scholarly journals The fate and lifespan of human monocyte subsets in steady state and systemic inflammation

2017 ◽  
Vol 214 (7) ◽  
pp. 1913-1923 ◽  
Author(s):  
Amit A. Patel ◽  
Yan Zhang ◽  
James N. Fullerton ◽  
Lies Boelen ◽  
Anthony Rongvaux ◽  
...  
Keyword(s):  
Steady State ◽  
Inflammatory Responses ◽  
Dynamic Equilibrium ◽  
Human Monocyte ◽  
Monocyte Subsets ◽  
Deuterium Labeling ◽  
Developmental Relationship ◽  
Endotoxemia Model ◽  
Classical Monocytes

In humans, the monocyte pool comprises three subsets (classical, intermediate, and nonclassical) that circulate in dynamic equilibrium. The kinetics underlying their generation, differentiation, and disappearance are critical to understanding both steady-state homeostasis and inflammatory responses. Here, using human in vivo deuterium labeling, we demonstrate that classical monocytes emerge first from marrow, after a postmitotic interval of 1.6 d, and circulate for a day. Subsequent labeling of intermediate and nonclassical monocytes is consistent with a model of sequential transition. Intermediate and nonclassical monocytes have longer circulating lifespans (∼4 and ∼7 d, respectively). In a human experimental endotoxemia model, a transient but profound monocytopenia was observed; restoration of circulating monocytes was achieved by the early release of classical monocytes from bone marrow. The sequence of repopulation recapitulated the order of maturation in healthy homeostasis. This developmental relationship between monocyte subsets was verified by fate mapping grafted human classical monocytes into humanized mice, which were able to differentiate sequentially into intermediate and nonclassical cells.

scholarly journals Monocyte Subsets Are Differentially Lost from the Circulation during Acute Inflammation Induced by Human Experimental Endotoxemia

2017 ◽  
Vol 9 (5) ◽  
pp. 464-474 ◽  
Author(s):  
Tamar Tak ◽  
Roger van Groenendael ◽  
Peter Pickkers ◽  
Leo Koenderman
Keyword(s):  
Acute Inflammation ◽  
Human Monocyte ◽  
Monocyte Subset ◽  
Monocyte Subsets ◽  
Human Endotoxemia ◽  
Cd38 Expression ◽  
Inflammatory Conditions ◽  
Endotoxemia Model ◽  
Differentiation Status ◽  
Classical Monocytes

Three human monocyte subsets are recognized with different functions in the immune system: CD14++/CD16- classical monocytes (CM), CD14++/CD16+ intermediate monocytes (IM) and CD14+/CD16++ non-classical monocytes (NCM). Increased IM and NCM percentages have been reported under inflammatory conditions, yet little is known about monocyte subsets at the onset of inflammation. The human endotoxemia model is uniquely capable of studying the first phases of acute inflammation induced by intravenous injection of 2 ng/kg bodyweight lipopolysaccharide (LPS) into healthy volunteers. After that, monocyte subset counts, activation/differentiation status and chemokine levels were studied over 24 h. The numbers of all subsets were decreased by >95% after LPS injection. CM numbers recovered first (3- 6 h), followed by IM (6-8 h) and NCM numbers (8-24 h). Similarly, increased monocyte counts were observed first in CM (8 h), followed by IM and NCM (24 h). Monocytes did not display a clear activated phenotype (minor increase in CD11b and CD38 expression). Plasma levels of CCL2, CCL4 and CX3CL1 closely resembled the cell numbers of CM, IM and NCM, respectively. Our study provides critical insights into the earliest stages of acute inflammation and emphasizes the necessity to stain for different monocyte subsets when studying the role of monocytes in disease, as neither function nor kinetics of the subsets overlap.

scholarly journals Protease-Activated Receptors 1 and 3 are Differentially Expressed on Human Monocyte Subsets and are Upregulated by Lipopolysaccharide Ex Vivo and In Vivo

2019 ◽  
Vol 119 (09) ◽  
pp. 1394-1402 ◽  
Author(s):  
Barbara Thaler ◽  
Philipp J. Hohensinner ◽  
Johanna Baumgartner ◽  
Patrick Haider ◽  
Konstantin A. Krychtiuk ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Critical Role ◽  
Human Monocyte ◽  
In Vivo Model ◽  
Monocyte Subsets ◽  
Plasminogen Activator Inhibitor 1 ◽  
Protease Activated Receptors ◽  
Messenger Ribonucleic Acid ◽  
Pai 1

AbstractMonocytes are activated in inflammatory conditions via a variety of cytokine receptors as well as in a procoagulatory setting through thrombin, acting upon protease-activated receptors (PARs). This study investigated the expression pattern of PAR1 and PAR3 on human monocyte subsets. Furthermore, a possible regulation of the expression of PAR1 and PAR3 in these cells by inflammatory activation were studied. CD16+ monocytes showed significantly higher levels of PAR1 and PAR3 as compared with CD16− monocytes. Ex vivo treatment of whole blood with lipopolysaccharide (LPS) increased PAR1 and PAR3 messenger ribonucleic acid (mRNA) in human monocytes. In addition, increase of PAR1 was seen in all three subsets upon LPS treatment, whereas PAR3 increased significantly only in CD16− monocytes and nonclassical CD16+ monocytes. Protein levels of PAR1 and PAR3 significantly increased on monocytes in vivo in human endotoxemia 1 hour after LPS infusion. PAR1 increased significantly in CD16− monocytes and nonclassical CD16+ monocytes. In this in vivo model, PAR3 was also significantly elevated in CD16− monocytes and increased slightly albeit not significantly in CD16+ monocytes. Endotoxemia increased plasminogen activator inhibitor-1 (PAI-1) and tissue factor (TF) expression in monocytes in humans. Pretreatment of healthy volunteers with the PAR1 antagonist vorapaxar blocked the increase in PAI-1 but not the increase in TF. We here provide new evidence for a critical role for monocytes as cellular mediators that contribute to the activation of coagulation in diseases characterized by an inflammatory state.

Abstract P786: Effect of Age and Stroke on Purinergic Receptor P2x4 (p2x4r) Expression in Human Monocyte Subsets

Stroke ◽  
2021 ◽  
Vol 52 (Suppl_1) ◽  
Author(s):  
Rajkumar Verma ◽  
Sharon E DiMauro ◽  
Leslie Blumenfeld ◽  
Pranay Srivastava ◽  
Sanjay Mittal ◽  
...  
Keyword(s):  
Therapeutic Potential ◽  
Purinergic Receptor ◽  
Flow Cytometric Analysis ◽  
Human Monocyte ◽  
Stroke Recovery ◽  
Monocyte Count ◽  
Monocyte Subsets ◽  
Fluorescent Intensity ◽  
Healthy Control ◽  
Classical Monocytes

Backgrounds: An acute ischemic stroke (AIS) triggers rapid infiltration of circulating immune cells in the brain. P2X4R, a receptor for adenosine triphosphate ATP, regulate activation of circulating monocytes after stroke injury. Over-stimulation of P2X4R contributes to ischemic injury. CD14 ++ CD16 – classical, CD14 ++ CD16 + intermediate, and CD14 + CD16 ++ non-classical monocytes are three primary subsets of monocytes. Alterations in activity of circulating monocyte subsets may independently predict pathogenesis of AIS, however, the role of P2X4R in the activation of these monocyte subsets is not known. Methods: Consecutive AIS patients (60-90 years) undergoing endovascular clot retrieval and healthy control subjects both young (18-45 years) and aged (60-90 years) of both sexes were recruited and informed consent obtained. Flow cytometric analysis of whole blood derived monocytes at 0-2 days (acute, n=10), 3-7 days (subacute, n=9), and 65±20 days (chronic, n=7) after stroke onset were compared with healthy subjects (n=9-10/ age group). Results: Both number of total monocyte counts and P2X4R intensity significantly increase with age when compared between healthy young and aged control (P<0.05). Total monocyte count progressively increased during recovery in AIS patient (P<0.05). No. of CD14 ++ CD16 + intermediate monocytes were significantly reduced after stroke ( p <0.05). Both CD14 ++ CD16 + intermediate, and CD14 + CD16 ++ non-classical monocytes showed a significant increased median fluorescent intensity (P<0.01) of P2X4R at subacute and chronic time after AIS. Conclusions: P2X4R expression increases with age and after stroke. Disappearance of the CD14 + CD16 ++ non-classical monocyte subpopulation from circulation during stroke recovery suggests potential migration of these cells to the site of injury, consistent with their potential role in inflammation/phagocytosis. Increased P2X4R expression in intermediate and non-classical monocytes subpopulation suggest its specific role in selective activation of these monocytes subtype. Detailed molecular characterization of P2X4R response in intermediate and non-classical monocyte subpopulations may provide novel insights into P2X4R’s therapeutic potential during AIS.

In Vivo Visualization of Stat3 Activation in Myeloid Cells during Inflammation and Tumor Growth.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3434-3434
Author(s):  
Pedro Horna ◽  
Fengdong Cheng ◽  
Richard Jove ◽  
Linda Mora ◽  
Eduardo M. Sotomayor
Keyword(s):  
Steady State ◽  
Immune Responses ◽  
Tumor Growth ◽  
Peripheral Blood ◽  
Inflammatory Responses ◽  
Myeloid Cells ◽  
Tumor Bearing ◽  
Inflammatory Site ◽  
Number Of Cells

Abstract Signal transducer and activator of transcription 3 (Stat3) is a key mediator of several cytokine and growth factor signaling pathways. On myeloid cells, activation of Stat3 to its phosphorylated form (pStat3) has been shown to negatively regulate inflammatory responses and play a central role in the decision leading to immune activation versus immune tolerance of antigen-specific T-cells1. Little is still known however, about the status of Stat3 signaling in myeloid cells in the steady state and during ongoing immune responses in vivo. To address this question we recently developed flow-cytometric and immuno-histochemistry assays that have allowed us to visualize the in vivo dynamics of Stat3 activation in myeloid cells during immune responses leading to divergent outcomes: productive inflammatory response to adjuvant immunization and tumor-induced unresponsiveness or tolerance. In the steady state we found that in peripheral blood only Ly6G+ polymorphonuclear cells display a positive nuclear staining for pStat3. Analysis of lymphoid organs revealed that although Stat3 protein was expressed almost ubiquitously on spleen sections of normal mice, only a small number of cells were positive for pStat3. Following immunization with complete Freund adjuvant (CFA) a dramatic increase in the number of cells expressing pStat3 was observed in the peripheral blood and spleen of treated animals. Ly6G+ pStat3+ were rapidly recruited from the blood to the inflammatory site where they now displayed significantly decreased levels of pStat3. During the growth of a subcutaneous tumor, a similar increase in the number of cells expressing pStat3 was observed in the blood and spleen of tumor-bearing mice. Further analysis by flow cytometry revealed that pStat3 expression was restricted to two sub-populations: a) CD11b+ myeloid cells expressing the lineage marker Gr-1 and b) Ly6G− mononuclear cells unable to down-regulate Stat3 activity following their migration from the blood into peripheral tissues. In vivo depletion of Gr-1+ cells eliminated most of the pStat3+ cells in tumor bearing mice. The immunoregulatory properties of these Gr-1+ cells was highlighted by the demonstration that in their absence, in vivo immunization with a peptide derived from influenza hemagglutinin (HA) in CFA markedly enhanced the priming of anti-HA specific CD4+ T-cells. More importantly, in animals depleted of Gr-1+ cells, the outcome in response to a tolerogenic stimuli was T-cell activation rather than tolerance induction. Taken together, although similar changes in the number of cells expressing pStat3 was observed in response to adjuvant immunization and during tumor progression, an important difference might relate to the extent of Stat3 activation in myeloid cells following their migration to the site of stimuli. While down-regulation of Stat3 in myeloid cells at the inflammatory site is an early event during productive inflammatory responses, a sustained Stat3 activation in myeloid cells such as that observed during tumor growth may provide an explanation for the state of immune unresponsiveness associated with malignancies.

scholarly journals Circulatory and maturation kinetics of human monocyte subsets in vivo

Blood ◽  
2017 ◽  
Vol 130 (12) ◽  
pp. 1474-1477 ◽  
Author(s):  
Tamar Tak ◽  
Julia Drylewicz ◽  
Lennart Conemans ◽  
Rob J. de Boer ◽  
Leo Koenderman ◽  
...  
Keyword(s):  
Human Monocyte ◽  
Monocyte Subsets ◽  
Kinetics Of

Gene expression profiling reveals the defining features of the classical, intermediate, and nonclassical human monocyte subsets

Blood ◽  
2011 ◽  
Vol 118 (5) ◽  
pp. e16-e31 ◽  
Author(s):  
Kok Loon Wong ◽  
June Jing-Yi Tai ◽  
Wing-Cheong Wong ◽  
Hao Han ◽  
Xiaohui Sem ◽  
...  
Keyword(s):  
Cytokine Production ◽  
Target Identification ◽  
Human Monocyte ◽  
Surface Expression ◽  
Surface Proteins ◽  
Monocyte Subsets ◽  
Developmental Relationship ◽  
Close Relationship ◽  
Transcription Factor Genes ◽  
Intermediate Monocytes

Abstract New official nomenclature subdivides human monocytes into 3 subsets: the classical (CD14++CD16−), intermediate (CD14++CD16+), and nonclassical (CD14+CD16++) monocytes. This introduces new challenges, as monocyte heterogeneity is mostly understood based on 2 subsets, the CD16− and CD16+ monocytes. Here, we comprehensively defined the 3 circulating human monocyte subsets using microarray, flow cytometry, and cytokine production analysis. We find that intermediate monocytes expressed a large majority (87%) of genes and surface proteins at levels between classical and nonclassical monocytes. This establishes their intermediary nature at the molecular level. We unveil the close relationship between the intermediate and nonclassic monocytes, along with features that separate them. Intermediate monocytes expressed highest levels of major histocompatibility complex class II, GFRα2 and CLEC10A, whereas nonclassic monocytes were distinguished by cytoskeleton rearrangement genes, inflammatory cytokine production, and CD294 and Siglec10 surface expression. In addition, we identify new features for classic monocytes, including AP-1 transcription factor genes, CLEC4D and IL-13Rα1 surface expression. We also find circumstantial evidence supporting the developmental relationship between the 3 subsets, including gradual changes in maturation genes and surface markers. By comprehensively defining the 3 monocyte subsets during healthy conditions, we facilitate target identification and detailed analyses of aberrations that may occur to monocyte subsets during diseases.

scholarly journals Thrombospondin 1 Is an Autocrine Negative Regulator of Human Dendritic Cell Activation

2003 ◽  
Vol 198 (8) ◽  
pp. 1277-1283 ◽  
Author(s):  
Virginie Doyen ◽  
Manuel Rubio ◽  
Deborah Braun ◽  
Toshiaru Nakajima ◽  
Jun Abe ◽  
...  
Keyword(s):  
Steady State ◽  
Cell Activation ◽  
Cytokine Production ◽  
Transforming Growth Factor ◽  
Human Monocyte ◽  
Negative Regulator ◽  
Dendritic Cell Activation ◽  
Tnf Α ◽  
Thrombospondin 1

Thrombospondin 1 (TSP) elicits potent antiinflammatory activities in vivo, as evidenced by persistent, multiorgan inflammation in TSP null mice. Herein, we report that DCs represent an abundant source of TSP at steady state and during activation. Human monocyte-derived immature dendritic cells (iDCs) spontaneously produce TSP, which is strongly enhanced by PGE2 and to a lesser extent by transforming growth factor (TGF) β, two soluble mediators secreted by macrophages after engulfment of damaged tissues. Shortly after activation via danger signals, DCs transiently produce interleukin (IL) 12 and tumor necrosis factor (TNF) α, thereby eliciting protective and inflammatory immune responses. Microbial stimuli increase TSP production, which is further enhanced by IL-10 or TGF-β. The endogenous TSP produced during early DC activation negatively regulates IL-12, TNF-α, and IL-10 release through its interactions with CD47 and CD36. After prolonged activation, DCs extinguish their cytokine synthesis and become refractory to subsequent stimulation, thereby favoring the return to steady state. Such “exhausted” DCs continue to release TSP but not IL-10. Disrupting TSP–CD47 interactions during their restimulation restores their cytokine production. We conclude that DC-derived TSP serves as a previously unappreciated negative regulator contributing to arrest of cytokine production, further supporting its fundamental role in vivo in the active resolution of inflammation and maintenance of steady state.

scholarly journals Distinct immune regulatory receptor profiles linked to altered monocyte subsets in sarcoidosis

2020 ◽  
pp. 00804-2020
Author(s):  
Simon D. Fraser ◽  
Michael G. Crooks ◽  
Paul M. Kaye ◽  
Simon P. Hart
Keyword(s):  
Inflammatory Responses ◽  
Pulmonary Sarcoidosis ◽  
Receptor Expression ◽  
Healthy Controls ◽  
Blood Monocytes ◽  
Monocyte Subsets ◽  
Oral Corticosteroids ◽  
Duration Of Disease ◽  
Classical Monocytes ◽  
Intermediate Monocytes

BackgroundIn sarcoidosis, blood monocytes, circulating precursors of granuloma macrophages, display enhanced inflammatory cytokine production, reduced expression of the regulatory (inhibitory) receptor CD200R, and altered subsets defined by CD14 and CD16. Regulatory receptors serve to dampen monocyte and macrophage inflammatory responses. We investigated the relationship between monocyte subsets and regulatory receptor expression in sarcoidosis.MethodsMulti-parameter flow cytometry was used to perform detailed analyses of cell surface regulatory molecules on freshly isolated blood immune cells from patients with chronic pulmonary sarcoidosis and age-matched healthy controls.ResultsTwenty-five patients with chronic pulmonary sarcoidosis (median duration of disease 22 months) who were not taking oral corticosteroids or other immunomodulators were recruited. Non-classical monocytes were expanded in sarcoidosis and exhibited significantly lower expression of regulatory receptors CD200R, SIRP-α, and CD47 than classical or intermediate monocytes. In sarcoidosis, all three monocyte subsets had significantly reduced CD200R and CD47 expression compared with healthy controls. A dichotomous distribution of CD200R was seen on classical and intermediate monocytes in the sarcoidosis population, with 14/25 (56%) sarcoidosis patients having a CD200R-low phenotype, and 11/25 (44%) CD200R-high. These distinct sarcoidosis monocyte phenotypes remained consistent over time.ConclusionNon-classical monocytes, which are expanded in sarcoidosis, express very low levels of regulatory receptors. Two distinct and persistent phenotypes of CD200R expression in classical and intermediate monocytes could be evaluated as sarcoidosis biomarkers.

MIR150 Is Involved in the Monocyte Subset Differentiation and Its Down-Regulation Leads to Classical Monocyte Accumulation in Chronic Myelomonocytic Leukemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1133-1133
Author(s):  
Dorothee Selimoglu-Buet ◽  
Julie Riviere ◽  
Margot Morabito ◽  
Catherine Lacout ◽  
Aurelie Chauveau ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Peripheral Blood ◽  
Human Monocyte ◽  
Chronic Myelomonocytic Leukemia ◽  
Monocyte Subset ◽  
Monocyte Subsets ◽  
Down Regulation ◽  
Cd34 Cells ◽  
Myelomonocytic Leukemia ◽  
Classical Monocytes

Abstract Background. Monocytes are a heterogeneous population of peripheral blood leukocytes. The expression of CD14 and CD16 distinguishes CD14+/CD16- classical from CD14+/CD16+ intermediate and CD14low/CD16+ non-classical monocytes. We have shown (Selimoglu-Buet D et al, Blood 2015) that monocytes that accumulate in the peripheral blood of patients with chronic myelomonocytic leukemia (CMML) are predominantly CD14+/CD16- classical monocytes that typically represent more than 94% of total blood monocytes. Strikingly, this phenotypic signature efficiently distinguishes CMML from a reactive monocytosis. Importantly, the CMML-associated increase in classical monocyte fraction disappears in patients who respond to hypomethylating drugs. Whereas in the mouse, the transcription factor Nr4a1 is required for the development of the Ly6Clowmonocytes, the molecular mechanisms that regulate the formation of the three human monocyte populations remain poorly understood. Analysis of the classical monocytes accumulation in CMML may provide insights into the regulation of monocyte subset differentiation. Methods. A microarray screen of miRNA expression was performed in monocytes sorted from 33 CMML and 5 healthy donor blood samples. Validation was performed by qRT-PCR, in monocytes of a cohort of 160 CMML patients and 20 controls, and in CD34+ cells from 44 CMML patients and 19 controls. A mouse model of MIR150-knock-out (Mir150-/-) was used to examine the consequences of the miRNA down-regulation. Multi-color flow cytometry assays were designed to explore mouse and human monocyte subsets. Results. Microarray analyses and validation experiments identified a decreased expression of miR150 in monocytes and CD34+cells from CMML patients compared to controls. Mir150-/- mouse model does not generate monocytosis even in ageing animals. However, an increase in Ly6Chigh inflammatory monocyte fraction at the expense of Ly6Clowpatrolling monocytes was observed in the bone marrow and peripheral blood, leading to further explore the link between MIR150 and monocyte populations. The abnormal repartition of monocyte populations in Mir150-/- mice is a cell-autonomous phenotype as wild-type (WT) mice receiving bone marrow from Mir150-/-mice demonstrated a reduced fraction of Ly6Clow monocytes. This phenotype was rescued by re-expression of MIR150 in LIN- cells of Mir150-/-mice before engraftment. The number of myeloid progenitors was normal in Mir150-/-mice, and the remaining Ly6Clow monocytes did not demonstrate an increased sensitivity to apoptosis. Competitive reconstitution experiments combining WT and Mir150-/-LIN- cells did not identify any significant fitness advantage to Mir150-/-cells, but Mir150-/-donor cells developed reduced numbers of Ly6Clow monocytes than cells from WT donors. These data suggest that MIR150 is involved during late stages of monocyte development and has a key role in the generation of Ly6Clowmonocytes. Finally, TET2 is the main gene mutated in CMML, and Tet2-/- animals develop a monocytosis. Mir150-/- crossed with Tet2-/-mice developed a CMML-like phenotype. In human, the expression of MIR150 decreases along myeloid differentiation and is low in classical compared to intermediate and non-classical monocytes. Depletion or overexpression of MIR150 in human CD34+ cells alters the repartition of CD14+/CD16- and CD14+/CD16+ cells generated in culture. In CMML patients who respond to hypomethylating agents, the normalization of monocyte subset repartition correlates with an increased expression of MIR150, suggesting an epigenetic regulation. MIR150 has several promoters. By combining ChIP-Seq and DNA methylation analyses, a differentially methylated region was detected in one of the MIR150 promoters in CMML patients compared to controls. Using monocyte differentiation conditions, RNA Sequencing performed in CD34+cells overexpressing MIR150, identified ID1 gene as a potential MIR150 target. Conclusion: We demonstrate a role for MIR150 in the generation of intermediate and non-classical monocyte subsets, and its down-regulation in CMML accounts for the characteristic accumulation of classical monocytes. Disclosures Fenaux: Celgene, Janssen,Novartis, Astex, Teva: Honoraria, Research Funding.

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