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MiR-16 regulates mouse peritoneal macrophage polarization and affects T-cell activation.

ABSTRACT: MiR-16 is a tumour suppressor that is down-regulated in certain human cancers. However, little is known on its activity in other cell types. In this study, we examined the biological significance and underlying mechanisms of miR-16 on macrophage polarization and subsequent T-cell activation. Mouse peritoneal macrophages were isolated and induced to undergo either M1 polarization with 100 ng/ml of interferon-? and 20 ng/ml of lipopolysaccharide, or M2 polarization with 20 ng/ml of interleukin (IL)-4. The identity of polarized macrophages was determined by profiling cell-surface markers by flow cytometry and cytokine production by ELISA. Macrophages were infected with lentivirus-expressing miR-16 to assess the effects of miR-16. Effects on macrophage-T cell interactions were analysed by co-culturing purified CD4(+) T cells with miR-16-expressing peritoneal macrophages, and measuring activation marker CD69 by flow cytometry and cytokine secretion by ELISA. Bioinformatics analysis was applied to search for potential miR-16 targets and understand its underlying mechanisms. MiR-16-induced M1 differentiation of mouse peritoneal macrophages from either the basal M0- or M2-polarized state is indicated by the significant up-regulation of M1 marker CD16/32, repression of M2 marker CD206 and Dectin-1, and increased secretion of M1 cytokine IL-12 and nitric oxide. Consistently, miR-16-expressing macrophages stimulate the activation of purified CD4(+) T cells. Mechanistically, miR-16 significantly down-regulates the expression of PD-L1, a critical immune suppressor that controls macrophage-T cell interaction and T-cell activation. MiR-16 plays an important role in shifting macrophage polarization from M2 to M1 status, and functionally activating CD4(+) T cells. This effect is potentially mediated through the down-regulation of immune suppressor PD-L1.

SUBMITTER: Jia X

PROVIDER: S-EPMC5020626 | biostudies-literature | 2016 Oct

REPOSITORIES: biostudies-literature

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MiR-16 regulates mouse peritoneal macrophage polarization and affects T-cell activation.

Jia Xiaoqin X Li Xiaomin X Shen Yating Y Miao Junjun J Liu Hao H Li Guoli G Wang Zhengbing Z

Journal of cellular and molecular medicine 20160531 10

MiR-16 is a tumour suppressor that is down-regulated in certain human cancers. However, little is known on its activity in other cell types. In this study, we examined the biological significance and underlying mechanisms of miR-16 on macrophage polarization and subsequent T-cell activation. Mouse peritoneal macrophages were isolated and induced to undergo either M1 polarization with 100 ng/ml of interferon-γ and 20 ng/ml of lipopolysaccharide, or M2 polarization with 20 ng/ml of interleukin (IL ...[more]

PMID: 27241533

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Project description:Eosinophils are terminally differentiated granulocytes that have long been considered as destructive cells associated with Th2 type immune responses such as allergic inflammation and helminth infections. Recently, eosinophils have been actively studied as multifunctional leukocytes regulating an array of physiological responses through interaction with other immune cells. In this study, we examined the expression and function of Toll-like receptors (TLRs) in eosinophilic EoL-1 cells and demonstrated the expression of a number of immune mediators in activated EoL-1 cells and their interaction with the macrophage cell line THP-1 upon TLR4 ligand stimulation. EoL-1 cells differentiated with butyrate increased expression of TLR3, TLR4, and TLR7 at mRNA and protein level with flow cytometry analysis. Mature eosinophils derived from human cord blood CD34+ cells were subjected to RNA-sequencing, and showed the expression of a panel of TLR transcripts and TLR4 was the most highly expressed TLR. Among the cognate ligands of TLR3, TLR4, and TLR7, lipopolysaccharide (LPS) or palmitic acid significantly increased mRNA expression of immune mediators in differentiated EoL-1 cells. Notably, Western blot analysis of palmitic acid-treated differentiated EoL-1 cells showed significantly up-regulated expression of Th2 type cytokines and transcription factors driving eosinophil differentiation. To evaluate functional significance of TLR4 ligand-stimulated eosinophils, we added conditioned media (CM) from EoL-1 cells to differentiated THP-1 cells and assessed the expression of M1 macrophage or M2 macrophage-related markers. M1 and M2 macrophage markers were significantly upregulated by CM from LPS and palmitic acid stimulated EoL-1 cells, respectively. In addition, the adipose tissue of obese mice, where eosinophils are decreased due to obesity-induced inflammation, showed significantly decreased frequency of M2 macrophages, despite an increase in the total macrophage numbers. Based on these collective data, we proposed that eosinophils regulate both inflammatory and anti-inflammatory polarization of macrophages through functional changes induced by different TLR4 ligands.

Project description:BackgroundAsymmetric dimethylarginine (ADMA), which is significantly elevated in the plasma of cancer patients, is formed via intracellular recycling of methylated proteins and serves as a precursor for resynthesis of arginine. However, the cause of ADMA elevation in cancers and its impact on the regulation of tumor immunity is not known.MethodsThree mouse breast cell lines (normal breast epithelial HC11, breast cancer EMT6 and triple negative breast cancer 4T1) and their equivalent 3D stem cell culture were used to analyze the secretion of ADMA using ELISA and their responses to ADMA. Bone marrow-derived macrophages and/or RAW264.7 cells were used to determine the impact of increased extracellular ADMA on macrophage-tumor interactions. Gene/protein expression was analyzed through RNAseq, qPCR and flow cytometry. Protein functional analyses were conducted via fluorescent imaging (arginine uptake, tumor phagocytosis) and enzymatic assay (arginase activity). Cell viability was measured via MTS assay and/or direct cell counting using Countess III FL system.ResultsFor macrophages, ADMA impaired proliferation and phagocytosis of tumor cells, and even caused death in cultures incubated without arginine. ADMA also led to an unusual macrophage phenotype, with increased expression of arginase, cd163 and cd206 but decreased expression of il10 and dectin-1. In contrast to the severely negative impacts on macrophages, ADMA had relatively minor effects on proliferation and survival of mouse normal epithelial HC11 cells, mouse breast cancer EMT6 and 4T1 cells, but there was increased expression of the mesenchymal markers, vimentin and snail2, and decreased expression of the epithelial marker, mucin-1 in EMT6 cells. When tumor cells were co-cultured ex vivo with tumor antigen in vivo-primed splenocytes, the tumor cells secreted more ADMA and there were alterations in the tumor cell arginine metabolic landscape, including increased expression of genes involved in arginine uptake, metabolism and methylation, and decreased expression of a gene that is responsible for arginine demethylation. Additionally, interferon-gamma, a cytokine involved in immune challenge, increased secretion of ADMA in tumor cells, a process attenuated by an autophagy inhibitor.ConclusionOur results suggest initial immune attack promotes autophagy in tumor cells, which then secrete ADMA to manipulate macrophage polarization favoring tumor tolerance.

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MiR-16 regulates mouse peritoneal macrophage polarization and affects T-cell activation.

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MiR-16 regulates mouse peritoneal macrophage polarization and affects T-cell activation.

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