Dataset Information

A Novel, Five-Marker Alternative to CD16-CD14 Gating to Identify the Three Human Monocyte Subsets.

ABSTRACT: Human primary monocytes are heterogeneous in terms of phenotype and function, but are sub-divided only based on CD16 and CD14 expression. CD16 expression distinguishes a subset of monocytes with highly pro-inflammatory properties from non-CD16 expressing "classical" monocytes. CD14 expression further subdivides the CD16⁺ monocytes into non-classical CD14^low and intermediate CD14^high subsets. This long-standing CD16-CD14 classification system, however, has limitations as CD14 is expressed in a continuum, leading to subjectivity in delineating the non-classical and intermediate subsets; in addition, CD16 expression is unstable, making identification of the subsets impossible after in vitro culture or during inflammatory conditions in vivo. Hence, we aimed to identify the three monocyte subsets using an alternative combination of markers. Additionally, we wanted to address whether the monocyte subset perturbations observed during infection is real or an artifact of differential CD16 and/or CD14 regulation. Using cytometry by time-of-flight (CyTOF), we studied the simultaneous expression of 34 monocyte markers on total monocytes, and derived a combination of five markers (CD33, CD86, CD64, HLA-DR, and CCR2), that could objectively delineate the three subsets. Using these markers, we could also distinguish CD16⁺ monocytes from CD16^- monocytes after in vitro stimulation. Finally, we found that the observed expansion of intermediate (CD14^high) monocytes in dengue virus-infected patients was due to up-regulated CD16 expression on classical monocytes. With our new combination of markers, we can now identify monocyte subsets without CD16 and CD14, and accurately re-examine monocyte subset perturbations in diseases.

SUBMITTER: Ong SM

PROVIDER: S-EPMC6676221 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

A Novel, Five-Marker Alternative to CD16-CD14 Gating to Identify the Three Human Monocyte Subsets.

Ong Siew-Min SM Teng Karen K Newell Evan E Chen Hao H Chen Jinmiao J Loy Thomas T Yeo Tsin-Wen TW Fink Katja K Wong Siew-Cheng SC

Frontiers in immunology 20190726

Human primary monocytes are heterogeneous in terms of phenotype and function, but are sub-divided only based on CD16 and CD14 expression. CD16 expression distinguishes a subset of monocytes with highly pro-inflammatory properties from non-CD16 expressing "classical" monocytes. CD14 expression further subdivides the CD16+ monocytes into non-classical CD14low and intermediate CD14high subsets. This long-standing CD16-CD14 classification system, however, has limitat ...[more]

PMID: 31402918

Similar Datasets

Project description:BackgroundHuman peripheral blood monocytes (Mo) consist of subsets distinguished by expression of CD16 (FCgammaRIII) and chemokine receptors. Classical CD16- Mo express CCR2 and migrate in response to CCL2, while a minor CD16+ Mo subset expresses CD16 and CX3CR1 and migrates into tissues expressing CX3CL1. CD16+ Mo produce pro-inflammatory cytokines and are expanded in certain inflammatory conditions including sepsis and HIV infection.ResultsTo gain insight into the developmental relationship and functions of CD16+ and CD16- Mo, we examined transcriptional profiles of these Mo subsets in peripheral blood from healthy individuals. Of 16,328 expressed genes, 2,759 genes were differentially expressed and 228 and 250 were >2-fold upregulated and downregulated, respectively, in CD16+ compared to CD16- Mo. CD16+ Mo were distinguished by upregulation of transcripts for dendritic cell (DC) (SIGLEC10, CD43, RARA) and macrophage (MPhi) (CSF1R/CD115, MafB, CD97, C3aR) markers together with transcripts relevant for DC-T cell interaction (CXCL16, ICAM-2, LFA-1), cell activation (LTB, TNFRSF8, LST1, IFITM1-3, HMOX1, SOD-1, WARS, MGLL), and negative regulation of the cell cycle (CDKN1C, MTSS1), whereas CD16- Mo were distinguished by upregulation of transcripts for myeloid (CD14, MNDA, TREM1, CD1d, C1qR/CD93) and granulocyte markers (FPR1, GCSFR/CD114, S100A8-9/12). Differential expression of CSF1R, CSF3R, C1QR1, C3AR1, CD1d, CD43, CXCL16, and CX3CR1 was confirmed by flow cytometry. Furthermore, increased expression of RARA and KLF2 transcripts in CD16+ Mo coincided with absence of cell surface cutaneous lymphocyte associated antigen (CLA) expression, indicating potential imprinting for non-skin homing.ConclusionThese results suggest that CD16+ and CD16- Mo originate from a common myeloid precursor, with CD16+ Mo having a more MPhi - and DC-like transcription program suggesting a more advanced stage of differentiation. Distinct transcriptional programs, together with their recruitment into tissues via different mechanisms, also suggest that CD16+ and CD16- Mo give rise to functionally distinct DC and MPhi in vivo.

Project description:Acute myocardial infarction (AMI) evokes a temporally coordinated immune response, in which monocytes are critically involved in the clearance of cell debris; however, excessive inflammation induced by the classical sub-population of monocytes frequently limits the endogenous reparative process. In the present study, the potential of the anti-inflammatory adipokine complement C1q tumor necrosis factor (TNF)-related protein-3 (CTRP3) to induce intermediate switch of monocytes to an anti-inflammatory phenotype was explored. Circulating monocytes were isolated from patients with AMI at various time-points (3-5 h, 3 days and 7 days) and categorized by flow cytometry/immunostaining into three sub-divisions based on the expression of CD14 and CD16 epitopes: Classical (CD14++/CD16-), non-classical (CD14+/CD16++) and intermediate populations (CD14++/CD16+). The phagocytic activity was evaluated by the ingestion of FITC-Zymosan and 19F-nanoemulsion and the migratory activity using Thin Cert™ Transwell assay. Monocytes were cultured using autologous serum in the presence of CTRP3 (1 µg/ml) for 24 h and the expression of interleukin 6 (IL-6) and TNF-α was quantified by reverse-transcription quantitative PCR. In addition, SB203580, a p38 mitogen-activated protein kinase (MAPK)/ERK inhibitor, was used to examine the downstream pathways of CTRP3. AMI evoked a transient increase in monocyte counts of the classical subset after onset of the ischemic insult, while the non-classical and intermediate subsets persistently expanded (P<0.01). The monocytes from patients at 3 days after AMI displayed enhanced phagocytic and migratory activities in comparison with those from healthy volunteers (P<0.01). Of note, addition of CTRP3 induced an intermediate switch of monocyte subsets and antagonized the enhanced expression of cytokines, particularly IL-6, in monocytes stressed by lipopolysaccharides, likely by blunting the ERK1/2 and P38 MAPK signaling pathway. In conclusion, the present study demonstrated a dynamic fluctuation of monocyte subsets and enhanced phagocytic and migratory activities in patients with AMI. Furthermore, the 'proof-of-concept' evidence pinpoints CTRP3 as an alternative candidate to modulate the 'uncontrolled' inflammatory response and thus to augment cardiac reparative processes in patients with AMI.

Dataset Information

A Novel, Five-Marker Alternative to CD16-CD14 Gating to Identify the Three Human Monocyte Subsets.

Publications

A Novel, Five-Marker Alternative to CD16-CD14 Gating to Identify the Three Human Monocyte Subsets.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure