Basic Study
Copyright ©The Author(s) 2019. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Dec 7, 2019; 25(45): 6634-6652
Published online Dec 7, 2019. doi: 10.3748/wjg.v25.i45.6634
Toxoplasma ROP16I/III ameliorated inflammatory bowel diseases via inducing M2 phenotype of macrophages
Yong-Wei Xu, Rui-Xin Xing, Wen-Hui Zhang, Lu Li, Yi Wu, Jing Hu, Cong Wang, Qing-Li Luo, Ji-Long Shen, Xi Chen
Yong-Wei Xu, Rui-Xin Xing, Wen-Hui Zhang, Lu Li, Yi Wu, Jing Hu, Xi Chen, Department of Gastroenterology, the First Affiliated Hospital of Anhui Medical University, Hefei 230022, Anhui Province, China
Cong Wang, Qing-Li Luo, Ji-Long Shen, Department of Pathogen Biology, Provincial Laboratory of Pathogen Biology and Zoonoses Anhui, Anhui Medical University, Hefei 230032, Anhui Province, China
Author contributions: Chen X, Sheng JL and Xu YW conceived and designed the trial; Xu YW, Li L, Xing RX and Wu Y performed the experiments; Xu YW, Zhang WH, Hu J, Wang C and Luo QL analysed the data; Xu YW wrote the manuscript; Chen X, Sheng JL and Xu YW critically revised the manuscript; All authors have read and approved the final manuscript.
Supported by the National Natural Science Foundation of China, No. 81471983; the Key Research and Development Plan Project of Anhui Province, Department of Science and Technology 2019, No. 201904a07020043; the Key Project of Natural Science Research in the Universities of Anhui Provence, No. KJ2017A202; and the Research Fund Project of Anhui Institute of Transforming Medicine, No. 2017zhyx04.
Institutional review board statement: This study does not involve human and animal subjects.
Institutional animal care and use committee statement: No animal models were used in this study.
Conflict-of-interest statement: None of the authors has any conflicts of interest to declare.
Data sharing statement: No additional data are available.
ARRIVE guidelines statement: The ARRIVE guidelines have been adopted.
Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See:
Corresponding author: Xi Chen, MD, PhD, Chief Doctor, Dean, Director, Professor, Senior Researcher, Department of Gastroenterology, the First Affiliated Hospital of Anhui Medical University, No. 218 Ji Xi Road, Hefei 230022, Anhui Province, China.
Telephone: +86-551-65908802 Fax: +86-551-65908802
Received: August 9, 2019
Peer-review started: August 9, 2019
First decision: September 19, 2019
Revised: October 3, 2019
Accepted: November 13, 2019
Article in press: November 13, 2019
Published online: December 7, 2019

Inflammatory bowel disease (IBD) is characterized by chronic and non-specific inflammation of the intestinal mucosa and mainly includes ulcerative colitis and Crohn's disease.


To explore the beneficial effect of ToxoROP16I/III-induced M2 phynotype macrophages in homeostasis of IBDs through downregulation of M1 inflammatory cells.


RAW264.7 macrophages stimulated by lipopolysaccharide (LPS) (M1 cells) were co-cultured with Caco-2 cells as an inflammatory model of IBD in vitro. The expression of ToxoROP16I/III was observed in RAW264.7 macrophages that were transfected with pEGFP-rop16I/III. The phenotypes of M2 and M1 macrophage cells were assessed by quantitative real-time reverse transcriptase polymerase chain reaction and the expression of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, transforming growth factor (TGF)-β1, IL-10, inducible nitric oxide synthase (iNOS), and arginase-1 (Arg-1) was detected. The expression of iNOS, Arg-1, signal transducer and activator of transcription 3 (Stat3), p-Stat3, Stat6, p-Stat6, programmed death ligand-2 (PD-L2), caspase-3, -8, and -9 was analyzed by Western blotting, and Griess assays were performed to detect nitric oxide (NO). TNF-α, IL-1β, IL-6, TGF-β1, and IL-10 expression in the supernatants was detected by enzyme-linked immunosorbent assay, and Caco-2 cell apoptosis was determined by flow cytometry after mixing M1 cells with M2 cells in a Caco-2 cell co-culture system.


M1 cells exhibited significantly increased production of iNOS, NO, TNF-α, IL-1β, and IL-6, while ToxoROP16I/III induced macrophage bias to M2 cells in vitro, showing increased expression of Arg-1, IL-10 and TGF-β1 and elevated production of p-Stat3 and p-Stat6. The mixed M1 and M2 cell culture induced by ToxoROP16I/III exhibited decreased production of NO and iNOS and upregulated expression of Arg-1 and PD-L2. Accordingly, Caco-2 cells became apoptotic, and apoptosis-associated proteins such as caspase-3, -8 and -9 were dampened during co-culture of M1 and M2 cells. Flow cytometry analysis showed that co-culture of M1 cells with Caco-2 cells facilitated the apoptosis of Caco-2 cells, but co-culture of M1 and M2 cells alleviated Caco-2 cell apoptosis.


ToxoROP16I/III-induced M2 macrophages inhibited apoptosis of Caco-2 cells caused by M1 macrophages. This finding may help gain a better understanding of the underlying mechanism and represent a promising therapeutic strategy for IBDs.

Keywords: Toxoplasma ROP16I/III, Caco-2, Inflammatory bowel disease, Immunity, Classically activated macrophages, Alternatively activated macrophages

Core tip:Toxoplasma ROP16I/III (ToxoROP16I/III) induced RAW264.7 polarization to M2 macrophage, down-regulated the M1-associated inflammation response and protective Caco-2 intestinal epithelial cells. ToxoROP16I/III can phosphorylate and activate the transcription factors signal transducer and activator of signal transducer and activator of transcription (Stat) 3 and Stat6, promote the polarization of M2 cells, and enhance the synthesis of arginase-1, interleukin (IL)-10, transforming growth factor-β1, and IL-13. The IL-1β, TNF-α, IL-6, nitric oxide (NO), and inducible nitric oxide synthase(iNOS)produced by M1 cells were notably downregulated when the ToxoROP16I/III-induced M2 macrophages were added to the mixture of culture.Co-culture with Caco-2 cells through transwell alleviated Caco-2 cell apoptosis and caspase-3, -8, and -9 associated proteins. This study aims to confirm that ToxoROP16I/III may provide a novel strategy for IBD immuno-therapy with parasite-derived effector molecules.