Resveratrol Prevents Endothelial Cells Injury in High- Dose Interleukin-2 Therapy against Melanoma Hongbing Guan, Narendra P. Singh, Udai P. Singh, Prakash S. Nagarkatti, Mitzi Nagarkatti* Department of Pathology, Microbiology and Immunology, University of South Carolina School of Medicine, Columbia, South Carolina, United States of America Abstract Immunotherapy with high-dose interleukin-2 (HDIL-2) is an effective treatment for patients with metastatic melanoma and renal cell carcinoma. However, it is accompanied by severe toxicity involving endothelial cell injury and induction of vascular leak syndrome (VLS). In this study, we found that resveratrol, a plant polyphenol with anti-inflammatory and anti-cancer properties, was able to prevent the endothelial cell injury and inhibit the development of VLS while improving the efficacy of HDIL-2 therapy in the killing of metastasized melanoma. Specifically, C57BL/6 mice were injected with B16F10 cells followed by resveratrol by gavage the next day and continued treatment with resveratrol once a day. On day 9, mice received HDIL-2. On day 12, mice were evaluated for VLS and tumor metastasis. We found that resveratrol significantly inhibited the development of VLS in lung and liver by protecting endothelial cell integrity and preventing endothelial cells from undergoing apoptosis. The metastasis and growth of the tumor in lung were significantly inhibited by HDIL-2 and HDIL-2 + resveratrol treatment. Notably, HDIL-2 + resveratrol co-treatment was more effective in inhibiting tumor metastasis and growth than HDIL-2 treatment alone. We also analyzed the immune status of Gr-1+CD11b+ myeloid-derived suppressor cells (MDSC) and FoxP3+CD4+ regulatory T cells (Treg). We found that resveratrol induced expansion and suppressive function of MDSC which inhibited the development of VLS after adoptive transfer. However, resveratrol suppressed the HDIL-2-induced expansion of Treg cells. We also found that resveratrol enhanced the susceptibility of melanoma to the cytotoxicity of IL-2-activated killer cells, and induced the expression of the tumor suppressor gene FoxO1. Our results suggested the potential use of resveratrol in HDIL-2 treatment against melanoma. We also demonstrated, for the first time, that MDSC is the dominant suppressor cell than regulatory T cell in the development of VLS. Citation: Guan H, Singh NP, Singh UP, Nagarkatti PS, Nagarkatti M (2012) Resveratrol Prevents Endothelial Cells Injury in High-Dose Interleukin-2 Therapy against Melanoma. PLoS ONE 7(4): e35650. doi:10.1371/journal.pone.0035650 Editor: Ali A. Ashkar, McMaster University, Canada Received January 2, 2012; Accepted March 19, 2012; Published April 20, 2012 Copyright: 2012 Guan et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported in part by National Institute of Health Grants R01 AI053703, R01 AI058300, R01 HL058641, and P01 AT003961. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: Mitzi.Nagarkatti@uscmed.sc.edu Introduction High-dose interleukin-2 (HDIL-2) therapy which produces overall response rates of 15% to 23% remains an effective and long-lasting treatment for metastatic renal cell carcinoma and melanoma [1,2]. However, it is associated with significant systemic toxicity, mainly, vascular leak syndrome (VLS), characterized by increased vascular permeability and decreased microcirculatory perfusion that causes extensive fluid retention in multiple organs and can lead to pulmonary and cardiovascular failure [3,4,5] Although it is dose-dependent and reversible with therapy discontinuation, there is no specific and effective treatment regimen. The endothelial cell damage is the major feature of the vascular pathology. Direct effects of IL-2 on endothelial cells [6,7], or through induction of inflammatory cytokines such as TNF-a, IL-1 and IFN-c [3], have been previously reported. Otherwise, the cytotoxic effects by lymphokine-activated killer cells (LAK) are the main cause of vascular injury [8,9,10,11]. Immunoregulation associated with the development of IL-2- induced VLS is not known. Our lab has reported that T regulatory cells (Treg) were amplified in vivo by IL-2 treatment; they suppress the cytolytic killing of endothelial cells by LAK cells in the in vitro experiments, thereby suggesting that Treg play a role in the negative regulation on the development of HDIL-2-induced VLS [12]. Lately, functional importance of myeloid-derived suppressor cells (MDSC) in immune responses has been appreciated. MDSC are a heterogeneous population of cells consisting of myeloid progenitor cells and immature myeloid cells. They are character- ized by the co-expression of the myeloid lineage differentiation molecule Gr1 and CD11b. These cells markedly expand systemically in pathological conditions, such as cancer, various infectious diseases and some autoimmune diseases [13,14]. Accumulating evidence demonstrates that their suppressive functionality contributes to the negative regulation of immune responses including adaptive and innate immunity, such as suppressing various T-cell functions, and NK and CTL cytotox- icity [13,14,15,16,17,18]. However, the role of MDSC in HDIL-2- induced inflammation and the development of VLS have not been elucidated. Resveratrol, a naturally occurring polyphenol found in grapes and red wine, is widely used in animal models and possesses broad-spectrum of beneficial health effects including anti-infective, anti-inflammatory, and antioxidant properties. These interesting properties confer the cardiovascular protective capacity and ability to protect endothelial function on resveratrol [19,20]. In cancer patients, resveratrol exhibits anticancer properties, such as suppression of tumor cell proliferation, induction of tumor cell apoptosis, increase in chemosensitization of tumor cells, and exerts PLoS ONE | www.plosone.org 1 April 2012 | Volume 7 | Issue 4 | e35650