The Expression Of IL-35 In Serum And Intestinal Mucosa Of Inflammatory Bowel Disease Patients

Objective:Inflammatory bowel disease (IBD), including Crohn’s disease (CD) and ulcerative colitis (UC), is a kind of chronic inflammatory disorder of the gastrointestinal tract. The disease is difficult to cure, easy to relapse and affecting the quality life of patients seriously. Although the etiology is not completely understood, initiation and exacerbation of the inflammatory process seem to be due to a massive local mucosal immune response. Analysis of immunoinflammatory pathways in the gut of patients with UC or CD has shown that tissue damage is driven by a complex and dynamic crosstalk between immune and nonimmune cells, and that cytokines are key mediators of this interplay. Interluken (IL)-35 is a newly discovered immume-suppressing cytokine belonging to the IL-12 family, which contains IL-12, IL-23, IL-27. It is composed of two subunits, p35 (IL-12a) and Epstein-Barr virus-induced gene 3 (Ebi3). Using experimental database mining and statistical analysis methods, Li reports that IL-35 is not constitutively expressed in human tissues but it is inducible in response to inflammatory stimuli. However, its expression in inflammatory bowel disease (IBD) patients has not been yet explored. Our aim was to evaluate serum and inflamed mucosal levels in IBD patients.Methods:20 UC and 7 CD patients were included at Shandong University Qilu Hospital from September 2013 to April 2014. Diagnosis was performed by the presence of history of abdomen pain, diarrhea or blood in stool, and macroscopic appearance by colonoscopy and pathology compatible with IBD. UC activity was assessed by Mayo Score Activity Index and CD activity was assessed by the Crohn’s disease activity index (CDAI). Additionally,15 non-inflamed controls were recruited among healthy subjects undergoing a colonoscopy because of screening for colorectal cancer or polyp surveillance. A fasting blood sample was taken from all patients and healthy subjects. It was centrifuged and serum was collected for enzyme-linked immunosorbent assay (ELISA). During endoscopy, biopsies from colon or ileocecum or small intestine were obtained from patients and healthy subjects. Two biopsies were to be used for RNA and protein assessment, were snap-frozen in liquid nitrogen, and then transferred to -80℃ for storage until processing. One biopsy was placed in formalin for pathology and immunohistochemical staining. Compare the two groups and analyze the expression of IL-35.Results:Serum IL-35 concentration was significantly reduced in the active UC patients and active CD patients compared with healthy controls. There were also significant differences between mild UC and moderate UC, mild UC and severe UC (P<0.05). In contrast, UC and CD group, moderate and mild UC group seem to be statistically meaningless. We also assessed whether the serum cytokine levels were associated with the Mayo score in UC patients. The results showed that lower IL-35 levels were moderately negative correlated Mayo Score (R=-0.636, P< 0.05). IL-35 was expressed in infiltrating immune cells but not in epithelial cells. Normal colon tissue from healthy subjects had no IL-35 expression at all. IL-35 positive cells were localized mainly in inflammatory infiltrates, predominantly mononuclear cell (lymphocytes). The number of IL-35-expressing cells in inflamed colonic tissue of patients with UC was higher than in CD patients’tissue. IBD patients had significantly higher Ebi3 and IL-12p35 gene expression compared with healthy control group (P< 0.0001). And the difference in Ebi3 and IL-12p35 mRNA expression in UC and CD biopsies did not reach statistical significance (p= 0.116; p= 0.779). Western blot analysis showed a significant upregulation of IL-35(P<0.05) in inflamed mucosa of patients as compared to controls. We found IL-35 protein expression in UC biopsies was higher than that in CD biopsies. No relationship between IL-35 mucosal expression and the activity was observed in UC and CD patients.Conclusions:The overexpression and enhanced production of IL-35 in colonic mucosa may play a role in the inflammatory process of IBD. IL-35 is a brand new potential detected and therapeutic cytokine for IBD. Howerer, our experiment group includes 27 cases, and large-scale testing needs to be performed to make it available and useful in the future.