Monocyte Subpopulations Research Articles

Abstract 4137829: Comprehensive Single-Cell Atlas of the Human Lung in Pulmonary Arterial Hypertension

Introduction: Pulmonary arterial hypertension (PAH) is a complex cardiopulmonary disease characterized by progressive vascular remodeling. Although various immune and vascular cell populations have been implicated in the pathobiology of PAH, a comprehensive assessment of the various cell types in human PAH lungs is still lacking. Methods: Single-nucleus RNA sequencing was performed on lung samples from the Pulmonary Hypertension Breakthrough Initiative using the 10x Genomics Chromium Fixed RNA Profiling workflow. Rigorous quality control measures were implemented, including the detection and removal of ambient RNA using CellBender and doublets using scDblFinder. Data normalization and dimensionality reduction were performed using Seurat, and alignment by sample and sequencing batch was conducted using Harmony. Cells were annotated by mapping onto the Human Lung Cell Atlas using Azimuth. Differential gene expression analysis was conducted using a robust pseudobulk approach with edgeR to limit false positive findings. Gene set enrichment analysis was performed using the Hallmark pathways. Results: Lung samples from a total of 67 individuals were profiled, including 42 PAH and 25 age- and sex-matched control lungs. After quality control filtering, over 800,000 cells were captured, with a median of 12,000 cells per sample. Clustering uncovered over 30 cell types, representing endothelial, stromal, immune, and epithelial lineages of the lung. A median of 2,500 transcripts and 1,500 genes were detected per cell. Differentially expressed genes between PAH and control were detected in all major cell populations (FDR < 0.05). Comparative analysis across all cell types revealed interferon response in monocyte subpopulations and endothelial-to-mesenchymal transition in endothelial subpopulations as top upregulated pathways. Conclusion: Comprehensive single-nucleus profiling of human lungs in PAH revealed extensive gene and pathway dysregulation across diverse cell types. This rich dataset provides a valuable resource for the research community, offering opportunities to advance and refine our understanding of the cellular pathobiology of PAH.

VSIG4 inhibits the anti-inflammatory reaction of immune cells after cardiogenic shock

Abstract Cardiogenic shock (CS) still has a mortality rate of approximately 50%. CS leads to a significant activation of the immune system. The aim of this study was to characterize the immune cell populations and the immune cell-specific changes in CS on admission to the clinic and after revascularization. We developed a web-based platform named 'IRCaS - Immune Cell Regulations in Cardiogenic Shock' to map the temporal course of immune cell-specific processes of CS after acute myocardial infarction (AMI) from admission to 3 days after revascularization. Our tool revealed an endogenous anti-inflammatory response after CS, which is inhibited by the cell-cell receptor VSIG4. The web platform contains newly acquired single-cell sequencing data from patients with CS after AMI on admission and 24, 48, and 72 hours after revascularisation, as well as controls. In total, 171,962 immune cells derived from peripheral blood mononuclear cells (PBMCs) were individually sequenced. Analysis of the immune cell populations revealed, that 24 hours after revascularisation, monocytes increased to 62.4% (vs. 30.4% in controls), while T cells decreased from 46.3% to 25.0%. On the immune cell expression level, we observed an inflammatory response indicated by IL1B which continuously increased from 24 h (1.6+fold), over 48h (3.1+fold) to 72 h (3.5+fold) after revascularization. Interestingly, there was a significant anti-inflammatory response shortly after revascularization, as evidenced by a transient increase in the anti-inflammatory CD16+ monocyte subpopulation from 12.1% to 26.7% at 24 hours and an increase in THBS1 after revascularization, which was only short-lived. We identified a negative feedback in the increasing anti-inflammatory CD16+ cells caused by a subsequent VSIG4 (+5.2-fold) increase. Indeed, overexpression of VSIG4 in monocytes resulted in a significant decrease of anti-inflammatory CD16+ monocytes by 33.9% and a decrease in anti-inflammatory transcripts. Our data show that in addition to the strong inflammatory response in CS, there is also an opposing anti-inflammatory response, which is however only short-lived, as it is attenuated by a negative feedback via an increase in VSIG4. In addition, our webplatform offers time-dynamic gene expression profiles of immune cells, allowing users to select and visualize specific genes during the course of CS after AMI.

Acute myocardial infarction leads to distinct sex differences in the coding transcriptome of human monocytes with a potential impact in disease aetiopathology

Abstract Background Immune responses are intricately linked to human disease, not only during pathogenic infections but also in sterile injury events such as in cardiovascular disease. Atherosclerosis is the main driver leading to coronary artery disease (CAD) and subsequent development of acute myocardial infarction (AMI) and heart failure. The vital role of monocytes in the pathophysiology of atherosclerosis is well established. In the onset of AMI, monocytes act as first responders, both in the inflammatory and tissue repair phases. It is now apparent that sex-specific differences in the inflammatory responses leading to AMI impact the severity and outcome of patients. Yet, the immune responses during AMI have been under-investigated in women, often under-represented in clinical studies, and more research in this topic is required. Purpose Identification of sex-specific differences in the coding transcriptome of monocytes subpopulations in the onset of AMI compared to matched healthy subjects and CAD patients. Methods We isolated monocytes from male and female AMI patients, CAD patients and healthy controls. FACS sorting of monocytes (CD14 and CD16) resulted into classical, intermediate, and non-classical subpopulations. We performed ribosomal depletion and bulk Illumina RNA sequencing for each monocyte subpopulation. Differentially expressed genes (DEG) were calculated using specific R packages. Pathway enrichment analysis was performed with Gene set enrichment analysis (GSEA). Other computational tools were used to determine enrichment of genes linked to GWAS identified in cardiovascular diseases or to infer pathway responsive genes upon perturbation (Progeny). Network comparison and visualization were performed using Cytoscape platform. Results Whereas observing a clear overlap of most pathways such as homeostasis, regulation of signalling cascades and inflammatory response, we also identified other pathways contrasting strongly between both sexes. In fact, TGF beta/ SMAD signalling, wound healing and epithelial to mesenchymal transition are increased in female cells. On the contrast the more pro-inflammatory BMP/SMAD signalling is increased in male cells accompanied by decreased respiratory electron transport and translation. Additionally, when comparing our data to signatures identified in trauma patients, we observe clear mapping of our gene sets, which are upregulated in AMI, with the first 3 trauma associated signatures, whereas one of the signatures associated to homeostasis associates clearly with our CAD data as well as the healthy subjects. Conclusions Human monocytes display a specific coding transcriptome and certain pathways are differentially expressed in AMI and differ greatly between male and female cohorts. Interestingly, our DEG map closely to trauma associated signatures, highlighting potential similarities in disease aetiopathology between trauma and AMI patients.

Comprehensive single-cell profiling of monocytes in HLA-B27-positive ankylosing spondylitis with acute anterior uveitis.

Acute anterior uveitis (AAU) is a common extra-articular manifestation of ankylosing spondylitis (AS), particularly in patients positive for the human leucocyte antigen (HLA)-B27 genetic marker. To explore the underlying mechanisms of HLA-B27+ AS-associated AAU, we employed single-cell RNA sequencing to profile the transcriptomes of peripheral blood mononuclear cells in three HLA-B27+ AS-associated AAU patients and three healthy controls (HCs). We identified 11 distinct immune cell clusters, with a particular focus on monocytes, revealing six subsets, including three previously unidentified subsets, namely, GTPase immune-associated proteins, Th17-related, and lncRNA monocytes, with unique gene expression patterns. Significant differences in monocyte composition, activation states, and gene expression were observed between patients and HCs, particularly within HLA monocyte subpopulations. Notably, enhanced expression of X-inactive specific transcript and myeloid cell nuclear differentiation antigen genes was validated across monocyte subclusters in patients. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis highlighted significant enrichment in antigen processing and presentation pathways, shedding light on the disease's molecular mechanisms. These findings provide novel insights into the molecular mechanisms of HLA-B27+ AS-associated AAU and may contribute to the development of targeted diagnostic and therapeutic strategies. Further clinical validation is essential.

Patients with coronary atherosclerosis had an altered ratio of monocyte subpopulations in the blood, which was associated with an inflammatory cell response

Coronary artery disease (CAD) is one of the leading causes of death in developed countries. Experimental data confirm the role of monocytes in the development of CAD. Three main subpopulations of circulating blood monocytes are known: classical CD14++CD16- (~80%), intermediate CD14+CD16+ (~5%) and non-classical CD14+CD16++(~15%). It is believed that each of the subpopulations of monocytes performs different functions. There are studies that demonstrate that classical monocytes respond more to the bacterial presence, whereas non-classical ones respond to the viral one. However, the functions of monocyte subpopulations have not been fully studied. Previously, we demonstrated that circulating monocytes of the blood of patients with atherosclerosis had increased proinflammatory activity. We decided to find out how the ratio of monocyte subpopulations in the blood is related to the inflammatory response of cells in atherosclerosis. The aim of our work was to investigate the relationship of the inflammatory response of primary monocytes with the distribution of monocyte subpopulations relative to the total pool of monocytes in healthy and sick CAD. The study included 20 men aged 46 to 70 years, 10 of them without CAD and 10 patients with CAD. A coronary angiography was performed, according to the results of which the patients were divided into patients with coronary atherosclerosis with detected stenosis in 2 or more arteries (CAD) and healthy ones without stenosis in the arteries. Next, blood was taken to study circulating monocytes. Monocyte subpopulations were detected by flow cytometry from the leukocyte fraction using antibodies against CD14- FITC and CD16-PB450-A. Immediately after isolation, monocytes were stimulated with LPS with a final concentration of 1 ug/mL, and a supernatant was selected 24 hours later for further enzyme immunoassay. The inflammatory response was assessed by the secretion of cytokines TNFα, IL-1β, IL-6, IL-8, IL-10, and CCL2 using ELISA. The monocytes of CAD patients had an altered inflammatory response in response to LPS stimulation compared to patients without CAD. This was manifested in increased secretion of IL-1β and TNF, and decreased secretion of CCL2 and IL-6. An increase in the number of intermediate and non-classical monocytes in patients with CAD was associated with changes in the inflammatory response of cells to the secretion of cytokines IL-1β and CCL2. It can be assumed that pathological changes in the representation of monocyte subpopulations in the bloodstream may be one of the causes of chronic inflammation in the walls of the arteries, contributing to the progression of atherosclerotic lesions.

The Modulation of Septic Shock: A Proteomic Approach.

Sepsis poses a significant challenge due its lethality, involving multiple organ dysfunction and impaired immune responses. Among several factors affecting sepsis, monocytes play a crucial role; however, their phenotype, proteomic profile, and function in septic shock remain unclear. Our aim was to fully characterize the subpopulations and proteomic profiles of monocytes seen in septic shock cases and discuss their possible impact on the disease. Peripheral blood monocyte subpopulations were phenotype based on CD14/CD16 expression by flow cytometry, and proteins were extracted from the monocytes of individuals with septic shock and healthy controls to identify changes in the global protein expression in these cells. Analysis using 2D-nanoUPLC-UDMSE identified 67 differentially expressed proteins in shock patients compared to controls, in which 44 were upregulated and 23 downregulated. These proteins are involved in monocyte reprogramming, immune dysfunction, severe hypotension, hypo-responsiveness to vasoconstrictors, vasodilation, endothelial dysfunction, vascular injury, and blood clotting, elucidating the disease severity and therapeutic challenges of septic shock. This study identified critical biological targets in monocytes that could serve as potential biomarkers for the diagnosis, prognosis, and treatment of septic shock, providing new insights into the pathophysiology of the disease.

Monocyte subpopulations in children with autoimmune liver disease

BackgroundPatients with autoimmune liver diseases require individualized long-term immunosuppressive therapy, whose discontinuation is possible after complete histological remission and that requires repeated liver biopsy. In view of this, the search for non-invasive markers is essential for patients with autoimmune liver disease. PurposeThe purpose of this research is to assess the possibility of predicting the recurrence of autoimmune liver disease in children. MethodThe biological material used in the study was blood serum from 80 children diagnosed with autoimmune hepatitis and autoimmune sclerosing cholangitis. Patients were divided into four groups according to disease activity and therapeutic approach. ResultsThe percentage of monocyte subpopulations was determined by flow cytometry, and disease activity, inflammation, and fibrosis markers were analyzed to study the relationship and diagnostic value of the parameters studied in detail. The results of the study indicate a significant relationship between disease activity and changes in the distribution of the percentage of monocyte subpopulations in the blood. The percentage of intermediate CD14++/CD16+ monocytes was found to correlate with disease activity, and non-classical CD14lowCD16+ monocytes were found to be of high diagnostic value in the diagnosis of disease relapse. ConclusionsThese findings not only expand the understanding of the pathogenesis of autoimmune liver disease but also point to the prospects of using monocyte subpopulations as potential biomarkers for predicting relapse, contributing to the development of more effective clinical management strategies.

Peculiarities of granulocyte-macrophage and macrophage colony-stimulating factors receptors expression in subpopulations of peripheral blood monocytes of patients with chronic obstructive pulmonary disease

Introduction. To analyze of the severity of respiratory symptoms in patients with chronic obstructive pulmonary disease (COPD), depending on the presence of an exacerbation or novel coronavirus infection (NCVI), taking into account the activity of acute phase blood parameters.Materials and methods. The medical documentation of 162 patients with COPD was studied, which were divided into 3 groups: group 1 (n=61) ‒ COPD and NCVI, group 2 (n=53) – stable COPD, group 3 (n=48) ‒ COPD exacerbation. The severity of respiratory symptoms was assessed using points. To assess the activity of inflammation the following biochemical indicators were used ‒ C-reactive protein (CRP) and fibrinogen (g/L).Results. According to the severity of cough and the intensity of dyspnea on the mMRC scale, the first, second and third groups did not differ statistically (p=0.07). Patients of the first group (82.5%) characterized by the absence of classical criteria for exacerbation of COPD. In terms of the severity of sputum production, the first, second and third groups are statistically different (p=0.0001). The first, second and third groups differ significantly in the level of serum CRP (p=0.0001) and fibrinogen (p=0.009). According to the results of the correlation analysis, some relationships found between respiratory symptoms and the level of CRP and fibrinogen.Conclusion. The clinical feature of the associated course of stable COPD and NCVI is the presence of severe dyspnea and the absence of classic criteria for exacerbation of COPD. Systemic inflammation in NCVI and stable COPD are more pronounced than in isolated stable COPD or exacerbation and correlates with cough and dyspnea. Practitioners for the differential diagnosis of NCVI in stable COPD can use the data obtained.

Immunologic mediators profile in COVID-19 convalescence

SARS-CoV-2 caused the pandemic situation experienced since the beginning of 2020, and many countries faced the rapid spread and severe form of the disease. Mechanisms of interaction between the virus and the host were observed during acute phase, but few data are available when related to immunity dynamics in convalescents. We conducted a longitudinal study, with 51 healthy donors and 62 COVID-19 convalescent patients, which these had a 2-month follow-up after symptoms recovery. Venous blood sample was obtained from all participants to measure blood count, subpopulations of monocytes, lymphocytes, natural killer cells and dendritic cells. Serum was used to measure cytokines, chemokines, growth factors, anti-N IgG and anti-S IgG/IgM antibodies. Statistic was performed by Kruskal–Wallis test, and linear regression with days post symptoms and antibody titers. All analysis had confidence interval of 95%. Less than 35% of convalescents were anti-S IgM+, while more than 80% were IgG+ in D30. Anti-N IgG decreased along time, with loss of seroreactivity of 13%. Eosinophil count played a distinct role on both antibodies during all study, and the convalescence was orchestrated by higher neutrophil-to-lymphocyte ratio and IL-15, but initial stages were marked by increase in myeloid DCs, B1 lymphocytes, inflammatory and patrolling monocytes, G-CSF and IL-2. Later convalescence seemed to change to cytotoxicity mediated by T lymphocytes, plasmacytoid DCs, VEGF, IL-9 and CXCL10. Anti-S IgG antibodies showed the longest perseverance and may be a better option for diagnosis. The inflammatory pattern is yet present on initial stage of convalescence, but quickly shifts to a reparative dynamic. Meanwhile eosinophils seem to play a role on anti-N levels in convalescence, although may not be the major causative agent. We must highlight the importance of immunological markers on acute clinical outcomes, but their comprehension to potentialize adaptive system must be explored to improve immunizations and further preventive policies.

Platinum-based chemotherapy promotes antigen presenting potential in monocytes of patients with high-grade serous ovarian carcinoma.

Ovarian cancer (OC) is the most lethal gynecologic malignancy worldwide. The major clinical challenge includes the asymptomatic state of the disease, making diagnosis possible only at advanced stages. Another OC complication is the high relapse rate and poor prognosis following the standard first-line treatment with platinum-based chemotherapy. At present, numerous clinical trials are being conducted focusing on immunotherapy in OC; nevertheless, there are still no FDA-approved indications. Personalized decision regarding the immunotherapy, including immune checkpoint blockade and immune cell-based immunotherapies, can depend on the effective antigen presentation required for the cytotoxic immune response. The major aim of our study was to uncover tumor-specific transcriptional and epigenetic changes in peripheral blood monocytes in patients with high-grade serous ovarian cancer (HGSOC). Another key point was to elucidate how chemotherapy can reprogram monocytes and how that relates to changes in other immune subpopulations in the blood. To this end, we performed single-cell RNA sequencing of peripheral blood mononuclear cells (PBMCs) from patients with HGSOC who underwent neoadjuvant chemotherapeutic treatment (NACT) and in treatment-naïve patients. Monocyte cluster was significantly affected by tumor-derived factors as well as by chemotherapeutic treatment. Bioinformatical analysis revealed three distinct monocyte subpopulations within PBMCs based on feature gene expression - CD14.Mn.S100A8.9hi, CD14.Mn.MHC2hi and CD16.Mn subsets. The intriguing result was that NACT induced antigen presentation in monocytes by the transcriptional upregulation of MHC class II molecules, but not by epigenetic changes. Increased MHC class II gene expression was a feature observed across all three monocyte subpopulations after chemotherapy. Our data also demonstrated that chemotherapy inhibited interferon-dependent signaling pathways, but activated some TGFb-related genes. Our results can enable personalized decision regarding the necessity to systemically re-educate immune cells to prime ovarian cancer to respond to anti-cancer therapy or to improve personalized prescription of existing immunotherapy in either combination with chemotherapy or a monotherapy regimen.

Dengue NS1 interaction with lipids alters its pathogenic effects on monocyte derived macrophages

BackgroundWhile dengue NS1 antigen has been shown to be associated with disease pathogenesis in some studies, it has not been linked in other studies, with the reasons remaining unclear. NS1 antigen levels in acute dengue are often associated with increased disease severity, but there has been a wide variation in results based on past dengue infection and infecting dengue virus (DENV) serotype. As NS1 engages with many host lipids, we hypothesize that the type of NS1-lipid interactions alters its pathogenicity.MethodsPrimary human monocyte derived macrophages (MDMs) were co-cultured with NS1 alone or with HDL, LDL, LPS and/or platelet activating factor (PAF) from individuals with a history of past dengue fever (DF = 8) or dengue haemorrhagic fever (DHF = 8). IL-1β levels were measured in culture supernatants, and gene expression analysis carried out in MDMs. Monocyte subpopulations were assessed by flow cytometry. Hierarchical cluster analysis with Euclidean distance calculations were used to differentiate clusters. Differentially expressed variables were extracted and a classifier model was developed to differentiate between past DF and DHF.ResultsSignificantly higher levels of IL-1β were seen in culture supernatants when NS1 was co-cultured with LDL (p = 0.01, median = 45.69 pg/ml), but lower levels when NS1 was co-cultured with HDL (p = 0.05, median = 4.617 pg/ml). MDMs of those with past DHF produced higher levels of IL-1β when NS1 was co-cultured with PAF (p = 0.02). MDMs of individuals with past DHF, were significantly more likely to down-regulate RPLP2 gene expression when macrophages were co-cultured with either PAF alone, or NS1 combined with PAF, or NS1 combined with LDL. When NS1 was co-cultured with PAF, HDL or LDL two clusters were detected based on IL10 expression, but these did not differentiate those with past DF or DHF.ConclusionsAs RPLP2 is important in DENV replication, regulating cellular stress responses and immune responses and IL-10 is associated with severe disease, it would be important to further explore how differential expression of RPLP2 and IL-10 could lead to disease pathogenesis based on NS1 and lipid interactions.

Characteristics of monocyte subpopulations in peripheral blood of the patients with chronic obstructive pulmonary disease

Rationale. As the precursors of macrophages, monocytes play an important role in the pathogenesis of chronic obstructive pulmonary disease (COPD). Traditionally, classical (CD14++CD16–), intermediate (CD14++CD16+) and non-classical (CD14+CD16++) subpopulations of monocytes are distinguished, which differ in their functional characteristics.Aim: To study the relative amount of circulating subpopulations of monocytes in patients with COPD and to identify their possible relationship with pulmonary function and humoral inflammatory markers.Methodology and Research Methods. The study enrolled 47 patients with COPD, predominantly GOLD II-III, and 25 individuals without bronchial obstruction (control group). Monocyte subpopulations were determined by flow cytometry. Plasma cytokine concentrations were measured using a multiplex assay on a flow cytometer. Pulmonary function was assessed by spirometry.Results. A reduced number of non-classical monocytes was observed in COPD patients as compared to the control group (10.5 (6.7–15.1)% vs. 14.4 (8.3–18.4)%, p = 0.04). Higher content of classical monocytes was associated with a more pronounced decrease in bronchial patency (FEV1 ρ = –0.37, p = 0.007), while intermediate monocytes were characterized by a direct relationship with FEV1 (ρ = 0.42, p = 0.003). The number of non-classical monocytes in the main group had an inverse correlation with cytokine concentrations (IL-4 ρ = –0.30, p = 0.04; IL-2 ρ = –0.36, p = 0.01; IL-1β ρ = –0.35, p = 0.02; TNF-α ρ = –0.47, p < 0.001; IL-17A ρ = –0.34, p = 0.02; IL-6 ρ = –0.32, p = 0.03; IL-10 ρ = –0.34, p = 0.02; IFN-γ ρ = – 0.35, p = 0.01; IL-12p70 ρ = –0.30, p = 0.04; IL-8 ρ = –0.40, p = 0.004).Conclusion. The obtained results indicate a deficiency of non-classical monocytes in COPD patients, which may contribute to systemic inflammatory response, while classical forms of monocytes may be involved in the formation of bronchial obstruction.

The Association between IL-1β and IL-18 Levels, Gut Barrier Disruption, and Monocyte Activation during Chronic Simian Immunodeficiency Virus Infection and Long-Term Suppressive Antiretroviral Therapy.

HIV-induced persistent immune activation is a key mediator of inflammatory comorbidities such as cardiovascular disease (CVD) and neurocognitive disorders. While a preponderance of data indicate that gut barrier disruption and microbial translocation are drivers of chronic immune activation, the molecular mechanisms of this persistent inflammatory state remain poorly understood. Here, utilizing the nonhuman primate model of Human Immunodeficiency Virus (HIV) infection with suppressive antiretroviral therapy (ART), we investigated activation of inflammasome pathways and their association with intestinal epithelial barrier disruption (IEBD). Longitudinal blood samples obtained from rhesus macaques with chronic SIV infection and long-term suppressive ART were evaluated for IEBD biomarkers, inflammasome activation (IL-1β and IL-18), inflammatory cytokines, and triglyceride (TG) levels. Activated monocyte subpopulations and glycolytic potential were investigated in peripheral blood mononuclear cells (PBMCs). During the chronic phase of treated SIV infection, elevated levels of plasma IL-1β and IL-18 were observed following the hallmark increase in IEBD biomarkers, intestinal fatty acid-binding protein (IFABP) and LPS-binding protein (LBP). Further, significant correlations of plasma IFABP levels with IL-1β and IL-18 were observed between 10 and 12 months of ART. Higher levels of sCD14, IL-6, and GM-CSF, among other inflammatory mediators, were also observed only during the long-term SIV + ART phase along with a trend of increase in the frequencies of activated CD14+CD16+ intermediate monocyte subpopulations. Lastly, we found elevated levels of blood TG and higher glycolytic capacity in PBMCs of chronic SIV-infected macaques with long-term ART. The increase in circulating IL-18 and IL-1β following IEBD and their significant positive correlation with IFABP suggest a connection between gut barrier disruption and inflammasome activation during chronic SIV infection, despite viral suppression with ART. Additionally, the increase in markers of monocyte activation, along with elevated TG and enhanced glycolytic pathway activity, indicates metabolic remodeling that could fuel metabolic syndrome. Further research is needed to understand the mechanisms by which gut dysfunction and inflammasome activation contribute to HIV-associated metabolic complications, enabling targeted interventions in people with HIV.

Monocyte Single-Cell Multimodal Profiling in Cardiovascular Disease Risk States.

Monocytes are a critical innate immune system cell type that serves homeostatic and immunoregulatory functions. They have been identified historically by the cell surface expression of CD14 and CD16. However, recent single-cell studies have revealed that they are much more heterogeneous than previously realized. We utilized cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) and single-cell RNA sequencing to describe the comprehensive transcriptional and phenotypic landscape of 437 126 monocytes. This high-dimensional multimodal approach identified vast phenotypic diversity and functionally distinct subsets, including IFN-responsive, MHCIIhi (major histocompatibility complex class II), monocyte-platelet aggregates, as well as nonclassical, and several subpopulations of classical monocytes. Using flow cytometry, we validated the existence of MHCII+CD275+ MHCIIhi, CD42b+ monocyte-platelet aggregates, CD16+CD99- nonclassical monocytes, and CD99+ classical monocytes. Each subpopulation exhibited unique characteristics, developmental trajectories, transcriptional regulation, and tissue distribution. In addition, alterations associated with cardiovascular disease risk factors, including race, smoking, and hyperlipidemia were identified. Moreover, the effect of hyperlipidemia was recapitulated in mouse models of elevated cholesterol. This integrative and cross-species comparative analysis provides a new perspective on the comparison of alterations in monocytes in pathological conditions and offers insights into monocyte-driven mechanisms in cardiovascular disease and the potential for monocyte subpopulation targeted therapies.

Neutrophil-like Monocytes Increase in Patients with Colon Cancer and Induce Dysfunctional TIGIT+ NK Cells.

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous family of immune cells including granulocytic (CD14neg/CD15+/HLA-DRneg) and monocytic subtypes (CD14+/CD15neg/HLA-DRneg). In the present study, we found a population of monocytes expressing the granulocyte marker CD15 that significantly increased in both peripheral blood (PB) and tumoral tissues of patients with colorectal cancer (CRC). Further phenotypical analysis confirmed the granulocytic-like features of this monocyte subpopulation that is associated with an increase in granulocyte-monocyte precursors (GMPs) in the PB of these patients (pts). Mechanistically, this granulocyte-like monocyte population suppressed NK cell activity by inducing TIGIT and engaging NKp30. Accordingly, an increased frequency of TIGIT+ NK cells with impaired functions was found in both the PB and tumoral tissue of CRC pts. Collectively, we provided new mechanistic explanations for tumor immune escape occurring in CRC by showing the increase in this new kind of MDSC, in both PB and CRC tissue, which is able to significantly impair the effector functions of NK cells, thereby representing a potential therapeutic target for cancer immunotherapy.

Understanding the relevance of immunological markers in severe multisystem inflammatory syndrome in children through machine learning

Objectives: Multisystem inflammatory syndrome in children (MIS-C) is a late manifestation of SARS-CoV-2 infection, which presents with symptoms ranging from milder mucocutaneous and gastrointestinal symptoms to severe cardiovascular and neurological manifestations. We studied the clinical, biochemical, hematological, and immunological characteristics of MIS-C patients to understand this disease entity and to identify markers of severe disease. Materials and Methods: Twenty-four MIS-C patients, four acute COVID-19 infections, and ten healthy controls (HC) from a tertiary care pediatric hospital in Mumbai were enrolled in the study. Clinical, biochemical, hematological, and immunological parameters comprising major lymphocyte, neutrophil, and monocyte subpopulations and key activator and inhibitory markers were studied. Statistical Analysis: All parameters were compared between the healthy, COVID and MIS-C groups at Day 0, 7 and 14 using non-parametric statistical tests. Machine learning tools were used for multivariate data analysis to identify the immunological parameters that could help predict severe disease. Results: NKp46pos NK cell (%), CD11 positive eosinophil (%), D-dimer, and Tim3pos Tc (%) were identified as the most important markers that could help predict severe disease, with NKp46pos NK cells as the top contributor. A disease severity metric utilizing these markers can be used to identify patients who are likely to have a severe course of disease. Conclusions: NK cells directly contribute to disease severity in MIS-C. As the JAK-STAT pathway is known to be important for NK cell development, maturation, and function, ruxolitinib, which is a JAK1/JAK2 inhibitor, might be beneficial in the management of this condition.

PD-1 AND Tim-3 Expression on different subpopulations of monocytes in chronic often recurrent herpesvirus infection

More than half of the world’s population is infected with the herpes simplex virus. In most cases, infection is not accompanied by symptoms, but in some people the disease occurs as a chronic infection with frequent and severe relapses. One of the most likely reasons for this may be a dysregulation of the immune system. In recent years, the role of checkpoint molecules, in particular PD-1 and Tim-3, in the regulation of the immune response and the functions of immunocompetent cells has been actively studied. Activation of PD-1 and Tim-3 on T cells has previously been shown to suppress the immune response. PD-1 and Tim-3 are also expressed on other immune cells, in particular monocytes. However, the expression of these molecules on monocytes during chronic viral infections has not been previously studied. The study was aimed at assessing the level of PD-1 and Tim-3 expression on various populations of monocytes in patients with chronic often recurrent herpesvirus infection. Twenty-six patients were recruited into the study. All patients received antiviral and immunomodulatory therapy in the immunological department. The number of classical, intermediate, and non-classical monocytes and the expression of PD-1 and Tim-3 on monocytes, were assessed by flow cytometry before and after the therapy. Monocytes were isolated from peripheral blood, and subpopulations were divided according to the level of expression of CD14 and CD16. In patients with herpes, a reduced number of monocytes was observed in comparison with healthy donors. The relative number of PD-1-positive monocytes, the mean fluorescence intensity of PD-1 and Tim-3, and the number of double-positive cells were reduced in herpes patients in all three monocyte subpopulations examined. Three months after therapy, the response to the therapy was assessed; patients who did not have a single recurrence of herpes within 3 months were considered to respond. Responding patients had a lower initial content of double-positive cells among intermediate and non-classical monocytes. The decrease in the level of PD-1 and Tim-3 positive monocytes during herpesvirus infection revealed in the present study may indicate the involvement of monocytes deficient in the expression of checkpoint molecules in the pathogenesis of the disease.

Morphofunctional changes in brain and peripheral blood in aged Wistar rats due to AlCl3 exposure

The purpose of the study: To examine morphofunctional changes of brain and peripheral blood in aged Wistar rats to observe adaptive reactions as a response on AlCl3 exposure. Methods: The work was performed on male Wistar rats, 24 months of age. Animals consumed a solution of AlCl3 100 mg/kg per day for 60 days. Morphological changes of neurons and microglia, mRNA expression levels of pro- and anti-inflammatory cytokines, microglia activation markers, amyloid-related and hypoxia-related proteins, as well as monocyte and lymphocyte subpopulations in peripheral blood, were examined. Results: AlCl3-treated old rats showed the increasing of hyperchromic neurons in 2 out of 3 examined regions of the hippocampus; morphological features of microglia cells’ dystrophy; the upregulation of pro-inflammatory cytokine Il-18 and the downregulation of anti-inflammatory cytokine Il-10, as well as App, Bace1, and Hif-1a; the decreasing of percentage of B-cells, general CD3+ lymphocyte population, including CD4+ T-helpers and CD8+ cytotoxic cells, but the increasing of CD4+/CD8+ ratio in peripheral blood. Conclusion: AlCl3-treated aged rats demonstrated systemic maladaptation to AlCl3 impact. Unlike adult rodents, aged ones have the background of inflammaging, as well as elderly people. The exposure of AlCl3 could potentially be a replacement of integral cellular and molecular processes accompanying age-related diseases, presenting in most elderly people, as an enhancer of inflammaging and hypoxia. These conditions make this model of neurodegeneration a reliable one to explore the initial mechanisms of such detrimental process, as well as prove aged rats more suitable subjects to perform future researches in this field.

Alveolar macrophages and monocyte subpopulations during Plasmodium berghei NK65 experimental malaria-associated acute respiratory distress syndrome

Alveolar macrophages (AM) and monocytes (MO) are myeloid cells that play a substantial role in the development and establishment of the innate and adaptive immune response. These cells are crucial for host defense against various pathogens, but their role in malaria is poorly understood. Here, we characterize the dynamics of AMs and recruited leukocytes subpopulations in the airways during experimental Plasmodium berghei NK65-NY (PbNK65). We show that PbNK65 infection induces an increased pulmonary vascular permeability that provides Ly6Clow MOs, neutrophils (NEU), CD4+ and CD8+ lymphocytes in the airways. This inflammatory environment resulted in an increase in the population and alteration of the activation state of the AMs. Taken together, the data presented provide new insights into airway inflammation associated with pulmonary malaria.

Dynamics of peripheral T cell exhaustion and monocyte subpopulations in neurocognitive impairment and brain atrophy in chronic HIV infection

Abstract Background HIV-associated neurocognitive disorders (HAND) is hypothesized to be a result of myeloid cell-induced neuro-inflammation in the central nervous system that may be initiated in the periphery, but the contribution of peripheral T cells in HAND pathogenesis remains poorly understood. Methods We assessed markers of T cell activation (HLA-DR + CD38+), immunosenescence (CD57 + CD28-), and immune-exhaustion (TIM-3, PD-1 and TIGIT) as well as monocyte subsets (classical, intermediate, and non-classical) by flow cytometry in peripheral blood derived from individuals with HIV on long-term stable anti-retroviral therapy (ART). Additionally, normalized neuropsychological (NP) composite test z-scores were obtained and regional brain volumes were assessed by magnetic resonance imaging (MRI). Relationships between proportions of immune phenotypes (of T-cells and monocytes), NP z-scores, and brain volumes were analyzed using Pearson correlations and multiple linear regression models. Results Of N = 51 participants, 84.3% were male, 86.3% had undetectable HIV RNA < 50 copies/ml, median age was 52 [47, 57] years and median CD4 T cell count was 479 [376, 717] cells/uL. Higher CD4 T cells expressing PD-1 + and/or TIM-3 + were associated with lower executive function and working memory and higher CD8 T cells expressing PD-1+ and/or TIM-3+ were associated with reduced brain volumes in multiple regions (putamen, nucleus accumbens, cerebellar cortex, and subcortical gray matter). Furthermore, higher single or dual frequencies of PD-1 + and TIM-3 + expressing CD4 and CD8 T-cells correlated with higher CD16 + monocyte numbers. Conclusions This study reinforces evidence that T cells, particularly those with immune exhaustion phenotypes, are associated with neurocognitive impairment and brain atrophy in people living with HIV on ART. Relationships revealed between T-cell immune exhaustion and inflammatory in CD16+ monocytes uncover interrelated cellular processes likely involved in the immunopathogenesis of HAND.