Virulizin induces production of IL-17E to enhance antitumor activity by recruitment of eosinophils into tumors

Abstract

Virulizin has demonstrated strong antitumor efficacy in a variety of human tumor xenograft models including melanoma, pancreatic cancer, breast cancer, ovarian cancer and prostate cancer. Our previous studies have demonstrated that macrophages, NK cells, and cytokines are important in the antitumor mechanism of Virulizin. Virulizin treatment of tumor bearing mice results in the expansion as well as increased activity of monocytes/macrophages and production of cytokines IL-12 and TNFalpha and activation of NK cells. In this study we show that the inflammatory cytokine IL-17E (IL-25) is induced by Virulizin treatment and is part of its antitumor mechanism. IL-17E is a proinflammatory cytokine, which induces a T(H)2 type immune response, associated with eosinophil expansion and infiltration into mucosal tissues. IL-17E was increased in sera of Virulizin-treated mice bearing human melanoma xenografts, compared to saline-treated controls, as shown by 2D gel electrophoresis and ELISA. Treatment of splenocytes in vitro with Virulizin resulted in increased IL-17E mRNA expression, which peaked between 24 and 32 h post-stimulation. Both in vitro and in vivo experiments demonstrated that B cells produced IL-17E in response to Virulizin treatment. Furthermore, Virulizin treatment in vivo resulted in increased blood eosinophilia and eosinophil infiltration into tumors. Finally, injection of recombinant IL-17E showed antitumor activity towards xenografted tumors, which correlated with increased eosinophilia in blood and tumors. Taken together, these results support another antitumor mechanism mediated by Virulizin, through induction of IL-17E by B cells, leading to recruitment of eosinophils into tumors, which may function in parallel with macrophages and NK cells in mediating tumor destruction.

Fig. 1

Identification of IL-17E in mouse serum following treatment with Virulizin^®. Human melanoma C8161 cells were subcutaneously injected into the right flank of CD-1 nude mice. The mice were then injected intraperitoneally with 0.2 ml of PBS or Virulizin^® daily for 4 weeks. Serum was then collected and analyzed by 2D gel electrophoresis and silver staining. Stained 2D gels are shown from mice treated with (a) PBS and (b) Virulizin^®. One spot (circle in b) that had higher expression in Virulizin^®-treated versus PBS-treated mice was excised and sequenced. c SDS-PAGE and Western blot analysis of serum from treated mice. The serum samples were run onto SDS-PAGE gels and proteins were immunoblotted with antibodies against IL-17E (c). Western blots of sera from three mice selected at random from each group (Virulizin^® or PBS) are shown. Blots were probed with antibodies to β-actin to standardize levels of serum protein among wells

Fig. 2

Increased serum IL-17E in mice treated with Virulizin^®. Nude mice bearing human melanoma C8161 xenografts were injected with 0.2 ml of PBS (n = 8) or Virulizin^® (n = 9) daily for 4 weeks. At the end of the experiment serum was collected from mice. IL-17E levels in the sera were determined using ELISA as described in the text. The data represent 3 independent experiments. The detection limit for mouse IL-17E in this assay was <2 pg/μl. *P < 0.05 compared to PBS control

Fig. 3

Quantitation of IL-17E mRNA in splenocytes following treatment with Virulizin^® in vitro. Splenocytes were isolated from C57BL/6 mice, and plated in triplicate in 6 well tissue culture plates after red blood cell lysis. The cells were treated with or without 5% Virulizin^® for various time points. At each time point, adherent and nonadherent cells were harvested and pooled, RNA was extracted by Trizol method, followed by cDNA preparation. Real time PCR was subsequently performed from the cDNA. Values were normalized to β-actin controls, and then compared to unstimulated samples. Results represent 5–6 experiments per time point. *P < 0.05 compared to PBS control

Fig. 4

Virulizin^® induces IL-17E expression in splenic B cells in vitro. Splenocytes were isolated and plated in 6 well culture with a no stimulation or b 5% Virulizin. After 72 h, cells were harvested, surface stained with anti-IgM antibodies, followed by intracellular staining using anti-IL-17E coupled to biotin, followed by phycoerythrin (PE)-Cy5.5-conjugated streptavidin. Samples were then analyzed by flow cytometry. Percentages of double positive IgM and IL-17E cells are shown in the top right quadrant of each dot plot. The average of 3 separate wells per group was tabulated (c). *P < 0.05

Fig. 5

Virulizin^® induces IL-17E expression in splenic B cells in tumor-bearing nude mice. CD1 nude mice bearing human melanoma C8161 xenograft were treated with either PBS or Virulizin^® (undiluted) daily for 4 weeks (0.2 ml/day). Splenocytes were isolated and surface stained with anti-IgM followed by intracellular staining for IL-17E. Representative dot plots from flow cytometry analysis of splenocytes from mice treated with PBS (a) and Virulizin (b) are shown. Percentages of double positive IgM and IL-17E cells are shown in the top right quadrant of each plot. The mean percentage of IL-17E positive stained cells from IgM+ gated cells was tabulated (c) (*P = 0.04; n = 5)

Fig. 6

Virulizin^® treatment results in expansion and activation of splenic B cells. Splenocytes from tumor-bearing mice treated daily for 4 weeks with undiluted Virulizin^® or PBS (0.2 ml/day) were isolated and surface stained with either a anti-IgM FITC for total B cell counts, or b anti-IgM FITC + anti-CD80 PE + anti-CD86-PE to measure numbers of activation B cells. As shown in both panels, the percentage of B cells (both total and activated) was significantly increased (P < 0.05) in Virulizin^® treated mice

Fig. 7

Virulizin^® induces blood eosinophilia. Peripheral blood from tumor-bearing nude mice treated daily with either undiluted Virulizin^® or PBS (0.2 ml/day) was collected and red blood cells were removed. The remaining cells were surface stained with anti-CCR3 antibody conjugated with PE and analyzed by flow cytometry. **P < 0.001; n = 31

Fig. 8

Virulizin^® treatment results in increased recruitment of eosinophils into tumors. CD1 nude mice bearing C8161 xenografts were treated with either PBS or undiluted Virulizin^® daily (0.2 ml/day) for 4 weeks. C8161 tumors from treated mice were collected and paraffin sections of tumors were prepared and stained for eosinophils using Sirius Red. a Representative micrographs of sections show stained eosinophils in tumors from mice treated with PBS or Virulizin^®. A scale bar in each panel represents 25 μm. The number of eosinophils per square millimeter of tumor tissue (b) was determined by computer-assisted image analysis. Each bar in the graph represents the mean ± SEM of determinations in six samples of the same treatment. ***P < 0.05 compared to PBS control

Fig. 9

Quantitation of cytokines IL-5 and eotaxin-1 in human tumor xenografts in mice. Nude mice bearing C8161 melanoma xenografts were treated daily with either undiluted Virulizin^® or PBS (0.2 ml/day) for 4 weeks. C8161 xenograft tumors from treated mice were excised and total RNA was prepared from tumor tissues, followed by preparation of cDNAs and quantitative PCR to measure levels of cytokine mRNAs. Levels of IL-5 mRNA (a) and eotaxin-1 mRNA (b) in tumor tissues were both significantly higher in tumors from mice treated with Virulizin^®. Mean values are shown, normalized to levels of β-actin. *P < 0.05 compared to PBS control

Fig. 10

Quantitation of cytotoxic eosinophil granule proteins in human tumor xenografts in mice following treatment with Virulizin^®. C8161 tumor xenografts were excised from mice treated daily with either undiluted Virulizin^® or PBS (0.2 ml/day) for 4 weeks, and total RNA was prepared and used for cDNA preparation. Real-time PCR was performed to quantitate eosinophil peroxidase (EPO) (a) EPO or major basic protein (MBP) (b). Levels of both EPO and MBP were increased in tumors from Virulizin^® treated mice. Mean values are shown, normalized to levels of β-actin. *P < 0.05 compared to PBS control

Fig. 11

IL-17E has anticancer activity in vivo. CD-1 nude mice bearing human melanoma C8161 xenografts were treated for 4 weeks with either PBS or Virulizin^® daily (0.2 ml/day), or 10 days with recombinant mouse IL-17E (1 μg/mouse), or recombinant mouse IL-17E plus Virulizin^® for the first 10 days followed by Virulizin^® daily for the remainder of the treatment period (4 weeks). Tumor weights were measured at the endpoint of the experiment. Weights of tumors from mice treated with Virulizin, IL-17E, or a combination of both were significantly reduced compared to those from PBS-treated mice [P values: 0.02, 0.02 and 0.004 for Virulizin^®, IL-17E, and IL-17E plus Virulizin^®, respectively. (n = 9)]. Tumor weights from mice treated with both Virulizin^® and IL-17E were decreased compared to tumors from mice treated with either agent alone, although this difference was not statistically significant