The immune-stimulating peptide WKYMVm has therapeutic effects against ulcerative colitis

OPEN Experimental & Molecular Medicine (2013) 45, e40; doi:10.1038/emm.2013.77 & 2013 KSBMB. All rights reserved 2092-6413/13 www.nature.com/emm ORIGINAL ARTICLE The immune-stimulating peptide WKYMVm has therapeutic effects against ulcerative colitis Sang Doo Kim1,2, Soonil Kwon1,2, Sung Kyun Lee1,2, Minsoo Kook1,2, Ha Young Lee1,2, Ki-Duk Song3, Hak-Kyo Lee3, Suk-Hwan Baek4, Chan Bae Park2,5 and Yoe-Sik Bae1,2,6 In this study, we examined the therapeutic effects of an immune-stimulating peptide, WKYMVm, in ulcerative colitis. The administration of WKYMVm to dextran sodium sulfate (DSS)-treated mice reversed decreases in body weight, bleeding score and stool score in addition to reversing DSS-induced mucosa destruction and shortened colon. The WKYMVm-induced therapeutic effect against ulcerative colitis was strongly inhibited by a formyl peptide receptor (FPR) 2 antagonist, WRWWWW, indicating the crucial role of FPR2 in this effect. Mechanistically, WKYMVm effectively decreases intestinal permeability by stimulating colon epithelial cell proliferation. WKYMVm also strongly decreases interleukin-23 and transforming growth factor-b production in the colon of DSS-treated mice. We suggest that the potent immune-modulating peptide WKYMVm and its receptor FPR2 may be useful in the development of efficient therapeutic agents against chronic intestinal inflammatory diseases. Experimental & Molecular Medicine (2013) 45, e40; doi:10.1038/emm.2013.77; published online 13 September 2013 Keywords: therapeutics; ulcerative colitis; WKYMVm INTRODUCTION Inflammatory bowel diseases (IBDs) such as Crohn’s disease or ulcerative colitis are chronic diseases that cause inflammation of the intestine.1,2 Although the incidence of IBD varies in different countries, a gradual increase has been noted recently, and the disease is a major health problem worldwide.2,3 Although the search for the cause of IBD has been a hot issue for several decades, it is still not clear what causes IBD. Currently, various factors, including environment, diet and genetic makeup, have been suggested to be associated with IBD pathogenesis.1–3 Among these, a genetic defect that affects the response of the human immune system to offending agents, such as bacteria, viruses or proteins in food, has been associated with IBD.1–3 The host immune system is closely associated with the pathogenesis and progress of IBD.4 Both innate and adaptive immune systems are critically involved in the response to intestinal microbiota.4,5 Pattern recognition receptors, such as the toll-like receptor, have roles in sensing conserved microbial molecules in the intestinal environment.4 Despite the need for efficient therapeutic molecules to treat human IBD, no cure has yet been developed. Clinically, some immunosuppressive agents that target tumor necrosis factor (TNF) are currently used to treat IBD.6 WKYMVm is a synthetic peptide that was identified by screening a peptide library.7,8 The peptide binds to at least three formyl peptide receptors (FPRs): FPR1, FPR2 and FPR3.9–11 WKYMVm stimulates the chemotactic migration of leukocytes such as neutrophils, monocytes, dendritic cells and natural killer cells.12–15 It also stimulates superoxide anion production in phagocytes including neutrophils and monocytes.12,16 Recently, we demonstrated that WKYMVm administered to a polymicrobial sepsis model had potent therapeutic activity in cecal ligation and puncture mice.17 The peptide was shown to inhibit the production of inflammatory cytokines, such as TNF-a and interleukin (IL)-1b, and augment the production of Th1 cytokines (IFN-g (interferon-g) and IL-12) to achieve this therapeutic effect against sepsis.17 Here, we investigate the effects of WKYMVm on dextran sodium sulfate (DSS)-induced ulcerative colitis, including its effects on cytokine production. 1Department of Biological Science, Sungkyunkwan University, Suwon, Korea; 2Mitochondria Hub Regulation Center, College of Medicine, Dong-A University, Busan, Korea; 3Genomic Informatics Center, Hankyong National University, Anseong, Korea; 4Department of Biochemistry and Molecular Biology, College of Medicine, Yeungnam University, Daegu, Korea; 5Institute for Medical Sciences, Ajou University School of Medicine, Suwon, Korea and 6Samsung Advanced Institute for Health Sciences and Technology, Sungkyunkwan University, Seoul, Korea Correspondence: Professor Y-S Bae, Department of Biological Science, Sungkyunkwan University, Suwon 440-746, Korea. E-mail: Received 20 May 2013; revised 18 June 2013; accepted 24 June 2013 Therapeutic effects of WKYMVm on colitis SD Kim et al 2 MATERIALS AND METHODS Animals and DSS-treated ulcerative colitis model Six-week-old C57BL/6 mice were obtained from Orient Bio Inc. (Seongnam, Korea). After adapting for 1 week following arrival, the mice were given drinking water containing 3% DSS (m.w. 36 000– 50 000; Sigma-Aldrich, St Louis, MO, USA) w/v for 5 days followed by fresh water until the end of the experiment on the 8th day. WKYMVm (Anygen, Gwangju, Korea) or vehicle (phosphate-buffered saline) was subcutaneously injected into DSS mice six times (at 0, 12, 24, 36, 48 and 60 h after DSS treatment). Body weight, rectal bleeding and stool score (stool consistency or diarrhea) were measured daily according to a previous report.18 Histology The mice were subjected to DSS treatment and were administered with phosphate-buffered saline or WKYMVm at a dose of 8 mg kg 1. The mice were euthanized 8 days after DSS treatment, and the intestines were fixed, sectioned and stained with hematoxylin and eosin for morphological analysis. Measurement of intestine permeability Food was withdrawn from the mice for 6 h, and the animals were gavaged with fluorescein isothiocyanate–dextran (10 mg per head; Sigma-Aldrich). Serum was collected retro-orbitally 4 h after the gavage. Fluorescein isothiocyanate–fluorescence was measured using the Gemini XPS fluorescence microplate reader (Molecular Devices, Sunnyvale, CA, USA). Cell proliferation assay Caco-2 human epithelial colorectal adenocarcinoma cells were cultured with Dulbecco’s modified Eagle’s medium containing 20% fetal bovine serum (Life Technologies, Grand Island, NY, USA). Healthy cultured cells were seeded at 4  103 cells per well. After 24 h, WKYMVm was added at several concentrations (0, 10, 100 and 1000 nM) in the absence or presence of cyclosporine H (CsH, 1 mM) or WRWWWW (WRW4, 10 mM) for 24 h. Quantification of cell proliferation was performed using the Cell Counting Kit-8 (Dojindo Molecular Technologies Inc., Rockville, MD, USA). Wound healing assay Caco-2 human epithelial colorectal adenocarcinoma cells were cultured with Dulbecco’s modified Eagle’s medium containing 20% fetal bovine serum. Vehicle or WKYMVm (1 mM) was added into a scratched Caco-2 cell layer for 0, 12 or 24 h. Images were obtained using a digital camera attached to a light microscope. Measurement of colon cytokines To measure DSS treatmen (...truncated)