The Expression and Significance of Interleukin-9, Interleukin-6 and Tumor Necrosis Factor-α in Colonic Mucosa of Patients with Ulcerative Colitis
QU Dong-dong1, JIN Shi-lu2,*, LIU Bao-zhen2, ZHANG Jian3, XUE Chun-xia2
1. School of Clinical Medicine, Binzhou Medical College, Yantai, Shandong, 264003, China
2. Department of Gastroenterology, Binzhou People’s Hospital, Binzhou, Shandong, 256610, China;
3. Department of Pathology, Binzhou People's Hospital, Binzhou, Shandong, 256610, China;
*Corresponding Author: JIN Shi-lu, E-mail: jinshiluluck@163.com
Abstract

Objective: To investigate the expression and significance of interleukin (IL)-9, IL-6 and tumor necrosis factor-α (TNF-α) in colonic mucosa of patients with ulcerative colitis (UC). Methods: The expression levels of IL-9, IL-6 and TNF-α in colonic mucosa were detected by immunohistochemistry in 60 patients with UC (38 with mild UC and 22 with moderate-severe UC), and in 20 controls. To explore the influence of the age and gender, we further divided these 60 patients into <20 years group, 20-49 years group, and >49 years. Results: The percentage of positive cells of IL-9 in colonic mucosa of moderate-severe UC group was significantly higher than that in mild UC group ( P<0.05), IL-9 of mild group was higher than that of control group ( P<0.01), and the expression levels of IL-6 and TNF-α had the same tendency as that of IL-9. For UC patients, the percentage of IL-9 positive cells in 20-49 years old group (85.0%) was higher than that in ≤20 years group (0.0%) or ≥49 years group (58.8%), and significant differences were shown. The percentage of the TNF-α positive cells in moderate-severe UC group (81.8%) was higher than that in mild UC group (51.3%) ( P<0.05), which was also evidently higher in mild group than that in control group (5.0%) ( P<0.01). The percentage of TNF-α positive cells in 20-49 years UC group (75.0%) was higher than that in <20 years UC group (67.6%), and the difference was statistically significant. There was no significant difference in the percentage of IL-9, IL-6, and TNF-α positive cells between females (75.0%) and males (72.7%) ( P>0.05).Conclusion: The expression levels of IL-9, IL-6 and TNF-α in colonic mucosa of UC are higher than those in the controls, and are all related to the severity of UC. It is speculated that IL-9, IL-6 and TNF-α play important roles in UC occurrence and development.

Key words: Interleukin-9; Interleukin-6; Tumor necrosis factor-α; Colonic mucosa
Introduction

Ulcerative colitis (UC) is a chronic inflammatory bowel disease, whose etiology and pathogenesis is multifactorial, such as heredity, environment, gut microbiome and immune factor [1]. In 2016, a report from Lancet has identified 163 susceptibility loci for UC [2]. However, UC is a chronic inflammatory bowel disease, and its exact etiology and pathogenesis is complex. Therefore, the targeted treatment for UC seems to be very difficult. The traditional routine treatment is to take inflammation suppressing drug such as sulfasalazine (SASP), 5-Aminosalicylic acid, which may have side effects for our health [3, 4].

In order to provide basis for treatment, it is urgent to find out the etiology and pathogenesis of UC. Obviously, inflammation is the central pathogenesis of UC [5], which involves various cytokines. Reports showed that a variety of cytokines involved in the occurrence and development of UC, such as Interleukin (IL)-6 and tumor necrosis factor (TNF-α ) [6]. However, there are not so many reports on the expression of IL-9, IL-6 and TNF-α in colonic mucosa of different stage of UC. This study aimed to detect the expression levels of IL-9, IL-6 and TNF-α in colonic mucosa by immunohistochemistry, and analyze its significance for clinical pathology, so as to investigate the roles of IL-9, IL-6 and TNF-a in the occurrence and development of UC, and provide reference for clinical treatment.

Materials and Methods
Materials

From December 2013 to December 2015, a total of 60 patients with UC were selected from Binzhou People’ s Hospital, and they all conformed to the diagnostic criteria of UC in Consensus of Diagnosis and Treatment of Inflammatory Bowel Diseases established by Study Group for Inflammatory Bowel Diseases, Chinese Society of Gastroenterology, Chinese Medical Association in 2012, They were divided into mild group (n=38) and severe group (n=22) according to their disease conditions, whereas 20 healthy people served as control group. There was no significant difference in age and gender between among the groups (P> 0.05) (Table 1). The study was approved by Ethics Committee of Binzhou People’ s Hospital, and all the patients signed the informed consent form.

Table1 Patients Characteristics
Methods

Specimen collection: All the enrolled subjects were examined by colonoscopy. UC patients with obvious 3-5 lesions in colonic mucosa were selected as observation group, while people with 3-5 blocks in colonic mucosa as control group.

Immunohistochemical staining: All specimens were processed by paraffin embedding, and sliced into continuous sections 4 μ m in thickness routinely, after routine dewaxing and hydration. Antigen in specimens was repaired by hot citric acid salt buffer. 3% hydrogen peroxide was dropped on the specimens to block endogenous peroxidase activity at room temperature. And then all specimens were incubated overnight at 4℃ after dropping the rabbit anti-human primary antibody of IL-9 (1:400), IL-6 antibody (1:100), TNF-α (1:200), respectively.

Then the specimens were incubated with secondary antibody at room temperature for 15 min, and then received DAB coloration, hematoxylin staining, gradient alcohol dehydration and drying, vitrification by dimethylbenzene, and neutral gum sealing piece. Rabbit anti-human antibody IL-9, IL-6 and TNF-α were all purchased from Abcam (Shanghai) Trading Co., Ltd., and rapid immunohistochemical kit MaxVisionTM was bought from Fuzhou Maixin Biotechnology Development Co., Ltd.

Evaluation criteria

The evaluation criterion of immunohistochemistry: the percentage of positive cells and the intensity of staining were scored respectively. (1) Positive cell percentage: five random visual fields were observed in each slice by high power microscope (× 200), and then the percentage of positive cells (number of positive cells/total number of cells) in each high-power field was recorded. The percentage of positive cells ≤ 5% was recorded as 0 point, 6%-25% for 1 point, 26%-50% for 2 points, 51%-75% for 3 points, > 75% for 4 scores; (2) Coloring degree: no coloring is 0 point, light yellow for 1 point, brown for 2 points, dark brown for 3 points. (3) Multiplying the two together, and the result ≤ 4 points was considered as negative expression (-), while that > 4 points as positive expression (+).

Results

The cytoplasm and (or) cell membrane of IL-9, IL-6 and TNF-positive cells was brownish yellow or light yellow in color. The percentage of IL-9 positive cells in moderate-severe UC group (81.8%) was higher than that in mild UC group (50.0%) (P< 0.05), which was higher in mild UC group than that in control group (10.0%), and the differences were statistically significant (P< 0.01). For UC patients, the percentage of IL-9 positive cells in 20-49 years old group (85.0%) was higher than that in ≤ 20 years group (0.0%) or ≥ 49 years group (58.8%), and significant differences were shown. There was no significant difference in UC group between females (62.5%) and males (61.4%) (P> 0.05).

Similarly, the percentage of IL-6 positive cells in moderate-severe UC group (90.9%) was higher than that in mild UC group (63.2%) (P< 0.05), which was higher in mild UC than that in control group (20.0%) (P< 0.01). The percentage in 20-49 years UC group (90.0%) was higher than that in ≤ 20 years UC group (16.7%) or ≥ 49 years UC group (73.5%) (P< 0.01). There was no statistical significance in the percentage of IL-6 positive cells of UC group between females (75.0%) and males (72.7%) (P> 0.05).

The percentage of the TNF-α positive cells in severe UC group (81.8%) was higher than that in mild UC group (51.3%) (P< 0.05), which was also evidently higher in mild group than that in control group (5.0%) (P< 0.01). The percentage of TNF-α positive cells in 20-49 years UC group (75.0%) was higher than that in < 20 years UC group (67.6%), and the difference was statistically significant. There was no statistical significance in UC group between females (68.6%) and males (63.6%) (P> 0.05). The expressions of IL-9, IL-6 and TNF-α in each group are shown in Tables 2-4, Figures 1-3.

Table 2 Expression of IL-9 in Colonic Mucosa of Each Group
Table 3 Expression of IL-6 in Colonic Mucosa of Each Group
Table 4 Expression of TNF-α in Colonic Mucosa of Each Group

Figure 1 Expression of IL-9, IL-6 and TNF-α in Colonic Mucosa of Patients with Different Severity UC Showed by Immunohistochemistry (× 200).

Figure 2 Expression of IL-9, IL-6 and TNF-α in Colonic Mucosa of Different-age Patients with UC Showed by Immunohistochemistry (× 200). Group A, age < 20 years; Group B, age 20-49 years; Group C, age > 49 years.

Figure 3 Expression of IL-9, IL-6 and TNF-α in Colonic Mucosa of Males and Females with UC Showed by Immunohistochemistry (× 200)

Discussion

UC is a lifelong illness, which significantly worsens the quality of life (QOL) and increases personal burden through a reduction in the ability in work and an enormous cost in endless treatment [7]. UC is a kind of chronic nonspecific colitis with unknown etiology [8], currently its diagnosis mainly depends on the clinical manifestation combined with endoscopic, histopathological and imaging examination, and the differential diagnosis to exclude infectious or non-inflammatory colonic infection[9]. With the further research on the etiology of UC, mounting evidences showed that immune factors played important roles in the pathogenesis of UC, which involved various cytokines [7, 8, 9, 10].

Previous reports showed that IL-9 could act on different types of immune cells, and exert multiple function of immune regulation [11, 12]. It plays an important role in the occurrence and development of autoimmune diseases, allergic diseases, tumor immunity and so on, and is involved in the development of many kinds of inflammatory diseases [13, 14, 15]. IL-9 can not only be secreted by CD4+T cells “ Th9 cells” [16, 17], but also by other CD4+T cell subgroups such as Th17, regulatory T cells (Treg cells) [18]. IL-9 receptor (IL-9R), as a heterodimer composed of anα chain and aγ chain, is widely distributed in the cell surface of Th9, Th17 and Treg cells, mast cells, macrophages, dendritic cells, natural killer (NK) cells and other cell surface [19].

IL-6 is a kind of single chain glycoprotein with multifunction, produced by fibroblast, monocytes, macrophages, and T cells. It plays biological function by binding to specific receptors on the surface of target cells through STAT3 to activate NF-κ B signal pathway, promote the expression of intercellular adhesion molecule, and mediate adhesion between lymphocytes, or via lymphocytes and target cells to promote the interaction between neutral granular cell and epithelial cells in patients with inflammatory bowel disease, and increases the intestinal mucosa tissue damage [20]. Report showed that IL-6 level was associated with steroid resistance and reflects disease activity in severe UC [21].

TNF is mainly secreted by activated macrophages, NK cells and T cells, including three categories TNF-α , TNF-β and TNF-γ [22]. TNF is a product of T cells and can act on T cells. TNF is both a pro-inflammatory and anti-inflammatory cytokine that is critical to the development of autoimmune diseases, cancers, and protection against infectious pathogens [23]. A report showed that TNF-related apoptosis inducing ligand (TRAIL) may play a role in T-cell apoptosis in Chinese Han UC population [24]. TNF-α is a kind of inflammatory mediator which can exert a variety of biological effects. It can increase leukocytes accumulation and activate them in the inflammatory local through stimulating expression of intercellular adhesion molecule. It changes the intestinal epithelial cell barrier function and morphology through collaborating with TNF-γ . Eventually, the permeability of the intestinal mucosa and the vessel wall is increased, and the integrity of the intestinal mucosa is destroyed, resulting in ulcer formation. Or it stimulates the expression of some pro-inflammatory cytokines to start the inflammatory cascade reaction, which leads to the intestinal mucosal injury [25].

Until now clinical trials for UC still have faced many challenges [26]. There is a study that determined the cytokines of patients’ plasma [27], but this study directly analyzed the clinical pathologic materials colonic mucosa of patients, which might provide some useful information for analyzing the disease itself. The results of this study showed that the expression levels of IL-9, IL-6 and TNF-α in colonic mucosa of UC patients were all higher than those in control group, and the levels in the colonic mucosa of moderate-severe UC group were also significantly higher than those in mild UC group, which were consistent with the previous study results by Gerlach et al.and Nalleweget al.[28, 29].

In addition, in UC group, the results of this study found that the positive expression levels of IL-9, IL-6 and TNF-α in the 20-49 years UC group were the highest, which showed that the 20-49 years old was the high-risk age of UC, which might be related to the higher expression levels of related cytokines. In addition, this study also compared the relationship between UC and gender, and the results revealed that IL-9, IL-6 and TNF-α expression levels were independent of gender. The results were consistent with the relevant study [30], which further verified the relationship between these three cytokines and the etiology of UC. However, a Japanese study including 343 patients demonstrated that a bimodal distribution of onset age in Japanese UC patients was similar to that in western countries, with a large peak at 10-20 years old, and a small peak at 40-44 years old[31]. Maybe there are some different influencing factors, such as racial gene, smoking cessation, and circumstance, ect. [1, 7, 32].

To sum up, the levels of IL-9, IL-6 and TNF-α have been obviously increased in colonic mucosa of patients with UC, and the expression levels are all positively correlated with the disease severity. These three cytokines can be indicators to monitorUC disease severity and efficacy, which contribute to the clinical diagnosis and treatment options of UC, especially to the supply a reliable basis for the diagnosis of patients who have not yet suffered from obvious clinical symptoms. However, due to the small sample size of this experiment, the results still need to be confirmed by repeated experiments with large-scale samples.

Declaration

The authors of this manuscript declare that they have no conflict of interest.

Reference
[1] Childers RE, Eluri S, Vazquez C, et al. Family history of inflammatory bowel disease among patients with ulcerative colitis: a systematic review and meta-analysis. J Crohns Colitis, 2014, 8(11): 1480-1497. doi: 10.1016/j.crohns.2014.05.008. [本文引用:2]
[2] Cleynen I, Boucher G, Jostins L, et al. Inherited determinants of Crohn’s disease and ulcerative colitis phenotypes: a genetic association study. Lancet, 2016, 387(10014): 156-167. doi: 10.1016/S0140-6736(15)00465-1. [本文引用:1]
[3] Wang Y, Parker CE, Feagan BG, et al. Oral 5-aminosalicylic acid for maintenance of remission in ulcerative colitis. Cochrane Database Syst Rev, 2016( 5): CD000544. doi: 10.1002/14651858.CD000544.pub4. [本文引用:1]
[4] Hauso Ø, Martinsen TC, Waldum H. 5-Aminosalicylic acid, a specific drug for ulcerative colitis. Scand J Gastroenterol, 2015, 50(8): 933-941. doi: 10.3109/00365521.2015.1018937. [本文引用:1]
[5] Derikx LA, Dieleman LA, Hoentjen F. Probiotics and prebiotics in ulcerative colitis. Best Pract Res Clin Gastroenterol, 2016, 30(1): 55-71. doi: 10.1016/j.bpg.2016.02.005. [本文引用:1]
[6] Guerra GC, Araújo AA, Lira GA, et al. Telmisartan decreases inflammation by modulating TNF-α, IL-10and RANK/RANKL in a rat model of ulcerative colitis. Pharmacol Rep, 2015, 67(3): 520-526. doi: 10.1016/j.pharep.2014.12.011. [本文引用:1]
[7] Kawalec P. Indirect costs of inflammatory bowel diseases: Crohn’s disease and ulcerative colitis. A systematic review. Arch Med Sci, 2016, 12(2): 295-302. doi: 10.5114/aoms.2016.59254. [本文引用:3]
[8] Roose L, D’cunja J, Biedermann L. Ulcerative colitis. Praxis (Bern 1994), 2016, 105(11): 607-615. doi: 10.1024/1661-8157/a002358. [本文引用:2]
[9] Kuno T, Kojima Y, Mochizuki H, et al. Factors Predicting Subsequent Hospitalization in Patients with Ulcerative Colitis: Total Colonoscopic Findings are the Strongest Predictor. Hepatogastroenterology, 2015, 62(140): 821-824. doi: 10.1016/s0016-5085(12)60978-0. [本文引用:2]
[10] Moldoveanu AC, Diculescu M, Braticevici CF. Cytokines in inflammatory bowel disease. Revue roumaine de médecine interne, 2015, 53(2): 118-127. doi: 10.1515/rjim-2015-0016. [本文引用:1]
[11] Nalleweg N, Chiriac MT, Podstawa E, et al. IL-9 and its receptor are predominantly involved in the pathogenesis of UC. Gut, 2015, 64(5): 743-755. doi: 10.1136/gutjnl-2013-305947. [本文引用:1]
[12] Knoops L, Renauld JC. IL-9 and its receptor: from signal transduction to tumorigenesis. Growth Factors, 2004, 22(4): 207-215. doi: 10.1080/08977190410001720879. [本文引用:1]
[13] Louahed J, Zhou Y, Maloy WL, et al. Interleukin 9 promotes influx and local maturation of eosinophils. Blood, 2001, 97(4): 1035-1042. doi: 10.1182/blood.v97.4.1035. [本文引用:1]
[14] 14 Nowak EC, Weaver CT, Turner H, et al. IL-9 as a mediator of Th17-driven inflammatory disease. J Exp Med, 2009, 206(8): 1653-1660. doi: 10.1084/jem.20090246. [本文引用:1]
[15] Feng LL, Gao JM, Li PP, et al. IL-9 contributes to immunosuppression mediated by regulatory T cells and mast cells in B-cell non-hodgkin’s lymphoma. J Clin Immunol, 2011, 31(6): 1084-1094. doi: 10.1007/s10875-011-9584-9. [本文引用:1]
[16] Veldhoen M, Uyttenhove C, van Snick J, et al. Transforming growth factor-beta ‘reprograms’ the differentiation of T helper 2 cells and promotes an interleukin 9-producing subset. Nat Immunol, 2008, 9(12): 1341-1346. doi: 10.1038/ni.1659. [本文引用:1]
[17] Dardalhon V, Awasthi A, Kwon H, et al. IL-4 inhibits TGF-beta-induced Foxp3+ T cells and , together with TGF-beta, generates IL-9+ IL-10Foxp3(-) effector T cells. Nat Immunol, 2008, 9(12): 1347-1355. doi: 10.1038/ni.1677. [本文引用:1]
[18] Goswami R, Kaplan MH. A brief history of IL-9. J Immunol, 2011, 186(6): 3283-3288. doi: 10.4049/jimmunol.1003049. [本文引用:1]
[19] Li H, Rostami A. IL-9: basic biology, signaling pathways in CD4+ T cells and implications for autoimmunity. J Neuroimmune Pharmacol, 2010, 5(2): 198-209. doi: 10.1007/s11481-009-9186-y. [本文引用:1]
[20] Mao F, Xu M, Zuo X, et al. 15-Lipoxygenase-1 suppression of colitis-associated colon cancer through inhibition of the IL-6/STAT3 signaling pathway. FASEB J, 2015, 29(6): 2359-2370. doi: 10.1096/fj.14-264515. [本文引用:1]
[21] Wine E, Mack DR, Hyams J, et al. Interleukin-6 is associated with steroid resistance and reflects disease activity in severe pediatric ulcerative colitis. J Crohns Colitis, 2013, 7(11): 916-922. doi: 10.1016/j.crohns.2012.12.012. [本文引用:1]
[22] Navarrete S, Alarcón M, Palomo I. Aqueous Extract of Tomato (Solanum lycopersicum L. ) and Ferulic Acid Reduce the Expression of TNF-α and IL-1β in LPS-Activated Macrophages. Molecules, 2015, 20(8): 15319-15329. doi: 10.3390/molecules200815319. [本文引用:1]
[23] Mehta AK, Gracias DT, Croft M. TNF activity and T cells. Cytokine, 2016, [Epub ahead of print]. doi: 10.1016/j.cyto.2016.08.003. [本文引用:1]
[24] Hu D, Xia SL, Shao XX, et al. Association of ulcerative colitis with TNF-related apoptosis inducing ligand (TRAIL) gene polymorphisms and plasma soluble TRAIL levels in Chinese Han population. Eur Rev Med Pharmacol Sci, 2015, 19(3): 467-476. [本文引用:1]
[25] Watson AJ, Hughes KR. TNF-α-induced intestinal epithelial cell shedding: implications for intestinal barrier function. Ann N Y Acad Sci, 2012, 1258: 1-8. doi: 10.1111/j.1749-6632.2012.06523.x. [本文引用:1]
[26] Hindryckx P, Baert F, Hart A, et al. Clinical trials in ulcerative colitis: a historical perspective. J Crohns Colitis, 2015, 9(7): 580-588. doi: 10.1093/ecco-jcc/jjv074. [本文引用:1]
[27] Hagel AF, de Rossi T, Konturek PC, et al. Plasma histamine and tumour necrosis factor-alpha levels in Crohn’s disease and ulcerative colitis at various stages of disease. J Physiol Pharmacol, 2015, 66(4): 549-556. [本文引用:1]
[28] Gerlach K, Hwang Y, Nikolaev A, et al. TH9 cells that express the transcription factor PU. 1 drive T cell-mediated colitis via IL-9 receptor signaling in intestinal epithelial cells. Nat Immunol, 2014, 15(7): 676-686. doi: 10.1038/ni.2920. [本文引用:1]
[29] Nalleweg N, Chiriac MT, Podstawa E, et al. IL-9 and its receptor are predominantly involved in the pathogenesis of UC. Gut, 2015, 64(5): 743-755. doi: 10.1136/gutjnl-2013-305947. [本文引用:1]
[30] Wang Y, Ouyang Q . Ulcerative colitis in China: retrospective analysis of 310hospitalized patients. J Gastroenterol Hepatol, 2007, 22(9): 1450-1455. doi: 10.1111/j.1440-1746.2007.04873.x. [本文引用:1]
[31] Takahashi H, Matsui T, Hisabe T, et al. Second peak in the distribution of age at onset of ulcerative colitis in relation to smoking cessation. J Gastroenterol Hepatol, 2014, 29(8): 1603-1608. doi: 10.1111/jgh.12616. [本文引用:1]
[32] Walker DG, Williams HR, Bancil AS, et al. Ethnicity differences in genetic susceptibility to ulcerative colitis: a comparison of Indian asians and white northern Europeans. Inflamm Bowel Dis, 2013, 19(13): 2888-2894. doi: 10.1097/01.MIB.0000437567.12067.e7. [本文引用:1]