CD14 Monocytes Research Articles

Integration of single-cell and bulk RNA sequencing revealed immune heterogeneity and its association with disease activity in rheumatoid arthritis patients.

Rheumatoid arthritis (RA) is a chronic, inflammatory, systemic autoimmune disease characterized by cartilage, bone damage, synovial inflammation, hyperplasia, autoantibody production, and systemic features. To obtain an overall profile of the immune environment in RA patients and its association with clinical features, we performed single-cell transcriptome and T-cell receptor sequencing of mononuclear cells from peripheral blood (PBMC) and synovial fluid (SF) from RA patients, integrated with two large cohorts with bulk RNA sequencing for further validation and investigation. Dendritic cells (DCs) exhibited relatively high functional heterogeneity and tissue specificity in relation to both antigen presentation and proinflammatory functions. Peripheral helper T cells (TPHs) are likely to originate from synovial tissue, undergo activation and exhaustion, and are subsequently released into the peripheral blood. Notably, among all immune cell types, TPHs were found to have the most intense associations with disease activity. In addition, CD8 effector T cells could be clustered into two groups with different cytokine expressions and play distinct roles in RA development. By integrating single-cell data with bulk sequencing from two large cohorts, we identified interactions among TPHs, CD8 cells, CD16 monocytes, and DCs that strongly contribute to the proinflammatory local environment in RA joints. Of note, the swollen 28-joint counts exhibited a more pronounced association with this immune environment compared to other disease activity indexes. The immune environment alternated significantly from PBMCs to SF, which indicated that a series of immune cells was involved in proinflammatory responses in the local joints of RA patients. By integrating single-cell data with two large cohorts, we have uncovered associations between specific immune cell populations and clinical features. This integration provides a rapid and precise methodology for assessing local immune activation, offering valuable insights into the pathophysiological mechanisms at play in RA.

Association of elevated circulating monocyte-platelet aggregates with hypercoagulability in patients with nephrotic syndrome

BackgroundHypercoagulability emerges as a central pathological feature and clinical complication in nephrotic syndrome. Increased platelet activation and aggregability are closely related to hypercoagulability in nephrotic syndrome. Monocyte-platelet aggregates (MPAs) have been proposed to represent a robust biomarker of platelet activation. The aim of this study was to investigate levels of the circulating MPAs and MPAs with the different monocyte subsets to evaluate the association of MPAs with hypercoagulability in nephrotic syndrome.MethodsThirty-two patients with nephrotic syndrome were enrolled. In addition, thirty-two healthy age and sex matched adult volunteers served as healthy controls. MPAs were identified by CD14 monocytes positive for CD41a platelets. The classical (CD14 + + CD16-, CM), the intermediate (CD14 + + CD16+, IM) and the non-classical (CD14 + CD16++, NCM) monocytes, as well as subset specific MPAs, were measured by flow cytometry.ResultsPatients with nephrotic syndrome showed a higher percentage of circulating MPAs as compared with healthy controls (p < 0.001). The percentages of MPAs with CM, IM, and NCM were higher than those of healthy controls (p = 0.012, p < 0.001 and p < 0.001, respectively). Circulating MPAs showed correlations with hypoalbuminemia (r=-0.85; p < 0.001), hypercholesterolemia (r = 0.54; p < 0.001), fibrinogen (r = 0.70; p < 0.001) and D-dimer (r = 0.37; p = 0.003), but not with hypertriglyceridemia in nephrotic syndrome. The AUC for the prediction of hypercoagulability in nephrotic syndrome using MPAs was 0.79 (95% CI 0.68–0.90, p < 0.001). The sensitivity of MPAs in predicting hypercoagulability was 0.71, and the specificity was 0.78.ConclusionIncreased MPAs were correlated with hypercoagulability in nephrotic syndrome. MPAs may serve as a potential biomarker for thrombophilic or hypercoagulable state and provide novel insight into the mechanisms of anticoagulation in nephrotic syndrome.

Tofaticinib as a potential therapeutic agent: a review

Tofacitinib is a Food and Drug Administration (FDA) approved second generation immunosuppressive disease-modifying anti-rheumatic drug (DMARD) that is used in the treatment of conditions like rheumatoid arthritis, ulcerative colitis polyarticular course juvenile idiopathic arthritis and psoriatic arthritis. Tofacitinib can be used to treat adult RA patients who are intolerant to methotrexate. At cellular level tofacitinib selectively inhibits Janus kinase (JAKs) in human genome and thereby stops the cytokine receptor-based signalling of interleukins viz., IL2, IL4, IL6, IL7, IL15, IL21, interferon alpha (IFNa) and IFNc in synovial fibroblasts and CD14 monocytes, thereby leading to disruption of immune and inflammatory responses. Tofacitinib is marketed as orally administered conventional tablets (5 mg and 10 mg doses) extended-release tablets (11 mg dose) and oral solutions (1 mg/ml dose). Tofacitinib is quickly absorbed after oral administration with systemic bioavailability of 74%. Tofacitinib is metabolized majorly by CYP3A4 and clearance is 70% via hepatic metabolism and 30% via renal excretion. Half-life of orally administered tofacitinib was observed to be 3 hours. Patients receiving tofacitinib therapy should be monitored for TB, renal impairment, hepatic impairment or any kind of bacterial, viral or fungal infections before initiating or during therapy. Most common reported adverse events of tofacitinib are headache, diarrhoea, nasopharyngitis, sore throat, hypertension and respiratory tract infections. Tofacitinib therapy should be cautiously used in females of reproductive age and in patients receiving renal transplant. Tofacitinib is not recommended with other immunosuppressants and vaccines. Thus, tofacitinib being a potential therapeutic agent the current review elaborates the history, detailed pharmacology, dosing, adverse events, interactions and contraindications of tofacitinib.

Predicting immunotherapy toxicity through single-cell sequencing of peripheral immune cells: A pilot study.

e14538 Background: Immune checkpoint inhibitors are widely used in oncology. However, immune-related adverse events (iRAEs) are idiosyncratic and can be severe. The overall incidence is 27%, with severe iRAEs occurring in 6% of patients. We hypothesised that distinct immune cell phenotypes (gene expression) correlate with immune-related toxicity from checkpoint inhibitor immunotherapy. Single-cell RNA sequencing (scRNA-seq) is a powerful tool that can assay the gene expression of thousands of individual cells. RPS26 expression mediates CD4/CD8 T cell activation through a known trans-expressive quantitative trait locus at rs1131017 and is linked with autoimmunity. FOS and JUN encode for c-Fos and c-Jun, which dimerise to create the activator protein-1 complex, a well-established transcriptional factor of the immune system. c-Fos is an immunoregulator known to suppress pro-inflammatory cytokines, suggesting that c-FOS deficiency plays a crucial role in developing autoimmunity. We aimed to analyse the transcriptomic differences of peripheral immune cells between patients who experienced severe checkpoint inhibitor therapy iRAE and those who did not. Methods: We identified a pilot cohort of eight patients who developed grade three or above toxicity due to immune checkpoint inhibitors and eight controls who were matched for age, cancer and treatment type. Peripheral blood mononuclear cells were collected prospectively at baseline and two subsequent treatment cycles. Cells were captured on the 10x Chromium platform, and libraries were sequenced to 30,000 reads per cell. Samples were demultiplexed, and the data was analysed with the Seurat package on R statistical software (version 4.3.0). Results: Five patients had baseline samples only, two patients had two samples from two cycles, and one patient had samples from three cycles. After quality control filtering, we identified 36,288 cells from 16 individuals and 24 samples. Immune cells were annotated with the Azimuth package into 29 subtypes. We identified differences in cell proportions of immune cell subsets in the toxicity vs control group, including B naive cells (5.57% vs 3.71% p=0.01) and reduced cytotoxic CD4 T lymphocyte cells (0.66% vs 2.26%, p=0.005). Differential gene expression analysis identified a significantly high expression of ribosomal genes, particularly RPS26, in multiple cell types at baseline (p&lt;0.01) and reduced expression of FOS and JUN-related genes in CD14 and CD16 monocytes, CD4 and CD8 T cells in the toxicity cohort (p&lt;0.01). Conclusions: Overexpression of ribosomal genes and reduced expression of FOS/ JUN genes amongst PBMCs at baseline are potentially predictive biomarkers of severe checkpoint inhibitor immunotherapy toxicity. Validation of these findings in a larger cohort is in progress.

Single-cell transcriptional profiling in osteosarcoma and the effect of neoadjuvant chemotherapy on the tumor microenvironment

Osteosarcoma (OS), a malignant tumor, originates from the bone marrow. Currently, treatment for OS remains limited, making it urgent to understand the immune response in the tumor microenvironment of patients with OS. A comprehensive bioinformatics analysis was performed, including cell clustering subgroups, differential expression genes screening, proposed temporal order, and genomic variant analysis on single-cell RNA-sequencing data, from ten pre-chemotherapy patients and eleven post-chemotherapy patients. Subsequently, we analyzed the differentiation trajectories of osteoblasts, osteoclasts, fibroblasts, myeloid cells, and tumor-infiltrating lymphocytes (TILs) in detail to compare the changes in cell proportions and differential genes pre- and post-chemotherapy. The nine cell types were identified, including fibroblasts, myeloid cells, osteoblasts, TILs, osteoclasts, proliferative osteoblasts, pericytes, endothelial cells, and B cells. Post-chemotherapy treatment, the proportions of myeloid cells and TILs in OS were declined, while the number of osteoblasts was elevated. Besides, a decrease was observed in CD74, FTL, FTH1, MT1X and MT2A, and an increase in PTN, COL3A1, COL1A1, IGFBP7 and FN1. Meanwhile, EMT, DNA repair, G2M checkpoint, and E2F targets were highly enriched post-chemotherapy. Furthermore, there was a down-regulation in the proportions of CD14 monocytes, Tregs, NK cells and CD1C-/CD141-DCs, while an up-regulation was observed in the proportions of SELENOP macrophages, IL7R macrophages, COL1A1 macrophages, CD1C DCs, CD4+ T cells and CD8+ T cells. Overall, these findings revealed changes in the tumor microenvironment of OS post-chemotherapy treatment, providing a new direction for investigating OS treatment.

Abstract 1036: Mechanisms Of Foamy Monocyte Formation In Atherosclerosis

Diseases linked to atherosclerosis are the leading cause of death in the United States. Transmigration of monocytes across the endothelial layer and their differentiation into macrophages play an important role in the initiation and progression of atherosclerosis. These monocyte-derived macrophages internalize modified LDL in the arterial wall and drive the pathogenesis of atherosclerosis. Recent studies have shown that pharmacological depletion of foamy monocytes inhibits atherosclerosis development in mice, identifying monocyte uptake of plasma LDL as a therapeutic target in atherosclerosis. However, the precise mechanisms by which monocytes internalize plasma LDL remain unknown. The goal of our study was to investigate the role, and relative contribution, of macropinocytosis and scavenger receptors ( Cd36 and Sr-a ) to foamy monocyte formation. The presence of foamy monocytes was confirmed in hypercholesterolemic mice and humans via flow cytometry analysis of Nile Red fluorescence. High resolution scanning electron microscopy and flow cytometry analysis of dextran internalization confirmed macropinocytosis stimulation in THP-1 and primary murine monocytes. Stimulation of macropinocytosis in monocytes induced uptake of both native LDL and oxLDL, leading to foamy monocyte formation in vitro . Pharmacological (EIPA) and genetic inhibition of macropinocytosis ( Lysm - CreER T2+/- Nhe1 -/- ) inhibited foamy monocyte formation (58.9%) in hypercholesterolemic mice in vivo . RT-PCR confirmed expression of Cd36 and Sr-a in monocytes and deletion of Cd36 and Sr-a inhibited foamy monocyte formation in hypercholesterolemic mice (10.3%) compared to wild type controls. FACS and bulk RNA sequencing characterized monocyte subsets including classical, intermediate, and non-classical monocytes in hypercholesterolemic Cd36 -/- / Sr-a -/- and Lysm - CreER T2+/- Nhe1 -/- mice and their respective wild type controls. Mechanistic studies identified NADPH oxidase 2 (Nox2) and downstream redox regulation of actin-cytoskeleton as a major stimulator of monocyte macropinocytosis. These results provide novel insights into the mechanisms of foamy monocyte formation and potentially identify new therapeutic targets in the treatment of atherosclerosis.

Comprehensive analysis of juvenile idiopathic arthritis patients' immune characteristics based on bulk and single-cell sequencing data.

The pathogenesis of juvenile idiopathic arthritis (JIA) is strongly influenced by an impaired immune system. However, the molecular mechanisms underlying its development and progression have not been elucidated. In this study, the computational methods TRUST4 were used to construct a T-cell receptor (TCR) and B-cell receptor (BCR) repertoire from the peripheral blood of JIA patients via bulk RNA-seq data, after which the clonality and diversity of the immune repertoire were analyzed. Our findings revealed significant differences in the frequency of clonotypes between the JIA and healthy control groups in terms of the TCR and BCR repertoires. This work identified specific V genes and J genes in TCRs and BCRs that could be used to expand our understanding of JIA. After single-cell RNA analysis, the relative percentages of CD14 monocytes were significantly greater in the JIA group. Cell-cell communication analysis revealed the significant role of the MIF signaling pathway in JIA. In conclusion, this work describes the immune features of both the TCR and BCR repertoires under JIA conditions and provides novel insight into immunotherapy for JIA.

CITEViz: interactively classify cell populations in CITE-Seq via a flow cytometry-like gating workflow using R-Shiny

BackgroundThe rapid advancement of new genomic sequencing technology has enabled the development of multi-omic single-cell sequencing assays. These assays profile multiple modalities in the same cell and can often yield new insights not revealed with a single modality. For example, Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-Seq) simultaneously profiles the RNA transcriptome and the surface protein expression. The surface protein markers in CITE-Seq can be used to identify cell populations similar to the iterative filtration process in flow cytometry, also called “gating”, and is an essential step for downstream analyses and data interpretation. While several packages allow users to interactively gate cells, they often do not process multi-omic sequencing datasets and may require writing redundant code to specify gate boundaries. To streamline the gating process, we developed CITEViz which allows users to interactively gate cells in Seurat-processed CITE-Seq data. CITEViz can also visualize basic quality control (QC) metrics allowing for a rapid and holistic evaluation of CITE-Seq data.ResultsWe applied CITEViz to a peripheral blood mononuclear cell CITE-Seq dataset and gated for several major blood cell populations (CD14 monocytes, CD4 T cells, CD8 T cells, NK cells, B cells, and platelets) using canonical surface protein markers. The visualization features of CITEViz were used to investigate cellular heterogeneity in CD14 and CD16-expressing monocytes and to detect differential numbers of detected antibodies per patient donor. These results highlight the utility of CITEViz to enable the robust classification of single cell populations.ConclusionsCITEViz is an R-Shiny app that standardizes the gating workflow in CITE-Seq data for efficient classification of cell populations. Its secondary function is to generate basic feature plots and QC figures specific to multi-omic data. The user interface and internal workflow of CITEViz uniquely work together to produce an organized workflow and sensible data structures for easy data retrieval. This package leverages the strengths of biologists and computational scientists to assess and analyze multi-omic single-cell datasets. In conclusion, CITEViz streamlines the flow cytometry gating workflow in CITE-Seq data to help facilitate novel hypothesis generation.

Abstract 6853: Spatial proteomic and transcriptomic characterization of the inflammatory landscape of formalin fixed paraffin embedded Barrett’s esophagus tissues

Abstract Esophageal adenocarcinoma (EAC), a significant cause of morbidity and mortality, develops from its metaplastic precursor lesion termed Barrett’s esophagus (BE). BE progresses to EAC within an inflammation rich environment, however a detailed understanding of the types of inflammatory cells present, how that population changes over time or diagnosis, and the related immunomodulatory pathways that may mediate BEs progression is still understudied. Traditional immunohistochemistry is often inadequate to provide detailed information about a broad number of cells, local cellular proportions, and cellular heterogeneity; while bulk or single cell RNA sequencing lose the spatial context and relationships between the stroma and epithelium. Utilizing modern multiplex profiling technologies such as Nanostring’s GeoMx digital Profiler (DSP) aid in identifying both cellular composition and distribution of cells within the epithelium and stroma. Therefore, our aim of this study was to determine the suitability of the DSP platform for analyzing formalin fixed paraffin embedded (FFPE) endoscopic samples of BE and determine any differences between non-dysplastic BE (NDBE) samples from patients who eventually progressed (progressors) compared to those who did not (non-progressors), with the goal to gain deeper insights about cellular composition and spatial distribution of immune cells that may dictate progression of BE. Our secondary goal was to investigate the relationship of these cells to the BE epithelial cells through understanding the immune signaling and regulatory pathways. Our pilot study utilizing a 49-plex antibody panel on 7 NDBE samples from progressors and 8 NDBE samples from non-progressors showed good quality metrics and deciphered an immune rich microenvironment in BE with several immune markers including CD66b (neutrophils), CD14 (monocytes), CD68 (macrophages) and CD56 (NK cells) being overexpressed in the stroma of progressors compared with non-progressors, corrected p value less than 0.05. The myeloid activation marker CD80 was overexpressed in the BE epithelial cells of progressors. To determine how spatial transcriptomic profiling performed in FFPE endoscopic samples, 1,812 transcripts within 48 regions of interest of four BE, HGD, and EAC samples was interrogated. Only 36 (2%) probes fell below the limit of detection quality threshold. Thus, our pilot results show that robust proteomic and transcriptomic profiling can be obtained from endoscopic BE biopsies using this system. Based on these results, we systematically interrogated the immune cell makeup and distribution as well as their related inflammatory and immunoregulatory pathways in 40 NDBE biopsies (20 progressors and 20 with stable non-progressing disease) within discrete compartments (stroma and epithelium) through spatial whole transcriptome profiling. Citation Format: Qurat ul Ain, Nicola Frei, Amir M. Khoshiwal, Pim Stougie, Robert Odze, Sophie Camilleri-Broet, Lorenzo Ferri, Lucas C. Duits, Jacques Bergman, Matthew D. Stachler. Spatial proteomic and transcriptomic characterization of the inflammatory landscape of formalin fixed paraffin embedded Barrett’s esophagus tissues [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6853.

Non-fatal outcomes of COVID-19 disease in pediatric organ transplantation associates with down-regulation of senescence pathways

This is a cross-sectional study examining kinetics and durability of immune response in children with solid organ transplants (SOTs) who had COVID-19 disease between November 2020 through June 2022, who were followed for 60-days at a single transplant center. Blood was collected between 1–14 (acute infection), and 15–60 days of a positive PCR (convalescence). SOT children with peripheral blood mononuclear cells (PBMC) cryopreserved before 2019 were non-infected controls (ctrls). PBMCs stimulated with 15-mer peptides from spike protein and anti-CD49d/anti-CD28. Testing done included mass cytometry, mi-RNA sequencing with confirmatory qPCR. 38 children formed the study cohort, 10 in the acute phase and 8 in the convalescence phase. 20 subjects were non-infected controls. Two subjects had severe disease. Subjects in the acute and convalescent phases were different subjects. The median age and tacrolimus level at blood draw was not significantly different. There was no death, and no subject was lost to follow-up. During acute infection CD57 expression was low in NKT, Th17 effector memory, memory Treg, CD4−CD8−, and γδT cells (p = 0.01, p = 0.04, p = 0.03, p = 0.03, p = 0.004 respectively). The frequencies of NK and Th2 effector memory cells increased (p = 0.01, p = 0.02) during acute infection. Non-switched memory B and CD8 central memory cell frequencies were decreased during acute infection (p = 0.02; p = 0.02), but the decrease in CD8 central memory cells did not persist. CD4−CD8− and CD14 monocyte frequencies increased during recovery (p = 0.03; p = 0.007). Our observations suggest down regulation of CD57 with absence of NK cell contraction protect against death from COVID-19 disease in children with SOTs.

Fc-gamma receptor expression and cytokine responses to intravenous human immunoglobulin in whole blood from non-pregnant and pregnant women and newborns.

Intravenous immunoglobulin (IVIG) can suppress the inflammatory response in adults, but its role in pregnant women and newborns is poorly studied. While the adult immune system is considered mature, it is immature in neonates and suppressed in pregnancy. Since the immune response differs in these 3 groups, the use of IVIG could differentially modulate the immune response. We aimed to explore the effect of IVIG on myeloid blood cells from non-pregnant women, pregnant women and newborns. Whole blood from healthy donors was incubated with lipopolysaccharide (LPS) and/or IVIG. After 0 h, 24 h and 48 h of culture, Fc-gamma receptor (CD16, CD32 and CD64) expression, monocyte and neutrophil bacterial phagocytosis, and cytokine and chemokine concentrations were determined in the supernatant. The baseline expression of monocyte CD16 was higher in newborns than in adult women, but the expression of CD32 and CD64 was similar between groups. Furthermore, LPS and IVIG stimulation, together or separately, did not change Fc-gamma receptor expression in monocytes or neutrophils and did not modify their phagocytosis capacity. On the other hand, IVIG did not downregulate the proinflammatory cytokine response induced by LPS in any group. Interestingly, IVIG induced a strong interleukin 8 (IL-8) response in neonates but not in non-pregnant or pregnant women. Our results show that IVIG did not induce changes in Fc-gamma receptor expression, phagocytic ability, or the cytokine response to LPS in blood cells from neonates, non-pregnant or pregnant women. However, IVIG induced a strong IL-8 response in neonates that could improve immunity.

Single-cell analysis of human PBMCs in healthy and type 2 diabetes populations: dysregulated immune networks in type 2 diabetes unveiled through single-cell profiling.

Abnormalities in glucose metabolism that precede the onset of type 2 diabetes (T2D) activate immune cells, leading to elevated inflammatory factors and chronic inflammation. However, no single-cell RNA sequencing (scRNA-seq) studies have characterized the properties and networks of individual immune cells in T2D. Here, we analyzed peripheral blood mononuclear cells (PBMCs) from non-diabetes and T2D patients by scRNA-seq. We found that CD14 monocytes in T2D patients were in a pro-inflammatory state and intermediate monocytes expressed more MHC class II genes. In T2D patients, cytotoxic CD4 T cells, effector memory CD8 T cells, and γδ T cells have increased cytotoxicity and clonal expansion. B cells were characterized by increased differentiation into intermediate B cells, plasma cells, and isotype class switching with increased expression of soluble antibody genes. These results suggest that monocytes, T cells, and B cells could interact to induce chronic inflammation in T2D patients with pro-inflammatory characteristics.

The Delayed Presentation of Achilles Tendon Ruptures Is Associated With Marked Alterations in the Gene Expression of COL1A1, MMPs, TIMPs, and IL-6

Background: Both acute and chronic Achilles tendon ruptures are affected by alterations in the extracellular matrix during the healing process of the tendon. Yet, these alterations in gene expression patterns are not well characterized. Purpose: To characterize temporal and spatial differences in gene expression patterns after an Achilles tendon rupture and to evaluate if cells from chronic Achilles tendon ruptures have the same ability to form new tendon tissue (tendon constructs) as healthy tendon cells. Study Design: Controlled laboratory study. Methods: A total of 35 patients with surgically treated Achilles tendon ruptures were included in the study and divided into 3 groups: acute (<4 weeks), short-term chronic (1-6 months), and long-term chronic (>6 months). Biopsy specimens were collected during surgical repair and were used to analyze the gene expression within the different groups and to compare mRNA levels in the proximal and distal tendon ends. A complementary in vitro experiment was performed to evaluate if cells from chronic Achilles tendon ruptures can form tendon constructs. Results: The mRNA levels for COL1A1 and COL3A1 were significantly higher in the short-term chronic group compared with the acute group (P < .05). Both MMP-1 and MMP-13 had the highest mRNA levels in the acute group (P < .01) compared with the long-term chronic group, while MMP-2 had the highest mRNA level in the short-term chronic group. Significant differences between the proximal and distal tendon ends were only detected for the monocyte and macrophage marker CD163 (P < .05), which was more expressed proximally. Cells extracted from chronic Achilles tendon ruptures displayed a similar ability and effectiveness to form tendon constructs as healthy tendon cells. Conclusion: A high collagenase gene activity after an Achilles tendon rupture indicated possible rapid matrix degradation in the acute phase. Chronic ruptures appeared to initiate the healing process even before treatment, indicated by the higher expression of collagen in the short-term chronic group. Cells from chronic Achilles tendon ruptures also displayed an ability to form new tendon tissue in vitro. Clinical Relevance: The study shows a rapid increase in collagenase gene expression, which could lead to matrix degradation that continues for months after an Achilles tendon rupture.

Circulating intermediate monocytes CD14++CD16+ are increased after elective percutaneous coronary intervention

Aim Inflammation plays a central role in the pathogenesis of atherosclerosis and in the sequelae of percutaneous coronary intervention (PCI). Previous work demonstrated that intermediate monocytes (CD14++CD16+) are associated with adverse cardiovascular events, yet monocyte subset response following elective PCI has not been described. This article explores the changes in monocyte subset and humoral response after elective PCI. Methods This prospective study included 30 patients without inflammatory diseases being referred for elective PCI. We included patients treated with drug coated balloons or 2nd generation drug eluting stents. Patients underwent blood tests at baseline (prior to PCI), four hours, two weeks and two months later. Analyses were performed in terms of monocyte subsets (classical CD14++CD16-, intermediate CD14++CD16+ and non-classical CD14+CD16++), gene expression of CD14+ leucocytes and humoral biomarkers. Results Intermediate monocytes decreased significantly four hours after PCI, were recovered at two weeks, and increased significantly at two months post elective, uncomplicated PCI. They remain significantly elevated in the DES group but not in the DCB group. Gene expression analysis of CD14+ leucocytes showed IL18 had decreased expression at two weeks, CXCR4 and IL1β decreased at two months, while pentraxin 3 increased at two weeks and two months. In terms of humoral biomarkers, hsTnI remains elevated up to two weeks post PCI while IL6 and TNFα remain elevated till two months post PCI. Conclusion Intermediate monocytes increase significantly two months following elective, uncomplicated PCI. They remain significantly elevated in the DES group but not in the DCB group suggesting that the PCI strategy could be one of the ways to modulate the inflammatory response post PCI.

Single-cell RNA-seq analyses inform necroptosis-associated myeloid lineages influence the immune landscape of pancreas cancer.

Tumor-infiltrating myeloid cells (TIMs) are key regulators in tumor progression, but the similarity and distinction of their fundamental properties in pancreatic ductal adenocarcinoma (PDAC) remain elusive. In this study, we conducted scRNA-seq data analysis of cells from 12 primary tumor (PT) tissues, 4 metastatic (Met) tumor tissues, 3 adjacent normal pancreas tissues (Para), and PBMC samples across 16 PDAC patients, and revealed a heterogeneous TIMs environment in PDAC. Systematic comparisons between tumor and non-tumor samples of myeloid lineages identified 10 necroptosis-associated genes upregulated in PDAC tumors compared to 5 upregulated in paratumor or healthy peripheral blood. A novel RTM (resident tissue macrophages), GLUL-SQSTM1- RTM, was found to act as a positive regulator of immunity. Additionally, HSP90AA1+HSP90AB1+ mast cells exhibited pro-immune characteristics, and JAK3+TLR4+ CD16 monocytes were found to be anti-immune. The findings were validated through clinical outcomes and cytokines analyses. Lastly, intercellular network reconstruction supported the associations between the identified novel clusters, cancer cells, and immune cell populations. Our analysis comprehensively characterized major myeloid cell lineages and identified three subsets of myeloid-derived cells associated with necroptosis. These findings not only provide a valuable resource for understanding the multi-dimensional characterization of the tumor microenvironment in PDAC but also offer valuable mechanistic insights that can guide the design of effective immuno-oncology treatment strategies.

Single-Cell Metabolic Profiling Integrated with Extracellular Vesicle Analysis Reveals Novel Metabolic Vulnerabilities and Prognostic Biomarkers in Acute Myeloid Leukemia

Introduction Acute myeloid leukemia (AML) is an aggressive disease for which less invasive tools are needed to explore disease dynamics. Changes in cell metabolism and metabolic adaptation are hallmark features for AML, thus emerging as promising therapeutic targets. However, it is still poorly elucidated whether and which extrinsic microenvironment signals may regulate the metabolic reprogramming occurring in AML. In this regard, Extracellular Vesicles (EVs) represent a promising tool for non-invasive liquid biopsy and may reveal a novel role in energy metabolism as well as clinically relevant biomarkers for AML. Along with EV profiling from peripheral blood (PB), we mapped the metabolic signature of leukemic stem cells (LSCs) and immune cell subsets to identify metabolic vulnerabilities and novel prognostic biomarkers. Methods PB (and paired bone marrow) were collected from AML patients at diagnosis (n=98) and healthy donors (HD, n=12). EVs were purified from platelet-free PB plasma of AML patients by size-exclusion chromatography followed by ultrafiltration. We have submitted all relevant data of our EV experiments to the EV-TRACK knowledgebase (EV-TRACK ID: EV230004). MACSPlex Exosome Kit was used for screening up to 37 surface EV markers. Western blot analysis was also performed. Energy cell metabolism was explored by Single Cell ENergetic metabolism by profilIng Translation inHibition (SCENITH TM) in LSC-like cells (CD34 +CD38 low/-)and immune cell subsets (CD3 +/CD4 +/CD8 +/T regulatory cells) using conventional/spectral flow-cytometry. Results Here, we developed a novel method exploiting both LSCs and EVs from AML patients using fresh whole blood for metabolic exploration. Firstly, we observed that LSC-like cells display low reactive oxygen species (ROS) levels with both high glutathione (GSH, as antioxidant molecule) levels and mitochondrial functionality suggesting a more resistant phenotype. Consistently, poor clinical outcomes were experienced by AML patients treated with conventional chemotherapy and with high GSH levels and mitochondrial potential in LSC-like cells (log-rank p=0.04). We also found high dependence on glucose and a preferential skewing toward glycolysis in LSC-like cells revealing a distinct metabolic status for LSCs in circulation. However, according to European LeukemiaNet (ELN) 2022 risk classification, a more prominent mitochondrial dependence on LSC-like cells was reported for intermediate- or adverse-risk AML patients revealing a prognostic relevance. By contrast, exploring the immunometabolic reprogramming in circulation, we revealed a significant decrease in glucose dependence for immune cells (CD4 + and CD8 + T cells) in comparison to LSCs proving the presence of a complex metabolic network in AML. Interestingly, we investigated the potential metabolic effects of EVs on LSCs. Indeed, CD34 + LSC-like cells co-cultured with circulating EVs enhanced their GSH levels and mitochondrial functionality towards a more aggressive phenotype. Accordingly, we discovered the presence of the antioxidant enzyme, glutathione peroxidases 3 (GPx-3), in AML EVs. Then, we profiled the surface proteins of isolated EVs to detect their cell origins and provide a reference map for clinical prognosis. We found that circulating AML EVs were significantly enriched in CD4 (helper T cell marker), CD14 (monocyte marker), CD40 (B-cell associated marker), CD44 and CD133-1 (stem cell markers), CD105 (stromal cell marker) and HLA-II (antigen-presenting cell marker) compared to HD EVs. In particular, we found that intermediate-risk AML patients showed a strong depletion in the expression of EV markers including T cell markers (CD2 and CD8) and mesenchymal markers (CD49e and CD146) in comparison to favorable and adverse-risk patients. Interestingly, AML patients who had been treated not intensively and showed low expression of CD40 (HR=0.23, 95% CI, 0.05−0,99, p=0.04) or CD62P (platelet activation marker, HR=0.24, 95% CI, 0.05−1.02, p=0.05) on EVs experienced poor outcomes suggesting that dedifferentiated phenotype on EVs might be associated with worse prognosis. Conclusion Overall, towards a new redefined ‘metabolic’ stratification for AML patients, we identified novel prognostic biomarkers for AML patients that might reveal novel metabolic vulnerabilities by exploiting in parallel single-cell metabolic profile and EV-based liquid biopsy.

Assessment of inter-operator variability in peripheral monocyte subset gating strategy using flow cytometry in patients with suspected acute stroke.

Innovative tools to reliably identify patients with acute stroke are needed. Peripheral monocyte subsets, that is, classical-Mon1, intermediate-Mon2, and non-classical-Mon3, with their activation marker expression analyzed using flow-cytometry (FCM) could be interesting cell biomarker candidates. To assess the inter-operator variability in a new peripheral monocyte subset gating strategy using FCM in patients with suspected acute stroke. In BOOST-study ("Biomarkers-algOrithm-for-strOke-diagnoSis-and Treatment-resistance-prediction," NCT04726839), patients ≥18 years with symptoms suggesting acute stroke within the last 24 h were included. Blood was collected upon admission to emergency unit. FCM analysis was performed using the FACS-CANTO-II® flow-cytometer and Flow-Jo™-software. Analyzed markers were CD45/CD91/CD14/CD16 (monocyte backbone) and CD62L/CD11b/HLA-DR/CD86/CCR2/ICAM-1/CX3CR1/TF (activation markers). Inter-operator agreement (starting from raw-data files) was quantified by the measure distribution and, for each patient, the coefficient of variation (CV). Three operators analyzed 20 patient blood samples. Median inter-operator CVs were below the pre-specified tolerance limits (10% [for Mon1 counts], 20% [Mon2, Mon3 counts], 15% [activation marker median-fluorescence-intensities]). We observed a slight, but systematic, inter-operator effect. Overall, absolute inter-operator differences in fractions of monocyte subsets were <0.03. Our gating strategy allowed monocyte subset gating with an acceptable inter-operator variability. Although low, the inter-operator effect should be considered in monocyte data analysis of BOOST-patients.

Single-Cell Multiomics Analysis Reveals Potential Drivers of Response to the Anti-TIM3 Inhibitor Sabatolimab Combined with Azacitidine in MDS and CMML

Background The median survival for patients with newly diagnosed (ND) higher risk myelodysplastic syndrome (MDS) and chronic myelomonocytic leukemia (CMML) remains poor. The potential curability of MDS and CMML with allogeneic hematopoietic stem cell transplantation supports the concept of anti-tumor immunity and has led to interest in evaluating immune-based therapeutic approaches in myeloid neoplasms. The immune checkpoint molecule TIM-3 (encoded by the gene HAVCR2) is a target of interest in myeloid neoplasms given its expression on leukemic stem cells as well as several subsets of immune cells including T cells, monocytes, dendritic cells and NK cells. Sabatolimab (MBG453) is an investigational IgG4 anti-TIM-3 antibody currently under evaluation for myeloid neoplasms. In this exploratory study, we sought to characterize the effects of sabatolimab combined with the hypomethylating agent (HMA), azacitidine, on the immune landscape using single cell sequencing of samples from subjects with MDS and CMML treated with the combination from a previously reported phase 1b study (NCT03066648; Brunner et al 2022). Methods Following IRB approval, single-cell RNA sequencing (scRNA-seq) and associated proteomic cellular indexing of transcriptomes and epitopes sequencing (CITE-seq) was performed on both blood (BLD) and bone-marrow (BM) derived samples from subjects with: 1) MDS (n=3) or CMML (n=2) treated with HMA therapy alone as part of standard care; 2) ND MDS (n=5) or CMML (n=3) treated with azacitidine combined with sabatolimab; and 3) relapsed/refractory MDS (n=3) treated with sabatolimab alone. Paired BLD and BM samples as well as samples from serial treatment time points were selected whenever feasible (Fig 1). A total of 206,183 cells from BM and 172,421 cells from BLD post-quality control filtering were analyzed. Further, scRNAseq data from normal BLD(200,000 cells) and BM(240,650 cells) generated as part of the human cell atlas (https://data.humancellatlas.org/explore/projects/cc95ff89-2e68-4a08-a234-480eca21ce79) were integrated with this dataset to assist with immune cell subset characterization, enable downstream comparisons to healthy hematopoietic cells, and to help define cell subsets associated with disease states. Results In baseline samples from subjects with MDS treated with azacitidine-sabatolimab, we found that an increased baseline abundance of interferon-responsive CD8 T cells in both the BM and BLD (FDR&amp;lt;0.1) was associated with response to therapy. An increased baseline abundance of plasmacytoid dendritic cells and a granulocyte population also suggested an association with response to therapy. Differential gene expression analysis of baseline samples from responding versus non-responding subjects with MDS treated with azacitidine-sabatolimab showed a higher baseline expression of TNF and IFNG in BM CD8 T cells and up-regulated expression of MHC-II machinery ( HLA-DRB1, HLA-DPA1, CD74) in myeloid cell subsets in responding subjects (FDR &amp;lt;0.1), suggesting that pre-treatment ability of these cells to be involved in antigen-presentation may play a role in response. In subjects with CMML treated with azacitidine-sabatolimab, we observed dynamic changes in cellular abundances and gene expression when comparing baseline to post-therapy timepoints. Specifically, BM CD8 T cell subsets showed an up-regulation of cytotoxicity genes ( GZMA), interferon response genes ( IFIT2, IFITM1, IFITM2) and IL32 cytokine post-therapy (FDR &amp;lt;0.1). When evaluating myelomonocytic cell populations, we observed an increase in CD16 monocytes post-treatment. Additionally, dendritic cells and monocytes showed an up-regulation of interferon response genes ( IFIT1, OAS1, IFI27). Conversely, we observed a down-regulation of metallothionein genes ( MT1E, MT1G, MT2A) as well as a collection of transcription factors ( NR4A1, FOSL2, JUN, CEBPB, CEBPD), NF-κB inhibitors ( NFKBIZ, NFKBIA) and CXCL8 in the post-treatment samples (FDR &amp;lt;0.1). Conclusions Our study provides one of the most comprehensive evaluations of the cellular dynamics of anti-TIM3 immunotherapy in patients to date, allowing for the nomination of novel putative predictive biomarkers of response and identification of potential immunomodulatory mechanisms induced by the combination of sabatolimab with azacitidine in MDS and CMML for further future analysis.

Cerebral uptake of microvesicles occurs in normocapnic but not hypocapnic passive hyperthermia in young healthy male adults.

Passive hyperthermia causes cerebral hypoperfusion primarily from heat-induced respiratory alkalosis. However, despite the cerebral hypoperfusion, it is possible that the mild alkalosis might help to attenuate cerebral inflammation. In this study, the cerebral exchange of extracellular vesicles (microvesicles), which are known to elicit pro-inflammatory responses when released in conditions of stress, were examined in hyperthermia with and without respiratory alkalosis. Ten healthy male adults were heated passively, using a warm water-perfused suit, up to core temperature+2°C. Blood samples were taken from the radial artery and internal jugular bulb. Microvesicle concentrations were determined in platelet-poor plasma via cells expressing CD62E (activated endothelial cells), CD31+ /CD42b- (apoptotic endothelial cells), CD14 (monocytes) and CD45 (pan-leucocytes). Cerebral blood flow was measured via duplex ultrasound of the internal carotid and vertebral arteries to determine cerebral exchange kinetics. From baseline to poikilocapnic (alkalotic) hyperthermia, there was no change in microvesicle concentration from any cell origin measured (P-values all >0.05). However, when blood CO2 tension was normalized to baseline levels in hyperthermia, there was a marked increase in cerebral uptake of microvesicles expressing CD62E (P=0.028), CD31+ /CD42b- (P=0.003) and CD14 (P=0.031) compared with baseline, corresponding to large increases in arterial but not jugular venous concentrations. In a subset of seven participants who underwent hypercapnia and hypocapnia in the absence of heating, there was no change in microvesicle concentrations or cerebral exchange, suggesting that hyperthermia potentiated the CO2 /pH-mediated cerebral uptake of microvesicles. These data provide insight into a potential beneficial role of respiratory alkalosis in heat stress. KEY POINTS: The hyperthermia-induced hyperventilatory response is observed in most humans, despite causing potentially harmful reductions in cerebral blood flow. We tested the hypothesis that the respiratory-induced alkalosis is associated with lower circulating microvesicle concentrations, specifically in the brain, despite the reductions in blood flow. At core temperature+2°C with respiratory alkalosis, microvesicles derived from endothelial cells, monocytes and leucocytes were at concentrations similar to baseline in the arterial and cerebral venous circulation, with no changes in cross-brain microvesicle kinetics. However, when core temperature was increased by 2°C with CO2 /pH normalized to resting levels, there was a marked cerebral uptake of microvesicles derived from endothelial cells and monocytes. The CO2 /pH-mediated alteration in cerebral microvesicle uptake occurred only in hyperthermia. These new findings suggest that the heat-induced hyperventilatory response might serve a beneficial role by preventing potentially inflammatory microvesicle uptake in the brain.

Liquid Biopsy in Organ Damage: small extracellular vesicle chip-based assessment of polytrauma.

Despite major advances in medicine, blood-borne biomarkers are urgently needed to support decision-making, including polytrauma. Here, we assessed serum-derived extracellular vesicles (EVs) as potential markers of decision-making in polytrauma. Our Liquid Biopsy in Organ Damage (LiBOD) study aimed to differentiate polytrauma with organ injury from polytrauma without organ injury. We analysed of blood-borne small EVs at the individual level using a combination of immunocapture and high-resolution imaging. To this end, we isolated, purified, and characterized small EVs according to the latest Minimal Information for Studies of Extracellular Vesicles (MISEV) guidelines from human blood collected within 24h post-trauma and validated our results using a porcine polytrauma model. We found that small EVs derived from monocytes CD14+ and CD14+CD61+ were significantly elevated in polytrauma with organ damage. To be precise, our findings revealed that CD9+CD14+ and CD14+CD61+ small EVs exhibited superior performance compared to CD9+CD61+ small EVs in accurately indicating polytrauma with organ damage, reaching a sensitivity and a specificity of 0.81% and 0.97%, respectively. The results in humans were confirmed in an independent porcine model of polytrauma. These findings suggest that these specific types of small EVs may serve as valuable, non-invasive, and objective biomarkers for assessing and monitoring the severity of polytrauma and associated organ damage.