The GL261 murine glioma cell line was obtained from the American Type Culture Collection (Manassas, VA, USA). Cells were cultured in vitro at 37°C (5% CO₂) in Iscove's Modified Dulbecco's Medium (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal calf serum (Sigma-Aldrich; Merck Millipore, Darmstadt, Germany), 1% 100 U/ml penicillin and 1% 100 g/ml streptomycin (Invitrogen; Thermo Fisher Scientific, Inc.) and 20 M β-mercaptoethanol (complete medium). IL-22 protein was purchased from PeproTech, Inc. (Rocky Hill, NJ, USA). Anti-IL-22 neutralising polyclonal rabbit antibodies (ab109819) were purchased from Abcam (Cambridge, MA, USA).

A total of 50 female C57BL/6 mice (age, 6–12 weeks; weight, 20–25 g) were obtained from Charles River Laboratories (Wilmington, MA, USA). A brain tumor model was set up as described previously (11). A total of 1×10⁴ GL261 glioma cells were washed twice in phosphate-buffered saline (PBS) and adjusted to 5 µl PBS in a 26-gauge Hamilton syringe. The mice were anesthetized with 1.2% isoflurane (792632; Sigma-Aldrich). Following shaving, an incision was made in the scalp, and a burr hole was made in the skull 2 mm lateral to the midline and 2 mm anterior to the bregma using a dental drill. Subsequently, GL261 glioma cells were incubated with anti-IL-22 neutralising polyclonal rabbit antibodies at 4°C for 24 h. Following neutralization, GL261 glioma cells were injected over 1 min at a depth of 2.5 mm below the dura mater into the right cerebral hemisphere. The mice were observed daily and sacrificed by cervical dislocation when characteristic symptoms such as hunched posture, reduced mobility, and significant weight loss (20%) occurred within 10 days of glioma implantation. Animals without such symptoms were regarded as long-term survivors after 90 days. A total of 50 IL-22-deficient [knock-out (KO)] mice were generated by targeting exons 1–3 and backcrossed onto C57BL/6 >8 times, as described previously (16). The targeting vector was constructed to replace the exons 1a, 1b, 2, and a part of exon 3 of the IL-22a gene by a neomycin-resistant gene. A 5′ arm of 1,521 bp was amplified using a mutated sense primer with a XhoI site (5′-CTTCGGCTCGAGATGGCCAC-3′) and a mutated antisense primer also containing a XhoI site (5′-GCCCTCGAGACACCAGGGTT-3′) to allow the direct insertion into the pPNT vector. The 3′ arm consisted of a 3,559-bp KpnI fragment, containing the end of exon 3 and exon 4, and was cloned.

Mice were divided into GL261 glioma implantation + IL-22 and GL261 glioma implantation + vehicle groups (n=6 per group) and the brain tissues were harvested. The mice were bred under specific pathogen-free conditions, and all experimental protocols were approved by the Institutional Animal Care and Use Committee of Hubei Cancer Hospital (Wuhan, China).

GL261 glioma cells were analyzed for proliferation using a Cell Counting kit-8 (CCK8; Dojindo Molecular Technologies, Inc., Shanghai, China). Cells were seeded into 96-well plates at densities of 1×10⁴ cells/well, and incubated in a humidified atmosphere containing 5% CO₂ and 95% air overnight. Normal cell medium containing either IL-22 or 0.01 M PBS vehicle at the desired concentration were added to the cells. After 72 h incubation, 10 µl WST-8 from CCK8 (5 g/l in PBS) was added. The plates were incubated for 4 h and the blue dye formed was dissolved in 100 µl dimethyl sulfoxide. Absorbance at 450 nm was recorded using an ELISA reader.

The cells were stained with propidium iodide (PI; BD Biosciences, San Jose, CA, USA) and cell death was evaluated according to the manufacturer's instructions. Briefly, cells were collected, washed with cold PBS and suspended in binding buffer (0.1M Hepes (pH 7.4), 1.4M NaCl and 25 mM CaCl₂ in solution; BD Biosciences). Following staining with 10 µl PI, the cells were analyzed using a FACScan flow cytometer (BD Biosciences).

The levels of IL-6, IL-1β, and tumor necrosis factor (TNF)-α in the brains of the mice were measured in brain tissue using ELISA kits (R&D Systems, Inc., Minneapolis, MN, USA), according to the manufacturer's instructions.

GL261 glioma cells were treated with IL-22 in vitro and cultured for 8 h. IL-22 and IL-22 receptor (IL-22BP) mRNA expression levels in the brains of GL261 glioma-inoculated mice on days 0, 7 and 14 were evaluated by RT-qPCR. Total RNA was extracted from the cells using an RNeasy mini kit (Qiagen China Co., Ltd., Beijing, China) according to the manufacturer's instructions. RT to cDNA was carried out using a Superscript III First Strand Synthesis kit (Invitrogen; Thermo Fisher Scientific, Inc.). qPCR was performed on an amplifier using real time PCR mix (Bio-Rad Laboratories, Inc., Hercules, CA, USA), RT products, 7.5 µl 2X iQSYBR Green mix (Bio-Rad Laboratories, Inc.), 300 nM forward and reverse primers and nanopure water to a final volume of 15 µl. Primer sequences were as follows: IL-22, forward AAG CAT TGC CTT CTA GGT CTCC and reverse TCA GAG ATA CAC GAG CTG GTT; IL-22BP, forward CAT TGC CTT CTA GGT CTC CTCA and reverse CCT GCT TGC CAG TGC AAAAT; STAT3, forward CAA TAC CAT TGA CCT GCC GAT and reverse GAG CGA CTC AAA CTG CCCT; STAT4, forward GCA GCC AAC ATG CCT ATCCA and reverse TGG CAG ACA CTT TGT GTT CCA; STAT6, forward CTC TGT GGG GCC TAA TTT CCA and reverse CAT CTG AAC CGA CCA GGAAC; Ki67, forward CGC AGG AAG ACT CGC AGTTT and reverse CTG AAT CTG CTA ATG TCG CCAA; and GAPDH, forward AAT GGA TTT GGA CGC ATT GGT and reverse TTT GCA CTG GTA CGT GTT GAT. PCR cycling conditions were as follows: 3 min at 95°C for the polymerase activation, 45 cycles of 10 sec at 95°C for denaturation, and 30 sec at 60°C for annealing and extension, followed by a DNA dissociation curve for the determination of the amplicon specificity. Analyses were performed in triplicate and water was used to replace DNA in samples used as negative controls. Data were analyzed using the Cq value normalized to the GAPDH endogenous reference gene. Data was collected and computed by iQ5 BioRad software with an automated analysis of the baseline and threshold of each run, and then exported in Excel files for further analyses.

The data are presented as the means ± standard deviation. Statistical differences were determined using a two-tailed paired Student's t-test. SPSS software version 17.0 (SPSS, Inc., Chicago, IL, USA) was used to carry out the statistical analyses. P<0.05 was considered to indicate a statistically significant result.

To determine the efficacy of IL-22 cytokine as a therapeutic treatment in the murine model of glioma, the expression levels of IL-22 were measured in the brain of GL261 glioma cell-inoculation mice at days 7 and 14. Significantly increased mRNA expression levels of IL-22 and IL-22BP were detected at day 7 and 14 compared with day 0 in the mouse glioma model (IL-22: day 7 vs. day 0, P=0.001; day 14 vs. day 0, P<0.001; and IL-22BP: day 7 vs. day 0, P=0.009; day 14 vs. day 0, P<0.001; Fig. 1A). To detect the biological function of IL-22 in the brain, IL-22 or vehicle were directly injected into the brain of normal mice to exclude the cytotoxic effect of IL-22 on the brain, the mice survived after local IL-22 treatment (data not shown). To assess the cytotoxic of IL-22 in vivo, IL-22 was injected into the healthy mice without glioma implantation, and the mice did not die after the single IL-22 injection. However, when the GL261 glioma cell was implanted into the cerebral hemisphere, the mice displayed severe disease following IL-22 injection, and significantly increased numbers of mice died compared with the vehicle-treated group (P=0.008; Fig. 1B). In addition, treatment with IL-22 significantly increased the expression levels of IL-6 (P=0.011), IL-1β (P<0.001) and TNF-α (P=0.018) in the brains of the mice, suggesting that IL-22 amplifies the immune response which is responsible for the brain tumor development in vivo (Fig. 1C)

To elucidate the mechanism underlying the pathogenic role of IL-22 in glioma, the effect of IL-22 was examined in vitro. Firstly, the expression levels of the IL-22 receptor (IL-22BP) were determined. The expression of IL-22BP in the GL261 glioma cell line was detected, but IL-22 itself did not change the expression after IL-22 stimulation in vivo (Fig. 2A). The expression levels of the genes of the JAK/STAT signaling pathway were also investigated, and the cells were treated with 100 ng/ml IL-22 in vitro. IL-22 promoted the proliferation of glioma in vitro, which was indicated by the high expression levels of Ki67. As shown in Fig. 2B, IL-22 induced the expression of STAT3 (P=0.001) and STAT6 (P=0.006), but had no effect on STAT4 expression in GL261 cells, suggesting that IL-22 modulates neuronal inflammatory proteins through activation of STAT3/STAT6 in the JAK/STAT signaling pathway. Furthermore CCK8 staining demonstrated that IL-22 promoted cell proliferation (Fig. 2C), results which were supported by the increase in Ki67 expression observed using qPCR (P<0.001; Fig. 2D). In addition, the cell death of the IL-22-treated cells was examined, and no significant difference was observed (Fig. 2E).

To further evaluate the effect of IL-22 on glioma, the phenotype of IL-22 KO and IL-22 wild-type (WT) mice were compared in vivo. First, the expression levels of IL-22 in the glioma and brain of the recipient mice were investigated to confirm the efficacy of the IL-22 KO, which indicated that IL-22 KO mice exhibited significantly diminished IL-12 expression in the brain (P<0.001; Fig. 3A). The KO and WT mice were inoculated intracerebrally with GL261 glioma, and then the mice were observed for clinical symptoms. The survival of the IL-22 KO mice with glioma was significantly prolonged compared with the IL-22 WT mice (Fig. 3B). None of the surviving animals exhibited neurological disabilities. Tumor growth was observed to be the cause of death for all of the deceased animals. Furthermore, the inflammatory cytokines including IL-6 (P=0.004), IL-1β (P=0.001) and TNF-α (P<0.001) were significantly reduced in the IL-22 KO mice (Fig. 3C), suggesting that the absence of IL-22, inflammatory cytokines which may be pathogenic for tumor development have decreased in the brain of glioma mice.

In order to develop a therapeutic approach for the treatment of IL-22, anti-IL-22 mAb was utilized. Similar to IL-22 KO mice, the anti-IL-22 antibody alleviated the symptoms of mice glioma model, as identified by the higher survival percentage (Fig. 4A), and anti-IL-22 mAb reduced the levels of IL-6 (P<0.001), IL-1β (P<0.001) and TNF-α (P<0.001) inflammatory cytokines in the brain tissue, supporting the evidence for a role of anti-IL-22 mAb in glioma (Fig. 4B).