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Background The complement receptor immunoglobulin (CRIg), a key microbial pathogen phagocytosis-promoting receptor, responsible for intravascular clearance of bacteria, is purported to be expressed selectively on tissue-fixed macrophages such as Kupffer cells. However, recently it has been reported that neutrophils can also express functional CRIg following activation by inflammatory mediators. Monocytes have been reported not to express CRIg under non-activated conditions. Thus, investigations were undertaken to examine whether blood monocytes express CRIg under cell activation conditions and its role in anti-microbial immunity. Methods Monocytes CRIg expression in whole human and mouse blood or peripheral blood mononuclear cells and purified monocytes using density gradient centrifugation or an affinity purification kit was examined using PE/FITC-labelled anti-CRIg monoclonal antibody and flow cytometry. Characterization of CRIg isoforms in monocytes was determined by the detection of CRIg mRNA transcripts and protein using RT-PCR and Western blot, respectively. Gene-edited CRIg – and CD18 – monocytic THP-1 cell lines were generated to assess the role of CRIg and CD18 in cell adhesion, phagocytosis, and microbial killing. Functional assays were performed using Staphylococcus aureus as a model pathogen. Results CRIg was constitutively expressed, dynamically, on the surface of human and mouse blood monocytes. All three human monocyte subpopulations expressed CRIg, equally. The inability to demonstrate expression on monocytes cell surface by previous studies can be explained by its lability during blood storage and loss during monocyte isolation steps. Interestingly of the monocyte subpopulations only the classical and intermediate but not the non-classical showed a loss of CRIg expression. The data showed that loss from the surface was most likely due to relocation of the receptor intracellularly. Monocytes expressed 6 different CRIg mRNA transcripts and immunoreactive isoforms. Using CRIg – and CD18 – THP-1 monocytic cells, we found that both CRIg and CD18 (CR3/CR4) were critical for cell adhesion, but for phagocytosis and killing of S. aureus, either receptor was independently effective. Conclusion The data provide compelling evidence that monocytes express functional CRIg, relevant to the cells’ anti-microbial role of the ‘wandering’ phagocyte and consolidate a view that CRIg is widely expressed in our phagocytic cell system, similar to the classical complement receptors CR3 and CR4.

Classical and non-classical monocytes, and the macrophages and monocyte-derived dendritic cells they produce, play key roles in host defense against pathogens, immune regulation, tissue repair and many other processes throughout the body. Recent studies have revealed previously unappreciated heterogeneity among monocytes that may explain this functional diversity, but our understanding of mechanisms controlling the functional programming of distinct monocyte subsets remains incomplete. Resolving monocyte heterogeneity and understanding how their functional identity is determined holds great promise for therapeutic immune modulation. In this review, we examine how monocyte origins and developmental influences shape the phenotypic and functional characteristics of monocyte subsets during homeostasis and in the context of infection, inflammation, and cancer. We consider how extrinsic signals and transcriptional regulators impact monocyte production and functional programming, as well as the influence of epigenetic and metabolic mechanisms. We also examine the evidence that functionally distinct monocyte subsets are produced via different developmental pathways during homeostasis and that inflammatory stimuli differentially target progenitors during an emergency response. We highlight the need for a more comprehensive understanding of the relationship between monocyte ontogeny and heterogeneity, including multiparametric single-cell profiling and functional analyses. Studies definingmechanismsofmonocytesubsetproductionandmaintenanceofunique monocyte identities have the potential to facilitate the design of therapeutic interventions to target specific monocyte subsets in a variety of disease contexts, including infectious and inflammatory diseases, cancer, and aging.

In contrast to the past reliance on morphology, the identification and enumeration of blood monocytes are nowadays done with monoclonal antibodies and flow cytometry and this allows for subdivision into classical, intermediate, and non-classical monocytes. Using specific cell surface markers, dendritic cells in blood can be segregated from these monocytes. While in the past, changes in monocyte numbers as determined in standard hematology counters have not had any relevant clinical impact, the subset analysis now has uncovered informative changes that may be used in management of disease.

The three subsets of human monocytes, classical, intermediate, and nonclassical, show phenotypic heterogeneity, particularly in their expression of CD14 and CD16. This has enabled researchers to delve into the functions of each subset in the steady state as well as in disease. Studies have revealed that monocyte heterogeneity is multi-dimensional. In addition, that their phenotype and function differ between subsets is well established. However, it is becoming evident that heterogeneity also exists within each subset, between health and disease (current or past) states, and even between individuals. This realisation casts long shadows, impacting how we identify and classify the subsets, the functions we assign to them, and how they are examined for alterations in disease. Perhaps the most fascinating is evidence that, even in relative health, interindividual differences in monocyte subsets exist. It is proposed that the individual’s microenvironment could cause long-lasting or irreversible changes to monocyte precursors that echo to monocytes and through to their derived macrophages. Here, we will discuss the types of heterogeneity recognised in monocytes, the implications of these for monocyte research, and most importantly, the relevance of this heterogeneity for health and disease.

In a nomenclature proposal published in 2010 monocytes were subdivided into classical and non-classical cells and in addition an intermediate monocyte subset was proposed. Over the last couple of years many studies have analyzed these intermediate cells, their characteristics have been described, and their expansion has been documented in many clinical settings. While these cells appear to be in transition from classical to non-classical monocytes and hence may not form a distinct cell population in a strict sense, their separate analysis and enumeration is warranted in health and disease.

Monocytes are a highly plastic innate immune cell population that displays significant heterogeneity within the circulation. Distinct patterns of surface marker expression have become accepted as a basis for distinguishing three monocyte subsets in humans. These phenotypic subsets, termed classical, intermediate and nonclassical, have also been demonstrated to differ in regard to their functional properties and disease associations when studied and . Nonetheless, for the intermediate monocyte subset in particular, functional experiments have yielded conflicting results and some studies point to further levels of heterogeneity. Developments in genetic sequencing technology have provided opportunities to more comprehensively explore the phenotypic and functional differences among conventionally-recognized immune cell subtypes as well as the potential to identify novel subpopulations. In this review, we summarize the transcriptomic evidence in support of the existence of three separate monocyte subsets. We also critically evaluate the insights into subset functional distinctions that have been garnered from monocyte gene expression analysis and the potential utility of such studies to unravel subset-specific functional changes which arise in disease states.

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 and intermediate CD14 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 culture or during inflammatory conditions . 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 stimulation. Finally, we found that the observed expansion of intermediate (CD14 ) 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.

Human blood monocytes are heterogeneous and conventionally subdivided into two subsets based on CD16 expression. Recently, the official nomenclature subdivides monocytes into three subsets, the additional subset arising from the segregation of the CD16+ monocytes into two based on relative expression of CD14. Recent whole genome analysis reveal that specialized functions and phenotypes can be attributed to these newly defined monocyte subsets. In this review, we discuss these recent results, and also the description and utility of this new segregation in several disease conditions. We also discuss alternative markers for segregating the monocyte subsets, for example using Tie-2 and slan, which do not necessarily follow the official method of segregating monocyte subsets based on relative CD14 and CD16 expressions.

Purpose The authors aim to investigate the altered monocytes subsets distribution in liver cirrhosis (LC) and subsequent hepatocellular carcinoma (HCC) in association with the expression level of plasma Homo sapiens (has)-miR-21-5p and hsa-miR-155-5p. A step toward non-protein coding (nc) RNA precision medicine based on the immune perturbation manifested as altered monocytes distribution, on top of LC and HCC. Methods Seventy-nine patients diagnosed with chronic hepatitis C virus (CHCV) infection with LC were enrolled in the current study. Patients were sub-classified into LC group without HCC ( n  = 40), LC with HCC ( n  = 39), and 15 apparently healthy controls. Monocyte subsets frequencies were assessed by flow cytometry. Real-time quantitative PCR was used to measure plasma hsa-miR-21-5p and hsa-miR-155-5p expression. Results Hsa-miR-21-5p correlated with intermediate monocytes ( r  = 0.30, p  = 0.007), while hsa-miR-155-5p negatively correlated with non-classical monocytes ( r  = − 0.316, p  = 0.005). ROC curve analysis revealed that combining intermediate monocytes frequency and hsa-miR-21 yielded sensitivity = 79.5%, specificity = 75%, and AUC = 0.84. In comparison, AFP yielded a lower sensitivity = 69% and 100% specificity with AUC = 0.85. Logistic regression analysis proved that up-regulation of intermediate monocytes frequency and hsa-miR-21-5p were independent risk factors for LC progression to HCC, after adjustment for co-founders. Conclusion Monocyte subsets differentiation in HCC was linked to hsa-miR-21-5p and hsa-miR-155-5p. Combined up-regulation of intermediate monocytes frequency and hsa-miR-21-5p expression could be considered a sensitive indicator of LC progression to HCC. Circulating intermediate monocytes and hsa-miR-21-5p were independent risk factors for HCC evolution, clinically and in silico proved. Graphical abstract

[This corrects the article DOI: 10.3389/fimmu.2022.889175.].