FRET) microscopy or immunoblots. RESULTS: Nebulized HA reduced TNFα expression (P < 0.005); TNFα, MIP-2, and MPO protein levels (P < 0.05); MPO activity (P < 0.05); and CD68+ cells counts (P < 0.005) in lung tissues of Cftr(F508del) and Scnn1b-Tg mice, compared with saline-treated mice. HA reduced ROS, TG2 SUMOylation, TG2 activity, phospho-p42-44, and increased PPARγ protein in both IB3-1 and CFBE41o cells (P < 0.05). CONCLUSIONS: Nebulized HA is effective in controlling inflammation in vivo in mice CF airways and in vitro in human airway epithelial cells. We provide the proof of concept for the use of inhaled HA as a potential anti-inflammatory drug in CF therapy.

Cystic fibrosis: a disorder with defective autophagy.

The accumulation of misfolded and/or ubiquitinated protein aggregates with a perturbation of autophagy has been described in several human pathologies. A sequestration of misfolded cystic: fibrosis transmembrane conductance regulator (CFTR) and cross-linked PPARγ has been observed in airway epithelia of cystic fibrosis (CF) patients. CF airways are also characterized by chronic inflammation, pro-oxidative environment and increased transglutaminase 2 (TG2) levels. We showed that defective CFTR drives autophagy inhibition through reactive oxygen species (ROS)-TG2- mediated aggresome sequestration of the Beclin 1 interactome. Rescuing Beclin 1 at the level of the endoplasmic reticulum and autophagy favors clearance of aggresomes, improves CFTR trafficking and ameliorates CF lung inflammation both in vitro and in vivo. Therefore, rescuing autophagy interrupts the vicious cycle linking defective CFTR and lung inflammation and may pave the way to the development of a novel class of drugs for the treatment of CF.