Interleukin 23 P 19 Gene Expressions in Patients with Ulcerative Colitis and its Relation to Disease Severity

Background and Study Aims: The pur pose of This study Is to détermine whether mucosal expression of IL 23 p 19 has rôle in the pathogenèses of ulcerative colitis and its relation to dise ase severity. Materials and Methods: This study was carried out on 50 patients with ulcerative colitis and 10 normal individuals as control. They were divided into: Group I : 27 patients with mild to moderate disease. Group II : 23 patients with severe disease. Group III : 10 normal individuals. All patients and control were subjected to histopathological study, IL-23p19 immunohistochemical staining, IL-23R expression by flow cytometry and serum IL-23 by ELISA. Results: There is significant increased in IL-23p19 gene and IL-23R in ulcerative colitis patients compared with control. Significant positive correlations were detected between increased expression of IL-23 p19 gene, IL-23R, high serum IL-23 and severity of the disease. Conclusion: increased expression of IL-23 p19 gene has a role in the pathogenesis of ulcerative colitis and that targeted therapy directed against IL23 p19 may be effective in the treatment. Increased expression of IL-23p19 gene and IL-23R with high serum IL-23 correlate positively with disease severity.


Introduction
Inflammatory Bowel Disease (IBD) consists of two distinct diseases, Crohn's Disease (CD) and Ulcerative Colitis (UC). Both diseases are thought to arise due to combination of genetic variations and alteration in bacterial flora which can subsequently drive a deregulated immune response that results in chronic intestinal inflammation [1,2].
Interleukin 23 (IL-23) is a member of a small family of proinflammatory cytokine, consisting of a p19 subunit and a common p40 subunit [3]. The receptor for IL-23 (IL-23 R) consists of the IL-12R beta 1 subunit and a novel component termed IL-23R [4], which is expressed predominantly on T, NK, and NKT cells and to a smaller extent on monocytes, macrophages and DCs [5]. IL-23 plays a crucial role in the pathogenesis of a number of immune-mediated inflammatory diseases by recruitment of several inflammatory cells and Th17 cells [6,7]. IL-23 promotes Th17 cells producing TNF α, IL-17, IL-6, IL-22, GM-CSF, and other novel factors, which are associated with the induction of autoimmune inflammation [6,8,9]. This study was carried out to determine whether mucosal expression of IL 23 p 19 has a role in the pathogenesis of ulcerative colitis and to elucidate its relation to disease severity

Patients and methods
This study was carried out on 50 patients with ulcerative colitis and 10 individuals whose colonoscopic and histopathologic findings were normal as control. The studied subjects were selected from the

Interleukin 23 P 19 Gene Expressions in Patients with
Ulcerative Colitis and its Relation to Disease Severity tissue were cut and stained by Hematoxylin and Eosin for histopathological evaluation and grading of the groups.

IL-23p19 Immunohistochemical staining
4-μm-thick serial sections of formalin fixed, paraffin-embedded tissue were cut and mounted on positively charged glass slides. After incubation at 60°C overnight and deparaffinization, sections placed in 0.01 M sodium citrate buffer (pH 6.0) and heated twice for 5 minutes in a microwave oven. After inactivation of endogenous peroxidase with 0.5% metaperiodic acid in Phosphate-Buffered Saline (PBS) for 10 minutes, sections were incubated with 10% horse serum in PBS for 1 hour Sections were incubated at 4°C overnight with 100× diluted primary goat anti-mouse IL-23 p19 antibody (R&D Systems, Inc.). The standard Avidin-Biotinperoxidase Complex (ABC) technique was performed using the Lab Vision Secondary Detection Kit (Ultra Vision Detection System Anti-polyvalent, HRP). The color was visualized by incubation with chromogen 3, 3' diaminobenzidine for 5 minutes. The slides were then counterstained with Mayer hematoxylin and cover slipped with Per mount (Stat Lab, McKinney, TX). Negative controls were set for each test without the primary antibodies.

Immunohistochemical evaluation
Results were expressed semi-quantitatively. Positively stained cells were counted by examining at least 10 random fields (X200) in each section and expressed as the percentage of positive cells over total cell number [13].

Peripheral Blood Mononuclear Cells (PBMC)
CD4+ T isolation from blood samples: Lymphocytes were isolated from peripheral blood by incubation with Rosette Sep Human CD4+T cells enrichment cocktail (Stem Cells Technologies, Grenoble, France) followed by centrifugation on a density gradient (Lymphoprep, PAA, Pasching, Austria). Lymphocytes were purified by centrifugation through Lymphoprep.

Lamina Propria Mononuclear Cells (LPMC)
Isolation of intestinal lymphocytes: For IEL isolation, endoscopic procedure was done, tissue (dissected mucosa) was placed and stirred for 15 minutes at room temperature in prewar med (37°C) 1× HBSS containing 10% fetal bovine serum (Gibco catalog no. 10082), 0.015 M HEPES, and 5 mM EDTA and stirred for 15 minutes at 37°C, followed by three 15-minutes washes with buffer adjusted to room temperature. The supernatant from each wash was pooled and poured through a nylon wool column to enrich for T cells and remove mucus. The resulting cell suspension was used to analyze IEL.
For IEL: The IEL suspensions containing approximately 4 × 10 6 cells each were re suspended in cold phosphate-buffered saline and stained with Aqua Live/Dead cell discriminator (Invitrogen catalog no. L34597) according to the manufacturer's protocol. Cells were then stained for 1 hour in the dark at 4°C with optimized concentrations of anti-CD3 Alexa750-APC (eBioscience clone 17A2), anti-CD8 Alexa700 (eBioscience clone 53-6.7), anti-CD4 Pacific Blue (eBioscience clone RM4-5), anti-TCR GD R-PE (BD Pharmingen clone GL3), and biotinylated polyclonal anti-IL-23R (BAF1686; R&D Systems). Cells were washed twice with PBS containing 1% bovine serum albumin (fluorescence-activated cell sorting [FACS] buffer). Cells were then stained for 1 hour with streptavidin-conjugated Q dot 605 (Q10101MP; Invitrogen). Stained cells were washed once in FACS buffer and subsequently fixed in 4% formalin for 1 hour. Cells were then washed once and re suspended in FACS buffer and analyzed using an LSR II flow cytometer (Becton Dickinson, San Jose, CA). The data were analyzed by using Flow Jo software (Tree star, Inc., Ashland, OR). Gates were set on singlet's and then on live lymphocytes. Subsequent gates were based on Fluorescence-Minus-One and unstained controls.

Serum IL-23 by ELISA
IL-23 cytokine level was assayed using a commercially available IL-22 ELISA kit (R&D Systems) according to manufacturers' instructions. All patients gave their informed consents and the study was approved by Ethical, and Research Committee, Tanta Faculty of Medicine, Tanta, Egypt.

Statistical analysis
The statistical data are reported as the mean ± SD, frequencies (number) and percentages when appropriate. A comparison of numerical variables between the study groups was performed using Mann-Whitney U test to compare independent samples from two groups. One-way analysis of variance test was used to compare between more than two groups when data were normal and the Kruskal-Wallis test when the data were not normal while qualitative data compared with chi square test. Spearman's rank correlation was used to quantify the association between continuous or ordered categorical variables. A P-value less than 0.05 were considered statistically significant. All statistical calculations were performed using the computer program SPSS (Statistical Package for the Social Science; SPSS, Chicago, IL, USA) version 15 for Microsoft Windows.

Results
Clinical and laboratory data of the studied groups (Table  1) Significant higher levels of ESR and disease duration were detected in patients with severe disease compared to patients with mild to moderate ulcerative colitis disease P < 0. 05. Abdominal pain, diarrhea, blood in the stool and ESR were significantly higher in ulcerative colitis patients compared with control.

Discussion
Patients with UC are at increased risk of inflammation. IL-23 is a newly identified cytokine with increased expression in inflamed biopsies of colon mucosa in patients with CD; however there is inconsistent evidence on its role in ulcerative colitis [14]. IL-23 is a heterodimeric cytokine that shows similar function to IL-12 in promoting cellular immunity and enhancing lymphocyte proliferation [15].    This study showed significant increased expression of IL-23p 19 gene in patients with UC compared with control. This increase was significantly higher in patients with severe UC disease compared with mild to moderate disease. This was similar to the results reported by Schmidt et al [16], Zhanju et al [13] and Kobayashi et al [17]. We observed that IL-23 p19 positive cells by immunohistochemistry were mainly macrophages. This was in agreement with Zhanju et al [13]. These findings suggest that IL-23 is produced by intestinal mucosal macrophages in inflamed mucosa of ulcerative colitis patients [13]. Upon stimulation by bacterial ligand, IL-23 is produced by antigen presenting cells. After binding to appropriate receptor (IL-23 R), this cytokine can stimulate the production of IL-17, TNF alpha and IL-6 from T cells. IL-17 stimulates the expression of adhesion molecules like ICAM-1 on endothelial cells, as well as the release of IL-6 and IL-8 from myofibroblast and epithelial cells. IL-8 act as chemo tactic factor for neutrophil in flux in to the intestine. Inflammatory neutrophil release inflammatory mediators like matrix metalloproteinase and inducible nitric oxide. This sequel of pathogenic events leads to the chronic inflammation and epithelial cell damage associated with the disease [18]. Therefore, IL-23 was proposed to play an integral role in the pathogenesis of IBD [19].
This study showed the increased expression of IL-23 R in both peripheral blood and mucosal biopsy of ulcerative colitis patients were significant compared with control group. This increased expression was higher in patients with severe disease compared with those have mild to moderate disease. Also, Zhanju et al [13] demonstrated significant increased expression of IL-23 R in peripheral blood and mucosal lamina propria cells.
In accordance with the findings reported by Mohammadi et al [14] and Zheng et al [20], we demonstrated increased serum level of IL-23 in ulcerative colitis patients compared with control. In addition, the increased serum levels of IL-23 were more in patients with severe disease compared with mild to moderate disease. Moreover, this study reported significant positive correlation between high serum IL-23 levels and endoscopic severity of the disease. These findings support the hypothesis that, increased IL-23 levels reflect the activity of T helper 17 in patients with ulcerative colitis and that IL-23 participate in the pathogenesis of the disease [20]. This study noticed, the increased expression of IL-23p19 and IL-23 R were significantly positively correlated with endoscopic severity of the disease. These were consistent with Schmidt et al [16] and Zhanju et al [13]. In consistent with experimental studies of Daniel et al [21] and Ando et al [22], there were significant positive correlation between histopathological severity and both IL-23p19, IL-23R expression in the mucosa of ulcerative colitis patients. Furthermore the severity of histopathological lesions was significantly correlated with serum IL-23, similar to an experimental study of Sheikh et al [23].
It can be concluded that the increased expression of IL-23p19  has a role in the pathogenesis of ulcerative colitis; therefore targeted therapy directed against IL-23p19 may have a therapeutic role for the disease. The increased expression of IL-23p19, IL-23 R and serum IL-23 correlate with disease severity.