Hepatic Ly6CLo Non-Classical Monocytes Have Increased Nr4a1 (Nur77) in Murine Biliary Atresia

Mohamedaly, Sarah; Levy, Claire S.; Korsholm, Cathrine; Alkhani, Anas; Rosenberg, Katherine; Ashouri, Judith F.; Nijagal, Amar

doi:10.3390/jcm11185290

Cited by 3 publications

(1 citation statement)

References 40 publications

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“…As the first step, we aimed to set up a flow cytometry panel to characterize classical and non-classical monocytes based on Ly6C and CX3CR1, as suggested in the literature [1,[36][37][38]. Thus, we isolated cells from the peripheral blood of C57Bl/6 mice and analyzed them by flow cytometry.…”

Section: Monocytes From the Peripheral Blood And Spleen Of C57bl/6 Mi...mentioning

confidence: 99%

Discrepant Phenotyping of Monocytes Based on CX3CR1 and CCR2 Using Fluorescent Reporters and Antibodies

Sommer,

Garibagaoglu,

Paap

et al. 2024

Cells

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Monocytes, as well as downstream macrophages and dendritic cells, are essential players in the immune system, fulfilling key roles in homeostasis as well as in inflammatory conditions. Conventionally, driven by studies on reporter models, mouse monocytes are categorized into a classical and a non-classical subset based on their inversely correlated surface expression of Ly6C/CCR2 and CX3CR1. Here, we aimed to challenge this concept by antibody staining and reporter mouse models. Therefore, we took advantage of Cx3cr1GFP and Ccr2RFP reporter mice, in which the respective gene was replaced by a fluorescent reporter protein gene. We analyzed the expression of CX3CR1 and CCR2 by flow cytometry using several validated fluorochrome-coupled antibodies and compared them with the reporter gene signal in these reporter mouse strains. Although we were able to validate the specificity of the fluorochrome-coupled flow cytometry antibodies, mouse Ly6Chigh classical and Ly6Clow non-classical monocytes showed no differences in CX3CR1 expression levels in the peripheral blood and spleen when stained with these antibodies. On the contrary, in Cx3cr1GFP reporter mice, we were able to reproduce the inverse correlation of the CX3CR1 reporter gene signal and Ly6C surface expression. Furthermore, differential CCR2 surface expression correlating with the expression of Ly6C was observed by antibody staining, but not in Ccr2RFP reporter mice. In conclusion, our data suggest that phenotyping strategies for mouse monocyte subsets should be carefully selected. In accordance with the literature, the suitability of CX3CR1 antibody staining is limited, whereas for CCR2, caution should be applied when using reporter mice.

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Section: Monocytes From the Peripheral Blood And Spleen Of C57bl/6 Mi...mentioning

confidence: 99%

Discrepant Phenotyping of Monocytes Based on CX3CR1 and CCR2 Using Fluorescent Reporters and Antibodies

Sommer,

Garibagaoglu,

Paap

et al. 2024

Cells

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Changes in <i>in vitro</i> GM-CSF-exposured monocyte subset composition and phagocytic activity in children with infectious mononucleosis

Савченко

Мартынова

Ikkes

et al. 2023

Russian Journal of Infection and Immunity

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The aim of the study was to investigate the features of changes in the subset composition of monocytes and their phagocytic activity in children with infectious mononucleosis (IM) under the influence of granulocyte-macrophage colony-stimulating factor (GM-CSF) in vitro. We examined 84 children aged 3 to 11 years with Epstein-Barr virus (EBV) infection. EBV infection was diagnosed on the basis of clinical signs of IM, a positive test for EBV DNA in blood lymphocytes and results of ELISA tests (EBV-VCAIgM (+), EBV-EA-DIgG (+)). The control group consisted of 40 practically healthy children of the same age range. Monocytes were obtained by the standard method of adhesion to plastic from mononuclear cells isolated from heparinized venous blood by density gradient centrifugation. The isolated monocytes were divided into two samples: control (without GM-CSF) and experimental (50 ng of GM-CSF per 1 ml of cell suspension). The subset composition and phagocytic activity of monocytes in both samples were determined by flow cytometry after incubation for 1 hour at 37C in a CO2-incubator. It was found that in children against the background of the development of IM, the subpopulation composition of monocytes in the blood changes and their phagocytic activity is impaired. It was found that the subset composition and phagocytic activity of the blood monocytes changed in children against the background of the development of IM. Changes in the subset composition of monocytes against the background of acute IM did not depend on the age group of children (3-6 and 7-11 years) and were characterized by an increase in the number of pro-inflammatory (intermediate) monocytes and a decrease in the content of anti-inflammatory (non-classical) monocytes. Features of violation of the phagocytic activity of the monocytes in children with IM depended on age. The phagocytic activity of all three subsets of the monocytes was reduced in children with IM 3-6 years old while children with IM 7-11 years old had reduced phagocytic activity only of intermediate and non-classical monocytes. The effect of GM-CSF in vitro on monocytes in patients with IM, regardless of the age of children, led to a significant increase in the level of anti-inflammatory monocytes while the phagocytic activity of cells changed less. An increase in the phagocytic number for classical monocytes after incubation with GM-CSF in vitro was noted in children with IM at the age of 3-6 years while the phagocytic index of this fraction of monocytes remained unchanged. The level of the phagocytic index increased only in classical monocytes of children with IM aged 7-11 years. The presented results determine the scientific and clinical value of studying the mechanisms of the effect of GM-CSF on cells of the immune system and prove that this cytokine can be used in a new immunotherapeutic strategy for the treatment of IM.

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Discrepant phenotyping of monocytes based on CX3CR1 using fluorescent reporters and antibodies

Sommer,

Garibagaoglu,

Wiendl

et al. 2023

Preprint

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Monocytes as well as downstream macrophages and dendritic cells are essential players of the immune system fulfilling key roles in homeostasis as well as in inflammatory conditions. Conventionally, driven by studies in reporter models, mouse monocytes are divided into a classical and a non-classical subset based on their inversely correlating surface expression of Ly6C and CX3CR1. Here, we analysed the expression of CX3CR1 by flow cytometry using several validated fluorochrome-coupled CX3CR1 antibodies and compared them with the reporter gene signal of a Cx3cr1GFP reporter mouse strain as well as of tamoxifen-inducible Cx3cr1 reporter mice. Although we were able to validate the specificity of several fluorochrome-coupled CX3CR1 flow cytometry antibodies, mouse Ly6Chigh classical and Ly6Clow non-classical monocytes showed no differences in CX3CR1 expression levels in peripheral blood and spleen, when stained with these antibodies. To the contrary, in reporter mice, we were able to reproduce the inverse correlation of CX3CR1 reporter gene signal and Ly6C surface expression. As determined by qPCR, the Cx3cr1 mRNA expression correlated with the GFP-reporter gene expression as quantified by flow cytometry. In conclusion, our data suggest that there is differential transcription, but not surface expression of CX3CR1 between classical and non-classical monocytes, which limits the suitability of CX3CR1 for phenotyping monocyte subsets by antibody staining.

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Hepatic Ly6CLo Non-Classical Monocytes Have Increased Nr4a1 (Nur77) in Murine Biliary Atresia

Cited by 3 publications

References 40 publications

Discrepant Phenotyping of Monocytes Based on CX3CR1 and CCR2 Using Fluorescent Reporters and Antibodies

Discrepant Phenotyping of Monocytes Based on CX3CR1 and CCR2 Using Fluorescent Reporters and Antibodies

Changes in <i>in vitro</i> GM-CSF-exposured monocyte subset composition and phagocytic activity in children with infectious mononucleosis

Discrepant phenotyping of monocytes based on CX3CR1 using fluorescent reporters and antibodies

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