Co-expression of fibrotic genes in inflammatory bowel disease; A localized event?

IntroductionExtracellular matrix turnover, a ubiquitous dynamic biological process, can be diverted to fibrosis. The latter can affect the intestine as a serious complication of Inflammatory Bowel Diseases (IBD) and is resistant to current pharmacological interventions. It embosses the need for out-of-the-box approaches to identify and target molecular mechanisms of fibrosis.Methods and resultsIn this study, a novel mRNA sequencing dataset of 22 pairs of intestinal biopsies from the terminal ileum (TI) and the sigmoid of 7 patients with Crohn’s disease, 6 with ulcerative colitis and 9 control individuals (CI) served as a validation cohort of a core fibrotic transcriptomic signature (FIBSig), This signature, which was identified in publicly available data (839 samples from patients and healthy individuals) of 5 fibrotic disorders affecting different organs (GI tract, lung, skin, liver, kidney), encompasses 241 genes and the functional pathways which derive from their interactome. These genes were used in further bioinformatics co-expression analyses to elucidate the site-specific molecular background of intestinal fibrosis highlighting their involvement, particularly in the terminal ileum. We also confirmed different transcriptomic profiles of the sigmoid and terminal ileum in our validation cohort. Combining the results of these analyses we highlight 21 core hub genes within a larger single co-expression module, highly enriched in the terminal ileum of CD patients. Further pathway analysis revealed known and novel inflammation-regulated, fibrogenic pathways operating in the TI, such as IL-13 signaling and pyroptosis, respectively.DiscussionThese findings provide a rationale for the increased incidence of fibrosis at the terminal ileum of CD patients and highlight operating pathways in intestinal fibrosis for future evaluation with mechanistic and translational studies

The interaction between pro-inflammatory mediators and subepithelial myofibroblasts in fibrogenesis of inflammatory bowel diseases as a target of new therapeutic strategies

Introduction - Inflammatory Bowel Diseases (IBDs), Crohn’s Disease (CD) and Ulcerative Colitis (UC), are characterized by chronic inflammation of the gastrointestinal tract, with unknown etiopathogenesis and immunologic mechanisms involved. Transmural fibrosis development is one of the main characteristics of CD and it is characterized by an increased production of inflammatory and pro-fibrotic mediators that ultimately lead to the activation, proliferation and differentiation of intestinal fibroblasts to myofibroblasts, as well as infiltration of myofibroblasts of different origin. The etiopathogenesis of fibrosis is mostly unknown, but the current literature suggests that innate (TL1A) and adaptive (Th1, Th2, Th17 and Treg) immune responses play a significant role in intestinal fibrogenesis. Tumor necrosis factor (TNF)-like cytokine 1A (TL1A) is a member of TNF’s superfamily, binds to the receptors, DR3 and DcR3 and so far, there is enough evidence suggesting that both TL1A and its receptors are implicated in IBD. Pro-inflammatory mediators of the innate or adaptive immunity induce pro-fibrotic effects on myofibroblasts, driving them towards a fibrotic phenotype. In particular, Th2- and Th17- related cytokines, such as IL-13 and IL-17, respectively, induce mostly pro-fibrotic effects, whereas, Th1- related cytokines, such as IFN-γ, promote mostly anti-fibrotic effects. The expression of TL1A and its receptors, DR3 and DcR3, in colonic subepithelial myofibroblasts (cSEMFs) and in colonic epithelial cells HT-29, as well as the effect of immune responses, Th1, Th2, Th17 and Treg, in cSEMFs’ biologic activity and fibrosis, are poorly studied. Therefore, it would be of interest to investigate the effect of inflammatory mediators in these cells’ physiology and in fibrogenesis. Aim and Objectives - The aim of the present PhD thesis was to study the role and implication of cSEMFs in the inflammatory process, which characterizes IBD, and leads to the onset of fibrosis. For this purpose, we studied the pathogenetic mechanisms involved in the fibrotic process and their regulatory factors, which could serve as new pharmacological targets.In particular, during the first part of the current PhD thesis, one of our objectives was to study the expression of TL1A and its receptors, DR3 and DcR3, in primary human cSEMFs from healthy controls, under inflammatory conditions that resemble IBD and in primary human cSEMFs from patients with CD. In addition, we investigated whether colonic epithelial cells HT-29 express the receptors DR3 and DcR3, under inflammatory conditions and whether there is a crosstalk between HT-29 cells and cSEMFs. During the second part, one of our objectives was to perform an expression mapping of all interleukin receptors, implicated in IBD, in primary human cSEMFs, and to examine the way that pro-inflammatory cytokines of innate immunity, as well as Lipopolysaccharide (LPS) alter this expression. In addition, we studied the effect of adaptive immune responses (Th1, Th2, Th17 and Treg) in the expression of pro-fibrotic factors by cSEMFs, as well as, in their migratory capacity. The ultimate goal of the present PhD thesis was to better understand the etiopathogenetic mechanisms implicated in intestinal fibrosis, and to highlight possible new pharmacological targets that will be part of novel and effective therapeutic strategies for the treatment of intestinal fibrosis. Materials and Methods - In order to achieve our aims, we initially isolated cSEMFs from colonic biopsies obtained from healthy individuals and patients with CD. We also cultured the cell lines, HT-29 and 18CO, which are colonic epithelial cells and colonic embryonic fibroblasts, respectively. Regarding the first part of the present PhD thesis, we first stimulated cSEMFs and 18CO with IL-1α and/or TNF-α, in order to study the mRNA and protein expression of TL1A and its receptors, DR3 and DcR3. Next, we stimulated HT-29 cells with IL-1α, TNF-α and IFN-γ, cultured tissue biopsies from patients with CD and their supernatants were used as stimuli in cSEMFs for the study of mRNA and protein expression of TL1A and its receptors, DR3 and DcR3. We also studied the mRNA and protein expression of TL1A and its receptors, DR3 and DcR3 in cSEMFs from patients with CD. Next, HT-29 cells were stimulated with IL-1α, TNF-α and IFN-γ, in order to investigate the mRNA and protein expression of DR3 and DcR3 receptors. Finally, we stimulated HT-29 cells with TL1A and we examined its effect in the induction of IL-8 mRNA expression.Regarding the second part, we initially stimulated cSEMFs with IL-1α and/or TNF-α, as well as with LPS, in order to investigate the basal and the pro-inflammatory cytokine-induced mRNA and protein expression of interleukin receptors. Next, cSEMFs were stimulated with Th1- (TNF-α and/or IFN-γ), Th2- (IL-4 and/or IL-13), Th17- (IL-17 and/or IL-22 and/or IL-23) and Treg- (IL-10 ad/or TGF-β) related cytokines, in order to investigate the mRNA expression of pro-fibrotic factors, collagen type I and III, fibronectin, α-SMA, TF, metalloproteinase-1 (MMP-1), MMP-9, tissue inhibitor of metalloproteinase-1 (TIMP-1) and TIMP-2 and the protein expression of collagen and fibronectin. Finally, we stimulated cSEMFs with Th1- (TNF-α and/or IFN-γ), Th2- (IL-4 and/or IL-13), Th17- (IL-17 and/or IL-22 and/or IL-23) and Treg- (IL-10 ad/or TGF-β) related cytokines, in order to examine their effect in cSEMFs’ migratory capacity via scratch test assay. During the mRNA expression studies, total RNA was isolated from cultured cells, cDNA synthesis was performed and the real time rt-PCR method was used for the detection of mRNA expression levels. Protein expression studies were performed using either immunofluorescence, or the ELISA method. Collagen protein levels were measured using the Sircol method. Statistical analysis was performed with Student's t test. Results - Regarding the first part, cSEMFs and 18CO cells stimulated with the pro-inflammatory cytokines IL-1α and/or TNF-α, abundant in the inflamed intestinal mucosa of patients with IBD, expressed upregulated levels of TL1A mRNA and protein expression. Tissue supernatants from cultured biopsies of patients with CD induced the TL1A mRNA and protein expression in cSEMFs. cSEMFs isolated from patients with CD were found to express TL1A. Stimulated HT-29 cells with IL-1α, TNF-α and IFN-γ expressed upregulated mRNA and protein levels of DR3 receptor, and their supernatants induced the mRNA and protein expression of TL1A in cSEMFs. HT-29 cells stimulated with TL1A expressed upregulated mRNA levels of IL-8. Regarding the second part, a basal expression of interleukin receptors (Τh1: IFNGR1, IFNGR2, TNFRSF1A, IL12RB2, IL1R1 and IL1R2, Th2: IL4R and IL13RA2, Th17: IL22RA1, Treg: IL10RA, IL10RB, TGFBR1 and TGFBR2) was found in cSEMFs isolated from healthy individuals. Stimulation of cSEMFs with pro-inflammatory cytokines IL-1α and/or TNF-α had a positive or negative effect in interleukin receptor expression patterns. LPS stimulation did not cause any statistically significant upregulation or downregulation of interleukin receptors expression. cSEMFs stimulated with Th1- related cytokines expressed upregulated mRNA levels of TF and MMP-1, upregulated protein levels of collagen and fibronectin and reduced mRNA levels of α-SMA, MMP-9 and migration rate. Th2- related cytokines induced an upregulation of mRNA collagen, TF, α-SMA and MMP-1 expression and migration rate in cSEMFs and a downregulation of fibronectin protein and MMP-9 mRNA expression. Th17- related cytokines had a mixed effect in cSEMFs; IL-17 and IL-23 induced an upregulation of fibronectin expression, whereas IL-22 stimulation resulted in reduced fibronectin and TF expression. Similar results were obtained from scratch test assay; IL-17 and IL-22 reduced migratory rate, whereas IL-23 enhanced it. Additionally, IL-23 induced the mRNA expression of both MMP-1 and TIMP-1. Finally and as expected, TGF-β proved to be the most potent pro-fibrotic factor, as it upregulated all studied pro-fibrotic mediators, as well as migratory capacity of cSEMFs. Conclusions - In the present PhD thesis, we concluded that cSEMFs are directly implicated in intestinal inflammation and that innate and adaptive immune responses have a significant role in the development of intestinal fibrosis. Additionally, the current PhD thesis highlights new possible pharmacological targets, whose further study may lead to the development of novel and effective therapeutic strategies for the treatment of intestinal fibrosis.Εισαγωγή - Οι Ιδιοπαθείς Φλεγμονώδεις Νόσοι του Εντέρου (ΙΦΝΕ), Νόσος του Crohn (ΝC) και Ελκώδης Κολίτιδα (ΕΚ), αποτελούν χρόνια φλεγμονώδη νοσήματα του γαστρεντερικού συστήματος, αγνώστου αιτιολογίας, των οποίων η αιτιοπαθογένεια διέπεται από ανοσολογικούς μηχανισμούς. Η ανάπτυξη διατοιχωματικής ίνωσης του εντέρου αποτελεί ένα από τα κύρια χαρακτηριστικά της NC και χαρακτηρίζεται από αυξημένη παραγωγή μεταβιβαστών της φλεγμονής και προ-ινωτικών παραγόντων που οδηγούν στην ενεργοποίηση, πολλαπλασιασμό και διαφοροποίηση των ινοβλαστών σε μυοϊνοβλάστες στον εντερικό βλεννογόνο, καθώς και προσέλκυση νέων μυοϊνοβλαστών. Η αιτιοπαθογένεια της ίνωσης μέχρι στιγμής παραμένει άγνωστη, αλλά δεδομένα αναφέρουν πως οι ανοσολογικές αποκρίσεις της έμφυτης (TL1A) και της ειδικής ανοσίας (Th1, Th2, Th17 και Treg) συμβάλλουν σημαντικά στην ανάπτυξη εντερικής ίνωσης.Ο TL1A (tumor necrosis factor (TNF)-like cytokine 1A, TL1A) ανήκει στην υπεροικογένεια του TNF, προσδένεται στους υποδοχείς, death-domain receptor 3 (DR3) και decoy receptor 3 (DcR3) και μέχρι στιγμής, υπάρχουν αρκετές αναφορές στην βιβλιογραφία που συσχετίζουν τον TL1A και τους 2 υποδοχείς του με τις ΙΦΝΕ. Οι φλεγμονώδεις μεταβιβαστές της έμφυτης ή της ειδικής ανοσίας (Th1, Th2 και Th17) προκαλούν αντίστοιχες ινωτικές αντιδράσεις στους μυοϊνοβλάστες, οδηγώντας τους σε έναν ινωτικό φαινότυπο. Συγκεκριμένα, φλεγμονώδεις παράγοντες της Th2 και της Th17 ανοσολογικής απόκρισης, όπως η interleukin (IL)-13 και η IL-17 αντίστοιχα, οδηγούν κατά κύριο λόγο σε ινωτικές διεργασίες, ενώ αντίθετα, παράγοντες της Th1 ανοσολογικής απόκρισης φαίνεται να μην συνδράμουν ενεργά στη δημιουργία της ίνωσης, αλλά να δρουν προς την αντίθετη κατεύθυνση, με την IFN-γ να εμφανίζει αντι-ινωτικές ιδιότητες. Η μελέτη της έκφρασης του TL1A και των υποδοχέων του, DR3 και DcR3, στους εντερικούς υποεπιθηλιακούς μυοϊνοβλάστες (ΕΥΜ) και στα κολονικά επιθηλιακά κύτταρα ΗΤ-29, καθώς και η επίδραση των ανοσολογικών αποκρίσεων, Th1, Th2 και Th17, στη βιολογία των ΕΥΜ και στην εμπλοκή τους στην ιστική αναδιοργάνωση και στην ίνωση, αποτελούν πεδίο έρευνας ελάχιστα μελετημένο και επομένως, χρήζει ιδιαίτερου ενδιαφέροντος η διερεύνηση της επίδρασης των στοιχείων της φλεγμονής στη φυσιολογία αυτών των κυττάρων και στη διεργασία της ίνωσης. Σκοπός και Στόχοι - Σκοπός της παρούσας διδακτορικής διατριβής ήταν η μελέτη του ρόλου και της εμπλοκής των υποεπιθηλιακών μυοϊνοβλαστών στις φλεγμονώδεις διεργασίες που διέπουν τις ΙΦΝΕ και οδηγούν στην ανάπτυξη ίνωσης. Για τον σκοπό αυτό, διερευνήθηκαν οι παθογενετικοί μηχανισμοί που εμπλέκονται στην ινωτική διαδικασία και οι παράγοντες που τους ρυθμίζουν άμεσα ή έμμεσα, οι οποίοι θα μπορούσαν να αποτελέσουν νέους θεραπευτικούς στόχους.Συγκεκριμένα, κατά το πρώτο σκέλος της παρούσας διδακτορικής διατριβής, στόχος ήταν ο έλεγχος της έκφρασης του TL1A και των υποδοχέων του, DR3 και DcR3, σε πρωτογενείς ανθρώπειους ΕΥΜ φυσιολογικών μαρτύρων, υπό συνθήκες φλεγμονής που σχετίζονται με τις ΙΦΝΕ και σε ΕΥΜ ασθενών με NC. Επιπλέον, έγινε διερεύνηση της έκφρασης των υποδοχέων DR3 και DcR3 σε κολονικά επιθηλιακά κύτταρα ΗΤ-29, υπό συνθήκες φλεγμονής, καθώς και μελέτη της αλληλεπίδρασης μεταξύ των ΕΥΜ και των ΗΤ-29 κυττάρων. Κατά το δεύτερο σκέλος, στόχος ήταν ο έλεγχος και η χαρτογράφηση της έκφρασης όλων των υποδοχέων των ιντερλευκινών, που σχετίζονται με τις ΙΦΝΕ, σε πρωτογενείς ανθρώπειους ΕΥΜ φυσιολογικών μαρτύρων, καθώς και η μελέτη της επίδρασης των προ-φλεγμονωδών κυτταροκινών της έμφυτης ανοσίας στην τροποποίηση της έκφρασης των συγκεκριμένων υποδοχέων στους ΕΥΜ. Επιπλέον, μελετήθηκε η επίδραση των ανοσολογικών αποκρίσεων (Th1, Th2, Th17 και Treg) στην επαγόμενη έκφραση προ-ινωτικών μορίων από τους ΕΥΜ και στην μεταναστευτική τους ικανότητα. Απώτερος στόχος της παρούσας μελέτης ήταν η κατανόηση των αιτιοπαθογενετικών μηχανισμών που διέπουν την εντερική ίνωση, καθώς και η πιθανή ανάδειξη νέων φαρμακολογικών στόχων για τον σχεδιασμό καινοτόμων και αποτελεσματικών θεραπευτικών προσεγγίσεων για την αντιμετώπιση της εντερικής ίνωσης. Υλικά και Μέθοδοι - Προκειμένου να επιτευχθούν οι παραπάνω στόχοι, τόσο του πρώτου, όσο και του δεύτερου σκέλους, έγινε απομόνωση ΕΥΜ από κολονικές βιοψίες φυσιολογικών μαρτύρων και ασθενών με NC. Επιπλέον, καλλιεργήθηκαν οι κυτταρικές σειρές, ΗΤ-29 και 18CO, οι οποίες είναι κολονικά επιθηλιακά κύτταρα και κολονικοί εμβρυικοί ινοβλάστες, αντίστοιχα. Ως προς το πρώτο σκέλος της παρούσας διδακτορικής διατριβής, αρχικά έγινε διέγερση των ΕΥΜ και των 18CO με IL-1α ή/και tumor necrosis factor α (TNF-α) και μελετήθηκε η mRNA και πρωτεϊνική έκφραση του TL1A και των υποδοχέων του, DR3 και DcR3. Κατόπιν, έγινε διέγερση των ΗΤ-29 κυττάρων με IL-1α, TNF-α, και interferon γ (IFN-γ) και καλλιέργεια εντερικών ιστοτεμαχίων από ασθενείς με NC και το υπερκείμενο αυτών, χρησιμοποιήθηκε ως διεγέρτης σε καλλιέργειες ΕΥΜ για τον έλεγχο της mRNA και πρωτεϊνικής έκφρασης του TL1A και των υποδοχέων του, DR3 και DcR3. Παράλληλα, μελετήθηκε η mRNA και πρωτεϊνική έκφραση του TL1A και των υποδοχέων του, DR3 και DcR3 σε ΕΥΜ από ασθενείς με NC. Στη συνέχεια, έγινε διέγερση των ΗΤ29 κυττάρων με IL-1α, TNF-α και IFN-γ, προκειμένου να μελετηθεί η mRNA και η πρωτεϊνική έκφραση των υποδοχέων του TL1A, DR3 και DcR3. Τέλος, έγινε διέγερση των ΗΤ29 κυττάρων με TL1A, προκειμένου να μελετηθεί η mRNA έκφραση της IL-8.Ως προς την επίτευξη των στόχων του δεύτερου σκέλους της παρούσας διδακτορικής διατριβής, αρχικά, έγινε διέγερση των ΕΥΜ με IL-1α ή/και TNF-α, καθώς και με Lipopolysaccharide (LPS) προκειμένου να μελετηθεί η βασική και η επαγόμενη από τις προ-φλεγμονώδεις κυτταροκίνες, mRNA και πρωτεϊνική έκφραση των υποδοχέων των ιντερλευκινών. Στη συνέχεια, ακολούθησε διέγερση των ΕΥΜ με κυτταροκίνες της Th1 (TNF-α ή/και IFN-γ), της Th2 (IL-4 ή/και IL-13), της Th17 (IL-17 ή/και IL-22 ή/και IL-23) και της Treg (IL-10 ή/και transforming growth factor β (TGF-β)) ανοσοαπόκρισης, προκειμένου να μελετηθεί η mRNA έκφραση των προ-ινωτικών παραγόντων, κολλαγόνου τύπου Ι και ΙΙΙ, φιμπρονεκτίνης, α smooth muscle actin (α-SMA), tissue factor (TF), μεταλλοπρωτεϊνάσης-1 (metalloproteinase-1, MMP-1), MMP-9, αναστολέα των μεταλλοπρωτεϊνασών-1 (tissue inhibitor of metalloproteinase-1, TIMP-1) και ΤΙΜΡ-2 και η πρωτεϊνική έκφραση των προ-ινωτικών παραγόντων, κολλαγόνου και φιμπρονεκτίνης. Τέλος, έγινε διέγερση των ΕΥΜ με κυτταροκίνες της Th1 (TNF-α ή/και IFN-γ), της Th2 (IL-4 ή/και IL-13), της Th17 (IL-17 ή/και IL-22 ή/και IL-23) και της Treg (IL-10 ή/και TGF-β) ανοσοαπόκρισης, προκειμένου να διερευνηθεί η επίδρασή τους στην μεταναστευτική ικανότητα των ΕΥΜ, μέσω της δοκιμασίας επούλωσης τραύματος.Για τις mRNA μελέτες έκφρασης, έγινε απομόνωση ολικού RNA από τα κύτταρα, δημιουργία συμπληρωματικού DNA και μελέτη της έκφρασης με την χρήση της μεθόδου real time rt-PCR. Οι μελέτες πρωτεϊνικής έκφρασης έγιναν είτε με την μέθοδο του ανοσοφθορισμού, είτε με την μέθοδο της ELISA. Τα επίπεδα της πρωτεΐνης του κολλαγόνου μετρήθηκαν με την μέθοδο Sircol. Η στατιστική ανάλυση των αποτελεσμάτων έγινε με Student's t test. Αποτελέσματα - Ως προς το πρώτο σκέλος, η διέγερση των ΕΥΜ και των 18CO κυττάρων με τις προ-φλεγμονώδεις κυτταροκίνες IL-1α ή/και TNF-α, οι οποίες έχουν βρεθεί αυξημένες στον φλεγμαίνοντα εντερικό βλεννογόνο ασθενών με ΙΦΝΕ, οδήγησε στην αύξηση της mRNA και της πρωτεϊνικής έκφρασης του TL1A. Ιστικά υπερκείμενα από καλλιέργειες βιοψιών ασθενών με NC προκάλεσαν την mRNA και πρωτεϊνική έκφραση του TL1A στους ΕΥΜ. ΕΥΜ από ασθενείς με NC βρέθηκαν θετικοί στην έκφραση του TL1A. Η διέγερση των ΗΤ-29 κυττάρων με IL-1α, TNF-α και IFN-γ οδήγησε στην επαγωγή της έκφρασης του DR3 και τα υπερκείμενα αυτών, προκάλεσαν την mRNA και πρωτεϊνική έκφραση του TL1A στους ΕΥΜ. Η διέγερση των ΗΤ-29 κυττάρων με TL1A οδήγησε στην επαγωγή της mRNA έκφρασης της IL-8.Ως προς το δεύτερο σκέλος, σε ΕΥΜ από φυσιολογικούς μάρτυρες ανιχνεύθηκε μία βασική έκφραση των υποδοχέων ιντερλευκινών (Τh1: IFNGR1, IFNGR2, TNFRSF1A, IL12RB2, IL1R1 και IL1R2, Th2: IL4R και IL13RA2, Th17: IL22RA1, Treg: IL10RA, IL10RB, TGFBR1 και TGFBR2). Η διέγερση των ΕΥΜ με τις προ-φλεγμονώδεις κυτταροκίνες IL-1α ή/και TNF-α επηρέασε, είτε θετικά, είτε αρνητικά την έκφραση αυτή. Αντίθετα, η διέγερση των ΕΥΜ με LPS δεν προκάλεσε οποιοδήποτε στατιστικώς σημαντικό αποτέλεσμα στην έκφραση των υποδοχέων. Η διέγερση των ΕΥΜ με Th1 κυτταροκίνες είχε ως αποτέλεσμα την αύξηση της έκφρασης του mRNA του TF και της ΜΜP-1, της πρωτεΐνης του κολλαγόνου και της φιμπρονεκτίνης, καθώς και την μείωση της έκφρασης του mRNA της α-SMA και της ΜΜΡ-9 και του ρυθμού μετανάστευσης των ΕΥΜ. Η διέγερση των ΕΥΜ με Th2 κυτταροκίνες προκάλεσε την αύξηση του mRNA του κολλαγόνου, TF, α-SMA, ΜΜΡ-1 και του ρυθμού μετανάστευσης των ΕΥΜ και την μείωση της πρωτεΐνης της φιμπρονεκτίνης και του mRNA της ΜΜΡ-9. Οι Th17 κυτταροκίνες είχαν μεικτή επίδραση στους ΕΥΜ, καθώς οι IL-17 και IL-23 προκάλεσαν την αύξηση της φιμπρονεκτίνης, ενώ η IL-22, την μείωσή της και την ελάττωση της παραγωγής του TF. Παρόμοια ήταν και τα αποτελέσματα στην μεταναστευτική ικανότητα των μυοϊνοβλαστών: οι IL-17 και IL-22 προκάλεσαν την μείωση του ρυθμού μετανάστευσης, ενώ η IL-23, το αντίθετο. Επιπλέον, η IL-23 προκάλεσε την αύξηση της έκφρασης τόσο της MMP-1, όσο και της ΤΙΜΡ-1. Τέλος, ως αναμένετο, ο TGF-β αποδείχθηκε ως ο πιο αποτελεσματικός προ-ινωτικός παράγοντας, καθώς προκάλεσε την αύξηση όλων των υπό μελέτη προ-ινωτικών μορίων, καθώς και του ρυθμού μετανάστευσης των ΕΥΜ. Συμπεράσματα - Στην παρούσα διδακτορική διατριβή αναδεικνύεται πως οι ΕΥΜ εμπλέκονται άμεσα στην εντερική φλεγμονή και πως οι ανοσολογικές αποκρίσεις της έμφυτης και της ειδικής ανοσίας διαδραματίζουν ενεργό ρόλο στην ανάπτυξη εντερικής ίνωσης. Επιπλέον, η παρούσα διδακτορική διατριβή αναδεικνύει νέους πιθανούς θεραπευτικούς στόχους, η μελέτη των οποίων, ίσως, να οδηγήσει στην ανάπτυξη αποτελεσματικών θεραπευτικών στρατηγικών για την αντιμετώπιση της εντερικής ίνωσης

Probiotics in Intestinal Mucosal Healing: A New Therapy or an Old Friend?

Inflammatory bowel disease (IBD), Crohn’s disease, and ulcerative colitis are characterized by chronic and relapsing inflammation, while their pathogenesis remains mostly unelucidated. Gut commensal microbiota seem to be one of the various implicated factors, as several studies have shown a significant decrease in the microbiome diversity of patients with IBD. Although the question of whether microbiota dysbiosis is a causal factor or the result of chronic inflammation remains unanswered, one fact is clear; active inflammation in IBD results in the disruption of the mucus layer structure, barrier function, and also, colonization sites. Recently, many studies on IBD have been focusing on the interplay between mucosal and luminal microbiota, underlining their possible beneficial effect on mucosal healing. Regarding this notion, it has now been shown that specific probiotic strains, when administrated, lead to significantly decreased inflammation, amelioration of colitis, and improved mucosal healing. Probiotics are live microorganisms exerting beneficial effects on the host’s health when administered in adequate quantity. The aim of this review was to present and discuss the current findings on the role of gut microbiota and their metabolites in intestinal wound healing and the effects of probiotics on intestinal mucosal wound closure

Ad-Dressing Stem Cells: Hydrogels for Encapsulation

Regenerative medicine is a novel scientific field that employs the use of stem cells as cell-based therapy for the regeneration and functional restoration of damaged tissues and organs. Stem cells bear characteristics such as the capacity for self-renewal and differentiation towards specific lineages and, therefore, serve as a backup reservoir in case of tissue injuries. Therapeutically, they can be autologously or allogeneically transplanted for tissue regeneration; however, allogeneic stem cell transplantation can provoke host immune responses leading to a host-versus-transplant reaction. A probable solution to this problem is stem cell encapsulation, a technique that utilizes various biomaterials for the creation of a semi-permeable membrane that encases the stem cells. Stem cell encapsulation can be accomplished by employing a great variety of natural and/or synthetic hydrogels and offers many benefits in regenerative medicine, including protection from the host’s immune system and mechanical stress, improved cell viability, proliferation and differentiation, cryopreservation and controlled and continuous delivery of the stem-cell-secreted therapeutic agents. Here, in this review, we report and discuss almost all natural and synthetic hydrogels used in stem cell encapsulation, along with the benefits that these materials, alone or in combination, could offer to cell therapy through functional cell encapsulation

Melatonin and cortisol exhibit different circadian rhythm profiles during septic shock depending on timing of onset: a prospective observational study

Abstract Background Septic shock has been found to disrupt circadian rhythms. Moreover, timing of onset has been associated with different circadian profiles in experimental studies. Results In this prospective study, we enrolled 26 patients divided into two groups: Group A (N = 15) included subjects who had septic shock at the time of ICU admission and Group B (N = 11) included patients who developed septic shock during ICU admission. 6-Sulfatoxymelatonin (aMT6s) and cortisol levels were measured in urine samples every 4 h over a 24-h period. Two sets of samples were taken from Group A (entry/septic shock and exit) and three sets from Group B (entry, septic shock and exit). Mean, amplitude that is the difference between peak and mean values, as well as peak time, were estimated for both aMT6s and cortisol. In Group A, amplitude of aMT6s upon entry (septic shock) was reduced in relation to exit (437.2 ± 309.2 vs. 674.1 ± 657.6 ng/4 h, p < 0.05). Peak time occurred earlier (10:00 p.m. vs. 07:00 a.m, p < 0.05) and correlated with higher APACHE II score and longer ICU stay. In Group B, aMT6s mean values were significantly increased during septic shock (2492.2 ± 1709.1 ng/4 h) compared to both entry (895.4 ± 715.5 ng/4 h) and exit (1308.6 ± 1214.4 ng/4 h, p < 0.05 for all comparisons). Amplitude of aMT6s was also elevated during septic shock (794.8 ± 431.8 ng/4 h) in relation to entry (293.1 ± 275.9 ng/4 h, p < 0.05). Regarding cortisol rhythm in Group A, during septic shock amplitude was increased compared to exit (13.3 ± 31 ng/4 h vs. 8.7 ± 21.2 ng/4 h p < 0.05) and correlated with reduced hospital length of stay. In Group B, cortisol mean values and amplitude during septic shock (10 ± 5.3 and 3 ± 1.8 ng/4 h, respectively) were significantly reduced compared to both entry (30 ± 57.9 and 12.3 ± 27.3 ng/4 h) and exit (14.4 ± 20.7 and 6.6 ± 8.7 ng/4 h, p < 0.05 for all comparisons) and correlated with higher SOFA score and longer ICU and hospital stay. Conclusions Septic shock induced inverse changes of aMT6s and cortisol circadian rhythm profiles both within and between different groups of patients, depending on timing of onset. Reduced rhythmicity was correlated with severity of disease and longer ICU stay

The Probiotic Strains Bifid&omicron;bacterium lactis, Lactobacillus acidophilus,&nbsp;Lactiplantibacillus plantarum and Saccharomyces boulardii Regulate Wound Healing and Chemokine Responses in Human Intestinal Subepithelial Myofibroblasts

Bifidobacterium lactis,&nbsp;Lactobacillus acidophilus, Lactiplantibacillus plantarum and Saccharomyces boulardii are common probiotic supplements. Colonic subepithelial myofibroblasts (cSEMFs) are actively involved in mucosal wound healing and inflammation. cSEMFs, isolated from healthy individuals, were stimulated with 102 or 104 cfu/mL of these probiotic strains alone and in combination, and their effect on chemokine and wound healing factor expression was assessed by qRT-PCR, ELISA and Sircol Assay, and on cSEMFs migration, by Wound Healing Assay. These strains remained viable and altered cSEMFs&rsquo; inflammatory and wound healing behavior, depending on the strain and concentration. cSEMFs treated with a combination of the four probiotics had a moderate, but statistically significant, increase in the mRNA and/or protein expression of chemokines CXCL1, CXCL2, CXCL4, CXCL8, CXCL10, CCL2 and CCL5, and healing factors, collagen type I and III, fibronectin and tissue factor. In contrast, when each strain was administered alone, different effects were observed, with greater increase or decrease in chemokine and healing factor expression, which was balanced by the mixture. Overall, this study highlights that the use of multiple probiotic strains can potentially alert the gut mucosal immune system and promote wound healing, having a better effect on mucosal immunity than the use of single probiotics

Development of a Human Intestinal Organoid Model for In Vitro Studies on Gut Inflammation and Fibrosis

Inflammatory Bowel Diseases (IBDs) are characterized by chronic intestinal inflammation and fibrosis, the latter being the predominant denominator for long-term complications. Epithelial and mesenchymal 2D cultures are highly utilized in vitro models for the preclinical evaluation of anti-inflammatory and antifibrotic therapies. More recently, human intestinal organoids (HIOs), a new 3D in vitro model derived from pluripotent stem cells, have the advantage to closely resemble the architecture of the intestinal mucosa. However, the appropriate timing for the study of inflammatory and fibrotic responses, during HIO development, has not been adequately investigated. We developed HIOs from the human embryonic stem cell line, H1, and examined the expression of mesenchymal markers during their maturation process. We also investigated the effect of inflammatory stimuli on the expression of fibrotic and immunological mediators. Serial evaluation of the expression of mesenchymal and extracellular matrix (ECM) markers revealed that HIOs have an adequately developed mesenchymal component, which gradually declines through culture passages. Specifically, CD90, collagen type I, collagen type III, and fibronectin were highly expressed in early passages but gradually diminished in late passages. The proinflammatory cytokines IL-1 alpha and TNF-alpha induced the mRNA expression of fibronectin, collagen types I and III, tissue factor (TF), and alpha-smooth muscle actin (alpha-SMA) primarily in early passages. Similarly, HIOs elicited strong mRNA and protein mesenchymal (CXCL10) and epithelial (CXCL1, CCL2, CXCL8, and CCL20) chemokine responses in early but not late passages. In contrast, the epithelial tight junction components, CLDN1 and JAMA, responded to inflammatory stimulation independently of the culture passage. Our findings indicate that this HIO model contains a functional mesenchymal component, during early passages, and underline the significance of the mesenchymal cells&apos; fitness in inflammatory and fibrotic responses. Therefore, we propose that this model is suitable for the study of epithelial-mesenchymal interactions in early passages when the mesenchymal component is active

Role of <i>Lactiplantibacillus plantarum</i> UBLP-40, <i>Lactobacillus rhamnosus</i> UBLR-58 and <i>Bifidobacterium longum</i> UBBL-64 in the Wound Healing Process of the Excisional Skin

The probiotics Lactiplantibacillus plantarum UBLP-40, Lactobacillus rhamnosus UBLR-58 and Bifidobacterium longum UBBL-64 seem to promote wound healing when applied topically. Our aim was to investigate their effect on the mRNA expression of pro-inflammatory, healing and angiogenetic factors during the healing process of a standardized excisional wound model in rats. Rats subjected to six dorsal skin wounds were allocated to Control; L. plantarum; combined formula of L. rhamnosus plus B. longum; L. rhamnosus; and B. longum treatments, applied every two days, along with tissue collection. The pro-inflammatory, wound-healing, and angiogenetic factors of mRNA expression were assessed by qRT-PCR. We found that L. plantarum exerts a strong anti-inflammatory effect in relation to L. rhamnosus–B. longum, given alone or in combination; the combined regime of L. rhamnosus–B. longum, works better, greatly promoting the expression of healing and angiogenic factors than L. plantarum. When separately tested, L. rhamnosus was found to work better than B. longum in promoting the expression of healing factors, while B. longum seems stronger than L. rhamnosus in the expression of angiogenic factors. We, therefore, suggest that an ideal probiotic treatment should definitively contain more than one probiotic strain to speed up all three healing phases