Immunomodulatory role of Parkinson's disease 7 in inflammatory bowel disease.

Recently the role of Parkinson's disease 7 (PARK7) was studied in gastrointestinal diseases, however, the complex role of PARK7 in the intestinal inflammation is still not completely clear. Expression and localization of PARK7 were determined in the colon biopsies of children with inflammatory bowel disease (IBD), in the colon of dextran sodium sulphate (DSS) treated mice and in HT-29 colonic epithelial cells treated with interleukin (IL)-17, hydrogen peroxide (H2O2), tumor necrosis factor (TNF)-α, transforming growth factor (TGF)-ß or lipopolysaccharide (LPS). Effect of PARK7 on the synthesis of IBD related cytokines was determined using PARK7 gene silenced HT-29 cells and 3,4,5-trimethoxy-N-(4-(8-methylimidazo(1,2-a)pyridine-2-yl)phenyl)benzamide (Comp23)-compound increasing PARK7 activity-treated mice with DSS-colitis. PARK7 expression was higher in the mucosa of children with Crohn's disease compared to that of controls. While H2O2 and IL-17 treatment increased, LPS, TNF-α or TGF-ß treatment decreased the PARK7 synthesis of HT-29 cells. PARK7 gene silencing influenced the synthesis of IL1B, IL6, TNFA and TGFB1 in vitro. Comp23 treatment attenuated the ex vivo permeability of colonic sacs, the clinical symptoms, and mucosal expression of Tgfb1, Il1b, Il6 and Il10 of DSS-treated mice. Our study revealed the role of PARK7 in the regulation of IBD-related inflammation in vitro and in vivo, suggesting its importance as a future therapeutic target.

PARK7 Diminishes Oxidative Stress-Induced Mucosal Damage in Celiac Disease.

Coeliac disease (CD) is a chronic, immune-mediated small intestinal enteropathy, accompanied with gluten-triggered oxidative damage of duodenal mucosa. Previously, our research group reported an increased mucosal level of the antioxidant protein Parkinson's disease 7 (PARK7) in children with CD. In the present study, we investigated the role of increased PARK7 level on the epithelial cell and mucosal integrity of the small intestine. The presence of PARK7 was investigated using immunofluorescent staining on duodenal mucosa of children with CD and on FHs74Int duodenal epithelial cells. To investigate the role of oxidative stress, FHs74Int cells were treated with H2O2 in the absence or presence of Comp23, a PARK7-binding compound. Intracellular accumulation of reactive oxygen species (ROS) was determined by DCFDA-based assay. Cell viability was measured by MTT, LDH, and Annexin V apoptosis assays. Disruption of cytoskeleton and cell adhesion was investigated by immunofluorescence staining and by real-time RT PCR. Effect of PARK7 on mucosal permeability was investigated ex vivo using intestinal sacs derived from control and Comp-23-pretreated mice. Comp23 treatment reduced the H2O2-induced intracellular accumulation of ROS, thus preserving the integrity of the cytoskeleton and also the viability of the FHs74Int cells. Accordingly, Comp23 treatment increased the expression of antioxidants (NRF2, TRX1, GCLC, HMOX1, NQO1), cell-cycle regulators (TP53, CDKN1A, PCNA, BCL2, BAX), and cell adhesion molecules (ZO1, CDH1, VCL, ITGB5) of H2O2-treated cells. Pretreatment with Comp23 considerably decreased the small intestinal permeability. In this study, we demonstrate that PARK7-binding Comp23 reduces the oxidative damage of duodenal epithelial cells, via increased expression of NRF2- and P53-regulated genes. Our results suggest that PARK7 plays a significant role in the maintenance of mucosal integrity in CD.

Characterization of IL-19, -20, and -24 in acute and chronic kidney diseases reveals a pro-fibrotic role of IL-24.

BACKGROUND: Recently, the role of IL-19, IL-20 and IL-24 has been reported in renal disorders. However, still little is known about their biological role. METHODS: Localization of IL-20RB was determined in human biopsies and in the kidneys of mice that underwent unilateral ureteral obstruction (UUO). Renal Il19, Il20 and Il24 expression was determined in ischemia/reperfusion, lipopolysaccharide, streptozotocin, or UUO induced animal models of kidney diseases. The effects of H2O2, LPS, TGF-ß1, PDGF-B and IL-1ß on IL19, IL20 and IL24 expression was determined in peripheral blood mononuclear cells (PBMCs). The extents of extracellular matrix (ECM) and α-SMA, Tgfb1, Pdgfb, and Ctgf expression were determined in the kidneys of Il20rb knockout (KO) and wild type (WT) mice following UUO. The effect of IL-24 was also examined on HK-2 tubular epithelial cells and NRK49F renal fibroblasts. RESULTS: IL-20RB was present in the renal biopsies of patients with lupus nephritis, IgA and diabetic nephropathy. Amount of IL-20RB increased in the kidneys of mice underwent UUO. The expression of Il19, Il20 and Il24 increased in the animal models of various kidney diseases. IL-1ß, H2O2 and LPS induced the IL19, IL20 and IL24 expression of PBMCs. The extent of ECM, α-SMA, fibronectin, Tgfb1, Pdgfb, and Ctgf expression was lower in the kidney of Il20rb KO compared to WT mice following UUO. IL-24 treatment induced the apoptosis and TGF-ß1, PDGF-B, CTGF expression of HK-2 cells. CONCLUSIONS: Our data confirmed the significance of IL-19, IL-20 and IL-24 in the pathomechanism of renal diseases. Furthermore, we were the first to demonstrate the pro-fibrotic effect of IL-24.

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-1ß, TNF-α, TGF-ß 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-1ß 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.

Role of microRNA-223 in the regulation of poly(ADP-ribose) polymerase in pediatric patients with Crohn's disease.

OBJECTIVES: Crohn's disease (CD) is a multifactorial disease, characterized by oxidant-induced tissue injury with a possible activation of poly(ADP-ribose) polymerase (PARP)-1. MicroRNAs (miRs) can offer a potential link between the genetic susceptibility, environmental and immunologic factors in the pathogenesis of CD. Previously, PARP-1 was identified as a direct target gene of miR-223 in an epithelial cell line. Our aim was to examine PARP activation and miR-223 expression in colonic biopsies of pediatric CD. To support our in vivo findings, the effect of lipopolysaccharide (LPS) on same parameters was examined in HT-29 colonic epithelial cell line. METHODS: Colonic biopsies were taken from patients with macroscopically inflamed and intact mucosa with CD and controls. LPS treated HT-29 cells served as our in vitro model. To analyze the PARP-1 expression real-time PCR, Western blot and immunohistochemical analyses were used. PARP-1 enzymatic activity was assessed on the basis of poly(ADP-ribosyl)ated proteins. Expression of miR-223 was examined by real-time PCR. RESULTS: PARP-1 mRNA and miR-223 expression was significantly elevated, however, the amount of PARP-1 protein and poly(ADP-ribose) was reduced in pediatric CD compared to controls. LPS incubation did not affect the expression of PARP-1 mRNA, however, decreased miR-223 expression, and enhanced PARP-1 activity. CONCLUSIONS: In our study, we showed that the expression of miR-223 is up-regulated and poly(ADP-ribosyl)ation is reduced in pediatric patients with CD. Moreover, we confirmed their opposite change in LPS treated epithelial cells, too. These data suggest that the hypofunctionality of PARP-1 may play a potential role in the pathomechanism of CD.

Selective measurement of α smooth muscle actin: why ß-actin can not be used as a housekeeping gene when tissue fibrosis occurs.

BACKGROUND: Prevalence of fibroproliferative diseases, including chronic kidney disease is rapidly increasing and has become a major public health problem worldwide. Fibroproliferative diseases are characterized by increased expression of α smooth muscle actin (α-SMA) that belongs to the family of the six conserved actin isoforms showing high degree homology. The aim of the present study was to develop real-time PCRs that clearly discriminate α-SMA and ß-actin from other actin isoforms. RESULTS: Real-time PCRs using self-designed mouse, human and rat specific α-SMA or ß-actin primer pairs resulted in the specific amplification of the artificial DNA templates corresponding to mouse, human or rat α-SMA or ß-actin, however ß-actin showed cross-reaction with the housekeeping γ-cyto-actin. We have shown that the use of improperly designed literary primer pairs significantly affects the results of PCRs measuring mRNA expression of α-SMA or ß-actin in the kidney of mice underwent UUO. CONCLUSION: We developed a set of carefully designed primer pairs and PCR conditions to selectively determine the expression of mouse, human or rat α-SMA and ß-actin isoforms. We demonstrated the importance of primer specificity in experiments where the results are normalized to the expression of ß-actin especially when fibrosis and thus increased expression of α-SMA is occur.

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 >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-ß 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.

Fibrosis Related Inflammatory Mediators: Role of the IL-10 Cytokine Family.

Importance of chronic fibroproliferative diseases (FDs) including pulmonary fibrosis, chronic kidney diseases, inflammatory bowel disease, and cardiovascular or liver fibrosis is rapidly increasing and they have become a major public health problem. According to some estimates about 45% of all deaths are attributed to FDs in the developed world. Independently of their etiology the common hallmark of FDs is chronic inflammation. Infiltrating immune cells, endothelial, epithelial, and other resident cells of the injured organ release an orchestra of inflammatory mediators, which stimulate the proliferation and excessive extracellular matrix (ECM) production of myofibroblasts, the effector cells of organ fibrosis. Abnormal amount of ECM disturbs the original organ architecture leading to the decline of function. Although our knowledge is rapidly expanding, we still have neither a diagnostic tool to detect nor a drug to specifically target fibrosis. Therefore, there is an urgent need for the more comprehensive understanding of the pathomechanism of fibrosis and development of novel diagnostic and therapeutic strategies. In the present review we provide an overview of the common key mediators of organ fibrosis highlighting the role of interleukin-10 (IL-10) cytokine family members (IL-10, IL-19, IL-20, IL-22, IL-24, and IL-26), which recently came into focus as tissue remodeling-related inflammatory cytokines.

Peroxisome proliferator-activated receptor-γ and thymic stromal lymphopoietin are involved in the pathophysiology of childhood coeliac disease.

Celiac disease (CD) is a chronic autoimmune enteropathy caused by exposure to dietary gluten in genetically predisposed individuals. The transcription factor peroxisome proliferator-activated receptor gamma (PPARγ) was shown to exert protective effects in several immune-mediated disorders. Activation of PPARγ suppressed the expression of thymic stromal lymphopoietin (TSLP), an inducer of proinflammatory cytokines. Since the role of TSLP in gluten-sensitive enteropathy is completely unknown, we investigated the involvement of TSLP and its regulator PPARγ in childhood CD. We collected duodenal biopsy specimens from 19 children with newly diagnosed CD, 6 children with treated CD (gluten-free diet, GFD), and 10 controls. Expression of mRNA and protein levels of PPARγ, TSLP, and TSLP receptor were determined by real-time RT-PCR and Western blot, respectively. Duodenal localization of PPARγ and TSLP was studied by immunohistochemistry. In duodenal mucosa of children with CD, the amount of PPARγ was significantly lower and simultaneously that of TSLP significantly higher compared to controls (p < 0.05). In GFD-treated patients, the levels of PPARγ mRNA and protein were significantly higher while that of TSLP markedly lower compared to newly diagnosed CD (p < 0.05). Immunohistochemistry revealed PPARγ and TSLP expression in lamina propria immune cells and in enterocytes. Low expression of PPARγ and high expression of TSLP in the duodenal mucosa of children with newly diagnosed CD suggest that they are involved in the pathophysiology of CD. We hypothesize that PPARγ may be an inhibitory regulator of TSLP-stimulated inflammatory processes in CD.

Expression of PARK7 is increased in celiac disease.

Recently, it has been suggested that the gene called Parkinson's disease 7 (PARK7) might be an upstream activator of hypoxia-inducible factor (HIF)-1α, which plays a major role in sustaining intestinal barrier integrity. Furthermore, PARK7 has been proposed to participate in the Toll-like receptor (TLR)-dependent regulation of the innate immune system. Our aim was to investigate the involvement of PARK7 in the pathogenesis of coeliac disease (CD). Duodenal biopsy specimens were collected from 19 children with untreated CD, five children with treated CD (maintained on gluten-free diet), and ten children with histologically normal duodenal biopsies. PARK7 mRNA expression and protein level were determined by real-time polymerase chain reaction (PCR) and Western blot, respectively. Localization of PARK7 was visualized by immunofluorescence staining. Protein level of PARK7 increased in the duodenal mucosa of children with untreated CD compared to children with treated CD or to control biopsies (p <0.03). We detected intensive PARK7 staining in the epithelial cells and lamina propria of the duodenal mucosa of children with untreated CD compared with that in control biopsies. Our finding that mucosal expression of PARK7 is increased suggests that PARK7 is involved in the pathogenesis of gastrointestinal diseases, notably CD. Our results suggest that PARK7 may alter processes mediated by HIF-1α and TLR4, which supports a role for PARK7 in the maintenance of epithelial barrier integrity, immune homeostasis, or apoptosis.