MiR-146a modulates macrophage polarization by inhibiting Notch1 pathway in RAW264.7 macrophages
Graphical abstract
Proposed model of miR-146a in the regulation of macrophage polarization
Introduction
Macrophages are heterogeneous and versatile cells that function as immune effectors and regulators, tissue remodelers, or scavengers in both tissue homeostasis and lesions. Activation of macrophages is important to eliminate the invading pathogens or an insulting stimulation. Available evidence suggested that macrophages could influence the initiation and progression of various diseases [1], [2]. However, the inflammatory signaling must be resolved to prevent self-destruction while the infectious or injurious agents are cleared. Thus, the proper activation of macrophages and the transition from an inflammatory to immunosuppressive phenotype are equally important [3], [4].
It is generally believed that macrophages are highly heterogeneous cells which can rapidly change their phenotype and function in response to the microenvironment signals [5], [6]. Upon exposure to microbial products or pro-inflammatory cytokines (IFN-γ, TNF, or TLR ligands), macrophages undergo a phenotype switch referred to as classically activated macrophages or M1 macrophages. These macrophages produce pro-inflammatory cytokines, such as TNF-α, IL-6, IL-1β and nitric oxide (NO) which is induced by NO synthase (iNOS) [7]. In contrast, stimulation of macrophages with IL-4 and IL-13, immune complexes plus TLR ligands, IL-10, TGF-β or glucocorticoids induce the generation of alternatively activated macrophages or M2 macrophages. These macrophages produce high levels of M2-associated genes such as mannose receptor (Mrc) 1/2, Chitinase 3-like 3 (Chi3l3, also called Ym1), and arginase 1 (Arg1) [8], [9]. M1 macrophages play a crucial role in protecting the host against infection, like eliminating bacterial, viral and fungal infections, while they also contribute to the pathogenesis of inflammatory and degenerative diseases [10], [11]. In contrast, although M2 macrophages suppress T cells response and enhance tumor progression, they are beneficial to inflammation modulation and tissue remodeling [11], [12]. However, the underlying molecular mechanisms of macrophage polarization are still obscure.
Emerging evidence suggests that microRNAs (miRNA) play important roles in macrophage polarization and function [13], [14].These functional miRNAs like miR-155 and miR-21 have been identified to be associated with polarized macrophages via different pathways or signalings [14], [15]. Thus, the importance of miRNAs in determining macrophage polarization has been increasingly appreciated. A previous study by our group showed that miR-146a might function as a novel feedback negative regulator to LPS-induced production of inflammatory cytokines. MiR-146a, located in the second exon of the LOC285628 gene on human chromosome 5, is involved in human diseases such as the innate immune response, hemopoiesis, inflammatory disease and cancer [16], [17], [18], [19]. However, little is known about the potential role of miR-146a in the regulation of macrophage polarization.
The Notch signaling pathway is an evolutionarily conserved pathway that influences cell-to-cell communication in a variety of tissues and organs. It is implicated in many genetic and acquired diseases and conditions. Specifically, the Notch pathway is associated with various cell fate controls during cell differentiation, proliferation, and apoptosis [20], [21], [22]. It plays a critical role in macrophage modulation [23], [24]. A recent study provided evidence that miR-146a exerts an anti-tumor effect through downregulation of Notch1 in glioblastomas, suggesting that there is an intimate relationship between miR-146a and Notch1 [25]. Therefore the current study attempted to explore whether miR-146a participates in the regulation of macrophage polarization and the potential underlying mechanisms in the process.
Section snippets
Cell culture and macrophage polarization
The RAW264.7 macrophage cell lines (ATCC, USA) were maintained in DMEM (Gibco, USA) supplemented with 10% (v/v) FBS (Gibco, USA), 100 μg/ml streptomycin, 100 U/ml penicillin, and 2 μM l-glutamine (Beyotime, China) and incubated at 37 °C and an atmosphere of 5% CO2. M1 macrophages were generated by treatment with LPS (1000 ng/ml, Sigma, GER) and IFN-γ (10 ng/ml, Sigma, GER) for 12 h. M2 macrophages were generated by treatment with IL-4 (15 ng/ml, Sigma, GER) for 48 h.
Transient transfection of miR-146a mimic and miR-146a inhibitor
To overexpress and downregulate the
MiR-146a is highly expressed in M2 macrophages
To determine macrophage polarization and plasticity, we induced M1 and M2 polarization in RAW264.7 respectively. As confirmed by our laboratory, M1 phenotype was polarized by treatment with LPS (1000 ng/ml) and IFN-γ (10 ng/ml) for 12 h, and M2 macrophages were induced with IL-4 (15 ng/ml) for 48 h. As shown in Fig.1A, iNOS and TNF-α were highly expressed in M1 macrophages while Arg1 and IL-10 were highly expressed in M2 macrophages, confirming the M1 and M2 activating states respectively. One-step
Discussion
At present, the underlying molecular mechanisms of macrophage polarization remain largely unclear. In this study, we found that miR-146a could suppress the pro-inflammatory M1 macrophage switch and promote M2 macrophage polarization partially through Notch1-dependent mechanism. Interestingly, it was also demonstrated that miR-146a could increase the expression of PPARγ to promote M2 macrophage activation (Fig. 6). These findings were based on the facts that over-expression of miR-146a not only
Conflict of interest
The authors declare that there are no conflicts of interest.
Acknowledgments
This study was supported by the National Science Foundations of China (Nos. 81473268 and 81273526), the Specialized Research Fund for the Doctoral Program of Higher Education of China (No. 20123420120001), Anhui Provincial Natural Science Foundation (No. 1408085MKL31 and 1308085MH145), and the Foundation of Anhui Medical University (No. XJ201118).
References (47)
- et al.
Retinoid X receptors in macrophage biology
Trends Endocrinol. Metab.
(2013) - et al.
Differential macrophage programming in the tumor microenvironment
Trends Immunol.
(2012) - et al.
Identifying functional microRNAs in macrophages with polarized phenotypes
J. Biol. Chem.
(2012) - et al.
MicroRNA-mediated control of macrophages and its implications for cancer
Trends Immunol.
(2013) - et al.
An NF-kappaB-sensitive micro RNA-146a-mediated inflammatory circuit in Alzheimer disease and in stressed human brain cells
J. Biol. Chem.
(2008) - et al.
microRNAs and the immune response
Curr. Opin. Pharmacol.
(2009) - et al.
The canonical Notch signaling pathway: unfolding the activation mechanism
Cell
(2009) - et al.
Deadly crosstalk: Notch signaling at the intersection of EMT and cancer stem cells
Cancer Lett.
(2013) - et al.
Integrated regulation of Toll-like receptor responses by notch and interferon-gamma pathways
Immunity
(2008) - et al.
The Notch ligand, Delta-1, inhibits the differentiation of monocytes into macrophages but permits their differentiation into dendritic cells
Blood
(2001)
MiR-34a inhibits lipopolysaccharide-induced inflammatory response through targeting Notch1 in murine macrophages
Exp. Cell Res.
Macrophage polarization: tumor-associated macrophages as a paradigm for polarized M2 mononuclear phagocytes
Trends Immunol.
The interleukin 13 (IL-13) pathway in human macrophages is modulated by microRNA-155 via direct targeting of interleukin 13 receptor alpha1 (IL13Ralpha1)
J. Biol. Chem.
PPARgamma activation primes human monocytes into alternative M2 macrophages with anti-inflammatory properties
Cell Metab.
Macrophage function and polarization in cardiovascular disease: a role of estrogen signaling?
Arterioscler. Thromb. Vasc. Biol.
Protective and pathogenic functions of macrophage subsets
Nat. Rev. Immunol.
Macrophage biology in development, homeostasis and disease
Nature
Alveolar macrophages: plasticity in a tissue-specific context
Nat. Rev. Immunol.
Transcriptional regulation of macrophage polarization: enabling diversity with identity
Nat. Rev. Immunol.
An unrestrained proinflammatory M1 macrophage population induced by iron impairs wound healing in humans and mice
J. Clin. Invest.
The M1 and M2 paradigm of macrophage activation: time for reassessment
Macrophage plasticity and polarization: in vivo veritas
J. Clin. Invest.
Exploring the full spectrum of macrophage activation
Nat. Rev. Immunol.
Cited by (123)
The pathogenesis of food allergy and protection offered by dietary compounds from the perspective of epigenetics
2024, Journal of Nutritional BiochemistryThe role of the Notch signaling pathway in bacterial infectious diseases
2024, Microbial PathogenesisSilica nanoparticles promoted pro-inflammatory macrophage and foam cell transformation via ROS/PPARγ/NF-κB signaling
2023, Science of the Total EnvironmentThe miRNA neuroinflammatory biomarkers in COVID-19 patients with different severity of illness
2023, NeurologiaCitation Excerpt :In addition, Mir-146a targets other pro-inflammatory mediators including STAT-1,26,27 IRF-527 and CFH.28,29 The polarization of macrophages and microglia are also altered by mir-146a.30 mir-124 is also an anti-inflammatory miRNA and has a major role in neuronal differentiation31 and is highly expressed in microglia under normal conditions, but is not expressed in peripheral macrophages.32
- 1
This author contributed equally to this work with the first author.