Development of villous atrophy in potential celiac disease: search for new biomarkers

• Grant: FC 005/2016
• Title: Development of villous atrophy in potential celiac disease: search for new biomarkers
• Duration: Triennial Project
• Principal Investigator: Monia Porpora, European Laboratory for the Investigation of Food-Induced Diseases, Naples, Italy
• Tutor (Head Lab): Renata Auricchio, European Laboratory for the Investigation of Food-Induced Diseases, Naples, Italy

Publications originated from the Project:

• Porpora M, Conte M, Lania G, Bellomo C, Rapacciuolo L, Chirdo FG, Auricchio R, Troncone R, Auricchio S, Barone MV, Nanayakkara M. Inflammation Is Present, Persistent and More Sensitive to Proinflammatory Triggers in Celiac Disease Enterocytes. Int J Mol Sci. 2022 Feb 10;23(4):1973. doi: 10.3390/ijms23041973. PMID: 35216089; PMCID: PMC8880034. https://pubmed.ncbi.nlm.nih.gov/35216089/

Project rationale and aims

Celiac disease (CD) is a chronic inflammatory response caused by a genetic predisposition to an abnormal T-cell-mediated immune response to the presence of gluten in the diet. Several environmental proinflammatory factors can elicit and amplify the T-cell mediated response. The aim of this study was to define biomarkers that would allow us to predict the onset of the disease in potential celiac patients. Analysis by sequencing of RNA from biopsies of celiac disease patients on a gluten-containing (GCD-CD) or gluten-free (GFD-CD) diet, as well as biopsies of potential patients with celiac disease (Pot-CD) before onset of intestinal lesions and controls (CTR) has led to the identification of different proteins whose levels change in different classes of patients with a trend that seems to be indicative of the stage of the disease to which they belong. Many of these proteins are part of the inflammation pathway so we decided to study the role of enterocytes in intestinal inflammation of celiac disease and their response to various proinflammatory factors, such as gliadin and viruses. Organoids isolated from patient biopsies were used to assess NF-kB, ERK, IL-6, and IL-1b levels by Western blot (WB), ELISA, and quantitative PCR. Toll-like receptor ligand loxoribin (Lox) and gliadin peptide P31-43 were used as proinflammatory stimuli. In CD biopsies, inflammatory markers IL-1β and IL-6 increased in enterocytes, and also in Pot-CD before the onset of intestinal injury and in GFD-CD. The inflammatory markers pNF-κB, pERK, IL-1β, and IL-6 are increased in CD organoids; these organoids were more sensitive to P31-43 and Lox stimuli than CTR organoids. Taken together, these observations indicate constitutive inflammation in CD enterocytes, which are more sensitive to inflammatory stimuli such as food components and viruses.

Research plan and results obtained

In this study we identified two proteins IL-1β and IL-6 involved in inflammation whose levels increase in the enterocytes of patients with celiac disease, not only in the acute phase of the disease but also in the phase of remission and in potential patients before onset of intestinal disease. Both IL-1β and IL-6 levels are mainly altered in the epithelium at all stages of the disease. The presence of inflammation in the Pot-CD indicates that the inflammation of the epithelium precedes remodeling of the mucosa and indicates that the intestinal epithelium is a key component in the inflammatory response in the CD. In CD organoids, inflammation markers such as pNF-κB, pERK in addition to proteins, IL-1β and IL-6 are increased, making them more sensitive to P31-43 and Lox stimuli than CTR organoids. The factors that create a proinflammatory environment in the CD gut can be exogenous, such as food and viruses, but they can also be endogenous. Indeed, low-grade inflammation of the CD epithelium, probably constitutive, is present even before intestinal damage. Intestinal organoids reproduced this constitutive inflammation and thus represent a good model for the study of epithelial inflammation in CD. Furthermore, the intestinal epithelium in CD is more sensitive to proinflammatory stimuli, including gliadin and viruses. These results indicate constitutive alterations, possibly genetic or epigenetic, which make the CD epithelium more sensitive to inflammatory stimuli such as food components, viruses and microbiota.

Experimental design and methodologies

To study the RNA levels of IL1b and IL6 we used organoids isolated from duodenal biopsies per individual from CD patients and from controls were taken with standard endoscopic EGDS during routine gastroduodenoscopy and we extracted the total RNA, we also analyzed the protein levels IL-1β, IL-6, pNF-κB, pERK by western blot (WB). The mRNA expression of IL-1β and IL-6 in situ was analyzed in intestinal biopsies of patients with celiac disease in different stages of the disease and in those of control.

Potential pitfalls and caveats

The main problem could have been the lack of biopsies from CDpot/atr patients.  But thanks to the records kept in the bio-bank at the ELFID we were able to trace the destin of several potential patients that were followed on a gluten contain diet. At least 10 of them  (on average) become celiacs in 2,5 years e this allowed me to perform my research. As potential patients are currently followed in the clinic it is likely that more patients could become CD during the course of the fellowship. In fact 30% of potential patients become CD during the follow-up  at a gluten containing diet. So the main problem concerning this project, the lack of Cd-pot/atr patient, seems to overcome.

Conclusions and discussion

The identification of biological markers of inflammation in the very early stages of the disease could in the future be useful in defining the risk of developing the disease in a celiac family subject even in very early periods of life before the appearance of any symptoms of the disease. The factors that create a proinflammatory environment in the CD in‐ testine can be exogenous, such as food and viruses, but can also be endogenous. In fact, low‐grade inflammation of the CD epithelium, probably constitutive, is present even before intestinal damage. Intestinal organoids reproduced this constitutive inflammation and thus represent a good model for studying epithelial inflammation in CD. Moreover, the intestinal epithelium in CD is more sensitive to proinflammatory stimuli, including gliadin and viruses. Taken together, these observations point to constitutive alterations, probably genetic or epigenetic, which render the CD epithelium more sensitive to inflam‐ matory stimuli such as food components, virus, and microbiota.

These data show a constitutive difference that makes CD epithelium more sensitive to inflammatory stimuli. Once confirmed these data we will investigate the biological and immunological consequences of the alteration of the late endocytic compartment.

This will have 2 main implications:

1) The alterations of these proteins could be used as a “bio- marker” able to predict the disease.

2) A constitutive alteration of the inflammation components could represent a new approach to the pathogenesis of the disease, which could explain the susceptibility to gliadin of CD intestine. It could also justify the role of viral infections and probably the microbiota in the onset of celiac disease. Therefore, a pharmacological approach for CD could be hypothesized in the future capable of modulating the variations of the proteins responsible for the inflammatory response.