Bellomo, Claudia (2025) ORGANOIDS AS A MODEL OF INFLAMMATION IN INTESTINAL DISEASES: POSTBIOTICS AND PRECISION MEDICINE. [Tesi di dottorato]

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Abstract

Organoids are self-organised 3D tissues, obtained from stem cells. They can mimic the key functional, structural, and biological complexity of an organ1,2. Stem cells could be derived from induced pluripotent stem cells (iPSCs) or tissue-derived cells (TDCs), the second group include normal stem/progenitor cells, differentiated cells and cancer cells3. The use of organoids gives the possibility to better characterise the physio-pathological mechanisms of diseases. Animal models or secondary tumour-derived cultures cannot reproduce patient specificity, tissue-like structures and functions compared with organoids. They are useful in several diseases, to investigate the possible disease-related mechanisms, and they are more accessible for genetic manipulation, drug-personalised therapies and diagnosis2. The isolation of TDCs made it possible to investigate deeper diseases associated with the small intestine4, colon5, stomach6, tongue7, liver8, lung9, pancreas10, heart11, ear12, skin13 and brain14 . Intestinal organoids are a useful model of diseases to be used in research. They can be obtained from TDCs, more specifically from ASCs (adult stem cells), generated from LGR5+ stem cell populations present in crypts isolated from intestinal biopsies15. This kind of multipotent cells could differentiate toward a limited number of cell types in the presence of specific growth factors16. They are useful both to investigate intestinal epithelial physiology and most of the diseases related to it and phenotypic and/or genetic characteristics, becoming an optimal tool for the development of precision medicine1,17,18. The stem cells are grown in 3D and enclosed in a matrix, which serves as a basal membrane and by using appropriate stimuli they differentiate into epithelial cells. They can be grown and amplified for several weeks as spheroids. However, to know the response to stimuli of a pro inflammatory nature or to investigate the possible beneficial effect of pre-pro-postbiotics and nutraceuticals or drugs, it is necessary to create a 2D monolayer of intestinal epithelial cells. Two dimensional monolayers of intestinal organoids allow direct contact of the apical, adsorptive pole of the cells with the culture medium; this cannot occur in 3D culture as the apical sides of the cells are enclosed in the spherical organoids. A recent review demonstrated that intestinal epithelium was not only important as a target of proinflammatory stimuli, but it can also play a central role in the generation of inflammation and can induce and/or sustain an adaptive autoimmune response in several different settings19. The first purpose of this thesis was the collection of intestinal organoids, from control subjects (CTR), Celiac Disease (CeD) patients at different stages of the disease, Eosinophilic Esophagitis (EoE) 4 subjects, and subjects with co-morbidities related to CeD, such as patients with Type 1 Diabetes (T1D) and Down Syndrome (DS) (Chapter 1). 5 Aims To better characterised the role of intestinal organoids to study CeD patients the first aim of this thesis was to collect intestinal organoids from a cohort of paediatric patients, at different stages of celiac disease, its comorbidities respect to controls patients (Chapter 1). Simultaneously, the activity of milk-based postbiotics from Lactobacillus plantarum, with and without the addition sources of linoleic acid, was tested on CaCo-2 cells to evaluate the possible effect in preventing inflammation induced by gliadin peptide, P31-43, and the possible reduction on the constitutive inflammation in intestinal organoids from patients with CeD (Chapter 2). The third aim was to investigate the role of autophagy flux /mTOR pathway in intestinal biopsies and organoids from CeD patients in active and remission stages of the disease. Furthermore, the possible the crosstalk-between inflammation, delay of autophagy flux/the activation of the mTOR pathway was investigated (Chapter 3). Lastly, in eosinophilic esophagitis patients in which one of the incriminating antigens is gluten, the fourth aim was to investigate the grade of inflammation in the duodenum, and their sensitivity to gliadin peptide, P31-43, in intestinal organoids of these patients in remission stage of the disease (Chapter 4).

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