The Presence of IL-17A and T Helper 17 Cells in Experimental Mouse Brain Tumors and Human Glioma

Background Recently, CD4+IL-17A+ T helper 17 (Th17) cells were identified and reported in several diseased states, including autoimmunity, infection and various peripheral nervous system tumors. However, the presence of Th17 in glia-derived tumors of the central nervous system has not been studied. Methodology/Principal Findings In this report, we demonstrate that mRNA expression for the Th17 cell cytokine IL-17A, as well as Th17 cells, are present in human glioma. The mRNA expression for IL-17A in glioma was recapitulated in an immunocompetent mouse model of malignant glioma. Furthermore, the presence of Th17 cells was confirmed in both human and mouse glioma. Interestingly, some Th17 cells present in mouse glioma co-expressed the Th1 and Th2 lineage markers, IFN-γ and IL-4, respectively, but predominantly co-expressed the Treg lineage marker FoxP3. Conclusions These data confirm the presence of Th17 cells in glia-derived CNS tumors and provide the rationale for further investigation into the role of Th17 cells in malignant glioma.


Introduction
Glioma, which is a tumor that arises from a cell with glial lineage in the central nervous system (CNS), is a categorical designation for multiple types of glial tumors, including ependymomas, astrocytomas, oligodendrogliomas and glioblastoma multiforme (GBM). Of note, GBM is a highly malignant glioma with poor treatment options and an average lifespan of 15 months after diagnosis [1]. Recently, our lab demonstrated that a specific subset of T helper cells, CD4 + CD25 + T regulatory (Treg) cells, are increased in human GBM [2], as well as various grades of astrocytoma [3]. Furthermore, Treg accumulation is recapitulated in an experimental mouse model of malignant glioma [4,5]. Additionally, we demonstrated that Treg depletion in a mouse model of malignant glioma significantly increases lifespan [4]. Collectively, these data suggest that T helper subsets, such as Treg, are potential targets for future therapy of GBM. However, as additional T helper subsets are characterized, further investigation is required.
Recent work has identified the T helper 17 (Th17) cell subset, which is characterized as CD4 + T cells that produce large amounts of IL-17A (CD4 + IL-17A + ) [6]. Similar to Tregs, which differentiate in the presence of transforming growth factor-beta (TGF-b), Th17 differentiate in the combined presence of TGF-b and interleukin-6 (IL-6) [7]. In contrast to Tregs, Th17 require the additional cytokine, IL-23, for pathogenic activation [8]. The presence of Th17 cells has been demonstrated in autoimmune pathology [9], infectious inflammatory states [10] and tumor microenvironment [11]. Thus far, Th17 cells have not been reported to be exclusive to any one type of malignancy, as they have been observed in human ovarian-, colon-and breast-cancer, as well as melanoma [12]. However, the presence of Th17 cells in primary brain tumors has not been reported. This is particularly relevant since tumors that form in the CNS are shielded from effective T cell-mediated immunity [13], which is likely attributable due to the lack of a lymphatic system and presence of the blood-brain-barrier.
In this report, we use the GL261-cell based mouse model of malignant glioma, as well as human glioma (grade 2-4), to investigate the hypothesis that Th17 cells infiltrate brain tumors. We report that mRNA for the Th17 cytokine, IL-17A, is expressed in immunocompetent mice with glioma, but not in Rag1 2/2 immunodeficient mice with glioma. Furthermore, mRNA for the Th17-inducing cytokines, IL-6 and TGF-b, as well as the Th17-activating cytokine, IL-23, is expressed in immunocompetent mice with glioma. Additionally, Th17 were detectable in mouse draining lymph nodes (dLN) and glioma. Importantly, mRNA expression for the Th17 cytokine, IL-17A, is increased in human glioma relative to normal human brain and Th17 cells are present in GBM. Collectively, the results suggest that Th17 cells infiltrate human and mouse glioma.

Animals
Six to eight-week old male C57BL/6 (stock # 000664) and Rag1KO (stock # 002216) mice were obtained from Jackson Laboratories (Bar Harbor, ME) and maintained in a specific pathogen-free facility at the University of Chicago. All surgical procedures were completed in accordance with NIH (National Institutes of Health) guidelines on the care and use of laboratory animals for research purposes. The protocol was approved by the Institutional Committee on Animal Use at the University of Chicago.

GL261 cell line
GL261 murine glioma cells were cultured in Dulbecco's modified Eagle medium supplemented with 10% fetal calf serum, as well as streptomycin (100 mg/ml) and penicillin (100 U/ml) at 37uC in a humidified atmosphere of 95% air/5% CO2.

Mouse intracranial injection model
Mice were anesthetized with an intraperitoneal injection of 0.1 ml of a stock solution containing ketamine HCl (25 mg/ml), xylazine (2.5 mg/ml), and 14.25% ethyl alcohol (diluted 1:3 in 0.9% NaCl). For the stereotactic intracranial injection, the surgical site was shaved and prepared with 70% ethyl alcohol. A midline incision was made, and a 1 mm diameter right parietal burr hole, centered 2 mm posterior to the coronal suture and 2 mm lateral to the sagittal suture, was drilled. Mice were placed in a stereotactic frame and 2.5 mL PBS or 4610 5 GL261 cells in 2.5 mL PBS was injected intracranially with a 26-gauge needle at a depth of 3 mm. The needle was removed and the skin was sutured with 4-0 nylon thread.

Patient samples
Resected specimens from patients who underwent operations in the Section of Neurosurgery at the University of Chicago Medical Center between 2009 and 2010 were evaluated in this study. According to the WHO classification, samples included normal brain, grade II astrocytoma, grade III oligodendroglioma, and GBM. Normal brain control tissue consisted of non-malignant tissue obtained during resection from a patient with glioma. Histological confirmation of the diagnosis for tumors was obtained in all cases by an attending neuropathologist. The tissue was collected in accordance with a protocol approved by the Institutional Review Board (IRB) at the University of Chicago.

RNA isolation, semi-quantitative PCR and real-time PCR
Total cellular RNA from normal WT mouse brain (n = 3), 3 week post-operative (WPO) PBS-injected WT mouse brain (n = 6), 3 WPO WT mouse brain with glioma (n = 6) and 3 WPO Rag1 2/2 mouse brain with glioma (n = 3) or normal human brain (n = 3), human astrocytoma (grade 2; n = 3), human oligodendroglioma (grade 3; n = 1) and human glioblastoma multiforme (n = 12) was isolated using the RNeasy Mini kit (Qiagen; Valencia, CA) according to the manufacturer's protocol. Equivalent amounts of RNA were reverse-transcribed with the iScript cDNA Synthesis Kit (Bio-rad Laboratories; Hercules, CA). Semi-quantitative PCR was performed using 5 uL Taq PCR Master Mix, 2.5 mM forward primer, 2.5 mM reverse primer, 2 uL RNase-free H 2 O and 1 ng input cDNA per reaction. Amplification was performed using the iCycler Thermal Cycler (Bio-Rad Laboratories) under the following conditions: 3 min at 95uC, followed by 30 cycles of 30 sec at 95uC, 30 sec at 60uC and 30 sec at 72uC, followed by 5 min at 72uC. PCR products combined with 2 uL 6X DNA Loading Dye (Fermentas Life Sciences; Glen Burnie, MD) were ran on a 2% agarose gel with the GeneRuler 100 bp DNA ladder. The gel was visualized in the ChemiDoc XRS Universal Hood II (Bio-rad Laboratories). Realtime PCR was performed using 5 uL SYBR GreenER qPCR Supermix Universal (Invitrogen; Carlsbad, CA), 0.5 mM forward primer, 0.5 mM reverse primer, 3 uL RNase-free H 2 O and 1 ng input cDNA per reaction. Amplification and detection was performed using the Opticon 2 Real-Time PCR Detector (Biorad Laboratories) using Opticon Monitor software (version 3.1.32; Bio-rad Laboratories) under the following conditions: 15 min hot start at 95uC, 15 sec denaturation at 95uC, 20 sec annealing of primers at 60uC, and 15 sec elongation at 72uC, for 35 cycles. Triplicate reactions were performed for all cDNA samples. For mouse and human samples, percent change in mRNA levels was calculated using the formulas ((Experimental group/PBS group) 6100)) -100% and ((Glioma/Normal brain) 6100)) -100%, respectively. Mouse and human primer sequences used for PCR reactions are found in Tables 1 and 2, respectively.

Statistical analysis
All data are presented as means 6 standard error of measurement (SEM). The results from the experiments were analyzed using GB-STAT School Pak (Dynamic Microsystems, Inc.; Silver Spring, MD). For statistical comparisons, data were analyzed using the 2-way analysis of variance (ANOVA) method, followed by post hoc comparisons using the Newman-Keuls test.

An experimental mouse model of glioma
As shown in Fig. 1, GL261-cell based glioma takes up a large volume of intracranial space at 3 weeks post-operative (WPO) in the mouse brain. Additionally, reactive GFAP + astrocytes and CD11b + microglia/macrophages form a glial border around the glioma. Furthermore, CD11b + and CD11c + antigen presenting cells are found clustered together in the glioma. Similarly, Figure 2. Th17-related mRNA expression levels in mouse brain with or without a GL261 cell injection. (A) PCR products for IL-17A and GAPDH from control (Normal) mouse brain or 3 week post-operative wild-type (WT) mouse brain that received an intracranial (IC) injection of PBS, WT mouse brain that received an IC injection of 4610 5 GL261 cells (Glioma) or recombinase activating gene 1-deficient (Rag1 2/2 ) mouse brain that received an IC injection of 4610 5 GL261 cells (Rag1 2/2 ). Individual mice are differentiated by numbers 1-3. (B) IL-23 mRNA levels are displayed as the percent change between the experimental group (Exp.) and the mouse brain that received an IC injection of PBS (PBS), normalized to GAPDH. (C) TGF-b1 mRNA levels are displayed as the percent change between the experimental group (Exp.) and the mouse brain that received an IC injection of PBS (PBS), normalized to GAPDH. (D) IL-6 mRNA levels are displayed as the percent change between the experimental group (Exp.) and the mouse brain that received an IC injection of PBS (PBS), normalized to GAPDH. (E) CD3e mRNA levels are displayed as the percent change between the experimental group (Exp.) and the mouse brain that received an IC injection of PBS (PBS), normalized to GAPDH. Bar heights represent means (6SEM). ND = not detectable. doi:10.1371/journal.pone.0015390.g002 CD4 + FoxP3 2 T helper and CD4 + FoxP3 + T regulatory cells are found clustered together in the glioma. Thus, mouse glioma is comprised of many types of cells closely juxtaposed around-and intermixed throughout-the malignancy.

Th17-secreted, activating and inducing cytokine levels are increased in glioma
To determine if IL-17A mRNA is increased in mouse glioma, normal mouse brain, PBS-injected WT mouse brain, WT mouse brain with glioma and Rag1 2/2 (lack functional T and B cells) mouse brain with glioma were analyzed ( Fig. 2A). IL-17A mRNA is not detectable in normal mouse brain or mouse brain injected with PBS. However, IL-17A mRNA is detectable in glioma of WT mice, but not in glioma of Rag1 2/2 mice. Furthermore, mRNA levels are unchanged for the Th17-activating cytokine, IL-23, in normal, WTglioma and Rag1 2/2 -glioma mouse brain (33651%, 216.3665% and 102696%, respectively). In addition, although mRNA for the Th17-inducing cytokine, TGF-b, is not detectable in normal mouse brain, it is induced in WT-glioma and Rag1 2/2 -glioma mouse brain (5066171% and 6626319%, respectively). In contrast, mRNA levels for the Th17-inducing cytokine, IL-6, is 67653% in normal mouse brain and increases in WT-glioma and Rag1 2/2glioma mouse brain (6186316% and 356667%, respectively). Finally, although mRNA for the T cell-specific complex, CD3e, is not detectable in normal or Rag1 2/2 -glioma mouse brain, it is induced in WT-glioma mouse brain (736762570%). These data suggest that glioma-induced mRNA expression for the Th17 cytokine, IL-17A, as well as T cell infiltration represented by CD3e, requires both the presence of malignancy, as well as a functional immune system. In addition, the presence of glioma appears to increase the Th17-inducing cytokines, TGF-b and IL-6.

Th17 cells are present in glioma and draining lymph nodes
To determine if Th17 cells develop in glioma-draining lymph node (dLN) and are present in mouse glioma, PBS-dLN, glioma- dLN, PBS-brain and glioma-brain were analyzed at 3 weeks postoperative in WT mice. As shown in Fig. 3A-C, a two-way ANOVA for changes in CD4 + IL-17 + T cell frequency revealed a main effect between mice with or without glioma [F(1,11) = 8.55, p = 0.0192]. Isotype control cells are not detectable in mouse dLN and brain. The average CD4 + IL-17 + cell frequency is 1263.7%, 1261.1%, 0% and 1061.5% in PBS-dLN, glioma-dLN, PBSbrain and glioma-brain, respectively. These data suggest that Th17 cells are present in the mouse dLN in response to an injury to the brain and that the level of Th17 cells is not affected by the presence of glioma. Furthermore, these data suggest that Th17 cells are present in mouse brain due to the glioma and not in response to an injury to the brain.
To determine if brain-derived Th17 cells share lineage plasticity with Th1, Treg and Th2 cells, PBS-and glioma-mouse brains were analyzed at 3 weeks post-operative in WT mice. As shown in Fig. 3D-E The average frequency of Th17 that co-expressed IFN-c, FoxP3 and IL-4 increased from 0%, 0%, and 0% in PBSbrains to 1064%, 2265% and 463%, in glioma brains, respectively. These data suggest that glioma affects the lineage specificity of Th17 cells.

IL-17A mRNA and Th17 cells in human glioma
To determine if IL-17A mRNA is increased in human glioma, normal human brain, as well as astrocytoma (grade 2), oligodendroglioma (grade 3) and GBM were analyzed (Fig. 4A). The level of IL-17A mRNA in astrocytoma is 778627% and increases in oligodendroglioma and GBM (6658% and 498161344%, respectively). Additionally, the CD3e mRNA level in astrocytoma is 9416323% and increases in oligodendroglioma and GBM (7515% and 523161942%). When IL-17A mRNA levels are normalized to CD3e mRNA levels, there is no difference between astrocytoma, oligodendroglioma and GBM (20.7627%, 214% and 40635%, respectively). Finally, the frequency of CD4 + IL-17A + Th17 in GBM was determined (Fig. 4D). Isotype control cells are not detectable in GBM, while the CD4 + IL-17 + cell frequency is 17%. Collectively, the data indicate that Th17 are present in human glioma and that the expression of the Th17 cytokine, IL-17A, as well as the number of T cells, is increased in oligodendroglioma and GBM, compared to low-grade astrocytoma. However, IL-17A expression appears to be dependent on T cell accumulation, since IL-17A mRNA levels were not different between types of human glioma, when normalized to CD3e mRNA levels.

Discussion
Collectively, the data demonstrate the presence of Th17 in mouse and human glioma. However, the presence of Th17 that co-express cytokines from other T helper lineages poses the challenge of identifying the significance and role of multi-lineage CD4 + T cells. Recently, it was demonstrated in mouse tumordraining lymph nodes that the inhibition of indoleamine 2,3 dioxygenase (IDO) caused the conversion of Treg into a polyfunctional T helper phenotype with Th17 properties [17]. Additionally, Treg that expand in adenomatous polyps of colon cancer start to produce IL-17A, which results in simultaneous immunosuppression of T helper function, while promoting tumor progression through inflammation-driven mastocytosis [18]. Whether polyfunctional T helper cells have an equal level of pathogenesis in glioma has yet to be explored.
From a clinical perspective, determining the effector function of Th17 in glioma may bring novel therapeutic avenues in the field of brain tumor immunotherapy, just as determining the role of Treg in glioma did several years ago [2][3][4]. Currently, small molecule inhibitors of Th17 [19] or inhibitors of Th17 differentiation are currently being pursued [20]. In addition, Th17 inhibitors have seen mixed success in human studies, failing to control relapsing multiple sclerosis, while showing promise in patients with psoriasis and Crohn's disease [21]. Given the immunosuppressive nature of the CNS, combined with the tolerogenic [22] and multi-cellular nature of glioma, determining the role of the pro-inflammatory Th17 is important to address.
The presence of IL-17A mRNA in human glioma was recently identified through a large scale microarray analysis of Th1-, Th2and Th17-related genes [23]. However, it was neither established whether IL-17A mRNA levels were dependent on T cell levels nor was it identified that Th17 cells were present. This study confirmed the presence of the Th17 cytokine, IL-17A, and identified the presence of Th17 cells in both human and mouse glioma. Importantly, we could not detect mRNA for the Th17secreted cytokine, IL-17A, in Rag1 2/2 mice with glioma, suggesting that T or B cells are required for IL-17A expression. In future investigations, we intend to identify whether IL-17A expression in glioma is solely a CD4 + T cell cytokine or if B cells also produce this cytokine through analysis of IL-17A mRNA levels in CD4 T cell-and B cell-deficient mice with malignant glioma. Additionally, we will identify the in vivo role of Th17 cells using IL-17A 2/2 or Rag1 2/2 mice with malignant glioma, adoptively transferred CD4 + T cells from IL-17A 2/2 mice. Finally, we plan to compare the chemokine receptor repertoire of glioma-resident polyfunctional T helper cells with that of polarized T helper cell subsets. Collectively, these future experiments will determine the role of Th17 in a pre-clinical malignant glioma model, as well as determine whether these cells represent a future therapeutic target in human health.