Elsevier

Cellular Immunology

Volume 289, Issues 1–2, May–June 2014, Pages 42-48
Cellular Immunology

The role of indoleamine 2,3-dioxygenase (IDO) in immune tolerance: Focus on macrophage polarization of THP-1 cells

https://doi.org/10.1016/j.cellimm.2014.02.005Get rights and content

Highlights

  • IFN-γ differentiates THP-1 cells to M1-type macrophages and upregulates IDO expression.

  • Over expression of IDO promotes M2-polarized THP-1 macrophages.

  • Effects of IDO-knockdown on the macrophage polarization of THP-1 cells.

Abstract

Macrophages can be divided into two groups as M1 and M2 phenotype. Our results and other groups revealed that IFN-γ can up-regulate the IDO expression and differentiate THP-1 cells to M1 phenotype. Therefore we hypothesized that IDO may play potential roles in macrophage differentiation. Interesting, our results indicated that the ectopic IDO increases the expression of M2 markers such as IL-10 and CXCR4 while decreases the M1 markers such as CCR7 and IL-12p35. In contrast, the knockdown of IDO expression in THP-1 cells resulted in increased M1 markers and lower M2 markers. Our results suggested that the expression intensity of IDO modulates macrophages differentiation. These finding support the counter-regulatory role for IDO with regarding to the polarization of macrophages to restrain excessive or inappropriate immune activation in inflammatory or tumor microenvironment. It throws new light on the mechanisms about the immunosuppressive effect of IDO in tumor or inflammatory diseases.

Introduction

Mononuclear phagocytes will express specialized and polarized functional properties after they are stimulated by cytokines and microbial products [1], [2]. The stimulations lead to macrophages have a great deal of plasticity and therefore can be broadly divided into two groups as M1- and M2-type macrophages, which are recognized as classically activated and alternatively activated macrophages, respectively [3]. M1-type macrophages have known to be induced by granulocyte–macrophage colony-stimulating factor (GM-CSF), interferon gamma (IFN-γ), and/or lipopolysaccharide (LPS) and have an IL-12high, IL-23high, IL-10low phenotype [4]. At the opposite extreme, M2-type macrophages are various forms of macrophages other than the classic M1 including cells exposed to IL-4, IL-13, immune complexes, IL-10, glucocorticoids [5]. They share an IL-12low, IL-23low, IL-10high phenotype [2]. Generally, M1-type macrophages are considered as the effector cells defend the body against the pathogens and tumor cells, while M2-type macrophages suppress inflammatory responses and adaptive immunity and stimulate angiogenesis and tumor growth [6].

The enzyme indoleamine 2,3-dioxygenase (IDO) has been considered as an immunosuppressive and tolerogenic mechanism in cancer [7]. It catalyzes the initial and rate-limiting step of tryptophan degradation, reduces the local tryptophan concentration, produces immune modulatory tryptophan metabolites, and therefore exhibits immunosuppressive effects [8], [9]. Expression of IDO was detected in many different types of tumor cells and tumor-draining lymph node. It has been shown to correlate with poor prognosis in several cancers such as ovarian carcinoma, endometrial carcinoma, and colon carcinoma [10], [11], [12]. Previous studies suggested that IDO expressed in tumor microenvironment leads to immune escape and immunosuppression mainly through two aspects: (1) inhibition of effective T-cell priming by antigen presenting cells (APCs) derived IDO in tumor-draining lymph nodes [13]; (2) the effective phase of an anti-tumor immune response is hampered because many human solid tumors themselves express IDO [12], [14].

IDO is not constitutively expressed in APCs, epithelial cells, fibroblast, or tumor cells, but can be induced so by a variety of stimuli, of which IFN-γ is the most potent [15]. It was reported that IFN-γ regulates the proliferation and differentiation of mesenchymal stem cells (MSC) via activation of IDO [16], [17]. In the present study, we demonstrate a role for IDO in determining the phenotype of macrophage. IFN-γ can promote the macrophages differentiation to M1-type macrophages and up-regulate the expression of IDO in differentiated THP-1(dTHP-1) cells. However, forced expression of IDO in dTHP-1 cells drove increased expression of M2-specific markers. Conversely, the induction of M1-specific markers was up-regulated in dTHP-1 cells with knockdown of IDO expression mediated by small interfering RNA (siRNA). Our findings provide some new evidence about the role of IDO in macrophage differentiation and throw new light on the mechanisms about the tumor immune tolerance and the theoretical basis for IDO as a valuable therapeutic target.

Section snippets

Chemicals and reagents

Phorbol-12-myristate 13-acetate (PMA) and IFN-γ were purchased from Sigma–Aldrich (Deisenhofen, Germany). M-CSF was purchased from Peprotech (Rocky Hill, NJ, USA). The plasmid vectors pEGFP-N1 was purchased from Promega (Madison, WI, USA). The plasmid pEGFP-N1-IDO and rabbit anti-human IDO polyclonal antibody were generated in our laboratory [18]. β-tubulin was obtained from Cell Signaling Technology (MA, USA). Horseradish peroxidase (HRP)-conjugated secondary antibody and Lipofectamine 2000

IFN-γ treatment polarizes dTHP-1 cells to M1-type macrophages

THP-1 cells have been widely used as the model for monocytes/macrophages differentiation. When treated with PMA (320 nM) for 48 h, THP-1 cells quickly stopped proliferating, became attached and spread, differentiated to macrophages (Fig. 1A), and showed a significant induction of CD68 (marker for macrophage differentiation) in both mRNA and protein levels (Fig. 1B and C, respectively).

To investigate the polarization effect of IFN-γ in macrophages, dTHP-1 cells were incubated with IFN-γ (100 

Discussions

Macrophages have a high degree of functional plasticity: they can switch from an activated M1 state back to M2, and vice versa, depending on the cytokines (Th1 or Th2) to which they are exposed [21]. Considering that IFN-γ can stimulate the monocytes differentiated to M1-type macrophage and up-regulate the expression of IDO, we investigated whether IDO is involved in macrophage polarization. We found that IDO contributed to macrophage plasticity; that is, modulation of its expression led to the

Acknowledgments

This work was supported by grants from the National Basic Research Program of China (973 Program, No. 2011CB935800), and the Natural Science Fund of China (Nos. 30873032, 81071712, and 30973194) .

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