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Cellular Physiology and Biochemistry :... 2015The accumulation of free cholesterol in atherosclerotic lesions has been well documented in both animals and humans. In studying the relevance of free cholesterol...
BACKGROUND/AIMS
The accumulation of free cholesterol in atherosclerotic lesions has been well documented in both animals and humans. In studying the relevance of free cholesterol buildup in atherosclerosis, contradictory results have been generated, indicating that free cholesterol produces both pro- and anti-atherosclerosis effects in macrophages. This inconsistency might stem from the examination of only select concentrations of free cholesterol. In the present study, we sought to investigate the implication of excess free cholesterol loading in the pathophysiology of atherosclerosis across a broad concentration range from (in µg/ml) 0 to 60.
METHODS
Macrophage viability was determined by measuring formazan formation and flow cytometry viable cell counting. The polarization of M1 and M2 macrophages was differentiated by FACS (Fluorescence-Activated Cell Sorting) assay. The secretion of IL-1β in macrophage culture medium was measured by ELISA kit. Macrophage apoptosis was detected by flow cytometry using a TUNEL kit.
RESULTS
Macrophage viability was increased at the treatment of lower concentrations of free cholesterol from (in µg/ml) 0 to 20, but gradually decreased at higher concentrations from 20 to 60. Lower free cholesterol loading induced anti-inflammatory M2 macrophage polarization. The activation of the PPARx03B3; (Peroxisome Proliferator-Activated Receptor gamma) nuclear factor underscored the stimulation of this M2 phenotype. Nevertheless, higher levels of free cholesterol resulted in pro-inflammatory M1 activation. Moreover, with the application of higher free cholesterol concentrations, macrophage apoptosis and secretion of the inflammatory cytokine IL-1β increased significantly.
CONCLUSION
These results for the first time demonstrate that free cholesterol could render concentration-dependent diversification effects on macrophage viability, polarization, apoptosis and inflammatory cytokine secretions, thereby reconciling the pros and cons of free cholesterol buildup in macrophages to the pathophysiology of atherosclerosis.
Topics: Animals; Apoptosis; Cell Survival; Cells, Cultured; Cholesterol; Dose-Response Relationship, Drug; Interleukin-1beta; Macrophages; Mice; PPAR gamma
PubMed: 26314949
DOI: 10.1159/000430365 -
Stem Cell Research & Therapy Apr 2022The prevalence of osteoarthritis (OA) is increasing, yet clinically effective and economical treatments are unavailable. We have previously proposed a cell-free fat...
BACKGROUND
The prevalence of osteoarthritis (OA) is increasing, yet clinically effective and economical treatments are unavailable. We have previously proposed a cell-free fat extract (CEFFE) containing multiple cytokines, which possessed antiapoptotic, anti-oxidative, and proliferation promotion functions, as a "cell-free" strategy. In this study, we aimed to evaluate the therapeutic effect of CEFFE in vivo and in vitro.
METHODS
In vivo study, sodium iodoacetate-induced OA rats were treated with CEFFE by intra-articular injections for 8 weeks. Behavioral experiments were performed every two weeks. Histological analyses, anti-type II collagen, and toluidine staining provided structural evaluation. Macrophage infiltration was assessed by anti-CD68 and anti-CD206 staining. In vitro study, the effect of CEFFE on macrophage polarization and secretory factors was evaluated by flow cytometry, immunofluorescence, and quantitative reverse-transcription polymerase chain reaction (qRT-PCR). The effect of CEFFE on cartilage regeneration was accessed by cell counting kit-8 assay and qRT-PCR. The generation of reactive oxygen species (ROS) and levels of ROS-related enzymes were investigated by qRT-PCR and western blotting.
RESULTS
In rat models with sodium iodoacetate (MIA)-induced OA, CEFFE increased claw retraction pressure while decreasing bipedal pressure in a dose-dependent manner. Moreover, CEFFE promoted cartilage structure restoration and increased the proportion of CD206 macrophages in the synovium. In vitro, CEFFE decreased the proportion of CD86 cells and reduced the expression of pro-inflammatory factors in LPS + IFN-γ induced Raw 264.7. In addition, CEFFE decreased the expression of interleukin-6 and ADAMTs-5 and promoted the expression of SOX-9 in mouse primary chondrocytes. Besides, CEFFE reduced the intracellular levels of reactive oxygen species in both in vitro models through regulating ROS-related enzymes.
CONCLUSIONS
CEFFE inhibits the progression of OA by promoting cartilage regeneration and limiting low-grade joint inflammation.
Topics: Animals; Cell Extracts; Chondrocytes; Immunomodulation; Macrophages; Mice; Osteoarthritis; Rats
PubMed: 35365233
DOI: 10.1186/s13287-022-02813-3 -
Physiological Reports Oct 2022Moderate-intensity exercise performed during wound healing has been reported to decrease inflammatory cytokines and chemokines and accelerate wound healing. However, its...
Moderate-intensity exercise performed during wound healing has been reported to decrease inflammatory cytokines and chemokines and accelerate wound healing. However, its effect on macrophage phenotype and the mechanism by which exercise accelerates wound healing remain unclear. The purpose of this study was to investigate the effect of exercise on macrophage phenotype during wound healing and to clarify the relationship between angiogenesis and wound healing. 12-week-old male C57BL/6J mice were divided into sedentary (n = 6) and exercise groups (n = 6). The exercise group performed moderate-intensity treadmill running exercise (9.0 m/min, 60 min) for 10 days. Double immunofluorescence analysis was performed using F4/80 inducible nitric oxide synthase (iNOS) for M1 macrophages, F4/80 transforming growth factor-beta (TGF-β)1 for M2 macrophages, and CD31 alpha smooth muscle actin (α-SMA) for angiogenesis. The exercise group showed significantly accelerated wound healing compared with the sedentary group. From early wound healing onward, exercise significantly inhibited M1 macrophage infiltration and increased M2 macrophage count. Exercise also significantly increased angiogenesis. Furthermore, the M2 macrophage phenotype was significantly correlated with angiogenesis in the exercise group, indicating that M2 macrophages and angiogenesis are related to accelerated wound healing. These findings suggest that moderate-intensity exercise increases TGF-β1 derived from M2 macrophages, which may be associated with enhanced angiogenesis and wound healing in young mice.
Topics: Actins; Animals; Cytokines; Macrophages; Male; Mice; Mice, Inbred C57BL; Nitric Oxide Synthase Type II; Transforming Growth Factor beta1; Transforming Growth Factors; Wound Healing
PubMed: 36200164
DOI: 10.14814/phy2.15447 -
Frontiers in Immunology 2019Monocytes (Mo) and macrophages (Mϕ) are key components of the innate immune system and are involved in regulation of the initiation, development, and resolution of many... (Review)
Review
Monocytes (Mo) and macrophages (Mϕ) are key components of the innate immune system and are involved in regulation of the initiation, development, and resolution of many inflammatory disorders. In addition, these cells also play important immunoregulatory and tissue-repairing roles to decrease immune reactions and promote tissue regeneration. Several lines of evidence have suggested a causal link between the presence or activation of these cells and the development of autoimmune diseases. In addition, Mo or Mϕ infiltration in diseased tissues is a hallmark of several autoimmune diseases. However, the detailed contributions of these cells, whether they actually initiate disease or perpetuate disease progression, and whether their phenotype and functional alteration are merely epiphenomena are still unclear in many autoimmune diseases. Additionally, little is known about their heterogeneous populations in different autoimmune diseases. Elucidating the relevance of Mo and Mϕ in autoimmune diseases and the associated mechanisms could lead to the identification of more effective therapeutic strategies in the future.
Topics: Animals; Autoimmune Diseases; Biomarkers; Disease Susceptibility; Humans; Leukocyte Count; Macrophages; Monocytes
PubMed: 31178867
DOI: 10.3389/fimmu.2019.01140 -
Oncoimmunology 2020Early trials for immune checkpoint inhibitors in sarcomas have delivered mixed results, and efforts to improve outcomes now look to combinatorial strategies with novel...
Early trials for immune checkpoint inhibitors in sarcomas have delivered mixed results, and efforts to improve outcomes now look to combinatorial strategies with novel immunotherapeutics, including some that target macrophages. To enhance our understanding of the sarcoma immune landscape, we quantified and characterized tumor-associated macrophage infiltration and expression of the targetable macrophage-related immune checkpoint CD47/SIRPα across sarcoma types. We surveyed immunohistochemical expression of CD68, CD163, CD47, and SIRPα in tissue microarrays of 1242 sarcoma specimens (spanning 24 types). Non-translocation sarcomas, particularly undifferentiated pleomorphic sarcoma and dedifferentiated liposarcoma, had significantly higher counts of both CD68+ and CD163+ macrophages than translocation-associated sarcomas. Across nearly all sarcoma types, macrophages outnumbered tumor-infiltrating lymphocytes and CD163+ (M2-like) macrophages outnumbered CD68+ (M1-like) macrophages. These findings were supported by data from The Cancer Genome Atlas, which showed a correlation between increasing macrophage contributions to immune infiltration and several measures of DNA damage. CD47 expression was bimodal, with most cases showing either 0% or >90% tumor cell staining, and the highest CD47 scores were observed in chordoma, angiosarcoma, and pleomorphic liposarcoma. SIRPα scores correlated well with CD47 expression. Given the predominance of macrophage infiltrates over tumor-infiltrating lymphocytes, the bias toward M2-like (immunosuppressive) macrophage polarization, and the generally high scores for CD47 and SIRPα, macrophage-focused immunomodulatory agents, such as CD47 or IDO-1 inhibitors, may be particularly worthwhile to pursue in sarcoma patients, alone or in combination with lymphocyte-focused agents.
Topics: Humans; Lymphocytes, Tumor-Infiltrating; Macrophages; Receptors, Immunologic; Sarcoma; Tumor-Associated Macrophages
PubMed: 32313727
DOI: 10.1080/2162402X.2020.1747340 -
Molecular Oncology Dec 2022Gliomas cause high mortality around the world. The metabolic pattern of the tumor was previously suggested to be associated with the patient's survival outcome and...
Gliomas cause high mortality around the world. The metabolic pattern of the tumor was previously suggested to be associated with the patient's survival outcome and immune activity. Yet, this relationship in glioma remains unknown. This study systematically evaluated the immune landscape in different phenotypes classified by metabolic-related pathways of 3068 glioma samples and 33 glioblastoma single-cell sequencing samples. Machine learning prediction analysis of microarray with R (pamr) was used for validating clustering results. A total of 5842 pan-cancer samples were used for external validation of the metabolic clusters. Cell Counting Kit-8 (CCK8) assay, cell clone assay, EdU assay, wound healing assay, Transwell assay, and co-culture assay were performed to verify the distinction in molecular characteristics among metabolic clusters. Metabolomics and RNA sequencing were performed on HS683 and U251 cells to annotate potential hyaluronic acid (HA)-mediated pathways. Three distinct metabolic phenotypes were identified. Metabolic cluster 1 correlated with a high number of immune infiltrating cells and poor survival of glioma patients. Metabolic clusters were proved with different levels of the macrophage markers CD68 and CD163 by multiplex immunofluorescence staining. Glioma cells from other metabolic clusters also expressed various levels of HA. HA was further found to mediate glioma proliferation, progression, and invasion. Moreover, HA potentially promoted macrophage recruitment and M2 polarization through the IL-1/CHI3L1 and TGF-b/CHI3L1 axes. HA also regulated the expression of PD-L1. This work revealed the significant connection between metabolic patterns, especially HA, and tumor immune infiltration in gliomas.
Topics: Humans; Hyaluronic Acid; Tumor Microenvironment; Glioma; Macrophages; Phenotype
PubMed: 36134697
DOI: 10.1002/1878-0261.13315 -
Cancer Research Aug 2018Although radiotherapy (RT) decreases the incidence of locoregional recurrence in breast cancer, patients with triple-negative breast cancer (TNBC) have increased risk of...
Although radiotherapy (RT) decreases the incidence of locoregional recurrence in breast cancer, patients with triple-negative breast cancer (TNBC) have increased risk of local recurrence following breast-conserving therapy. The relationship between RT and local recurrence is unknown. Here, we tested the hypothesis that recurrence in some instances is due to the attraction of circulating tumor cells to irradiated tissues. To evaluate the effect of absolute lymphocyte count on local recurrence after RT in patients with TNBC, we analyzed radiation effects on tumor and immune cell recruitment to tissues in an orthotopic breast cancer model. Recurrent patients exhibited a prolonged low absolute lymphocyte count when compared with nonrecurrent patients following RT. Recruitment of tumor cells to irradiated normal tissues was enhanced in the absence of CD8 T cells. Macrophages (CD11bF480) preceded tumor cell infiltration and were recruited to tissues following RT. Tumor cell recruitment was mitigated by inhibiting macrophage infiltration using maraviroc, an FDA-approved CCR5 receptor antagonist. Our work poses the intriguing possibility that excessive macrophage infiltration in the absence of lymphocytes promotes local recurrence after RT. This combination thus defines a high-risk group of patients with TNBC. This study establishes the importance of macrophages in driving tumor cell recruitment to sites of local radiation therapy and suggests that this mechanism contributes to local recurrence in women with TNBC that are also immunosuppressed. http://cancerres.aacrjournals.org/content/canres/78/15/4241/F1.large.jpg .
Topics: Animals; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Cell Movement; Female; Humans; Macrophages; Mastectomy; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Recurrence, Local; Neoplastic Cells, Circulating; Receptors, CCR5; Retrospective Studies; Triple Negative Breast Neoplasms
PubMed: 29880480
DOI: 10.1158/0008-5472.CAN-17-3623 -
Placenta Dec 2022As the most common complication of pregnancy, hyperglycemia has a profound impact on maternal and fetal health and the long-term development of the offspring. Placental... (Review)
Review
As the most common complication of pregnancy, hyperglycemia has a profound impact on maternal and fetal health and the long-term development of the offspring. Placental macrophages, including fetal-derived macrophages also known as Hofbauer cells (HBCs) and placenta-associated maternal monocyte/macrophages (PAMMs), are thought to play a crucial role in regulating pregnancy and maintaining the internal environment, and are critical for fetal development. This review aims to profile existing knowledge on HBCs and PAMMs across gestation, and consider how hyperglycemia may impact their phenotype and function in pregnancy.
Topics: Pregnancy; Humans; Female; Placenta; Hyperglycemia; Leukocyte Count; Macrophages; Fetus
PubMed: 36417786
DOI: 10.1016/j.placenta.2022.11.004 -
Molecular Biology of the Cell Aug 2010S100A4, a member of the S100 family of Ca(2+)-binding proteins, is directly involved in tumor metastasis. In addition to its expression in tumor cells, S100A4 is...
S100A4, a member of the S100 family of Ca(2+)-binding proteins, is directly involved in tumor metastasis. In addition to its expression in tumor cells, S100A4 is expressed in normal cells and tissues, including fibroblasts and cells of the immune system. To examine the contribution of S100A4 to normal physiology, we established S100A4-deficient mice by gene targeting. Homozygous S100A4(-/-) mice are fertile, grow normally and exhibit no overt abnormalities; however, the loss of S100A4 results in impaired recruitment of macrophages to sites of inflammation in vivo. Consistent with these observations, primary bone marrow macrophages (BMMs) derived from S100A4(-/-) mice display defects in chemotactic motility in vitro. S100A4(-/-) BMMs form unstable protrusions, overassemble myosin-IIA, and exhibit altered colony-stimulating factor-1 receptor signaling. These studies establish S100A4 as a regulator of physiological macrophage motility and demonstrate that S100A4 mediates macrophage recruitment and chemotaxis in vivo.
Topics: Actomyosin; Animals; Bone Marrow Cells; Cell Count; Cell Surface Extensions; Chemotaxis; Cytoskeleton; Heterocyclic Compounds, 4 or More Rings; Humans; Inflammation; Macrophage Colony-Stimulating Factor; Macrophages; Mice; Mice, Knockout; Models, Biological; Receptor, Macrophage Colony-Stimulating Factor; S100 Calcium-Binding Protein A4; S100 Proteins; Signal Transduction
PubMed: 20519440
DOI: 10.1091/mbc.e09-07-0609 -
Journal of Innate Immunity 2022The pattern of immune cells infiltrating the corneal stroma has been extensively studied in mice, but data on human tissue have been far less elaborate. To further...
PURPOSE
The pattern of immune cells infiltrating the corneal stroma has been extensively studied in mice, but data on human tissue have been far less elaborate. To further characterize the number and differentiation state of resident immune cells in organ-cultured human corneal tissue, we employed a comprehensive bioinformatic deconvolution (xCell) of bulk RNA-sequencing (RNA-seq) data, immunohistochemistry (IHC), and flow cytometry (FC).
METHODS
A transcriptome-based analysis of immune cell types in human corneal samples was performed. The results were validated by IHC, focusing on the identification of pro-inflammatory (M1) and regulatory (M2) macrophages. A protocol was established to identify these 2 different macrophage populations in human corneal tissue by means of FC. Subsequently, corneal samples in organ culture were differentially stimulated by IL-10, IL-4 & IL-13, or LPS and macrophage populations were evaluated regarding their response to these stimuli. Furthermore, cell survival was analyzed in correlation with time in organ culture.
RESULTS
xCell-based mathematical deconvolution of bulk RNA-seq data revealed the presence of CD8 T cells, Th17 cells, dendritic cells, and macrophages as the predominant immune cell types in organ-cultured human corneal tissue. Furthermore, RNA-seq allowed the detection of different macrophage marker genes in corneal samples, including PTPRC (CD45), ITGAM (CD11b), CD14, and CD74. Our RNA-seq data showed no evidence of a relevant presence of monocytes in human corneal tissue. The presence of different macrophage subtypes was confirmed by IHC. The disintegration and subsequent FC analysis of human corneal samples showed the presence of both M1 (HLA-DR+, CD282+, CD86+, and CD284+) and M2 (CD163+ and CD206+) macrophage subtypes. Furthermore, we found that the total number of macrophages in corneal samples decreased more than the total cell count with increasing tissue culture time. Treatment with IL-10 led to higher total cell counts per cornea and to an increased expression of the M2 marker CD163 (p < 0.05) while expression levels of various M1 macrophage markers were not significantly reduced by interleukin treatment.
CONCLUSIONS
Regarding different macrophage populations, untreated human corneas showed more M1 than M2 macrophages. With increasing organ culture time, these macrophages decreased. In terms of cell dynamics, adding interleukins to the organ culture medium influenced the phenotype of macrophages within the cornea as detected by FC. Modifying the immunomodulatory properties of human grafts appears a promising approach to further reduce the risk of graft rejection in patients. In this context, treatment with interleukins was more effective in upregulating M2 macrophages than in suppressing M1 macrophages in corneal tissue.
Topics: Animals; Cornea; Humans; Immunity, Innate; Macrophages; Mice; Monocytes; Organ Culture Techniques
PubMed: 34182556
DOI: 10.1159/000516669