**3. Autophagy and autoimmune diseases**

*Celiac Disease - From the Bench to the Clinic*

**2. The autophagy process**

developing enteropathy-associated T cell lymphomas.

Parkinson's [7] and Huntington's disease [8].

lysosomes action.

ATG5, ATG7, and LC3.

delivered to the lysosome for degradation.

and (6) fusion of the autophagosome with the lysosome.

in lysosomes through membrane invagination processes.

the possibility of inadvertent gluten exposure. Second, it is important to exclude other causes of persistent symptoms. It is necessary to underline the hypothetic presence of alternative diagnosis (i.e., lactose or fructose intolerance, irritable bowel syndrome, microscopic colitis, pancreatic insufficiency, and small intestinal bacterial overgrowth). Another reason for the recurrence of clinical manifestations is refractory celiac disease, whereby CD patients present symptoms of malabsorption and villous atrophy despite a GFD for more than 12 months. In the primary form, there is no initial response to a diet, while in the secondary one, a relapse occurs after an initial response to a GFD. Patients with refractory CD are at risk of

Eukaryotic cells digest their cytoplasmic content through different processes that come under the general term autophagy (from the Greek words *auto* meaning "self" and *paghein* meaning "to eat"). Autophagy includes different forms of digestive pathways such as macroautophagy, microautophagy, chaperone-mediated autophagy, and noncanonical autophagy. Generally, the term autophagy refers to macroautophagy, and this process depends on specialized autophagy-related proteins (ATGs) to digest different targets, such as organelles, large aggregates of proteins, and microorganisms. Autophagy also plays a key role in direct microorganisms and virus clearance, in the control of inflammation through the inhibition of inflammasome, in antigen presentation, in regulating T cell homeostasis, and the secretion of immune mediators [6]. It is worth nothing that autophagy impairment plays a crucial role in several diseases, in particular proteopathies, such as

As analytically described by Codogno et al. [9], there are four types of autophagy:

• Macroautophagy: organelles or other cargos (proteins, lipids, or nucleic acids) are sequestered in the autophagosome, a double-membrane vesicle, and

• Microautophagy: small cytosolic materials are degraded after their engulfment

• Noncanonical autophagy: under specific circumstances autophagosome formation in macroautophagy can bypass the canonical steps. To date two noncanonical pathways have been described: Beclin-1-independent autophagy and a pathway that bypasses the action of specific autophagy-related proteins such as

To date, several autophagy-related genes (*ATG*s) have been described and exert a finely coordinated function at different stages of the pathway. It is widely accepted that autophagy consists of six sequential steps: (1) initiation; (2) nucleation or phagophore formation; (3) ATG5-ATG12 conjugation, interaction with ATG16L, and multimerization at the phagophore; (4) LC3 processing and insertion into the nascent autophagosome; (5) capture of random/selective targets for degradation;

• Chaperone-mediated autophagy: proteins with the specific KFERQ target sequence are recognized by chaperone Hsc70 protein and then degraded by

**56**

Autophagy plays a crucial role in the pathogenesis of several autoimmune diseases. In particular, in Crohn's disease, an inflammatory bowel disease is caused by a combination of environmental, immune, and bacterial factors in genetically susceptible individuals. It has been demonstrated that genetic polymorphisms in the *ATG16L* and *IRGM* autophagy-related genes lead to a strong predisposition for the development of Crohn's disease [10]. Despite this significant association, the role played by the ATG16L protein in the disease pathogenesis is still under debate. In particular, the ATG16L protein is known to suppress the inflammatory process, as demonstrated in Atg16L-deficient mice, which were find to be highly susceptible to colitis induced by dextran sulfate sodium. Subsequently the symptoms developed by these mice were effectively treated with injections of anti-IL-1β and IL-18 antibodies [11]. Recently, nucleotide polymorphisms within the key regulatory autophagy gene *ULK1* have been shown to increase susceptibility to Crohn's disease, thus demonstrating that autophagy might contribute to the pathogenesis of this disease [12]. Similarly, the ATG5 protein, another essential component of the autophagic machinery, is implicated in the development of systemic lupus erythematosus (SLE), multiple sclerosis (MS), and rheumatoid arthritis (RA). SLE is an autoimmune disease in which the patient's immune system attacks healthy tissues. Different single nucleotide polymorphisms (SNPs), identified near and within the *ATG5* locus, are associated with SLE initiation and/or development, although the pathogenetic mechanisms involved are still unknown. In another study, it was demonstrated that T cells in SLE patients are autoreactive and autophagy promotes their survival and contributes to their persistence in autoimmune conditions [13]. MS is a demyelinating disease that affects the brain and the spinal cord. Changes in the expression of the ATG5 protein correlates also with immune-mediated myelin injury in MS-derived mice and in affected patients. Specifically, ATG5 is overexpressed in circulating T cells of relapsing–remitting MS patients compared with healthy controls. ATG5 altered expression seems to extend T cell survival and proliferation during active disease; moreover, ATG5 expression profiles correlate with the severity of the disease in mice models [14]. In RA, anti-citrullinated protein antibodies are the most powerful biomarkers in the diagnosis of this disease. During inflammation, the arginine residues of self-proteins are converted to citrulline (a nonessential alpha amino acid) by the peptidylarginine deiminase enzyme, in a process known as citrullination, thus leading to an altered immune response. Presentation of these peptides is blocked after treatment with 3-methyladenine, an autophagy modulator drug, or by reducing ATG5 protein expression, confirming a key role of autophagy in RA pathogenesis [15].

The clinical spectrum of celiac disease is broad, and often it may be not so easy to discern between poor compliance, difficult acceptance of therapy, or presence of disease complications.

It is important to emphasize experimental therapies, in terms of alternative treatment (versus gluten-free diet) or GFD adjuvant.

Recent advances in the "non-dietary" treatment of CD include engineering gluten-free grains, degrading immunodominant gliadin peptides, decreasing intestinal permeability, and inducing oral tolerance to gluten with a therapeutic vaccine.

## **4. Non-dietary therapies**

As outlined above, there are many reasons behind the need to identify new therapeutic options for celiac disease, especially non-dietary therapies. The purpose is to offer a better quality of life to celiac patients.

Over the years several studies regarding alternative therapies have been conducted.

The aim of experimental research is to find a drug that reduces bowel inflammation despite gluten exposure. Evaluation of mucosal damage is the best way to verify the efficacy of alternative drugs, but it is an invasive procedure and especially distressing for children. Noninvasive markers of efficacy could be serological normalization (using tTG-IgA title) or the improvement of clinical symptoms.

In celiac patients exposed to gluten, increased intestinal permeability has been observed and is due to defects in tight junctions, which are structures involved in regulating the passage through the paracellular space. Increased permeability determines the passage of gluten peptides, which reach lamina propria and stimulate inflammatory response [16].

*Larazotide acetate* is an oral peptide derived from the zonula occludens toxin secreted by *Vibrio cholerae*. It is believed to be involved in the modulation of tight junctions, consequently preventing gliadin passage through the epithelial barrier. Numerous trials have been conducted to verify the efficacy of larazotide. It has been shown to reduce symptoms in patients on a gluten-free diet [17].

Significant effects of larazotide on serological markers have not been demonstrated, and mucosal damage healing does not appear to be the focus of the evaluation of this drug's efficacy.

Another target of alternative drug is the degradation of toxic gluten peptides making them non-immunogenic. Several *endopeptidases* have been studied for this purpose.

*AN-PEP* (*Aspergillus niger* prolyl endoprotease) is an *Aspergillus niger*-derived endopeptidase which was studied in a randomized trial where celiac patients were divided into two groups: one of which received AN-PEP and the other a placebo. All patients followed a diet with gluten exposure (7 g of gluten daily) for 2 weeks. AN-PEP appeared to be well-tolerated, but no difference as regards mucosal damage was observed between two groups [18].

*ALV003* is a combination of two types of glutamine-specific endoprotease, EP-B2 from barley and a prolyl endopeptidase (SC PEP from *Sphingomonas capsulata*); it is active at gastric pH and able to detoxify gluten [19].

It has been observed that ALV003 can prevent mucosal damage, secondary to gluten exposure, in celiac patients with moderate gluten consumption [20].

Over the years, the emerging role of gut *microbiota* in different diseases has been noted. Variations in the composition of microbiota could play a causative role in the pathogenesis of inflammatory and autoimmune diseases.

The microbial community is composed of more than 1000 species of microbes which exert various functions on the immune system, including protecting the body against pathogens, harvesting nutrients and energy from diet, and fermenting nondigestible carbohydrates.

A specific role of intestinal microbiota in the development of celiac disease has been suggested.

Frequent infectious diseases and consequent antibiotic treatments (with secondary effects on the intestinal microbiota) have also been associated with the onset of celiac disease in genetically susceptible infants [21].

In celiac patients, a prevalence of *Bacteroides* spp. has been found [22] along with a reduction in numbers of *Bifidobacterium* species [23].

Furthermore, regardless of GFD, celiac patients presented less variability of *Bacteroides* species in biopsy samples of the duodenal microbiota in comparison with controls [24].

Several studies regarding the association of *Bifidobacterium* and celiac disease have been conducted. *Bifidobacterium infantis* and *Bifidobacterium lactis* were found to contrast the increase of permeability, secondary to gluten ingestion, in the celiac bowel. They are also thought to play a role as downregulators of immune response in celiac patients [25–27].

**59**

*The Emerging Role of the Autophagy Process in Children with Celiac Disease: Current Status…*

Therefore, a therapeutic function of *Bifidobacteria* may be considered and it

*BL-7010* is an orally available polymer with a high affinity for gliadins. Polymeric binders are used to reduce the intestinal absorption of endogenous or exogenous

BL-7010 sequesters gliadin and masks it from enzymatic degradation. Through this mechanism, it is thought to prevent the formation of immunogenic peptides

It is chemically stable at a wide pH range, is non-biodegradable, and is watersoluble. It exerts its function locally in the gastrointestinal tract and is not absorbed systemically; therefore, it has a very good safety profile as was demonstrated in preclinical studies. In vitro and in mouse models have shown that BL-7010 is able to bind gliadin and to prevent barrier dysfunction, changes in IELs, and villous/crypt

Another therapeutic target identified in recent years is a vaccine called *Nexvax2*. The aim of this drug is to restore immune tolerance to gluten using peptide-based immunotherapy to induce T cells to make a regulatory response. Murine models were used for the study, and three immunogenic peptides have been identified. These peptides elicit an immune response in patients with celiac disease who carry the HLA-DQ2 immune recognition gene. Tolerance is based on reduction of CD4 T cell proliferation, decreasing IL-2 and IFN-γ levels and promoting expression of T cells with regulatory functions. It was reported that patients treated with the vaccine (especially with high doses) presented gastrointestinal side effects similar to celiac symptoms. This finding corroborates the hypothesis that the vaccine has a

Another attempt at immuno-modulation was made using *CCX282-B* an antagonist of the CCR9 chemokine receptor. Increased blood levels of T cells expressing CCR9 have been found in celiac patients. It is thought that CCX282-B may prevent T cell migration from the blood to the intestinal mucosa. Unfortunately the results of

A further consideration is the hygiene hypothesis, whereby excess hygiene is thought to trigger the inflammatory process through immune imbalance resulting in autoimmune disorders. The effect of helminth infections on immunity has also been studied, in particular their role in celiac disease and in inflammatory bowel disease. Interestingly, it has been reported that the *Necator americanus* species of hookworm enabled individuals with celiac disease to tolerate escalating challenges with dietary gluten [30]. The exact mechanism responsible for tolerance is not known,

As previously described, reasons justifying the search for an alternative therapies for celiac disease are numerous, and several studies have been conducted to assess their efficacy. The aim is to identify a new drug and, at the same time, to define new

Over the years we have studied the autophagy process. Our results have highlighted the possible contribution of this process to the degradation and the reduction of extracellular release of gliadin peptides and suggest novel molecular targets

The primary link between autophagy and celiac disease is that autophagy is conventionally described as a catabolic pathway where the cytoplasmic material sequestered by autophagosomes is degraded. Therefore, the exogenous gliadin

hypothetical therapeutic targets to improve celiac patients' quality of life.

similar action to gluten ingestion as regards intestinal immunity [29].

*DOI: http://dx.doi.org/10.5772/intechopen.80692*

that trigger the immune system.

ratios induced by gliadin [28].

this study are not yet available.

and further studies are expected.

to counteract gliadin-induced toxicity in CD.

**5. Autophagy and celiac disease**

molecules.

could be investigated through further studies.

*The Emerging Role of the Autophagy Process in Children with Celiac Disease: Current Status… DOI: http://dx.doi.org/10.5772/intechopen.80692*

Therefore, a therapeutic function of *Bifidobacteria* may be considered and it could be investigated through further studies.

*BL-7010* is an orally available polymer with a high affinity for gliadins. Polymeric binders are used to reduce the intestinal absorption of endogenous or exogenous molecules.

BL-7010 sequesters gliadin and masks it from enzymatic degradation. Through this mechanism, it is thought to prevent the formation of immunogenic peptides that trigger the immune system.

It is chemically stable at a wide pH range, is non-biodegradable, and is watersoluble. It exerts its function locally in the gastrointestinal tract and is not absorbed systemically; therefore, it has a very good safety profile as was demonstrated in preclinical studies. In vitro and in mouse models have shown that BL-7010 is able to bind gliadin and to prevent barrier dysfunction, changes in IELs, and villous/crypt ratios induced by gliadin [28].

Another therapeutic target identified in recent years is a vaccine called *Nexvax2*. The aim of this drug is to restore immune tolerance to gluten using peptide-based immunotherapy to induce T cells to make a regulatory response. Murine models were used for the study, and three immunogenic peptides have been identified. These peptides elicit an immune response in patients with celiac disease who carry the HLA-DQ2 immune recognition gene. Tolerance is based on reduction of CD4 T cell proliferation, decreasing IL-2 and IFN-γ levels and promoting expression of T cells with regulatory functions. It was reported that patients treated with the vaccine (especially with high doses) presented gastrointestinal side effects similar to celiac symptoms. This finding corroborates the hypothesis that the vaccine has a similar action to gluten ingestion as regards intestinal immunity [29].

Another attempt at immuno-modulation was made using *CCX282-B* an antagonist of the CCR9 chemokine receptor. Increased blood levels of T cells expressing CCR9 have been found in celiac patients. It is thought that CCX282-B may prevent T cell migration from the blood to the intestinal mucosa. Unfortunately the results of this study are not yet available.

A further consideration is the hygiene hypothesis, whereby excess hygiene is thought to trigger the inflammatory process through immune imbalance resulting in autoimmune disorders. The effect of helminth infections on immunity has also been studied, in particular their role in celiac disease and in inflammatory bowel disease.

Interestingly, it has been reported that the *Necator americanus* species of hookworm enabled individuals with celiac disease to tolerate escalating challenges with dietary gluten [30]. The exact mechanism responsible for tolerance is not known, and further studies are expected.

As previously described, reasons justifying the search for an alternative therapies for celiac disease are numerous, and several studies have been conducted to assess their efficacy. The aim is to identify a new drug and, at the same time, to define new hypothetical therapeutic targets to improve celiac patients' quality of life.

Over the years we have studied the autophagy process. Our results have highlighted the possible contribution of this process to the degradation and the reduction of extracellular release of gliadin peptides and suggest novel molecular targets to counteract gliadin-induced toxicity in CD.

## **5. Autophagy and celiac disease**

The primary link between autophagy and celiac disease is that autophagy is conventionally described as a catabolic pathway where the cytoplasmic material sequestered by autophagosomes is degraded. Therefore, the exogenous gliadin

*Celiac Disease - From the Bench to the Clinic*

late inflammatory response [16].

tion of this drug's efficacy.

was observed between two groups [18].

nondigestible carbohydrates.

in celiac patients [25–27].

been suggested.

Over the years several studies regarding alternative therapies have been conducted. The aim of experimental research is to find a drug that reduces bowel inflammation despite gluten exposure. Evaluation of mucosal damage is the best way to verify the efficacy of alternative drugs, but it is an invasive procedure and especially distressing for children. Noninvasive markers of efficacy could be serological normalization (using tTG-IgA title) or the improvement of clinical symptoms. In celiac patients exposed to gluten, increased intestinal permeability has been observed and is due to defects in tight junctions, which are structures involved in regulating the passage through the paracellular space. Increased permeability determines the passage of gluten peptides, which reach lamina propria and stimu-

*Larazotide acetate* is an oral peptide derived from the zonula occludens toxin secreted by *Vibrio cholerae*. It is believed to be involved in the modulation of tight junctions, consequently preventing gliadin passage through the epithelial barrier. Numerous trials have been conducted to verify the efficacy of larazotide. It has been

Significant effects of larazotide on serological markers have not been demonstrated, and mucosal damage healing does not appear to be the focus of the evalua-

them non-immunogenic. Several *endopeptidases* have been studied for this purpose. *AN-PEP* (*Aspergillus niger* prolyl endoprotease) is an *Aspergillus niger*-derived endopeptidase which was studied in a randomized trial where celiac patients were divided into two groups: one of which received AN-PEP and the other a placebo. All patients followed a diet with gluten exposure (7 g of gluten daily) for 2 weeks. AN-PEP appeared to be well-tolerated, but no difference as regards mucosal damage

*ALV003* is a combination of two types of glutamine-specific endoprotease, EP-B2 from barley and a prolyl endopeptidase (SC PEP from *Sphingomonas* 

It has been observed that ALV003 can prevent mucosal damage, secondary to

Over the years, the emerging role of gut *microbiota* in different diseases has been noted. Variations in the composition of microbiota could play a causative role in the

The microbial community is composed of more than 1000 species of microbes which exert various functions on the immune system, including protecting the body against pathogens, harvesting nutrients and energy from diet, and fermenting

A specific role of intestinal microbiota in the development of celiac disease has

Frequent infectious diseases and consequent antibiotic treatments (with secondary effects on the intestinal microbiota) have also been associated with the onset of

In celiac patients, a prevalence of *Bacteroides* spp. has been found [22] along with

Furthermore, regardless of GFD, celiac patients presented less variability of *Bacteroides* species in biopsy samples of the duodenal microbiota in comparison with controls [24]. Several studies regarding the association of *Bifidobacterium* and celiac disease have been conducted. *Bifidobacterium infantis* and *Bifidobacterium lactis* were found to contrast the increase of permeability, secondary to gluten ingestion, in the celiac bowel. They are also thought to play a role as downregulators of immune response

gluten exposure, in celiac patients with moderate gluten consumption [20].

*capsulata*); it is active at gastric pH and able to detoxify gluten [19].

pathogenesis of inflammatory and autoimmune diseases.

celiac disease in genetically susceptible infants [21].

a reduction in numbers of *Bifidobacterium* species [23].

Another target of alternative drug is the degradation of toxic gluten peptides making

shown to reduce symptoms in patients on a gluten-free diet [17].

**58**

peptides content might be a potential target of the autophagy clearance process. The autophagy escape, on the other hand, might specifically lead to MHC antigen presentation by dendritic cells or to other unspecific exocytic/endocytic processes between different cells.

Autophagy is known to modulate two crucial aspects of the adaptive immune response involved in the pathological context of the celiac condition: it can enhance priming of CD4+ T cell responses, but at the same time, by allowing the presentation of self-peptides, it may also regulate the establishment of peripheral T cell tolerance [31]. Importantly, antigen presentation by MHC class I or II proteins is dependent on the activity of the proteasome or the endocytic/phagocytic system, respectively, and therefore associated with the functionality of the autophagy process.

To date, however, only a few scientific publications have attempted to investigate the functional role of the autophagy process in celiac disease.

Weersma and colleagues [32] first reported the absence of association of *IL23R* and *ATG16L1* mutations with celiac disease susceptibility, in contrast to that of Crohn's disease. This result was subsequently confirmed independently by genetic analysis in a different cohort of celiac disease patients [33].

More recently, Rajaguru and colleagues [34] evaluated the expression of the LC3 autophagy marker in duodenal biopsies of celiac patients at initial presentation and after 6 months of gluten-free diet, reporting a time-related reduction of LC3 expression in dendritic cells through immunohistochemical analysis. The authors concluded that the observed typical histological pathological hallmarks in duodenal biopsies were associated with a reduction in activated dendritic cells expressing autophagic proteins. This alteration within the autophagy executor organelles may well play an important role in the pathogenesis of autoimmune disorders like celiac disease.

Comincini and collaborators [35] have recently tackled a different question regarding celiac disease, i.e., the possibility to identify novel molecular markers in order to increase the sensitivity and specificity in the diagnosis of pediatric celiac disease patients. To this end, the expression levels of two key autophagy executor genes (*ATG7* and *BECN1*) and their regulatory validated miRNAs (miR-17 and miR-30a, respectively) were analyzed by relative quantitative real-time PCR on a cohort of confirmed celiac patients compared to age-related controls, analyzing peripheral blood, and corresponding duodenal specimens. Among the investigated gene/miRNA targets, statistical analysis indicated the highest significant association as that of *BECN1* expression profiles with the pathological status in the blood, while in intestinal biopsies, all the investigated sequences were positively associated with the celiac disease condition. The authors were also able to identify specific celiac/ control molecular subtypes based on specific genes and miRNA expression signatures. Overall, the authors described novel molecular markers that might be useful in increasing the accuracy in CD diagnosis and in molecular-based stratification of the patients, further reinforcing the functional involvement of the regulation of the autophagy process in digestive and autoimmune-related disorders such as CD.

In the latest PubMed contribution, Manai and collaborators [36] reported that in Caco-2 cells, a widely used in vitro model for celiac disease studies, the administration of enzymatically digested gliadin (PT-gliadin) peptides significantly reduced the expression of the LC3-II autophagy-related marker. Furthermore, electron and fluorescent microscope analysis suggested a compromised function of the autophagosome apparatus. The improvement of the dysregulated autophagy process, along with a reduction of PT-gliadin toxicity, was achieved by means of a starvation induction protocol and by 3-methyladenine administration, while rapamycin, a well-known autophagy inducer, did not produce significant improvement in the clearance of extra- and intracellular fluorescent PT-gliadin amounts. Importantly, these results highlighted the possible

**61**

*The Emerging Role of the Autophagy Process in Children with Celiac Disease: Current Status…*

contribution of the autophagy process to the degradation and the reduction of extracellular release of gliadin peptides and suggest novel molecular targets to counteract gliadin-induced toxicity in celiac disease. A schematized summary of the autophagy-modulation strategy to counteract the gluten-derived cellular

*Gluten degradation by autophagy process. Gluten digested peptides (1) can be internalized into different cell types, including human dendritic cells. These peptides form relatively large intracellular aggregates, partially resistant to intracellular catabolic process, and they can also be released to other surrounding cells by exocytic processes. The discussed approach, (2) based on the autophagy activation using drugs (rapamycyn, chloroquine, etc.) or by means of starvation induction, (3) leads to the fusion of autophagosomes and lysosomes to produce autophago-lysosomes, able to digest the gluten digested peptides. (4) this intracellular digestion produces a reduction of the exocytosis of the gluten peptides, and, mostly, (5) it might reduce the gluten-peptide presentation by MHC-II molecules (HLA-DQ2.5) and therefore the pathological activation of T cell in the* 

Celiac disease is an increasingly complex disease, with a well-established genetic background but with a plethora of molecular/cellular actors involved. Despite this emerging complexity, the cellular uptake of the digested gliadin components and their ultimate fate is the key determinant for this disease. Once within a cell, gliadin peptides, as with any exogenous components, undergo different catabolic processes, including the relatively low-energy consumption processes such as exocytosis. In this scenario, autophagy protein turnover might represent a pro-survival process to counteract a surge in potentially toxic gliadin. However, for reasons still unknown, the autophagy process seems to be impaired in the celiac condition: as a result, gliadin is easily internalized in different types of cells, but no marked signs of a prominent degradation are reported. On the other hand, more and more is being learned about the process of autophagy and its molecular players, and, consequently, a relatively large number of molecular and pharmacological modulators are being put on the market and assayed in clinical trials for different pathologies. Therefore, once the alterations of the steady-state status of the autophagy process are clarified by comparing physiological to celiac pathological conditions, one could realistically hope to counteract gliadin toxicity by improving its catabolism within the cells, bearing in mind however

*DOI: http://dx.doi.org/10.5772/intechopen.80692*

toxicity is illustrated in **Figure 1**.

**6. Concluding remarks**

**Figure 1.**

*celiac disease.*

*The Emerging Role of the Autophagy Process in Children with Celiac Disease: Current Status… DOI: http://dx.doi.org/10.5772/intechopen.80692*

#### **Figure 1.**

*Celiac Disease - From the Bench to the Clinic*

between different cells.

peptides content might be a potential target of the autophagy clearance process. The autophagy escape, on the other hand, might specifically lead to MHC antigen presentation by dendritic cells or to other unspecific exocytic/endocytic processes

Autophagy is known to modulate two crucial aspects of the adaptive immune response involved in the pathological context of the celiac condition: it can enhance priming of CD4+ T cell responses, but at the same time, by allowing the presentation of self-peptides, it may also regulate the establishment of peripheral T cell tolerance [31]. Importantly, antigen presentation by MHC class I or II proteins is dependent on the activity of the proteasome or the endocytic/phagocytic system, respectively, and

To date, however, only a few scientific publications have attempted to investigate

Weersma and colleagues [32] first reported the absence of association of *IL23R* and *ATG16L1* mutations with celiac disease susceptibility, in contrast to that of Crohn's disease. This result was subsequently confirmed independently by genetic

More recently, Rajaguru and colleagues [34] evaluated the expression of the LC3 autophagy marker in duodenal biopsies of celiac patients at initial presentation and after 6 months of gluten-free diet, reporting a time-related reduction of LC3 expression in dendritic cells through immunohistochemical analysis. The authors concluded that the observed typical histological pathological hallmarks in duodenal biopsies were associated with a reduction in activated dendritic cells expressing autophagic proteins. This alteration within the autophagy executor organelles may well play an important

Comincini and collaborators [35] have recently tackled a different question regarding celiac disease, i.e., the possibility to identify novel molecular markers in order to increase the sensitivity and specificity in the diagnosis of pediatric celiac disease patients. To this end, the expression levels of two key autophagy executor genes (*ATG7* and *BECN1*) and their regulatory validated miRNAs (miR-17 and miR-30a, respectively) were analyzed by relative quantitative real-time PCR on a cohort of confirmed celiac patients compared to age-related controls, analyzing peripheral blood, and corresponding duodenal specimens. Among the investigated gene/miRNA targets, statistical analysis indicated the highest significant association as that of *BECN1* expression profiles with the pathological status in the blood, while in intestinal biopsies, all the investigated sequences were positively associated with the celiac disease condition. The authors were also able to identify specific celiac/ control molecular subtypes based on specific genes and miRNA expression signatures. Overall, the authors described novel molecular markers that might be useful in increasing the accuracy in CD diagnosis and in molecular-based stratification of the patients, further reinforcing the functional involvement of the regulation of the autophagy process in digestive and autoimmune-related disorders such as CD. In the latest PubMed contribution, Manai and collaborators [36] reported that in Caco-2 cells, a widely used in vitro model for celiac disease studies, the administration of enzymatically digested gliadin (PT-gliadin) peptides significantly reduced the expression of the LC3-II autophagy-related marker. Furthermore, electron and fluorescent microscope analysis suggested a compromised function of the autophagosome apparatus. The improvement of the dysregulated autophagy process, along with a reduction of PT-gliadin toxicity, was achieved by means of a starvation induction protocol and by 3-methyladenine administration, while rapamycin, a well-known autophagy inducer, did not produce significant improvement in the clearance of extra- and intracellular fluorescent PT-gliadin amounts. Importantly, these results highlighted the possible

therefore associated with the functionality of the autophagy process.

the functional role of the autophagy process in celiac disease.

analysis in a different cohort of celiac disease patients [33].

role in the pathogenesis of autoimmune disorders like celiac disease.

**60**

*Gluten degradation by autophagy process. Gluten digested peptides (1) can be internalized into different cell types, including human dendritic cells. These peptides form relatively large intracellular aggregates, partially resistant to intracellular catabolic process, and they can also be released to other surrounding cells by exocytic processes. The discussed approach, (2) based on the autophagy activation using drugs (rapamycyn, chloroquine, etc.) or by means of starvation induction, (3) leads to the fusion of autophagosomes and lysosomes to produce autophago-lysosomes, able to digest the gluten digested peptides. (4) this intracellular digestion produces a reduction of the exocytosis of the gluten peptides, and, mostly, (5) it might reduce the gluten-peptide presentation by MHC-II molecules (HLA-DQ2.5) and therefore the pathological activation of T cell in the celiac disease.*

contribution of the autophagy process to the degradation and the reduction of extracellular release of gliadin peptides and suggest novel molecular targets to counteract gliadin-induced toxicity in celiac disease. A schematized summary of the autophagy-modulation strategy to counteract the gluten-derived cellular toxicity is illustrated in **Figure 1**.
