Indirubin Increases CD4+CD25+Foxp3+ Regulatory T Cells to Prevent Immune Thrombocytopenia in Mice

PLOS ONE, Dec 2019

Indirubin, a traditional Chinese medicine, is used to treat autoimmune diseases in clinics. However, the effects of indirubin on the immunosuppressive CD4+CD25+Foxp3+ regulatory T cells (Treg) have not been addressed. Thus, we aimed to investigate the effects of indirubin on CD4+CD25+Treg cells in immune thrombocytopenia (ITP) CBA mice, which were established by immunization with Wistar rat platelets. 50 mg/kg indirubin treatment daily for 4 weeks significantly decreased anti-platelet antibody production and prevented the decrease of platelets caused by immunization in ITP mice. Consistently, indirubin significantly enhanced the percentage and cell number of CD4+CD25+Foxp3+Treg cells in the peripheral blood, spleen and lymph nodes. We also observed a significant increase of the frequency and cell number of CD4+CD25+Foxp3+Treg cells in the thymus upon indirubin treatment. Furthermore, CD4+CD25+Treg cells from indirubin-treated mice showed similar immunosuppression on T effector cells as compared to those from control mice. Altogether, indirubin ameliorates ITP by enhancing CD4+CD25+Foxp3+Treg cell level with preserving immunosuppressive function.

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Indirubin Increases CD4+CD25+Foxp3+ Regulatory T Cells to Prevent Immune Thrombocytopenia in Mice

November + + + Indirubin Increases CD4 CD25 Foxp3 Regulatory T Cells to Prevent Immune Thrombocytopenia in Mice Aijun Zhang 0 1 Bin Ning 0 1 Nianzheng Sun 0 1 Jianlu Wei 0 1 Xiuli Ju 0 1 0 1 Department of Pediatrics, Qilu Hospital, Shandong University , Jinan , China , 2 Department of Orthopaedic, Jinan Central Hospital, Shandong University , Jinan , China 1 Editor: Guangwei Liu, Fudan University , CHINA Indirubin, a traditional Chinese medicine, is used to treat autoimmune diseases in clinics. However, the effects of indirubin on the immunosuppressive CD4+CD25+Foxp3+ regulatory T cells (Treg) have not been addressed. Thus, we aimed to investigate the effects of indirubin on CD4+CD25+Treg cells in immune thrombocytopenia (ITP) CBA mice, which were established by immunization with Wistar rat platelets. 50 mg/kg indirubin treatment daily for 4 weeks significantly decreased anti-platelet antibody production and prevented the decrease of platelets caused by immunization in ITP mice. Consistently, indirubin significantly enhanced the percentage and cell number of CD4+CD25+Foxp3+Treg cells in the peripheral blood, spleen and lymph nodes. We also observed a significant increase of the frequency and cell number of CD4+CD25+Foxp3+Treg cells in the thymus upon indirubin treatment. Furthermore, CD4+CD25+Treg cells from indirubin-treated mice showed similar immunosuppression on T effector cells as compared to those from control mice. Altogether, indirubin ameliorates ITP by enhancing CD4+CD25+Foxp3+Treg cell level with preserving immunosuppressive function. Data Availability Statement; All relevant data are within the paper - OPEN ACCESS Introduction Immune thrombocytopenia (ITP) is a common autoimmune bleeding disorder characterized by persistent thrombocytopenia in children, caused by the production of anti-platelet autoantibody against platelet membrane glycoproteins which mediates the destruction of platelets in the reticuloendothelial system, especially in the spleen [ 1 ]. It has been reported that decreased number and/or function of CD4+CD25+ Foxp3+ regulatory T (Treg) cells, which are crucial for self-tolerance, represent one possible mechanism leading to the development of ITP [ 2,3 ]. Indirubin is a traditional Chinese medicine currently used for the treatment of chronic myelocytic leukemia [ 4 ] and certain autoimmune conditions and anti-inflammatory therapy [ 5,6 ]. Indirubin is a potent cyclin-dependent kinases (CDKs) and glycogen synthetase kinase 3 (GSK-3) inhibitor and suppresses tumor necrosis factor (TNF)-induced NF-κB activation [7]. However, the effects of indirubin on the immunosuppressive CD4+CD25+ Treg cells have not Competing Interests: The authors have declared that no competing interests exist. Abbreviations: ITP, immune thrombocytopenia; CTLA4, cytotoxic T-lymphocyte–associated protein 4; FCM, flow cytometry; FITC, fluorescein isothiocyanate; Foxp3, forkhead box protein 3; GITR, glucocorticoid-induced tumor necrosis factor receptor; MLR, mixed leukocyte reactions; PBMCs, peripheral blood mononuclear cells; PE, phycoerythrin; PI, propidium iodide; Treg, regulatory T cells. been addressed. In the present study, we investigated the effect of indirubin on CD4+CD25+Treg cells in experimental ITP mice. Materials and Methods Animals Six-to-eight weeks old female CBA mice were purchased from Model Animal Research Center (Najing, China). Wistar rats were obtained from The Laboratory Animal Center Academy of Military Medical Sciences Genetics (Jinan, China). Mice were maintained for 2 weeks prior experimentation in a specific pathogen-free(SPF) animal facility and were housed in macroisolator cages containing sterilized feed, autoclaved bedding, and water at 20 temperature and 40% humidity condition in the Experimental Animal Centre of Qilu Hospital Shandong University. All the experimental procedures were approved by the Animal Care and Use Committee of Qilu Hospital and conducted under the guidelines for Animal Care and Use of Shandong University, China. All surgery was performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering. Monoclonal antibodies (mAbs) and reagents The following mAbs were purchased from BD Biosciences PharMingen (San Diego, CA): FITC-labeled rat anti-mouse CD25 mAb (7D4; IgM), Fluorescein isothiocyanate (FITC)conjugated anti-mouse CD4 mAb (RM4-5; rat IgG2a), FITC-labeled anti-mouse CD8 mAb (53–6.7; rat IgG2a), phycoerythrin (PE)-labeled rat anti-mouse CD4 mAb(clone GK1.5), PElabeled anti-mouse CD25 mAb, and PE-labeled anti-mouse CD8α mAb (53–6.7; rat IgG2a). In addition, PE-labeled anti-mouse Foxp3 mAb (FJK-16s) and its staining kit were obtained from eBiosciences (San Diego, CA). The culture medium used in the present study was RPMI 1640 (Hyclone, Logan, UT) supplemented with 10% heat-inactivated FCS (Hyclone, Logan, UT), 100U/ml penicillin, 100μg/ ml streptomycin, 2mM L-glutamine, 10mM HEPES, 1mM sodium pyruvate and (...truncated)


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Aijun Zhang, Bin Ning, Nianzheng Sun, Jianlu Wei, Xiuli Ju. Indirubin Increases CD4+CD25+Foxp3+ Regulatory T Cells to Prevent Immune Thrombocytopenia in Mice, PLOS ONE, 2015, Volume 10, Issue 11, DOI: 10.1371/journal.pone.0142634