Elsevier

Experimental Cell Research

Volume 357, Issue 2, 15 August 2017, Pages 155-162
Experimental Cell Research

Involvement of JNK signaling in IL4-induced M2 macrophage polarization

https://doi.org/10.1016/j.yexcr.2017.05.010Get rights and content

Highlights

  • IL-4 induced high expression of JNK and transcription c-Myc in M2 macrophages.

  • JNK inhibition in M2 macrophages blocked c-Myc and led to an impaired M2 status.

  • JNK inhibition leads to the reduction of wound healing capacity of M2 cells.

  • M2 macrophages had high ability of migration depending on cytokines production.

Abstract

It has been generally accepted that alternatively activated macrophages (M2), which can be induced by type 2 cytokines such as IL-4, is responsible for tissue repair. However, the function of JNK in IL-4-induced M2 macrophage polarization remains unclear. Here, we demonstrated that M0 macrophages can be polarized into M2 status in response to IL-4 stimulation with the increased expression of the M2-specific molecular markers. We also found that IL-4 induced higher expression of JNK and transcription factor c-Myc in M2 macrophages. Our Q-PCR and Western blot results showed that JNK increased the expression of c-Myc and M2 markers Arg1, Mrc1. We also demonstrated c-Myc was the downstream of IL-4-JNK pathway. Further, the depletion of c-Myc, Arg1 and Mrc1 could inhibit the migration ability of M2 macrophages. Taken together, our data establishes a new role for JNK signaling in IL-4-induced alternative activation of macrophages and may provide a novel strategy for immune therapy.

Introduction

Macrophages play different roles in diseases, depending on their diverse phenotypes. There are two major macrophage phenotypes, classically activated macrophages (M1) and alternatively activated macrophages (M2) which were widely accepted [1], [2]. M1 macrophages are associated with phagocytizing bacteria and damaging tissue, whereas M2 macrophages are the dominant subset in wound healing and tissue repair [3]. It has been reported that M1/M2 ratio is linked with inflammatory response [4]. M1 macrophages are induced by interferon (IFN)-γ or lipopolysaccharide (LPS) [2]. This phenotype expresses high levels of pro-inflammatory cytokines, such as interleukin-12 (IL12). By contrast, M2 macrophages develop in response to T helper type 2 (Th2) cytokines, such as IL-4 and IL-13. They predominantly produce higher levels of anti-inflammatory cytokines arginase-1 (Arg1), the mannose receptor-1 (Mrc1) and IL10 [5]. The macrophage phenotypes are mainly rely on cytokine production in the tissue environment [6]. Transformation from a macrophage producing inflammatory cytokine to producing anti-inflammatory cell may be pivotal to induced non-immune responses or peripheral tolerance [7].

IL-4 is a well-known anti-inflammatory cytokine produced by Th2 cells. It has been well established that IL-4 is the key stimuli which induces M2 macrophage polarization. This process has been shown to be dependent on activation of STAT-6 and PI3K signaling pathways [8]. In addition, mitogen-activated protein kinases (MAPKs) are highly conserved serine/threonine protein kinase that are pivotal for cellular responses such as regulating the proliferation, differentiation, activation and survival of immune cells [9]. There have been three major MAPK pathways identified to participate in the regulation of immune cell responses: the extracellular signal-regulated kinase-1/2 (ERK), c-Jun N-terminal kinase (JNK) and p38 pathways [10]. Recent studies have reported the critical role of p38 MAPK in IL-4-induced macrophage polarization [11]. Moreover, it has been shown that in a tumor microenvironment under stress conditions, IL-4 triggers a simultaneous activation of the JNK-pathway and the up-regulated proliferation in cancer cells [12]. However, the role of JNK pathway in IL-4-induced macrophage polarization is still unknown.

Here, we demonstrated that IL-4 induces JNK increasing in macrophages, leading to the subsequent increasing downstream transcription c-Myc. Furthermore, our results showed that up-regulation of typical M2 markers and higher ability of migration upon IL-4 stimulation is dependent on JNK pathway, suggesting that JNK plays a role in the polarization toward M2 phenotype.

Section snippets

Cell culture and transfection

The macrophage cell lines, RAW264.7 cells and THP-1 cells, were maintained in RPMI-1640 Medium (Gibco, Grand Island, NY, USA), supplemented with 10% fetal bovine serum (FBS, Gibco, Grand Island, NY, USA) and 1% penicillin/ streptomycin (Gibco, Grand Island, NY, USA). The cells were cultured at 37 C in a humidified 5% CO2 atmosphere. RAW264.7 macrophages can be differentiated into M2 macrophages by IL-4 (214-14, Peprotech, USA). THP-1 cells can be transferred into M0 macrophages by PMA (BS1819–1 

M0 status stimulated with IL-4 readily acquire M2 status

To construct the M2 macrophage cell model, IL4 was applied and the mRNA levels of M2 markers including IL10, Arg1, Mrc1 were detected to verify the success of the cell model [13]. As expected, IL-4-treated RAW264.7 cells demonstrated a significantly higher mRNA level of IL-10, Mrc1 and Arg1 compared with the unstimulated (BSA alone-treated) cells (Fig. 1A and B). What's more, the protein level of MRC1 and ARG1 was also incresed (Fig. 1C). However, M1 associated cytokine, IL12, was expressed at

Discussion

Macrophages, which exhibit plasticity in response to different environmental cues [1], [2],, play a critical role in variety of physiological and pathological processes [15]. It could be classified into M1 and M2 polarizing phenotype with different function in three critical phases of wounding healing progression, including inflammation, granulation tissue formation, matrix deposition and remodeling [16]. The inflammatory phase mostly involves with M1 pro-inflammatory macrophages which promote

Acknowledgements

This work was supported by grant from the National Natural Science Foundation of China (Grant No. 31401191) to Xiaoyan Lv.

References (21)

There are more references available in the full text version of this article.

Cited by (0)

View full text