Elsevier

International Immunopharmacology

Volume 39, October 2016, Pages 251-264
International Immunopharmacology

Depletion of T lymphocytes ameliorates cardiac fibrosis in streptozotocin-induced diabetic cardiomyopathy

https://doi.org/10.1016/j.intimp.2016.07.027Get rights and content

Highlights

  • Diabetic myocardium exhibited profibrotic milieu manifested by increased T cell infiltration with enhanced expression of TGF-β1 and CD34-expressing fibrocytes.

  • FTY720 inhibited profibrotic milieu in diabetic myocardium and exerted cardioprotection in diabetic C57BL/6 mice.

  • FTY720 augmented cardiac fibrosis in diabetic Rag1 knockout mice.

Abstract

T cell infiltration has been associated with increased coronary heart disease risk in patients with diabetes mellitus. Effect of modulation of T cell trafficking on diabetes-induced cardiac fibrosis has yet to be determined. Therefore, our aim was to investigate the circulatory T cell depletion-mediated cardioprotection in streptozotocin-induced diabetic cardiomyopathy. Fingolimod (FTY720), an immunomodulatory drug, was tested in wild-type (WT) C57BL/6 and recombination activating gene 1 (Rag1) knockout (KO) mice without mature lymphocytes in streptozotocin-induced type 1 diabetic model. FTY720 (0.3 mg/kg/day) was administered intraperitoneally daily for the first 4 weeks with interim 3 weeks then resumed for another 4 weeks in 11 weeks study period. T lymphocyte counts, cardiac histology, function, and fibrosis were examined in diabetic both WT and KO mice. FTY720 reduced both CD4+ and CD8+ T cells in diabetic WT mice. FTY720-treated diabetic WT mouse myocardium showed reduction in CD3 T cell infiltration and decreased expression of S1P1 and TGF-β1 in cardiac tissue. Fibrosis was reduced after FTY720 treatment in diabetic WT mice. Rag1 KO mice exhibited no CD4+ and CD8+ T cells in the blood and CD3 T cells in the heart. Diabetic Rag1 KO mouse hearts appeared no fibrosis and exhibited preserved myocardial contractility. FTY720-induced antifibrosis was abolished in diabetic Rag1 KO mice. These findings demonstrate that chronic administration with FTY720 induces lymphopenia and protects diabetic hearts in WT mice whereas FTY720 increases cardiac fibrosis and myocardial dysfunction in diabetic Rag1 KO mice without mature lymphocytes.

Introduction

Myocardial fibrosis, one of the cardinal features of diabetic cardiomyopathy, contributes to increased ventricular stiffness which is accountable for contractile dysfunction in failing diabetic hearts [1], [2]. Much progress on the cellular and molecular mechanisms of cardiac fibrosis has been achieved. Immune cells, cardiomyocytes, and endothelial cells have been proposed to contribute to the formation of fibrotic process by secreting pro-fibrotic mediators to modulate differentiation of fibroblasts to myofibroblasts [3]. Some neurohumoral factors, cytokines, and reactive oxygen species are recognized to play distinct but overlapping roles in the pathogenesis of fibrosis [4]. However, the underlying mechanism of fibrosis remains controversial.

Several patient-based studies have shown increased expansion of proinflammatory T cell subsets in diabetic patient blood that is correlated with adverse cardiac events including acute coronary syndrome [5], [6]. A recent study on murine pressure-overload-induced heart failure has shown that T lymphocyte presence in injured cardiac tissue is associated with exacerbated fibrosis [7]. T lymphocytes can activate profibrotic cells and are involved in fibrosis perpetuation by secreting proinflammatory cytokines [8]. T cells infiltration in diabetic myocardium has been observed [9]. Although results in different approaches including neutralizing antibodies or genetic knockout of signaling protein of T cells have shown reduced fibrosis in murine heart failure models, T cell-based therapeutic interventions to protect hearts against diabetic injury need to be explored. Modulation of T cell trafficking and its effect on long term diabetic fibrogenesis are still not known clearly. Further, crosstalk between T cells and other cellular components in myocardial fibrosis has not been addressed. Fibrocytes, blood derived monocyte-lineage cells that secrete increased extracellular matrix protein under chronic injury/inflammation or with appropriate external stimuli, e.g. transforming growth factor beta 1 (TGF-β1), can differentiate fibroblasts into myofibroblasts [10], [11].

Sphingosine 1-phosphate (S1P), a small bioactive lipid molecule, plays pivotal roles in many physiological processes, such as: cell migration, angiogenesis, cytoskeleton reorganization, and survival, by activating G protein-coupled S1P receptors or intracellular targets [12]. Clinical and experimental data have shown that circulating S1P is predictive for obstructive coronary artery disease and type 1 diabetes [13], [14]. S1P and S1P receptor 1 (S1P1) signaling have been implicated in inflammation-mediated myocardial injury [15]. Mature lymphocytes upregulate the expression of S1P1 for their egress from lymphoid organs to circulation [16]. FTY720 down-modulates lymphocytic S1P1 receptor, thus induces lymphopenia by sequestering them in secondary lymphoid organs [17]. FTY720 protects cardiac microvascular structure and function in diabetic rats [18], [19]. However, the role of T cells in FTY720-induced cardioprotection against diabetic injury was unknown.

We hypothesized that modulation of T cell trafficking through S1P1 functional antagonism could protect the heart from diabetes-associated fibrosis and cardiac dysfunction. In the present study, we investigated T cell trafficking modulation by using fingolimod (FTY720), an immunomodulatory drug, and recombination activating genes 1 (Rag1) knock-out mice without mature lymphocytes as a genetic approach in streptozotocin-induced type 1 diabetic cardiomyopathy model by quantifying T lymphocytes in the heart and circulation, identification of CD34-bearing fibrocyte localization in cardiac tissue, profibrotic TGF-β1 expression, S1P1 expression, heart histological study, fibrosis area measurement in heart sections, and ex vivo cardiac function evaluation.

Section snippets

Animals

This investigation was carried out in accordance with the Guide for the Care and Use of Laboratory Animals set forth by U.S. National Research Council (8th Edition, 2011). All experimental procedures involving mice in this study were approved by the Institutional Animal Care and Use Committee at South Dakota State University. Eight-week old male wild-type (WT) C57BL/6 mice were purchased from the Charles River Laboratories (Wilmington, MA, USA) and two breeding pairs of Rag1 knockout (KO) mice

Assessment of metabolic and physiological parameters under diabetes

In the present study, we used multiple low doses of STZ to induce type 1 diabetes in mice. Body weight, blood glucose levels and serum insulin levels were measured and analyzed in all groups of both WT and KO mice (Fig. 1). In WT mice, both untreated (STZ, n = 9) and FTY720-treated (FTY720 + STZ, n = 9) diabetic mice demonstrated lower body weight gain trend compared with only buffer treatment (Control, n = 8) mice throughout 11 weeks (Fig. 1B). At the end, control group gained 10% higher body weight

Discussion

The major findings of the present study indicate that chronic administration of FTY720 treatment induces sustained reduction of T lymphocytes in circulation with reduced CD3 T cell infiltration into heart tissue under hyperglycemic condition and exerts cardioprotection and anti-fibrosis in WT diabetic mice. Cardiac protection and anti-fibrosis are also observed in diabetic Rag1 KO mice without mature lymphocytes. Remarkably, FTY720 causes heart injury in diabetic Rag1 KO mice.

FTY720, a

Disclosures

No conflicts of interest, financial or otherwise are declared by the authors.

Acknowledgements

This project was supported by the Department of Pharmaceutical Sciences, College of Pharmacy, South Dakota State University and the Department of Pharmaceutical & Biomedical Sciences, College of Pharmacy, California Northstate University (Z. Jin).

References (34)

  • L. van Heerebeek et al.

    Diastolic stiffness of the failing diabetic heart: importance of fibrosis, advanced glycation end products, and myocyte resting tension

    Circulation

    (2008)
  • S.M. Grundy et al.

    Diabetes and cardiovascular disease: a statement for healthcare professionals from the American Heart Association

    Circulation

    (1990)
  • H. Bugger et al.

    Molecular mechanisms of diabetic cardiomyopathy

    Diabetologia

    (2014)
  • S. Giubilato et al.

    Expansion of CD4+ CD28null T-lymphocytes in diabetic patients: exploring new pathogenetic mechanisms of increased cardiovascular risk in diabetes mellitus

    Eur. Heart J.

    (2011)
  • R.X. Zhao et al.

    Increased peripheral proinflammatory T helper subsets contribute to cardiovascular complications in diabetic patients

    Mediat. Inflamm.

    (2014)
  • T. Nevers et al.

    Left ventricular T cell recruitment contributes to the pathogenesis of heart failure

    Circ. Heart Fail.

    (2015)
  • P.M. Becher et al.

    Assessment of cardiac inflammation and remodeling during the development of streptozotocin-induced diabetic cardiomyopathy in vivo: a time course analysis

    Int. J. Mol. Med.

    (2013)
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    1

    Current address: Center for Cardiovascular Diseases and Sciences, Department of Pathology, Louisiana State University Health Science Center, Shreveport, LA 71103, USA.

    2

    Current address: Divison of Cardiovascular Medicine, Department of Internal Medicine, University of Iowa, Iowa City, IA 52242, USA.

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