EFFECT OF M2 MACROPHAGE-DERIVED SOLUBLE FACTORS ON DIFFERENTIATION OF SH-SY5Y CELLS

Macrophages play a key role in triggering and regulation of neuroregeneration. The characteristic feature of macrophages is pronounced plasticity, which manifests itself in the ability of macrophages to change their functional phenotype depending on the micromilieu. Apoptotic cell clearance (efferocytosis) is an important inducer of a macrophage polarization to M2 phenotype under pathological settings. Previously, we have developed an original protocol for the generation of M2-like macrophages, polarized by efferocytosis under serum-deprived conditions (M2 (LS), Low Serum). The present study was aimed to assess a neuroregenerative potential of M2 (LS) macrophages. We studied their effect on the differentiation of SH-SY5Y cells in comparison with retinoic acid (RA). As the morphological criteria of differentiation we have assessed the relative content of differentiated cells, i.e., cells with a neurite length exceeding the cell body length, and the average neurite length on days 3, 7, and 13. The ratio of neuron-like (N-type) and epithelial-like (S-type) cells in cultures was also assessed. SH-SY5Y cells were characterized by a low level of spontaneous differentiation, both under standard conditions (10% FBS) and serum deprivation (1% FBS). Upon RA treatment, SH-SY5Y cells stopped proliferating and underwent neuronal differentiation. Cultivation of SH-SY5Y cells in the presence of M2 (LS) conditioned medium also led to a significant increase in the relative content of differentiated cells, the average length of neurite-like processes, as well as a change in the balance of S- and N-type cells towards a pronounced predominance of the latter. The morphological features of differentiation were significantly less pronounced at early stage (day 3) of differentiation as compared with the RA-induced changes and reached the level of positive control only at later stages (day 13) (p < 0.05). In contrast to retinoic acid, M2 (LS) conditioned medium induced neuronal differentiation of SH-SY5Y cells without suppressing their proliferative activity. The data obtained may indicate a high neuroregenerative potential of M2 macrophages in vitro, which is realized through soluble factors and manifests itself in promoting SH-SY5Y differentiation.

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Metformin Promotes Neuronal Differentiation via Crosstalk between Cdk5 and Sox6 in Neuroblastoma Cells

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2019/1765182 ◽

2019 ◽

Vol 2019 ◽

pp. 1-13 ◽

Cited By ~ 2

Author(s):

Thunwa Binlateh ◽

Supita Tanasawet ◽

Onnicha Rattanaporn ◽

Wanida Sukketsiri ◽

Pilaiwanwadee Hutamekalin

Keyword(s):

Cell Differentiation ◽

Neuronal Differentiation ◽

Molecular Mechanisms ◽

Neuroblastoma Cells ◽

Metformin Treatment ◽

Neurite Length ◽

Differentiation Markers ◽

Morphological Criteria ◽

Phosphorylated Akt ◽

Ros Scavenger

Metformin has recently emerged as a key player in promotion of neuroblastoma differentiation and neurite outgrowth. However, molecular mechanisms of how metformin promotes cellular differentiation have not yet been fully elucidated. In this study, we investigated how metformin promotes cell differentiation, via an inhibition of cell proliferation, by culturing SH-SY5Y neuroblastoma cells with or without metformin. Pretreatment with reactive oxygen species (ROS) scavenger, NAC, revealed that ROS plays a crucial role in induction of cell differentiation. Cell differentiation was observed under various morphological criteria: extension of neuritic processes and neuronal differentiation markers. Treatment with metformin significantly increased neurite length, number of cells with neurite, and expression of neuronal differentiation markers, β-tubulin III and tyrosine hydroxylase (TH) compared with untreated control. Further investigation found that metformin significantly decreased Cdk5 but increased Sox6 during cell differentiation. Analysis of the mechanism underlying these changes using Cdk5 inhibitor, roscovitine, indicated that expressions of Cdk5 and Sox6 corresponded to metformin treatment. These results suggested that metformin produces neuronal differentiation via Cdk5 and Sox6. In addition, phosphorylated Erk1/2 was decreased while phosphorylated Akt was increased in metformin treatment. Taken together, these findings suggest that metformin promotes neuronal differentiation via ROS activation through Cdk5/Sox6 crosstalk, relating to Erk1/2 and Akt signaling.

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Serum and Ectopic Endometrium from Women with Endometriosis Modulate Macrophage M1/M2 Polarization via the Smad2/Smad3 Pathway

Journal of Immunology Research ◽

10.1155/2018/6285813 ◽

2018 ◽

Vol 2018 ◽

pp. 1-14 ◽

Cited By ~ 9

Author(s):

Mei-Fang Nie ◽

Qi Xie ◽

Ya-Hong Wu ◽

Hua He ◽

Lu-Jie Zou ◽

...

Keyword(s):

Conditioned Medium ◽

Molecular Mechanisms ◽

Macrophage Polarization ◽

Leukemia Cell ◽

M2 Macrophage ◽

Monocytic Leukemia ◽

Serum Samples ◽

Endometrial Stromal Cells ◽

M2 Polarization ◽

M2 Macrophage Polarization

Objective. This study investigated the alterations in macrophage polarization in patients with endometriosis as well as the underlying molecular mechanisms. Methods. Peritoneal washings, serum samples, and endometrial tissues were collected from endometriosis patients and control subjects. Endometrial stromal cells (ESCs) were isolated from endometrial tissue, and conditioned medium was prepared by treating ESCs with or without various concentrations of interleukin- (IL-) 6, estrogen, or progestin. The frequencies of CD86+ and CD163+ cells and expression levels of these markers as well as the cytokines IL-12 and IL-10 were measured in THP-1- (human monocytic leukemia cell) derived macrophages. Results. There was a decrease in the percentage of CD86+ macrophages in the peritoneal wash solution of patients with endometriosis. Ectopic endometrial homogenates could promote M1 to M2 macrophage polarization in response to lipopolysaccharide (LPS), as evidenced by the increased percentage of CD163+ macrophages and increased IL-10 expression as well as a decreased percentage of CD86+ cells and lower IL-12 expression. In contrast, addition of serum from women with endometriosis to THP-1 cells resulted in the polarization of macrophages towards both M1 and M2 phenotypes. Upregulation of Smad2/Smad3 in macrophages upon exposure to eutopic and ectopic endometrial homogenates as well as serum of women with endometriosis was observed, and blockage of Smad2/Smad3 with their inhibitor SB431542 could reverse the macrophage polarization from M1 to M2. Conditioned medium induced by IL-6, but neither estrogen nor progestin, could facilitate M2 polarization. Neutralization of IL-6 diminished macrophage M2 polarization in endometriosis. Conclusion. This study provides detailed evidence supporting alterations in M1 to M2 macrophage polarization that may contribute to the initiation as well as progression of endometriosis.

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LncRNAs interacting with the translation machinery contribute to human neuronal differentiation

10.1101/2020.10.01.321919 ◽

2020 ◽

Author(s):

Katerina Douka ◽

Isabel Birds ◽

Dapeng Wang ◽

Sophie Clayton ◽

Abigail Byford ◽

...

Keyword(s):

Neuronal Differentiation ◽

Selective Constraint ◽

Ribosome Profiling ◽

Neuronal Function ◽

Neurite Length ◽

Translation Machinery ◽

Differentiated Cells ◽

High Sequence Conservation ◽

Functional Consequences ◽

Small Orfs

AbstractLncRNAs are less conserved, yet more tissue and developmental-stage specific than mRNAs and are particularly enriched in the nervous system of Drosophila melanogaster, mouse and human. The function of cytoplasmic lncRNAs and their potential translation remains poorly understood. Here we performed Poly-Ribo-Seq to understand the interaction of lncRNAs with the translation machinery and the functional consequences during neuronal differentiation of SH-SH5Y cells. We discovered 237 cytoplasmic lncRNAs upregulated during early neuronal differentiation, most of which are associated with polysome complexes. The majority are cytoplasmically enriched and are intergenic or anti-sense. In addition, we find 45 small ORFs in lncRNAs to be actively translated, 17 specifically upon differentiation. 11 of these smORFs exhibit high sequence conservation across Hominidae suggesting they are under strong selective constraint with putative function in this clade. We discover LINC01116 is induced upon differentiation and contains an 87 codon smORF, which we detect as translated, with increased ribosome profiling signal upon differentiation. The LINC01116 peptide exhibits a cytoplasmic distribution and is detected in neurites. Knockdown of LINC01116 results in significant reduction of neurite length in differentiated cells indicating it contributes to neuronal differentiation. Our findings indicate lncRNAs are a source of non-canonical peptides and contribute to neuronal function.

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Cervical Cancer Cell Supernatants Induce a Phenotypic Switch from U937-Derived Macrophage-Activated M1 State into M2-Like Suppressor Phenotype with Change in Toll-Like Receptor Profile

BioMed Research International ◽

10.1155/2014/683068 ◽

2014 ◽

Vol 2014 ◽

pp. 1-11 ◽

Cited By ~ 13

Author(s):

Karina Sánchez-Reyes ◽

Alejandro Bravo-Cuellar ◽

Georgina Hernández-Flores ◽

José Manuel Lerma-Díaz ◽

Luis Felipe Jave-Suárez ◽

...

Keyword(s):

Cervical Cancer ◽

Macrophage Polarization ◽

M2 Macrophage ◽

Toll Like Receptor ◽

Soluble Factors ◽

Immune Effector ◽

Effector Cells ◽

M1 Macrophage ◽

Receptor Profile ◽

Alternatively Activated

Cervical cancer (CC) is the second most common cancer among women worldwide. Infection with human papillomavirus (HPV) is the main risk factor for developing CC. Macrophages are important immune effector cells; they can be differentiated into two phenotypes, identified as M1 (classically activated) and M2 (alternatively activated). Macrophage polarization exerts profound effects on the Toll-like receptor (TLR) profile. In this study, we evaluated whether the supernatant of human CC cells HeLa, SiHa, and C-33A induces a shift of M1 macrophage toward M2 macrophage in U937-derived macrophages.Results. The results showed that soluble factors secreted by CC cells induce a change in the immunophenotype of macrophages from macrophage M1 into macrophage M2. U937-derived macrophages M1 released proinflammatory cytokines and nitric oxide; however, when these cells were treated with the supernatant of CC cell lines, we observed a turnover of M1 toward M2. These cells increased CD163 and IL-10 expression. The expression of TLR-3, -7, and -9 is increased when the macrophages were treated with the supernatant of CC cells.Conclusions. Our result strongly suggests that CC cells may, through the secretion of soluble factors, induce a change of immunophenotype M1 into M2 macrophages.

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Mechanistic and functional changes in Ca2+ entry after retinoic acid-induced differentiation of neuroblastoma cells

Biochemical Journal ◽

10.1042/bj20042127 ◽

2005 ◽

Vol 388 (3) ◽

pp. 941-948 ◽

Cited By ~ 8

Author(s):

Anna M. BROWN ◽

Fiona C. RIDDOCH ◽

Andrew ROBSON ◽

Christopher P. F. REDFERN ◽

Timothy R. CHEEK

Keyword(s):

Retinoic Acid ◽

Neuronal Differentiation ◽

Neuroblastoma Cells ◽

Functional Change ◽

Plateau Phase ◽

Functional Changes ◽

Differentiated Cells ◽

Fluorescence Measurements ◽

Undifferentiated Cells ◽

Ca2 Channels

We have investigated effects of neuronal differentiation on hormone-induced Ca2+ entry. Fura-2 fluorescence measurements of undifferentiated SH-SY5Y neuroblastoma cells, stimulated with methacholine, revealed the presence of voltage-operated Ca2+-permeable, Mn2+-impermeable entry pathways, and at least two voltage-independent Ca2+- and Mn2+-permeable entry pathways, all of which apparently contribute to both peak and plateau phases of the Ca2+ signal. Similar experiments using 9-cis retinoic acid-differentiated cells, however, revealed voltage-operated Ca2+-permeable, Mn2+-impermeable channels, and, more significantly, the absence or down-regulation of the most predominant of the voltage-independent entry pathways. This down-regulated pathway is probably due to CCE (capacitative Ca2+ entry), since thapsigargin also stimulated Ca2+ and Mn2+ entry in undifferentiated but not differentiated cells. The Ca2+ entry components remaining in methacholine-stimulated differentiated cells contributed to only the plateau phase of the Ca2+ signal. We conclude that differentiation of SH-SY5Y cells results in a mechanistic and functional change in hormone-stimulated Ca2+ entry. In undifferentiated cells, voltage-operated Ca2+ channels, CCE and NCCE (non-CCE) pathways are present. Of the voltage-independent pathways, the predominant one appears to be CCE. These pathways contribute to both peak and plateau phases of the Ca2+ signal. In differentiated cells, CCE is either absent or down-regulated, whereas voltage-operated entry and NCCE remain active and contribute to only the plateau phase of the Ca2+ signal.

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ANXA1 Contained in EVs Regulates Macrophage Polarization in Tumor Microenvironment and Promotes Pancreatic Cancer Progression and Metastasis

International Journal of Molecular Sciences ◽

10.3390/ijms222011018 ◽

2021 ◽

Vol 22 (20) ◽

pp. 11018

Author(s):

Nunzia Novizio ◽

Raffaella Belvedere ◽

Emanuela Pessolano ◽

Silvana Morello ◽

Alessandra Tosco ◽

...

Keyword(s):

Pancreatic Cancer ◽

Tumor Microenvironment ◽

Cancer Progression ◽

Macrophage Polarization ◽

M2 Macrophage ◽

Wild Type ◽

Soluble Factors ◽

Knock Out ◽

Orthotopic Xenografts ◽

M2 Phenotype

The tumor microenvironment (TME) is a dynamic system where nontumor and cancer cells intercommunicate through soluble factors and extracellular vesicles (EVs). The TME in pancreatic cancer (PC) is critical for its aggressiveness and the annexin A1 (ANXA1) has been identified as one of the oncogenic elements. Previously, we demonstrated that the autocrine/paracrine activities of extracellular ANXA1 depend on its presence in EVs. Here, we show that the complex ANXA1/EVs modulates the macrophage polarization further contributing to cancer progression. The EVs isolated from wild type (WT) and ANXA1 knock-out MIA PaCa-2 cells have been administrated to THP-1 macrophages finding that ANXA1 is crucial for the acquisition of a protumor M2 phenotype. The M2 macrophages activate endothelial cells and fibroblasts to induce angiogenesis and matrix degradation, respectively. We have also found a significantly increased presence of M2 macrophage in mice tumor and liver metastasis sections previously obtained by orthotopic xenografts with WT cells. Taken together, our data interestingly suggest the relevance of ANXA1 as potential diagnostic/prognostic and/or therapeutic PC marker.

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Polarization of M2 Macrophages by Interaction between Prostate Cancer Cells Treated with Trichomonas vaginalis and Adipocytes

The Korean Journal of Parasitology ◽

10.3347/kjp.2020.58.3.217 ◽

2020 ◽

Vol 58 (3) ◽

pp. 217-227

Author(s):

Hyo-Yeoung Chung ◽

Jung-Hyun Kim ◽

Ik-Hwan Han ◽

Jae-Sook Ryu

Keyword(s):

Conditioned Medium ◽

Immune Cells ◽

Trichomonas Vaginalis ◽

Macrophage Polarization ◽

Prostatic Hyperplasia ◽

M2 Macrophage ◽

M2 Macrophages ◽

Prostate Cancer Cells ◽

M2 Macrophage Polarization ◽

Prostate Cancers

Trichomonas vaginalis causes inflammation of the prostate and has been detected in tissues of prostate cancers (PCa), prostatitis and benign prostatic hyperplasia. Obesity is a risk factor for PCa and causes a chronic subclinical inflammation. This chronic inflammation further exacerbates adipose tissue inflammation as results of migration and activation of macrophages. Macrophages are the most abundant immune cells in the PCa microenvironment. M2 macrophages, known as Tumor-Associated Macrophages, are involved in increasing cancer malignancy. In this study, conditioned medium (TCM) of PCa cells infected with live trichomonads contained chemokines that stimulated migration of the mouse preadipocytes (3T3-L1 cells). Conditioned medium of adipocytes incubated with TCM (ATCM) contained Th2 cytokines (IL-4, IL-13). Macrophage migration was stimulated by ATCM. In macrophages treated with ATCM, expression of M2 markers increased, while M1 markers decreased. Therefore, it is suggested that ATCM induces polarization of M0 to M2 macrophages. In addition, conditioned medium from the macrophages incubated with ATCM stimulates the proliferation and invasiveness of PCa. Our findings suggest that interaction between inflamed PCa treated with T. vaginalis and adipocytes causes M2 macrophage polarization, so contributing to the progression of PCa.

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