Role of IL-10 in immune suppression in cervical cancer

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1 Indian Journal of Biochemistry & Biophysics Vol. 44, October 2007, pp Role of IL-10 in immune suppression in cervical cancer Ravi Kiran Bhairavabhotla 1, Veena Verma 1, Hemant Tongaonkar 2, Surendra Shastri 3, Ketayun Dinshaw 4 and Shubhada Chiplunkar 1 * 1 Immunology, Advanced Centre for Treatment, Research and Education in Cancer, (ACTREC) Tata Memorial Centre, Navi Mumbai , India 2 Gynecology and Urology, 3 Preventive Oncology, Tata Memorial Hospital, Tata Memorial Centre, Mumbai, India 4 Radiation Oncology, Tata Memorial Hospital, Tata Memorial Centre, Mumbai , India Received 29 May 2007; revised 29 August 2007 Cervical cancer is the second most common cancer in the women worldwide and the most frequent in developing countries, including India. Human papilloma virus (HPV) is the major etiological factor in cervical cancer patients. Host factors are also critical in regulating tumor growth and cytokines that modulate immunologic control may be of particular importance. In the present study, we investigated the correlation between the presence of HPV and type of cytokines expressed in cervical carcinomas and attempted to elucidate the possible reasons for the immune suppression. Cytokines investigated were type-1 cytokine IFN-γ (shows immunostimulatory function and capable of limiting tumor growth) and type-2 cytokines IL-4, IL-10 and IL-6 (show immunosuppressive function and capable of stimulating tumor growth). Our data demonstrated the presence of HPV sub-types 16 and 18 in 86% and 13.8% of cervical tumor biopsies, respectively. The cervical tumor biopsies showed increased presence for mrna for IL-10 and IL-1α, while none of the biopsies showed expression for IFN-γ. A correlation was observed between the presence of HPV in cervical tumor biopsies and mrna for IL-10. Increased percentages of CD4+CD25+ regulatory T cells (Tregs) were observed in circulation in cervical cancer patients, providing evidence for increased immune suppression. IL-10 may play a key role in maintenance of Tregs and explains the immunosuppressive state of cervical cancer patients. Keywords: Cervical cancer, Human papilloma virus, IL-10, Regulatory T cells (Tregs), Immune suppression Cervical cancer remains an important public health problem worldwide and India for one-fifth of the world's burden of cervical cancer 1. There are approximately 130,000 new cases of cervical cancer in India per year and the disease is reported to be responsible for almost 20 percent of all female deaths 2. The recognition of human papillomavirus (HPV) as the causative agent for 95% of cervical cancers has provided opportunities for prevention, treatment and therapeutic vaccinations 3,4. Genital infection with certain strains of HPV is associated with a high risk of malignant transformation. Cervical *Author for correspondence: schiplunkar@actrec.gov.in Tel: (022) ; Fax: (022) Abbreviations: CIN, cervical intraepithelial neoplasia; CTLA4, cytolytic T-lymphocyte-associated antigen-4; GITR, glucocorticoidinduced tumor-necrosis factor receptor related protein; G3PDH, glyceraldehyde-3-phosphate dehydrogenase; HPV, human papilloma virus; IFN, interferon; IL, interleukin; PBMC, peripheral blood mononuclear cells; PCR, polymerase chain reaction; RT-PCR, reverse transcriptase polymerase chain reaction; TGF, transforming growth factor; MHC, major histocompatibility complex; TNF, tumor necrosis factor; Tregs, regulatory T cells. cancer usually begins slowly with cervical intraepithelial neoplasia (CIN), showing precancerous abnormalities within the lining of the cervix, which can become invasive cancer 5. HPV is a non-lytic double-stranded DNA virus, with over 100 identified sub-types, almost half of which infect the anogenital region. Of these sub-types, a subset of oncogenic types which includes high-risk sub-types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73 and 82, probable high-risk sub-types 26, 53 and 66 and lowrisk sub-types 6, 11, 40, 43, 44, 54, 61, 70, 72 and 81 has been identified 6,7. The majority of cervical cancer cells contain HPV DNA integrated into the host cell genome and expresse two main viral oncoproteins E6 and E7, which interfere with cell-cycle progression 5. Although most women are capable of clearing HPV infection, some develop persistent infection, which may lead to cancer. Both humoral and cell-mediated responses have been shown to be important in the natural response to HPV infection Tumors have developed numerous mechanisms to evade immune response including down-regulation of major

2 BHAIRAVABHOTLA et al.: IL-10 IN CERVICAL CANCER 351 histocompatibility complex-i (MHC-I), loss of tumor antigens, absence of co-stimulatory signals and secretion of immunosuppressive cytokines, etc 10. One of the possible mechanisms by which tumor cells may limit an efficient immune response is the production of immunosuppressive cytokines IL-10 and TGF-β Regulatory T cells (Tregs) are specialized T cells characterized as CD4 + CD25 + T cells with suppressive function that contribute to the maintenance of immunologic self-tolerance. These cells typically express cytolytic T-lymphocyte-associated antigen-4 (CTLA-4), glucocorticoid-induced tumor-necrosis factor receptor related protein (GITR), CD45RO and a novel transcription factor Foxp3 18. One unique feature of these cells is their ability to secrete a large amount of IL-10 and TGFβ Studies demonstrate that tumor cells can recruit Tregs to inhibit antitumor immunity, thus limiting the efficiency of cancer immunotherapy 17,21,22. With this background, this study investigates whether there exists any correlation between presence of HPV and type of cytokines expressed in cervical carcinomas. The study attempts to elucidate the possible reasons for immune suppression observed in cervical cancer patients. Materials and Methods Subjects Cervical cancer patients referred to the Gynecologic Oncology Department of Tata Memorial Centre, Mumbai were included in the study after obtaining their informed ethical consent. Heparinized Table 1 Primer sequences for HPV and cytokines blood, punch biopsies of cervical tumor tissues and CIN lesions were collected from the patients. Punch biopsies of cervical tumors and CIN lesions were snap-frozen in liquid nitrogen after collection. The stages of cervical biopsies were confirmed by histopathological examinations by the extent of invasiveness size of cervical carcinoma according to classification of the International Federation of Gynecologists and Obstetricians (FIGO) 23. Cervical biopsies collected were processed further for DNA and RNA extraction by TRIZOL (Sigma, USA) according to manufacturer s instructions. Blood samples were collected from age-matched female healthy individuals after obtaining informed ethical consent. Detection of HPV DNA in cervical tumor biopsies DNA polymerase chain reaction (PCR) was performed on the extracted DNA from biopsies of cervical tumors and CIN lesions using primers from consensus sequence (spanning the E1 open-readingframe of the HPV genome to detect types 6, 11, 16, 18, 31 and 33) and HPV 16 and 18 sequences (Table 1). The reaction was carried out in a volume of 50 µl containing the following: 5 µl 10X PCR-Taq buffer, 2 mm MgCl 2, 25.0 pmol of each of the sense and anti-sense primers (Gibco BRL), 250 µm dntp mix, 1.0 unit Taq polymerase (Invitrogen, USA) 3 µl of template DNA and sterile distilled water was added to each reaction. Reaction was performed in a DNA thermal cycler (Eppendorf) as per the understated Primer Bases 5 to 3 Annealing temp ( 0 C) Product size HPV C Forward TTT GTT ACT GTG GTA GAT ACT AC Reverse CAA AAA TAA ACT GTA AAT CAT ATTC HPV 16 Forward AAG GCC AAC TAA ATG TCA C Reverse CTG CTT TTA TAC TAA CCG G HPV 18 Forward AAG GAT GCT GCA CCG GCT GA Reverse CAC GCA CAC GCT TGG CAG GT G3PDH Forward CAT GTG GGC CAT GAG GTC CAC CAC Reverse TGA AGG TCG GAG TCA ACG GAT TTG GT TNF-α Forward ATG AGC ACT GAA AGC ATG ATC CGG Reverse GCA ATG ATC CCA AAG TAG ACC TGC CC IL-6 Forward ATG AAC TCC TTC TCC ACA AGC GC Reverse GAA GAG CCC TCA GGC TGG ACT G IL-1-α Forward GTC TCT GAA TCA GAA ATC CTT CTA TC Reverse CAT GTC AAA TTT CAC TGC TTC ATC CC IFN-γ Forward ATG AAA TAT ACA AGT TAT ATC TTG GCT Reverse GCG ACA GTT CAG CCA TCA CTT G β Globin Forward GCT CAC TCA GTG TGG CAA Reverse GGT TGG CCA ATC TAC TCC CAG G IL-10 Forward ATG CCC CAA GCT GAG AAC CAA GAC CCA Reverse TCT CAA GGG GCT GGG TCA GCT ATC CCA bp bp bp bp bp bp bp bp bp bp

3 352 INDIAN J. BIOCHEM. BIOPHYS., VOL. 44, OCTOBER 2007 protocol. Denaturation was done for 5 min at 94 C for the first cycle, followed by 45 s each of denaturation at 94 C, annealing at respective temperatures (Table 1) and extension at 72 C for 34 cycles. The last cycle was extended for 10 min at 72 C. The electrophoresis of amplified products was done in 1.5% agarose gel. The gel was stained with ethidium bromide to visualize the amplified PCR product on a UV transilluminator. The β-globin gene was used as an internal control for PCR amplification. Detection of mrna cytokines in cervical tumor biopsies RT PCR were performed from total RNA extracted from cervical tumor biopsies and CIN lesions. cdna was synthesized from 1 µg RNA using Reverse transcriptase (Sigma, USA) with oligo dt primers (Gibco BRL). cdna was utilized for PCR amplification as mentioned above with primers for IL-1α, IL-6, IL-10, IFN-γ and TNF-α and glyceraldehyde-3-phosphate dehydrogenase (G3PDH) as house-keeping gene control (Table 1). Immunophenotyping of regulatory T cells (Tregs) Peripheral blood mononuclear cells (PBMC) isolated from blood by the Ficoll density gradient were stained with monoclonal antibodies anti-human CD4-FITC (BD Pharmingen) and anti-human CD25- PE (BD Pharmingen). The samples were acquired on flow cytometer (Becton Dickinson FACS Calibur) and results were analyzed with Cell Quest software. Statistical analysis Chi-square analysis was performed for calculation of significance between the HPV subtypes and cytokine profiles. Student s t test was carried out for calculation of significance of circulating Tregs between cervical cancer patients and healthy individuals. Results Presence of HPV in cervical tumors and CIN biopsies The presence of HPV and sub-types 16 and 18 in cervical tumor tissues was determined using PCR. The 88% (33/36) of cervical tumor biopsies showed presence of HPV, with 86% (31/36) HPV 16 and 13% (5/36) HPV 18 (Fig. 1). Some of the tumor biopsies also exhibited dual presence of HPV 16 and 18 (11%). The present findings showed an increased prevalence of HPV 16 in cervical tumors (Table 2). 11% (1/9) were positive for HPV 16. All the CIN lesions were negative for HPV 18 (Table 3). Fig. 1 HPV DNA in tumor biopsies of cervical cancer patients [Representative agarose gel images for PCR products using primers for HPV consensus (a), HPV16 (b) and HPV 18 (c). The 100 bp DNA ladder is shown in extreme right lane in Fig. a-c. Comparisons of HPV DNA subtypes in cervical tumor biopsies (d)] Determination of cytokine profiles in cervical tumors and CIN biopsies Cervical tumor biopsies (n = 36) were analyzed for expression of mrna for cytokines IL-10, IL-6, IL-1α, TNF-α and IFN-γ. The mrna expression for cytokines IL-1α, IL-6 and IL-10 were detected in 83.3%, 80.5% and 86.1% biopsies respectively (Fig. 2). On the contrary, no expression of IFN-γ was observed in cervical tumor biopsies, while TNF-α was observed in 8.3%. As positive control for IFN-γ, PBMC of two individuals stimulated with ril-2 (100 units) were used. The cdna was amplified using the IFN-γ primers and clear band of 485 bp was observed in these samples (Fig. 2). Results demonstrated high expression of IL-10, IL-1α and IL-6 in cervical tumors. CIN lesions showed presence of mrna for IL-1α and IL-6 in 33.3% of the biopsies while no expression for IL-10, IFN-γ and TNF-α mrna was observed (Table 2). The tumor biopsies analyzed showed presence of HPV 16 and high expression of

4 BHAIRAVABHOTLA et al.: IL-10 IN CERVICAL CANCER 353 Table 2 HPV sub-types and cytokine mrna expression in cervical tumor biopsies Stage HPV-C HPV-16 HPV-18 IL-1α IL-6 TNF-α IFN-γ IL-10 1 III b III b II a III b III b II b II b III b III b III b II a III b III b II a III b II a III b III b III b II a III b III b II a III b II a III b III b II b II b II a III b II a II a III b III b II b Total 33/36 31/36 5/36 30/36 29/36 3/36 0/36 31/36 % Positive Table 3 HPV sub-types and cytokine mrna expression in CIN biopsies Stage HPV-C HPV-16 HPV-18 IL-1α IL-6 TNF-α IFN-γ IL-10 1 CIN I CIN I CIN I CIN I CIN III CIN I CIN I CIN I CIN I Total 5/9 1/9 0/9 3/9 3/9 0/9 0/9 0/9 % Positive

An agarose gel image for PCR products using IFN-γ primers with two positive controls (PBMC from two individuals stimulated with ril-2 100 units) is shown in lanes on the left side (f).

5 354 INDIAN J. BIOCHEM. BIOPHYS., VOL. 44, OCTOBER 2007 Fig. 2 Expression of mrna for inflammatory cytokines in cervical tumor biopsies [Representative agarose gel images for PCR products using primers of IL-10 (a), IL-1α (b), IL-6 (c), TNF-α (d) and IFN-γ (e). An agarose gel image for PCR products using IFN-γ primers with two positive controls (PBMC from two individuals stimulated with ril units) is shown in lanes on the left side (f). The PCR products from cervical tumor biopsies (n=5) are shown along with the positive controls (f). The 100 bp DNA ladder is shown in extreme right lane in Fig. a-f. Comparisons of cytokine mrna expressions in cervical tumor biopsies (2g)] IL-10, IL-1α and IL-6 mrna. Chi square test demonstrated significant association between presence of HPV-16 and expression of mrna for IL-10 (p = 0.012) in cervical tumor biopsies. No Fig. 3 Increased circulating Tregs in cervical cancer patients [Representative plot of dual color flowcytometry staining for circulating Tregs (CD4 + CD25 + T cells) in PBMCs of cervical cancer patient and healthy individual. PBMCs were stained with CD4-PE and CD25-FITC (a). Comparisons of percentage circulating levels of CD4 + CD25 + Tcells in cervical cancer patients (n = 5) and healthy individuals (n = 5) (b). Each bar represents individual patient or healthy individual] significant association of HPV 16 was observed with other cytokines. Circulating levels of regulatory T cells Regulatory T cells (Tregs) were identified by immunostaining of PBMC from cervical cancer patients and healthy individuals using anti-cd4 and anti-cd25 monoclonal antibodies (Fig. 3a). High levels of circulating CD4 + CD25 + Tregs (p = ) were observed in cervical cancer patients (mean = 9.5%, n = 5), compared to healthy individuals (mean = 2.8%, n = 5) (Fig. 3b). Discussion In the present study, we observed dominant expression of HPV 16 DNA, compared to HPV 18 DNA in cervical tumor biopsies. Our results were in agreement with other published reports in Indian cervical cancer patients Although HPV association in cervical cancer is well documented, the immune scenario in these patients is not well understood. Cell-mediated immunity is thought to play an important role in control or progression of tumor growth, as active regression of papillomas is associated with infiltration of macrophages, NK and T cells in tumors 8,10,16. However, the cytokine profiles in cervical cancer patients are not well known.

6 BHAIRAVABHOTLA et al.: IL-10 IN CERVICAL CANCER 355 The cervical tumor biopsies exhibited high expression of mrna for IL-1α, IL-10 and IL-6 cytokines. CIN lesions showed low expression of IL- 1α and IL-6 with no expression for TNF-α, IFN γ and IL-10. A significant association was observed between presence of HPV 16 and expression of mrna for IL-10 in cervical tumor biopsies. None of the cervical tumor biopsies exhibited presence of IFN-γ. This study demonstrated a pronounced shift from type-1 to type-2 cytokine production, which was associated with HPV infection. The down-regulation of type-1 cytokines associated with an increase of type-2 cytokines was also reported in cancer in earlier studies This type-1 to type-2 cytokine shift might reflect reduced protective cell-mediated immunity against tumors 10,15,20. Our data demonstrated low IFN-γ mrna levels and high HPV 16 infection in cervical tumor biopsies. A significant decrease of IFN-γ gene expression during progression of cervical lesions from CIN to invasive cancer was reported earlier also 11,12,29. The present study showed that HPV infection was responsible for increased expression of IL-10 in cervical tumors and might explain the immunological unresponsiveness observed in cervical cancers patients. IL-10 is an immunosuppressive cytokine and is able to downregulate type-1 cytokines 15. The presence of IL-10 might enhance the persistence and progression of HPV related lesions 30. Treatment of HPV 16 positive cervical carcinoma cells with IL-10 in vitro increases the mrna levels of HPV E7 early gene transcription 30. Cervical cancer cells, but not normal cervical epithelial cells have been reported to produce IL-10 and TGF-β. Their expression also increases during the progression from carcinoma in situ to invasive cancer 15. These reports support the possible association of high IL-10 with increased prevalence of HPV in cervical cancer. The CD4 + CD25 + Tregs are known to play an important role in immune suppression observed in cancer patients IL-10 is required for the induction of Tregs in vitro and in vivo; IL-10 and TGF-β have been implicated in the maintenance of Tregs 19,20. Tumor cells not only secrete TGF-β and or IL-10, but also induce immature myeloid DCs to secrete TGF-β and IL-10 and help in maintenance of Tregs in the tumor microenvironment 21. Our study demonstrated increased levels of Tregs in cervical cancer patients compared to healthy individuals. However, the mechanism leading to accumulation of Tregs and their recruitment in tumor-bearing hosts is poorly understood 20. It may be argued that the increased Tregs in cervical cancer patients may lead to increased IL-10 production and thereby helps in their maintenance and immune suppression. Our study lends support to the important role of IL-10 in orchestring immune suppression in cervical cancer patients by increasing HPV persistence and also mediating immune suppression via maintenance of Tregs. Acknowledgements RKB thanks the Council of Scientific and Industrial Research, N. Delhi for award of Senior Research Fellowship. The project was partially funded by Department of Biotechnology, Govt. of India. References 1 Sankaranaryanan R, Buduk A M & Rajkumar R (2001) Bull World Health Organ 10, 79, Ferlay J, Bray F, Pisani P & Parkin D M (2004) Globocan In: IARC Cancer Base No. 5, IARC Press, Lyon 2 3 Durst M, Gissman L, Ikenberg H & Hausen H Z (1983) Proc Natl Acad Sci (USA) 80, Kremsdorf D, Jablonska S, Favre M & Orth G (1982) J Virol 43, Hausen H Z (1996) Biochim Biophys Acta 1288, 2, Clifford G M, Smith J S, Aguado T & Franceschi S (2003) Brit J Cancer 89, Munoz N, Bosch F X, desanjose S, Herrero R, Castellsague X, Shah K, Snijders P & Meijer C (2003) NEJM 348, Kast W M, Feltkamp M C, Ressing M E, Vierboom M P, Brandt R M & Melief C J (1996) Sem Virol 7, Davidson E J, Kitchener H C & Stern P L (2002) Clin Oncol 14, Tindle R (2002) Nat Rev Cancer 2, Clerici M, Merola M, Ferrario E, Trabattoni D, Villa M L, Stefanon B, Venzon D J & Clerici E (1997) J Natl Cancer Inst 89, 3, 101, De Gruijl T D, Bontkes H J & van den Muysenberg A J (1999) Eur J Cancer 35, El-Sherif A M, Seth R, Tighe P J & Jenkins D (2001) J Pathol 195, Pao C C, Lin C Y, Yao D S & Tseng C J (1995) Biochem Biophys Res Commun 214, Salazar O F, Lo pez N M & Mendoza N A (2007) Cytokine Growth Factor Rev 18, Onur B, Jared F P, Charles D S & Jonathan S (2007) Curr Opin Immunol 19, Sakaguchi S (2004) Annu Rev Immunol 22, Kretschmer K, Apostolou I, Jaeckel E, Khazaie K & Boehmer H V (2006) Immunol Rev 212,

7 356 INDIAN J. BIOCHEM. BIOPHYS., VOL. 44, OCTOBER Roncarolo M G, Gregori S, Battaglia M, Bacchetta R, Fleischhauer K & Levings M K (2006) Immunol Rev 212, Wang H Y & Wang R F (2007) Curr Opin Immunol 19, Yamazaki S, Inaba K, Tarbell K V & Steinman R M (2006) Immunol Rev 212, Gallimore A & Sakaguchi S (2002) Immunol 107, Shepherd J H (1996) Br J Obstet Gynaecol 103, 5, Rughooputh S, Kachaliya S, Jetly D & Greenwell P (2007) Br J Biomed Sci 64, 1, Payal R, Gupta S, Aggarwal R, Handa S, Radotra B D & Arora S K (2006) Dermatol J 31, Lalikangbam P, Sengupta S, Bhattacharya P, Duttagupta C, Singh D T, Y Verma, Roy S, Das R & Mukhopadhyaa S (2007) Int J Gynecol Cancer 17, Peedicayil A, Abraham P, Sathish N, John S, Shah K, Sridharan G & Gravitt P (2006) Int J Gynecol Cancer 16, 4, Aggarwal R, Gupta S, Nijhawan R, Suri V, Kaur A, Bhasin V & Arora S (2006) Indian J Cancer 43, 3, Srivani R, Pratibha K,12 Selvaluxmy G & Nagarajan B (2004) Curr Sci 84, 3, Istvan A, Kenneth G & Stephen K (2002) Antivir Res 55,

Supplementary Table 3. 3 UTR primer sequences. Primer sequences used to amplify and clone the 3 UTR of each indicated gene are listed.

Supplementary Table 3. 3 UTR primer sequences. Primer sequences used to amplify and clone the 3 UTR of each indicated gene are listed. Supplemental Figure 1. DLKI-DIO3 mirna/mrna complementarity. Complementarity between the indicated DLK1-DIO3 cluster mirnas and the UTR of SOX2, SOX9, HIF1A, ZEB1, ZEB2, STAT3 and CDH1with mirsvr and PhastCons