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Background: Monocytes are evolutionarily preserved innate immune cells that play essential roles in immune response regulation. Three activated monocyte subsets-classical (CD14++CD16-), intermediate (CD14++CD16+), and nonclassical (CD14+CD16++)-are associated with systemic lupus erythematosus (SLE) progression. This study aims to determine the association of monocyte subsets with SLE disease activity., Methods: A cross-sectional study involving 25 patients with SLE was conducted. Blood samples were collected, and monocyte subsets were identified using flow cytometry. Patients were grouped by disease activity using the Systemic Lupus Erythematosus Disease Activity Index 2000 (SLEDAI-2K) into inactive (SLEDAI-2K ≤ 4) and active (SLEDAI-2K > 4). The cutoff for monocyte subsets was determined using Receiver Operating Characteristic (ROC) analysis., Results: Nine active and 16 inactive subjects were identified. Compared with individuals without active disease, individuals with active disease had significantly lower mean classical monocyte subsets (71.9% vs 88%, p = 0.008), and higher median intermediate monocytes (29.1% vs 11.1%, p = 0.019). The median nonclassical monocyte subsets were not significantly different between the two groups. The cutoff for classical monocytes in active disease was ≤72.2%, AUC = 0.788, p = 0.021, with 66.7% sensitivity and 87.5% specificity; for intermediate monocytes, it was >22.3%, AUC = 0.788, p = 0.014, with 66.7% sensitivity and 100% specificity., Conclusion: Classical and intermediate monocytes could be considered as immune cellular markers for identifying active SLE.

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 Cx3cr1 GFP 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 Ly6C Ccr2 RFP 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 high classical and Ly6C low 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 Cx3cr1 GFP 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 Ccr2 RFP 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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Background: Interaction between multiple myeloma (MM) cells and proximal monocytes is expected during plasma cell proliferation. However, the role of monocyte subsets in the disease progression is unknown. Objective: This study evaluated circulating monocyte populations in MM patients and their correlation with disease severity. Methods: Peripheral monocytes from 20 patients with MM attending Assiut University Hospital in Assiut, Egypt, between October 2018 and August 2019 were processed using a flow cytometry procedure and stratified using the intensity of expression of CD14 and CD16 into classical (CD16−CD14++), intermediate (CD16+CD14++), and non-classical (CD16++CD14+) subsets. The data were compared with data from 20 healthy control participants with comparable age and sex. Results: In patients with MM, the percentage of classical monocytes was significantly lower (mean ± standard error: 77.24 ± 0.66 vs 83.75 ± 0.5), while those of non-classical (12.44 ± 0.5 vs 8.9 ± 0.34) and intermediate (10.3 ± 0.24 vs 7.4 ± 0.29) monocytes were significantly higher when compared with those of controls (all p 0.0001). Proportions of non-classical and intermediate monocytes correlated positively with serum levels of plasma cells, M-protein, calcium, creatinine and lactate dehydrogenase, and correlated negatively with the serum albumin level. Proportions of classical monocytes correlated positively with albumin level and negatively correlated with serum levels of M-protein, plasma cells, calcium, creatinine, and lactate dehydrogenase. Conclusion: Circulating monocyte subpopulations are skewed towards non-classical and intermediate monocytes in MM patients, and the intensity of this skewness increases with disease severity.

Background: Interaction between multiple myeloma (MM) cells and proximal monocytes is expected during plasma cell proliferation. However, the role of monocyte subsets in the disease progression is unknown. Objective: This study evaluated circulating monocyte populations in MM patients and their correlation with disease severity. Methods: Peripheral monocytes from 20 patients with MM attending Assiut University Hospital in Assiut, Egypt, between October 2018 and August 2019 were processed using a flow cytometry procedure and stratified using the intensity of expression of CD14 and CD16 into classical (CD16−CD14++), intermediate (CD16+CD14++), and non-classical (CD16++CD14+) subsets. The data were compared with data from 20 healthy control participants with comparable age and sex. Results: In patients with MM, the percentage of classical monocytes was significantly lower (mean ± standard error: 77.24 ± 0.66 vs 83.75 ± 0.5), while those of non-classical (12.44 ± 0.5 vs 8.9 ± 0.34) and intermediate (10.3 ± 0.24 vs 7.4 ± 0.29) monocytes were significantly higher when compared with those of controls (all p < 0.0001). Proportions of non-classical and intermediate monocytes correlated positively with serum levels of plasma cells, M-protein, calcium, creatinine and lactate dehydrogenase, and correlated negatively with the serum albumin level. Proportions of classical monocytes correlated positively with albumin level and negatively correlated with serum levels of M-protein, plasma cells, calcium, creatinine, and lactate dehydrogenase. Conclusion: Circulating monocyte subpopulations are skewed towards non-classical and intermediate monocytes in MM patients, and the intensity of this skewness increases with disease severity. [ABSTRACT FROM AUTHOR]

Sustained military operations (SUSOPs) result in psychological stress and cognitive dysfunction, which may be related to the recruitment of classical monocytes into the brain. This study examined the effect of beta-alanine (BA) on cognition and monocyte recruitment during a simulated 24-hour SUSOP. Nineteen healthy men ingested 12-g/d BA or placebo for 14 days before an SUSOP. Monocyte chemoattractant protein-1 (MCP-1), C-C chemokine receptor-2 (CCR2), and macrophage-1-antigen (CD11b) expression were assessed through multiplex assay and flow cytometry. Psychological stress and cognition were assessed through Automated Neuropsychological Assessment Metrics (ANAM). A composite measure of cognition (COGcomp) was generated from throughput scores extracted from 7 ANAM cognitive tests. Assessments occurred at baseline (0H), 12 hours (12H), 18 hours (18H), and 24 hours (24H). Significance was accepted at p ≤ 0.05. No significant effect of BA was noted for any variable (p's < 0.05). The frequency and severity of symptoms of psychological stress increased significantly at 18 and 24H compared with 0 and 12H (p's < 0.05). COGcomp decreased significantly at 18 and 24H compared with 0 and 12H (p's ≤ 0.001). MCP-1 peaked at 18H was significantly lower at 24H compared with 18H but remained elevated at 24H compared with 0H (p's < 0.001). CCR2 expression was significantly lower at 12 (p = 0.031), 18, and 24H (p's < 0.001). CD11b expression was significantly higher at 12H (p = 0.039) and 24H (p's = 0.003). MCP-1 was negatively associated with COGcomp (β = -0.395, p = 0.002, r2 = 0.174). Neither CCR2 or CD11b was related to COGcomp (p's > 0.05). Cognitive dysfunction during SUSOPs is related to serum concentrations of MCP-1 but is not influenced by BA supplementation. [ABSTRACT FROM AUTHOR]

Monocytes are important cells of the innate system. They are a heterogeneous type of cells consisting of phenotypically and functionally distinct subpopulations, which play a specific role in the control, development and escalation of the immunological processes. Based on the expression of superficial CD14 and CD16 in flow cytometry, they can be divided into three subsets: classical, intermediate and non‐classical. Variation in the levels of human monocyte subsets in the blood can be observed in patients in numerous pathological states, such as infections, cardiovascular and inflammatory diseases, cancer and autoimmune diseases. The aim of this review is to summarize current knowledge of human monocyte subsets and their significance in homeostasis and in pathological conditions. [ABSTRACT FROM AUTHOR]

Objective: To investigate the ex vivo pro-inflammatory properties of classical and non-classical monocytes as well as myeloid dendritic cells (mDCs) in systemic sclerosis (SSc) patients. Methods: Spontaneous production of CXCL10, CCL4, CXCL8 and IL-6 was intracellularly evaluated in classical, non-classical monocytes and Siglec-3-expressing mDCs from peripheral blood of SSc patients and healthy controls (HC) through flow cytometry. In addition, production of these cytokines was determined upon toll-like receptor (TLR) 4 plus Interferon-γ (IFN-γ) stimulation. Results: The frequency of non-classical monocytes spontaneously producing CXCL10 was increased in both limited (lcSSc) and diffuse cutaneous (dcSSC) subsets of SSc patients and CCL4 was augmented in dcSSc patients. The proportion of CCL4-producing mDCs was also elevated in dcSSc patients and the percentage of mDCS producing CXCL10 only in lcSSc patients. Upon stimulation, the frequency of non-classical monocytes expressing CXCL8 was increased in both patient groups and mDCs expressing CXCL8 only in lcSSc. Moreover, these parameters in unsupervised clustering analysis identify a subset of patients which are characterized by lung fibrosis and reduced pulmonary function. Conclusions: These data point towards a role of activated non-classical monocytes and mDCs producing enhanced levels of proinflammatory cytokines in SSc, potentially contributing to lung fibrosis. [ABSTRACT FROM AUTHOR]

Background: Systemic Lupus Erythematosus (SLE) is an auto-immune disease whose complex pathogenesis remains unraveled. Here we aim to explore the inflammatory ability of SLE patients' sera upon peripheral blood (PB) monocyte subsets and myeloid dendritic cells (mDCs) obtained from healthy donors. Methods: In this study we included 11 SLE patients with active disease (ASLE), 11 with inactive disease (ISLE) and 10 healthy controls (HC). PB from healthy donors was stimulated with patients' sera, toll-like receptor (TLR) 4 ligand -- lipopolysaccharide or both. The intracellular production of TNF-α was evaluated in classical, non-classical monocytes and mDCs, using flow cytometry. TNF-α mRNA expression was assessed in all these purified cells, after sera treatment. Results: We found that sera of SLE patients did not change spontaneous TNF-α production by monocytes or dendritic cells. However, upon stimulation of TLR4, the presence of sera from ASLE patients, but not ISLE, significantly increased the intracellular expression of TNF-α in classical and non-classical monocytes. This ability was related to titers anti-double stranded DNA antibodies in the serum. High levels of anti-TNF-α in the patients' sera were associated with increased TNF-α expression by co-cultured mDCs. No relationship was found with the levels of a wide variety of other pro-inflammatory cytokines. A slight increase of TNF-α mRNA expression was observed in these purified cells when they were cultured only in the presence of SLE serum. Conclusions: Our data suggest that SLE sera induce an abnormal in vitro TLR4 response in classical and non-classical monocytes, reflected by a higher TNF-α intracellular expression. These effects may be operative in the pathogenesis of SLE. [ABSTRACT FROM AUTHOR]

Reactive oxygen species (ROS) and the ability of immune cells to mount an oxidative burst represent an important defense during microbial invasion, but is also recognized for playing a significant role in the progression of inflammatory disorders and disease. Although neutrophils produce the strongest ROS-response, other leukocytes and their cell subsets could play a significant role. Isolation of specific cells for determining their ROS-response can affect their functionality and is laborious or hard to replicate in different settings. We have therefore established a whole blood assay, that only requires 100 μL heparinized blood and utilizes the dihydrorhodamine (DHR) 123 ROS-probe combined with cell surface antibody staining for the specific detection of ROS in several subsets of cells simultaneously using flow cytometry. Although the flow markers chosen are interchangeable with other direct conjugated and cell specific antibodies depending on the research question, we focused on neutrophils (SSChighCD16brightHLA-DRneg/low), eosinophils (SSChighCD16lowHLA-DRlow/negCD193positiveCD125positive) and monocyte subsets (SSCintermediateHLA-DRhighCD14low-positiveCD16negative-positive). As a RBC-lysis reagent we compared BD FACS Lysis Solution to the in-house prepared ammonium-chloride‑potassium based ACK Lysis Buffer, that does not fix or permeabilize the immune cells. We find that ACK-lysis of stimulated and stained samples results in superior surface staining, decreased loss of cell subsets, and enhanced resolution of the DHR-signal. Compared to the other cells analyzed in healthy blood donors, neutrophils responded with the highest ROS-response to all tested stimuli (fMLP (low stimuli), E. coli, and PMA (high stimuli)), where eosinophils and the three monocyte subsets also showed an extensive ROS-response when stimulated with E. coli or PMA. Our assay provides the possibility for researchers to examine the ROS-response of specific cell subsets in specific patient groups ex vivo and could also allow the analysis of pharmacological intervention studies targeting ROS, which ultimately can advance the field of immunological research. • Cell-specific production of reactive oxygen species in unprocessed whole blood. • Easy to establish flow cytometry protocol utilizing DHR-123 and antibody staining. • Simultaneous detection in neutrophils, eosinophils, and monocyte subsets. • Differentiates classical monocytes, intermediate monocytes, non-classical monocytes. • Flow cytometry panel with antibodies against CD14, CD16, CD125, CD193 and HLA-DR. [ABSTRACT FROM AUTHOR]

The article presents a definition of monocyte subsets. The availability of monoclonal reagents, particular fluorescent probes and compatible instrumentation over the previous years has led to the determination and characterization of many leukocyte subsets, including monocyte subsets. Human blood monocytes are characterized by forward and side scatter when using flow cytometry.

Acute exercise alters the surface expression of toll-like receptors (TLRs) and HLA.DR on blood monocytes, which could transiently compromise immunity. As serum factors might be responsible, we examined the effects of autologous post-exercise serum exposure on TLR2, TLR4 and HLA.DR expression on resting blood monocytes and their subtypes. Eight trained cyclists completed an ergometer 60 km time trial. PBMCs and serum were obtained before, immediately after and 1 h after exercise. TLR2, TLR4 or HLA.DR expression (gMFI) was determined on blood monocyte subtypes expressing combinations of CD14 and CD16 by flow cytometry, and on resting monocytes exposed to 50% autologous serum (pre, immediately after or 1 h after exercise) for 18 h in culture. Immediately after exercise, total monocyte expression of TLR2 and TLR4 increased by 41 and 27%, respectively, while HLA.DR expression was 39% lower than baseline. TLR2 and TLR4 was 53 and 84% greater 1 h after exercise, respectively, while HLA.DR was 48% lower. Changes in TLR2 and TLR4 expression occurred on the CD14(++bright)/CD16(+dim) monocyte subtype only, while HLA.DR expression changed on the CD14(+dim)/CD16(++bright) subtype. Serum did not affect monocyte TLR2 or TLR4 expression but 1 h post serum increased expression of HLA.DR on total monocytes and the CD14(+dim)/CD16(++bright) subtype, which was in contrast to the change observed at this time after exercise. We conclude that a bout of strenuous aerobic exercise alters the surface expression of TLR2, TLR4 and HLA.DR on blood monocytes and some of their subtypes, but these changes appear to be unrelated to blood serum factors. [ABSTRACT FROM AUTHOR]

Background: Leukocytes in peripheral blood (PB) are prognostic biomarkers in head and neck squamous cell carcinoma cancer patients (HNSCC-CPs), but differences between HNSCC-CPs and healthy adults (HAs) are insufficiently described. Methods: 10-color flow cytometry (FCM) was used for in-depth immunophenotyping of PB samples of 963 HAs and 101 therapy-naïve HNSCC-CPs. Absolute (AbsCC) and relative cell counts (RelCC) of leukocyte subsets were determined. A training cohort (TC) of 43 HNSCC-CPs and 43 HAs, propensity score (PS)-matched according to age, sex, alcohol, and smoking, was used to develop a score consecutively approved in a validation cohort (VC). Results: Differences in AbsCC were detected in leukocyte subsets (p < 0.001), but had low power in discriminating HNSCC-CPs and HAs. Consequently, RelCC of nine leukocyte subsets in the TC were used to calculate 36 ratios; receiver operating characteristic (ROC) curves defined optimum cut-off values. Binary classified data were combined in a score based on four ratios: monocytes-to-granulocytes (MGR), classical monocytes-to-monocytes (clMMR), monocytes-to-lymphocytes (MLR), and monocytes-to-T-lymphocytes (MTLR); ≥3 points accurately discriminate HNSCC-CPs and HAs in the PS-matched TC (p = 2.97 × 10−17), the VC (p = 4.404 × 10−178), and both combined (p = 7.74 × 10−199). Conclusions: RelCC of leukocyte subsets in PB of HNSCC-CPs differ significantly from those of HAs. A score based on MGR, clMMR, MLR, and MTLR allows for accurate discrimination. [ABSTRACT FROM AUTHOR]