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.

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.

Heat-shock factor 1 (HSF1) is the master transcription factor that regulates the response to proteotoxic stress by controlling the transcription of many stress-responsive genes including the heat-shock proteins. Here, we show a novel molecular mechanism controlling the activation of HSF1. We demonstrate that transglutaminase type 2 (TG2), dependent on its protein disulphide isomerase activity, triggers the trimerization and activation of HSF1 regulating adaptation to stress and proteostasis impairment. In particular, we find that TG2 loss of function correlates with a defect in the nuclear translocation of HSF1 and in its DNA-binding ability to the HSP70 promoter. We show that the inhibition of TG2 restores the unbalance in HSF1-HSP70 pathway in cystic fibrosis (CF), a human disorder characterized by deregulation of proteostasis. The absence of TG2 leads to an increase of about 40% in CFTR function in a new experimental CF mouse model lacking TG2. Altogether, these results indicate that TG2 plays a key role in the regulation of cellular proteostasis under stressful cellular conditions through the modulation of the heat-shock response.

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Inflammatory bowel disease (IBD) is a complex, chronic, and dysregulated inflammatory condition which etiology is still largely unknown. Its prognosis and disease progression are highly variable and unpredictable. IBD comprises several heterogeneous inflammatory conditions ranging from Ulcerative Colitis (UC) to Crohn’s Disease (CD). Importantly, a definite, well-established, and effective clinical treatment for these pathologies is still lacking. The urgent need for treatment is further supported by the notion that patients affected by UC or CD are also at risk of developing cancer. Therefore, a deeper understanding of the molecular mechanisms at the basis of IBD development and progression is strictly required to design new and efficient therapeutic regimens. Although the development of animal models has undoubtedly facilitated the study of IBD, such in vivo approaches are often expensive and time-consuming. Here we propose an organ ex vivo culture (Gut-Ex-Vivo system, GEVS) based on colon from Balb/c mice cultivated in a dynamic condition, able to model the biochemical and morphological features of the mouse models exposed to DNBS (5–12 days), in 5 h. Indeed, upon DNBS exposure, we observed a dose-dependent: (i) up-regulation of the stress-related protein transglutaminase 2 (TG2); (ii) increased intestinal permeability associated with deregulated tight junction protein expression; (iii) increased expression of pro-inflammatory cytokines, such as TNFα, IFNγ, IL1β, IL6, IL17A, and IL15; (iv) down-regulation of the anti-inflammatory IL10; and (v) induction of Endoplasmic Reticulum stress (ER stress), all markers of IBD. Altogether, these data indicate that the proposed model can be efficiently used to study the pathogenesis of IBD, in a time- and cost-effective manner.

Celiac disease (CD) is a complex immune-mediated chronic disease characterized by a consistent inflammation of the gastrointestinal tract induced by gluten intake in genetically predisposed individuals. Although initiated by the interaction between digestion-derived gliadin, a gluten component, peptides, and the intestinal epithelium, the disorder is highly complex and involving other components of the intestine, such as the immune system. Therefore, conventional model systems, mainly based on two- or three-dimension cell cultures and co-cultures, cannot fully recapitulate such a complex disease. The development of mouse models has facilitated the study of different interacting cell types involved in the disorder, together with the impact of environmental factors. However, such in vivo models are often expensive and time consuming. Here we propose an organ ex vivo culture (gut-ex-vivo system) based on small intestines from gluten-sensitive mice cultivated in a dynamic condition, able to fully recapitulate the biochemical and morphological features of the mouse model exposed to gliadin (4 weeks), in 16 h. Indeed, upon gliadin exposure, we observed: i) a down-regulation of cystic fibrosis transmembrane regulator (CFTR) and an up-regulation of transglutaminase 2 (TG2) at both mRNA and protein levels; ii) increased intestinal permeability associated with deregulated tight junction protein expression; iii) induction and production of pro-inflammatory cytokines such as interleukin (IL)-15, IL-17 and interferon gamma (IFNγ); and iv) consistent alteration of intestinal epithelium/villi morphology. Altogether, these data indicate that the proposed model can be efficiently used to study the pathogenesis of CD, test new or repurposed molecules to accelerate the search for new treatments, and to study the impact of the microbiome and derived metabolites, in a time- and cost- effective manner.

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.

Exposure to gluten, a protein present in wheat rye and barley, is the major inducer for human Celiac Disease (CD), a chronic autoimmune enteropathy. CD occurs in about 1% worldwide population, in genetically predisposed individuals bearing human leukocyte antigen (HLA) DQ2/DQ8. Gut epithelial cell stress and the innate immune activation are responsible for the breaking oral tolerance to gliadin, a gluten component. To date, the only treatment available for CD is a long-term gluten-free diet. Several studies have shown that an altered composition of the intestinal microbiota (dysbiosis) could play a key role in the pathogenesis of CD through the modulation of intestinal permeability and the regulation of the immune system. Here, we show that gliadin induces a chronic endoplasmic reticulum (ER) stress condition in the small intestine of a gluten-sensitive mouse model and that the coadministration of probiotics efficiently attenuates both the (unfolded protein response (UPR) and gut inflammation. Moreover, the composition of probiotics formulations might differ in their activity at molecular level, especially toward the three axes of the UPR. Therefore, probiotics administration might potentially represent a new valuable strategy to treat gluten-sensitive patients, such as those affected by CD.