Role of the Microbiome in Celiac Disease

Microbiome is the community of commensal, symbiotic and pathogenic microorganism that share our human body space. Intestinal microbiota has a defensive role in human health, it is implicated in metabolic and nutritional processes and plays an important role in the pathophysiology of several diseases. In recent years special attention has been paid to investigations targeting the changes of intestinal microbiome in various gastrointestinal disorders including inflammatory bowel disease, infectious colitis and celiac disease (CD). The aim of our present review is to summarize the role of the microbiome in CD and the changes of its composition in the intestine of patients suffering from CD

Role of IL-24 in the mucosal remodeling of children with coeliac disease

Background Recently, involvement of IL-19, IL-20 and IL-24 has been reported in inflammatory diseases associated with tissue remodeling. However, their impact on the pathomechanism of coeliac disease (CD) is still completely unknown. Methods Expression of IL19, IL20 and IL24 was measured by real-time RT-PCR, protein amount of IL-24, α smooth muscle actin (α-SMA) and fibronectin (FN) was determined by Western-blot analysis in the duodenal biopsies of therapy naive children with CD and controls. Localization of IL-24 and IL-20RB was investigated by immunofluorescent staining in the duodenal mucosa. Effect of recombinant IL-1β, TNF-α, TGF-β and IL-17 treatment on the expression of IL19, IL20, IL24 and their receptors was investigated by real-time RT-PCR in small intestinal epithelial cells (FHs74Int), in primary duodenal myofibroblasts (pdMFs) and in peripheral blood mononuclear cells (PBMCs). Effect of IL-24 on H2O2 treated FHs74Int cells and on pdMFs was measured by MTT, LDH, Annexin V assays, real-time RT-PCR and by fluorescent microscopy. Results We found increased level of IL-24 (3.3×, p < 0.05), α-SMA (2.4×, p < 0.05) and FN (2.3×, p < 0.05) in the duodenal mucosa and increased expression of IL19 (3.6×, p < 0.05) and IL24 (5.2×, p < 0.05) in the PBMCs of children with CD compared to that of controls. IL-1β was a strong inducer of IL24 expression of FHs74Int cells (9.9×, p < 0.05), pdMFs (552.9×, p < 0.05) or PBMCs (17.2×, p < 0.05), as well. IL-24 treatment reduced the number of apoptotic cells (0.5×, p < 0.05) and decreased the expression of inflammatory factors, including IL1A, IL6 and TNF of H2O2-treated FHs74Int cells. IL-24 decreased the proliferation (0.6×, p < 0.05) of PDGF-B treated pdMFs. Moreover, IL-24 treatment altered the morphology of pdMFs by influencing the size of the angles between stress fibers and the longitudinal axis of the cells (2.0×, p < 0.05) and the expression of cytoskeletal components, including ACTA2, ACTB, VIM, SNAI1 and SNAI2. Conclusion Our results suggest that IL-24 plays a significant role in the maintenance of duodenal mucosal integrity in CD

Microarray Analysis Reveals Increased Expression of Matrix Metalloproteases and Cytokines of Interleukin-20 Subfamily in the Kidneys of Neonate Rats Underwent Unilateral Ureteral Obstruction: A Potential Role of IL-24 in the Regulation of Inflammation and Tissue Remodeling

Background/Aims: Congenital obstructive nephropathy (CON) is the main cause of pediatric chronic kidney diseases leading to renal fibrosis. High morbidity and limited treatment opportunities of CON urge the better understanding of the underlying molecular mechanisms. Methods: To identify the differentially expressed genes, microarray analysis was performed on the kidney samples of neonatal rats underwent unilateral ureteral obstruction (UUO). Microarray results were then validated by real-time RT-PCR and bioinformatics analysis was carried out to identify the relevant genes, functional groups and pathways involved in the pathomechanism of CON. Renal expression of matrix metalloproteinase (MMP)-12 and interleukin (IL)-24 were evaluated by real-time RT-PCR, flow cytometry and immunohistochemical analysis. Effect of the main profibrotic factors on the expression of MMP-12 and IL-24 was investigated on HK-2 and HEK-293 cell lines. Finally, the effect of IL-24 treatment on the expression of pro-inflammatory cytokines and MMPs were tested in vitro. Results: Microarray analysis revealed 880 transcripts showing &#x3e;2.0-fold change following UUO, enriched mainly in immune response related processes. The most up-regulated genes were MMPs and members of IL-20 cytokine subfamily, including MMP-3, MMP-7, MMP-12, IL-19 and IL-24. We found that while TGF-β treatment inhibits the expression of MMP-12 and IL-24, H2O2 or PDGF-B treatment induce the epithelial expression of MMP-12. We demonstrated that IL-24 treatment decreases the expression of IL-6 and MMP-3 in the renal epithelial cells. Conclusions: This study provides an extensive view of UUO induced changes in the gene expression profile of the developing kidney and describes novel molecules, which may play significant role in the pathomechanism of CON

Az interleukin-24 citokin szerepe a krónikus vesebetegség patomechanizmusában

Tudományos háttér, célkitűzés: A kongenitális obstruktív nefropátia patomechanizmusát feltáró microarray vizsgálataink eredményei rávilágítottak, hogy az interleukin (IL)-20 citokin alcsalád tag IL-24 renális expressziója jelentősen fokozódik az urétert érintő obstrukciós inzultust követően. Mivel az IL-24 citokin funkciója nem ismert a krónikus vesebetegséghez (KVB) vezető patofiziológiás folyamatokban, célul tűztük ki szerepének vizsgálatát. Módszer: A kongenitális obstruktív nefropátia és vesefibrózis modellezéséhez újszölött patkányokon valamint vad típusú és IL-24 receptor (IL-20Rβ) génkiütött (KO) egereken unilaterális uréter obstrukciót (UUO) végeztünk. Az obstrukció hatására létrejövő genom-szintű génexpressziós változásokat microarray technika segítségével elemeztük. Méréseink során valós idejű RT-PCR, Western blot, valamint Masson festés segítségével határoztuk meg a vesefibrózis mértékét valamint a vizsgált molekulák mRNS és fehérje szintű expresszióját. Egészséges valamint KVB-s betegekből származó szérummintákban vizsgáltuk az IL-24 mennyiségét. Az IL-24 kezelés jelátviteli folyamatokra, valamint profibrotikus molekulák termelésére gyakorolt hatását in vitro kísérletekben tanulmányoztuk vese epitél sejteken. Eredmények: Újszülött állatok vesemintáin elvégzett microarray analízisünk adataival összhangban kimutattuk, hogy az IL-24 és receptorának mennyisége UUO-t követően felnőtt állatok veséiben is megemelkedik. A vad típusú állatokban tapasztaltakhoz képest az IL-20Rβ KO állatokban csökkent a fibrotikus markerek és növekedési faktorok (α-SMA, fibronectin, TGF-β, PDGF-B, kollagén 1,-3) mennyisége UUO-t követően. Eredményik szerint az IL-24 szérum szintje megemelkedik a KVB-ben szenvedő betegekben. In vitro vizsgálataink eredményei szerint az IL-24 kezelés profibrotikus jelátviteli útvonalak aktiválásán keresztül fokozza a vese epitél sejtek TGF-β valamint PDGF-B expresszióját. Következtetés: Az IL-24 KVB-ben emelkedett szérumszintje, profibrotikus molekulák termelését serkentő hatása, valamint az IL-20 citokin alcsalád receptorának hiányában kialakuló csökkent mértékű vesefibrózis arra utal, hogy az IL-24 szerepet játszik a krónikus veseelégtelenséghez vezető fibrotikus folyamatokban. Jelen eredményeink felvetik az IL-24 gátlásának, potenciális szerepét a krónikus vesebetegség terápiájában. Támogatás: K116928, VKE-2017-00006, A kutatás a Bolyai János Kutatási Ösztöndíj támogatásával készül

THE ROLE OF IL-20 CYTOKINE SUBFAMILY IN THE PATHOGENESIS OF CHRONIC KIDNEY DISEASE

Background: Regardless of the etiology kidney fibrosis is a common outcome of progressive chronic kidney diseases. Our recent study showed that levels of interleukin (IL)-20 subfamily members, including IL-19 and IL-24 significantly increased in kidneys underwent unilateral ureteral obstruction (UUO). However, their precise role in the pathomechanism of renal fibrosis is not clearly understood. Methods: To study the role of IL-20 cytokine subfamily we applied a mouse model of UUO induced kidney fibrosis on wild type and IL-20 receptor beta gene knockout (IL-20Rβ KO) mice. Masson’s trichrome and Picro-Sirius Red staining were used to investigate the renal accumulation of extracellular matrix proteins. Real-time RT-PCR and western blot method were performed to measure the renal expression of fibrosis associated molecules. We also investigated the in vitro effect of IL-24 treatment on transforming growth factor beta (TGF-β) and platelet derived growth factor B (PDGF-B) expression of human proximal tubular epithelial (HK-2) cells by real-time RT-PCR and flow cytometry. Results: We found elevated level of IL-19, IL-24 and IL-20Rβ in the fibrotic kidneys. IL-20Rβ KO mice showed reduced extracellular matrix deposition and decreased α-smooth muscle actin expression compared to wild-type mice following UUO. Treatment of renal epithelial cells with IL-24 increased their TGF-β and PDGF-B production. Conclusion: Our study provides direct evidence of the pathogenic role of IL-20 cytokine subfamily in the development of renal fibrosis, possibly through the IL-24 mediated production of pro-fibrotic factors. Therefore, inhibition of IL-24 may have therapeutic effect in treatment of chronic kidney diseases. Grants: OTKA K116928 and VKE-2017-00006. This project was supported by the János Bolyai Research Scholarship of the Hungarian Academy of Science

ROLE OF NANOVESICLES IN THE DEVELOPMENT OF PERITONEAL FIBROSIS DURING PERITONEAL DIALYSIS

Background: Nanovesicles (NVs) have significant therapeutic potential. They can protect against myocardial infarction, acute kidney injury or liver fibrosis as well. In 2017 Pearson et al successfully isolated extracellular vesicles from dialysis effluents (PDE) of PD patients, however their impact on the development of peritoneal fibrosis during PD remained unclear. Our aim was to determine, whether NVs isolated from PDE can affect/alter progression of peritoneal fibrosis. Methods: Three hundred ml of PDE have been collected from PD patients. NVs were isolated by different ways (ultrafiltration with MWCO filters or size exclusion chromatography) to obtain the most homogenous/concentrated NV fractions. NVs were analysed by dynamic light scattering (DLS) and Fourier transform infrared spectroscopy (FTIR). NRK-49F fibroblast cell line was treated with PDE derived NVs and their effect on the endogenous as well as platelet derived growth factor (PDGF) induced proliferation of this fibroblast cell line were measured by MTT test after 24h. Results: The average size and size distribution of NVs was about 80 [40-150] nm (DLS). FTIR analyses revealed that protein and lipid content and protein to lipid ratio of PDE derived NVs was typical for this type of extracellular vesicles. Incubation of NRK-49F with PDE derived NVs significantly reduced both the endogenous and PDGF induced proliferation rate of these fibroblast cells. Conclusion: NVs isolated from dialysis effluents of PD patients may inhibit peritoneal fibrosis contributing to the preservation of the peritoneal membrane structure. However, future in vivo and in vitro experiments are required for testing their therapeutic potential in PD and their impact as potential biomarker. This project was supported by ÚNKP-17- 4-IV- SE-60 New National Excellence Program of the Ministry of Human Capacities and by the János Bolyai Research Scholarship of the Hungarian Academy of Science

The role of interleukin-24 in the pathomechanism of IBD-associated tissue remodeling

Abstract: Introduction: Intestinal fibrosis is a serious complication of inflammatory bowel diseases (IBD). Interleukin(IL)-24 is a member of IL-20 cytokine subfamily, which regulatory effect is suspected in connection with inflammation, apoptosis or tissue remodeling in other organs. Increased level of IL-24 was described in the colon of patients with active IBD, however its biological role is still poorly understood. Methods: Colonic presence of IL-24 and its receptor IL-20RB was investigated in the dextran-sodium sulfate (DSS) induced mice model of IBD (n=8; C57BL/6J). Impact of IL-24 on colonic extracellular matrix (ECM) production was investigated in the DSS treated wild type and IL-20RB knockout (KO) mice. Effect of intracolonic injection of IL-24 was also investigated. The role of IL-24 treatment on the expression of fibrosis related genes was investigated in colonic epithelial (HT-29) and fibroblast (CCD-18Co) cells. Results: Expression of IL-24 increased in colonic tissue of DSS-treated mice compared to controls. Lack of IL-24 receptor resulted in reduced ECM deposition in IL-20RB KO mice compared to wild type group. Local administration of IL-24 increased the expression of the fibrosis associated genes in the colon. IL-24 treatment increased the expression of TGF-ß1 and PDGF-B in HT-29, and that of COL1, COL3, FN1, MMP2, -9, TIMP1, -2 in CCD-18Co cells. Discussion: IL-24 may promote tissue remodeling shifted toward an excessive deposition of ECM components directly by acting on fibroblast and indirectly via induction of pro-fibrotic factors on epithelial cells. Our data suggest that inhibition of IL-24 may have a significant anti-fibrotic effect. Support: OTKA-K116928, VKE-2017-00006,EFOP-3.6.3-VEKOP-16-2017-00009,MTA-SE Pediatrics and Nephrology Research Group, János Bolyai Research Scholarship of the Hungarian Academy of Science