Human Immunology 74 (2013) 1258–1262

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Involvement of Th17 cells in patients of urothelial carcinoma of bladder

0198-8859/$36.00 - see front matter � 2013 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.http://dx.doi.org/10.1016/j.humimm.2013.06.032

⇑ Corresponding author. Address: Department of Biochemistry, All India Instituteof Medical Sciences, New Delhi 110 029, India. Fax: +91 11 26588641.

E-mail address: dralpanasharma@gmail.com (A. Sharma).

Seema Chugh a, Vivek Anand a, Laxman Swaroop b, Manoj Sharma c, Amlesh Seth b, Alpana Sharma a,⇑a Department of Biochemistry, All India Institute of Medical Sciences, New Delhi, Indiab Department of Urology, All India Institute of Medical Sciences, New Delhi, Indiac Department of Radiation Oncology, Maulana Azad Medical College, New Delhi, India

a r t i c l e i n f o

Article history:Received 14 November 2012Accepted 14 June 2013Available online 1 July 2013

a b s t r a c t

Bladder cancer is the second most common urologic malignancy after prostate with urothelial carcinomaaccounting for 90% of all bladder cancers. Th17 cells representing novel subset of CD4+ cells have welldescribed roles in autoimmune diseases and inflammation. Recent studies suggest a potential impactof Th17 cells in tumor immunology. The implication of Th17 cells in bladder cancer can be judged bythe expression of their related cytokines and a key transcription factor, RORct which helps in the devel-opment of Th17 cells. Therefore, we aim to assess expression of Th17 related cytokines, RORct and dis-tribution of Th17 cells to understand its involvement in bladder cancer. In our study, frequency of Th17cells was significantly higher (p-value <0.001) in patients than controls. Circulating levels of pro-inflam-matory cytokines IL-17A, IL-23 and IL-6 were also significantly elevated in patients. Relative mRNAexpression of IL-17A and RORct in PBMCs and fold change in gene expression in tissues was found tobe significantly elevated. These findings indicate the possible involvement of Th17 cells in urothelial car-cinoma of bladder. Further the data can be validated to better understand the role of Th17 cells in thisdisease which might help in formulating targeted therapeutic strategy in future.� 2013 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights

reserved.

1. Introduction

Bladder cancer is the second most common urologic malig-nancy amongst the males after prostate cancer with most of thecases being diagnosed as urothelial carcinoma [1]. Bladder cancerbesides several physiological changes in and around bladder is alsoaccompanied by immunological changes. Th17 cells, a novel subsetof CD4+ cells have emerged as an important mediator in the devel-opment of inflammatory and autoimmune diseases such as graftversus host disease, inflammatory bowel disease, arthritis, multi-ple sclerosis, psoriasis, and lupus [2]. Recent studies indicate thatTh17 cells are also implicated in tumor immunology. Significant in-crease in the number of Th17 cells has been reported in peripheralblood and tumor tissues in various human malignancies includinggastric and pancreatic cancer [3,4].

Stimulation of naive CD4+T cells by TGF-b and IL-6 initiatesTh17 differentiation [5–7]. These cells are characterized by releaseof IL-17A, IL-17F, IL-21, and IL-22 cytokines [8]. RORct, member ofthe retinoic acid receptor-related orphan nuclear hormone recep-tor family, is a transcription factor expressed specifically in Th17cells which plays an important role in directing Th17 differentia-

tion and cytokine production [9]. IL-21 produced by Th17 cells actsin an autocrine manner amplifying production of Th17 cells [10]. Italso induces the expression of IL-23 receptor on Th17 cells to makethem responsive to IL-23 stimulation [11]. Major cytokines associ-ated with Th17 cells are IL17A, IL-23 and IL-6. IL-17A is a pro-inflammatory cytokine secreted by activated T cells. There are sev-eral roles attributed to this cytokine which includes regulation ofthe activity of NF-kB and mitogen-activated protein kinases[12,13]. It is also found to be associated with IL-6 and COX-2expression [14,15]. In inflammatory condition dendritic cells andmacrophages secrete IL-23 which promotes the development ofTh17 cells and helps in secretion of other pro-inflammatory cyto-kines in autocrine or paracrine manner [16]. IL-23 also activatessome of the STAT family transcription factor [17]. IL-6 is secretedat the site of acute or chronic inflammation and induces fever inan individual [18]. It is a multifunctional cytokine that is criticalfor the development and differentiation of T and B-cell [5,19]. IL-6 activates STAT3 and RORct expression and also involved ininducing human Th17 cell differentiation [11]. The enhanced pro-duction of IL-6 is also influenced by IL-17 cytokine by MAPK, NF-kB and PI3-kinase/Akt dependent signaling pathways [20,21].

There are various limitations of current cancer immunothera-pies for the treatment of established cancer. Herein, the presenceof Th17 cells in cancer patients might be of importance in diagno-sis, prognosis or cancer therapeutics which should be investigated

Table 1Circulatory levels of IL-17A, IL-6 and IL-23 in bladder cancer patients and in controls.

Th17 related cytokines Patients(n = 25)

Control(n = 15)

Significance

IL-17A (pg/ml) (mean ± SD) 2.92 ± 0.77 1.71 ± 0.42 p < 0.001IL-23 (pg/ml) (mean ± SD) 309.14 ± 53.48 237.26 ± 14.22 p < 0.001IL-6 (pg/ml) (mean ± SD) 22.7 ± 5.50 5.32 ± 0.98 p < 0.001

Fig. 1. Relative gene expression of IL-17A in PBMCs of patients and controls.⁄⁄Significance (p < 0.001) in controls versus patients.

S. Chugh et al. / Human Immunology 74 (2013) 1258–1262 1259

in detail. This makes it essential to understand the nature, regula-tion, and function of Th17 cells in peripheral blood and tumor tis-sues to study the interactions between these immune cells andcancer cells so that they can be of clinical relevance.

To the best of our knowledge the role of Th17 cells in Indian pa-tients of urothelial carcinoma of bladder have not been reported tilldate. Therefore in this study we aimed to investigate the circula-tory and molecular expression of Th17 related molecules and dis-tribution of these cells in bladder cancer patients.

2. Materials and methods

2.1. Patients

25 patients, all of which are diagnosed with histologically con-firmed muscle invasive, grade III, transitional cell carcinoma of uri-nary bladder and undergoing radical cystectomy were included inthe study. None of the patients received any chemotherapy/radio-therapy at the time of sampling. The mean ± SD of patient’s agewas 59.28 ± 10.93 with 84% and 16% of male to female ratio. Fif-teen healthy individuals with age and sex matched were taken asserum controls. This study was approved by ethics committee ofAIIMS (All India Institute of Medical Sciences) and written in-formed consent was obtained from all individuals.

2.2. Blood samples

Peripheral blood was collected from all the study subjects inEDTA vials and plain endotoxin free vials. Standard ficoll densitygradient centrifugation was used to isolate PBMCs (peripheralblood mononuclear cells) which were then used for analysis forthe presence of Th17 subpopulation and gene expression studiesby real time PCR. Serum was obtained after centrifugation andwas stored at �80 �C for ELISA estimations.

2.3. Tissue samples

Tumor tissues and adjacent normal tissues (verified histopatho-logically) that served as controls were collected from all the pa-tients after radical cystectomy in RNA later which wereincubated overnight at 2–8 �C and then kept at �80 �C for geneexpression studies.

2.4. Enzyme immunoassays

Levels of IL-17A, IL-23 and IL-6 were estimated in the serum ofpatients and healthy controls, with commercially available ELISAkits by Diaclone™ (Besancon Cedex, France).

2.5. Real-time quantitative PCR

RNA was extracted from tissue samples and PBMCs using TRI-ZOL method. After DNase treatment 1 lg of RNA was used to syn-thesize complementary DNA (cDNA) using MuLV ReverseTranscriptase (Fermentas), that was further used as template toanalyze the amplification (ABI 7500 real-time PCR) using primersspecific to the different molecules and Maxima SYBR green mastermix (Fermentas). The conditions used for PCR were as follows: ini-tial denaturation at 95 �C for 5 min, followed by 40 cycles at 95 �Cfor 15 s, 60 �C for 30 s, 72 �C for 30 s. Primers used were as follows– Beta-actin: AGA AAA TCT GGC ACC ACA CC (forward), TAG CACAGC CTG GAT AGC AA (reverse), IL-17A: CCC CTA GAC TCA GGCTTC CT (forward), TCA GCT CCT TTC TGG GTT GT (reverse), RORct:AGT CGG AAG GCA AGA TCA CGA (forward), CAA GAG AGG TTCTGG GCA AG (reverse).

2.6. Flow cytometry

Flow cytometric analysis was done to count Th17 cell subpopu-lation in the study subjects. Cells (PBMCs) were suspended in com-plete RPMI (Sigma Aldrich, St. Louis, MO, USA) and counted in aneubauer’s slide. One million cells were then stimulated by PMA(50 ng/ll, Sigma Aldrich, St. Louis, MO, USA) and Ionomycin(1 lM, Sigma Aldrich, St. Louis, MO, USA) in 24 well plate for 5 hat 37 �C in 5% CO2. 1 lM Brefeldin (BD GolgiPlug) was added after2 h. The cells were then centrifuged, washed and resuspended inFITC conjugated anti-human anti-CD4+ antibody (eBioscience) forsurface staining followed by incubation in dark. After permeabili-zation and fixation (Cytofix cytoperm kit-BD Biosciences) theywere stained intracellularly with PerCp-Cy5.5 conjugated anti-hu-man anti IL-17A (eBioscience) and incubated. Cells were thenwashed with Perm wash solution and resuspended in FACS stain-ing buffer. These are then acquired in BD FACS CANTO™ and ana-lyzed by BD Diva™ software.

2.7. Statistical analysis

Data were expressed as mean ± SD. Comparisons betweengroups were made using the Wilcoxon rank-sum (Mann–Whitney)test for nonparametric data and the Student’s t-test for parametricdata and significance was defined at a p value less than 0.05(p < 0.05). The correlation analysis of data of Th17 related mole-cules in circulation versus tissue was done by using Pearson’s cor-relation coefficient test.

3. Results

3.1. Increased level of cytokines-IL-17A, IL-6 and IL-23 in patients

Serum levels of cytokines IL-17A, IL-6 and IL-23 were signifi-cantly elevated in patients in comparison with the controls

Fig. 2. Relative gene expression of RORct in PBMCs of patients and controls.⁄⁄Significance (p < 0.001) in controls versus patients.

Fig. 3. Fold change of gene expression of RORct and IL-17A in tumor tissue relativeto uninvolved normal tissue.

1260 S. Chugh et al. / Human Immunology 74 (2013) 1258–1262

(p < 0.001), as shown in Table 1. Mean ± SD of IL-17A in patientsand control was 2.92 ± 0.77 and 1.71 ± 0.42, respectively, whereasit was 309.14 ± 53.48 and 237.26 ± 14.22 in case of IL-23 cytokine.

Fig. 4. Representative contour plots showing frequency o

The mean ± SD of IL-6 in patients and control was 22.7 ± 5.50 and5.32 ± 0.98, respectively.

3.2. Quantitative mRNA expression levels of IL-17A and RORct in blood

Relative mRNA expression of IL-17A, RORct in PBMC was foundto be significantly higher in patients in comparison with controls(p < 0.001) (Figs. 1 and 2). By using 2�dCt methods mean ± SD ofIL-17A in patients and control was found to be 2.45 ± 0.67 and0.03 ± 0.008, respectively. Similarly, the relative mRNA expressionof RORct was found to be 3.69 ± 1.46 and 0.104 ± 0.04 in patientsand controls respectively.

3.3. Fold change of gene expression in tissues

The fold change of RORct gene expression in tumor tissues wasfound to be 11 and in case of IL-17A the fold change of gene expres-sion were 5, as compared to normal uninvolved tissue (Fig. 3).

3.4. Th17 cells (CD4+IL17+) were significantly increased in peripheralblood of bladder cancer patients

The subpopulation of Th17 cells (CD4+IL-17A+) as a % of CD4+

population was observed to be higher in the blood of bladder can-cer patients as compared to healthy individuals (representativecontour plots shown in Fig. 4). The frequency of CD4+ IL 17A+

(Th17) cells in patients ranged from 0.8% to 3% (mean �1.39%),while the frequency in normal controls ranged from 0.06% to1.2% (mean �0.63%) of CD4+ population. The mean frequency ofdouble positive (CD4+ IL-17A+) Th17 cells was observed to be high-er in patients as compared to normal controls and this increase wasstatistically significant (p-value <0.001) (Fig. 5).

3.5. Correlation analysis of data

To measure the strength of association between Th17 relatedmolecules in circulation versus tissue, the Pearson’s correlationcoefficient was applied to our data. Serum IL-17A showed insignif-icant positive correlation with fold change of IL-17A (r = 0.305,p = 0.137) and RORct (r = 0.268, p = 0.195) in tumor tissue. How-ever, fold change of gene expression of IL-17A and RORct in tumortissue was found to be in significant positive correlation with ser-um level of IL-6 (r = 0.474, p = 0.01; r = 0.491, p = 0.01, respec-tively). The quantitative mRNA expression of RORct in circulation(PBMC) were also observed to be in significant positive correlation

f Th17 (CD4+IL17A+) cells in patient versus control.

Fig. 5. Mean frequency of Th17 cells in the peripheral blood of bladder cancerpatients and controls. ⁄⁄Significance (p < 0.001) in controls versus patients.

Table 2Pearson’s correlation coefficient (r) and its significance (p-value) between moleculesin circulation vs tissue expression of urothelial carcinoma of bladder.

Molecules inserum of patient’s

Fold expression ofmolecules in patient’stissue

Pearson’scorrelationcoefficient (r)

Significance(p-value)

IL-17A IL-17A 0.305 0.13IL-17A RORct 0.268 0.195IL-6 IL-17A 0.474 0.01IL-6 RORct 0.491 0.01

Q-PCR expression ofmolecule in PBMC

RORct IL-17A 0.621 0.001RORct RORct 0.386 0.05

S. Chugh et al. / Human Immunology 74 (2013) 1258–1262 1261

with IL-17A and RORct of tumor tissue of UBC patient’s (r = 0.621,p = 0.001; r = 0.386, p = 0.05, respectively) (Table 2).

4. Discussion

Inflammatory reactions that occur around tumor sites consti-tute tumor-associated immune response. T helper (Th17) cells, arecently discovered type of effector T cell are an important inflam-matory component and have been shown to play crucial role in anumber of autoimmune diseases [2]. Role of Th17 cells in cancerhas not been fully elucidated and there is some ambiguity regard-ing this [22–27]. In some cancers higher levels of Th17 cells havebeen found, which include pancreatic, gastric, ovarian and renalcell cancer [28,29]. There are few studies which correlate advancedcancer with higher levels of Th17 cells in tumor tissues or periph-eral blood [3]. On the contrary two studies describe the anti-tumoreffect of Th17 cells in patients who are long-term survivors or withlimited disease [30,31].

However, the distribution of these cells and expression of Th17related molecules in the bladder tumor microenvironment has notbeen well characterized. There is only one report showing theinvolvement of Th17 and Treg cells in tumor immunology of blad-der carcinoma [32]. Certain cytokines are found to be associatedwith these cells. IL-17 is a characteristic effector cytokine producedby Th17 cells and is associated with their biological function [33].IL-23 a member of IL-12 family is involved in promoting Th17 cellsdifferentiation and proliferation [34]. IL-6, by activating STAT3 and

RORct expression is also involved in inducing human Th17 cell dif-ferentiation [11]. Initially our lab has focused on biomarkers ofdiagnostic importance in bladder cancer patients [35,36]. Our pre-vious studies have shown the predominance of Th2 (IL-4, IL-5 andIL-10) cytokines while Th1 type cytokines [IL-2, IFNc] remain sup-pressed [37].

In this study using ELISA, circulatory levels of IL-17, IL-23 andIL-6 (Th17 related cytokines) in bladder cancer patients was foundto be higher which might suggests the prominent role of thesecytokines in differentiation and proliferation of Th17 cells in thisdisease. To further study the function of Th17 cells in bladder can-cer, the mRNA expression levels of Th17 related molecules, RORct(transcription factor) and its key cytokine IL-17A were examined intissues and PBMCs by real time PCR. The relative mRNA expressionof IL-17A and RORct in PBMCs was found to be significantly higherin patients as compared to controls. Also in case of tissues wherethe comparison of tumor tissues was made with adjacent normaltissue the fold expression of IL-17A and RORct was higher. Our re-sults are in concordance with earlier report in gastric cancer wherethe circulatory level and expression of IL-17A was elevated in pa-tients [3]. To measure the strength of association between variousmolecules in this study Pearson’s correlation coefficient was ap-plied. We observed that serum level of IL-17A was in positive cor-relation with tissue expression of IL-17A and RORct, but thiscorrelation was insignificant. Also, the fold change of IL-17A andRORct was in significant positive correlation with serum level ofIL-6 and real-time expression of RORct in circulation. These obser-vation shows that there might be some correlation between mole-cules related to Th17 in bladder cancer patients. However, thelarger sample size could provide the more rational correlation be-tween majority of Th17 related molecules and a better coordina-tion might be established amongst them.

In the present study, the PBMCs of patients and healthy individ-uals were also examined for the frequency of Th17 (CD4+IL-17+)cells by flow cytometry and the population of Th17 cells as a per-centage of total CD4+ cells was found to be significantly higher inthe patients as compared to controls. Zhang et al. [3] demonstratedin gastric cancer that there was significant increase in Th17 sub-population as a % of CD4+ in PBMCs of patients as compared tohealthy individuals. Also in case of pancreatic cancer patient’s fre-quency of Th17 cells was found to be significantly higher [4].Therefore our data corroborate from reports in gastric and pancre-atic cancers with limited number of subjects recruited in our study.

This study is a maiden attempt in which the circulatory levels ofIL-17A, IL-6, IL-23 cytokines, mRNA expression of Th17 relatedmolecules (RORct, IL-17A) in tumor tissues along with the distri-bution of Th17 population in the peripheral blood of the patientswith muscle invasive urothelial carcinoma of bladder was analyzedto investigate the relevance of Th17 cells in the bladder tumorimmunology. These findings indicate the possible involvement ofTh17 cells in urothelial carcinoma of bladder. Further the datacan be validated in larger patient cohort for better understandingthe role of these cells in this disease which might be an aide to for-mulate a more efficient therapeutic strategy with Th17 cells as atarget in future.

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