Gastroenterology and Hepatology From Bed to Bench.
©۲۰۱۷ RIGLD, Research Institute for Gastroenterology and Liver Diseases
۱ Gastroenterology and Liver Diseases Research Center, Research Institute for Gastroenterology and Liver Diseases, Shahid Beheshti University of Medical Sciences, Tehran, Iran ۲ Basic and Molecular Epidemiology of Gastrointestinal Disorders Research Center, Research Institute for Gastroenterology and Liver Diseases, Shahid Beheshti University of Medical Sciences, Tehran, Iran
Aim: In this study, we determined the gene expression analysis of IL-۱۷ gene family for early detection of subclinical inflammation among IBD patients
Background: Cytokines have a vital role in the pathogenesis of inflammatory bowel disease (IBD). Interleukin-۱۷ is the signature cytokine of the recently identified T helper ۱۷ (Th۱۷) cell subset. IL-۱۷F is mainly involved in mucosal host defense mechanisms whereas the functions of IL-۱۷B remain largely elusive
Methods: In this cross-sectional study, IBD patients divided into two active and inactive groups. Peripheral blood mononuclear cells (PBMCs) from ۳۸ IBD patients which ۲۰ inactive samples and ۱۸ active individuals were collected. Changes of IL-۱۷ F and IL-۱۷B mRNA expression level evaluated by quantitative-real time-PCR
Results: mRNA expression level of IL-۱۷B and IL-۱۷F in CD, UC, active and inactive groups have been assessed and there were no significant differences (P>۰.۰۵). Patients were classified into five different categories as follows: i) ۵-ASA; ii) ۵-ASA + Pred; iii) ۵-ASA + AZA; iv) ۵-ASA + Pred + AZA; v) ۵-ASA + Pred + AZA + IFX according to medication usage, expression of IL-۱۷F and IL-۱۷B had no differences (p>۰.۰۵).
Conclusion: Evaluation of IL-۱۷B and IL-۱۷F mRNA expression level illustrate no difference among active and inactive patients. Therefore, IL-۱۷B and IL-۱۷F are not biomarkers in an Iranian IBD patients.
Keywords: Inflammatory bowel disease, Crohn’s disease, Ulcerative colitis, Interleukin-۱۷, qPCR
(Please cite as: Safari MT, ChaleshiV, TarbanP, Nourian M, Balaii H, Shahrokh Sh, et al. Evaluation of IL-۱۷B and IL-۱۷F mRNA expression in peripheral blood mononuclear cells and association with clinical outcome of IBD patients. Gastroenterol Hepatol Bed Bench ۲۰۱۷;۱۰(Suppl. ۱):S۷۹-S۸۴)
Inflammatory bowel disease (IBD) is a multifactorial disorder that its symptoms include diarrhea with bleeding, fever and fatigue, Abdominal pain, reduced appetite, unintended weight loss in the
Received: ۲۹ June ۲۰۱۷ Accepted: ۱۸ September ۲۰۱۷ Reprint or Correspondence: Hamid Asadzadeh Aghdaei, MD. Basic and Molecular Epidemiology of Gastrointestinal Disorders Research Center, Research Institute for Gastroenterology and Liver Diseases, Shahid Beheshti University of Medical Sciences, Tehran, Iran. E-mail: hamid.assadzadeh@gmail.com
gastrointestinal tract (۱, ۲). Although the exact cause of IBD still remains unknown, various factors such as genetics factors, environmental factors, microbial intestinal flora and immunologic component can be involved for manifesting of IBD (۳). Ulcerative colitis (UC) and Crohn’s disease (CD) are two most common IBD entities that characterized by the location of inflammation in GI (۴). The rate of patients who suffer from IBD has increased in recent years. A number of studies have reported that more than three million patients in the United States and European countries discomfort of IBD, in IRAN the rate of this condition
has significantly increased in the last decade (۵, ۶). The role of genetics in IBD has been supported by studies on family history and monozygotic twins (۷, ۸). In recent investigations, about ۱۶۳ gene loci have been associated with IBD since ۱۱۰ genes have related to both UC and CD, ۳۰ specific genes are associated with CD and ۲۳ genes are considered as specific for UC (۹, ۱۰). The role of IL-۱۷ that is the ۲۰-kDa glycoprotein in inflammatory disease as a pro-inflammatory have been indicated (۸). IL-۱۷ is secreted exclusively by active T helper cells. Indeed, the past studies assessing sequence screening about IL-۱۷ illustrates six subgroups of IL-۱۷, from A to F (۹). IL-۱۷F isoform ۱ and ۲ have the highest degree of homology with IL۱۷A among all subgroups of IL-۱۷ and both have the vital role as a pro-inflammatory cytokine (۸, ۹). IL-۱۷B is expressed in various organs like trachea, prostate, lung, small intestine, testes, adrenal, and pancreas (۱۰). IL-۱۷B and IL-۱۷F can induce pro-inflammatory cytokines like tumor necrosis factor (TNF) (۱۱). Recent studies have investigated the role of IL-۱۷ in a number of diseases like Rheumatoid Arthritis as an autoimmune disease (۱۲). This paper will focus on evaluating mRNA expression level of IL-۱۷B and IL-۱۷F in PBMC of Iranian population patients with IBD. In addition, mRNA expression level of IL-۱۷B and IL۱۷F compared between active and inactive phases of IBD patients
In this cross-sectional study, ۳۸ participant subjects (۲۰ inactive, ۱۸ active) who referred to Gastroenterology and Liver Diseases Research Center, Research Institute for Gastroenterology and Liver Diseases, Shahid Beheshti University of Medical Sciences, Tehran, Iran from ۲۰۱۳ to ۲۰۱۶. A questionnaire including demographic and clinical features filled for all patients. In addition, ۶ ml whole blood collected in EDTA tubes and stored at ۴˚C until RNA extraction. The consent form was obtained from patients that ethics committee at the Research Center Gastroenterology and Liver Diseases (RCGLD) approved the protocol.
Total RNA was extracted from PBMC and YTA RNA Extraction (YEKTA TAJHIZ AZMA) was utilized. Spectrophotometric optical density measurement (۲۶۰ and ۲۸۰ nm) was used for evaluation of quality and quantity of RNA. cDNA synthesis used by Revertaid RT Reverse Transcription Kit (Thermo Fisher Scientific, Catalog number: K۱۶۹۱). For evaluation of IL-۱۷B and IL-۱۷F expression ABI ۷۵۰۰ real-time (۲.۳ version) PCR system (Applied Biosystems, Foster City, CA, USA) were employed with Takara SYBR Master Mix instructions (Shiga, Japan). Primers for IL-۱۷B, IL۱۷F, and Beta-۲-Microglobulin (as a housekeeping gene) were designed by using Gene Runner and Primer۳ online programs (Table ۱).
Determination of gene expression by Student t-test was apprised. One-way ANOVA and Student t-test were used to examine the IL-۱۷B and IL-۱۷F mRNA expression level between groups. Graph pad prism ۵ software (Graph Pad Software, Inc. La Jolla, CA, USA/ (https://www.graphpad.com/scientific-software/prism/) were utilized to draw the graphs. P < ۰.۰۵ regarded as an indication of statistically significant difference.
Thirty-eight patients have participated in the present study. Accurately, ۲۰ and ۱۸ individuals were in inactive and active phase respectively. To be more specific, ۲۰ patients were in inactive phase, including UC ۷۷.۳% and CD ۲۲.۷% and ۱۸ patients were in active phase which all of them were UC. In both groups, the rate of the male was ۳۶.۸%, the percentage of the female was ۶۳.۲%, and there was no significant association between them. The average of patients’ diagnosis age and BMI were ۳۰.۶۸ (minimum ۱۵, maximum ۵۵ ± ۱۲.۱۵۷) and ۲۵.۷۹۷۷(minimum ۱۱.۷۳, maximum ۳۸.۵۷± ۶.۵) respectively (Table ۲).
mRNA expression level of IL-۱۷B and IL-۱۷F in CD, UC, active and inactive groups have been assessed and there were no significant differences (P>۰.۰۵). Additionally, an evaluation of patients who had a history of taking medication was also conducted and there was no association among this groups (P>۰.۰۵). Patients were classified into five different categories as follows: i) ۵-ASA; ii) ۵-ASA + Pred; iii) ۵-ASA + AZA; iv) ۵-ASA + Pred + AZA; v) ۵-ASA + Pred + AZA + IFX according to medication usage (Figure II and I).
Revolution of the immune system in IBD patients has been investigated in many studies. It is addressed that impaired mucosal cytokine secretion has a direct effect on pathogenesis of IBD (۱۱, ۱۲). The presented study aimed to evaluate mRNA expression level of IL-۱۷B and IL-۱۷F in PBMC, which is the non-invasive and exclusive for patients of active and inactive IBD patients. Although the mRNA expression level of IL۱۷B was higher than IL-۱۷F in subjects, there was no significant association between expression of both genes and IBD. IL-۱۷ as a pro-inflammatory secretor, mainly derived from Th-۱۷ (۱۳). IL-۱۷ as a mediator producing cells can facilitate the development of inflammation and colorectal carcinoma by promoting angiogenesis and production of VEGF (vascular endothelial growth factor) by tumor cells. In addition, the modulation response of IL۱۷F may inhibit tumor angiogenesis and enhance the inflammatory response of the host to tumorigenesis (۱۴, ۱۵). Investigating on various subgroups of IL-۱۷ has been done in IBD. For instance, in ۲۰۱۴ study of IL-۱۷C in IBD patients in Germany by M Friedrich et.al, illustrated the increase in IL-۱۷C mRNA serum level of patients (۱۶). In ۲۰۰۸ Holtta Veera et al. evaluated mRNA expression level of cytokines such as IL-۱۷, IL-۲۳, TNF-a, etc in biopsy and stool of Crohn’s patients. Their results illustrate association in IL-۱۷ and IL-۲۳ connected to the etiology of patients (۱۷). Study mRNA expression level of IL-۱۲ and IL-۱۷ which conducted by Nielson and colleagues in biopsy of IBD patients in ۲۰۰۳ illustrated association of these genes in IBD. Their study suggested, these two genes might be a therapeutic target in future for IBD (۱۸). In ۲۰۰۷, Julia Seiderer studied mRNA expression level and p His۱۶۱Arg polymorphism of IL-۱۷F gene in biopsy of German IBD patients. Their results showed that association of mRNA expression level of IL-۱۷F by Crohn’s patients (۱۹). According to our knowledge, evaluation of IL۱۷B and IL-۱۷F mRNA expression in peripheral blood mononuclear cell (PBMC) was investigated for the first time in our study (۱۷, ۱۹). Indeed, several previously studies have shown significant results that is different from our results. The reason for these contradictory findings is not clear. Ethnic heterogeneity, sample type, gene environment interactions and different sample size may be the probable explanation for this discrepancy. Finally, we could not find any evidence to support an association between IL-۱۷B and IL-۱۷F mRNA expression in peripheral blood mononuclear cell (PBMC) and IBD patients. Therefore, in future studies, we suggest that cellular mechanisms and other subunits of IL-۱۷ be investigated. The present study examined association of IL-۱۷B and IL-۱۷F mRNA expression level illustrate no difference among active and inactive patients. In addition, expression of IL-۱۷F and IL-۱۷B had no differences (p>۰.۰۵) in patients who had a history of taking the four types of drug (۵-ASA, Pred, AZA or IFX). Thus, these analyses may be used in the prognosis, diagnosis and treatment of IBD.
The authors are grateful for support of Gastroenterology and Liver Diseases Research Center, Research Institute for Gastroenterology and Liver Diseases, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
The authors declare that they have no conflict of interest.
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