Mononuclear Cells Research Articles

Fecal microbiota transplantation against moderate-to-severe atopic dermatitis: A randomized, double-blind controlled explorer trial.

Fecal microbiota transplantation (FMT) is a novel treatment for inflammatory diseases. Herein, we assess its safety, efficacy, and immunological impact in patients with moderate-to-severe atopic dermatitis (AD). In this randomized, double-blind, placebo-controlled clinical trial, we performed the efficacy and safety assessment of FMT for moderate-to-severe adult patients with AD. All patients received FMT or placebo once a week for 3 weeks, in addition to their standard background treatments. Patients underwent disease severity assessments at weeks 0, 1, 2, 4, 8, 12, and 16, and blood and fecal samples were collected for immunologic analysis and metagenomic shotgun sequencing, respectively. Safety was monitored throughout the trial. Improvements in eczema area and severity index (EASI) scores and percentage of patients achieving EASI 50 (50% reduction in EASI score) were greater in patients treated with FMT than in placebo-treated patients. No serious adverse reactions occurred during the trial. FMT treatment decreased the Th2 and Th17 cell proportions among the peripheral blood mononuclear cells, and the levels of TNF-α, and total IgE in serum. By contrast, the expression levels of IL-12p70 and perforin on NK cells were increased. Moreover, FMT altered the abundance of species and functional pathways of the gut microbiota in the patients, especially the abundance of Megamonas funiformis and the pathway for 1,4-dihydroxy-6-naphthoate biosynthesis II. FMT was a safe and effective therapy in moderate-to-severe adult patients with AD; the treatment changed the gut microbiota compositions and functions.

Impact of proNGF/NGF-NGFR-axis on the regulation of arterial remodelling

Abstract Background Peripheral blood mononuclear cells (MNCs) contribute to the pathogenesis of arteriosclerosis. Nerve growth factor receptor (NGFR) is present in peripheral blood and the ischemic coronary artery. NGFR transduces neurotrophin signals towards cell survival or apoptosis in different cell types dependent on coupling co-receptors. However, the role of NGFR-positive (NGFR+) MNCs in arterial remodelling is unknown. Purpose To investigate the functional mechanisms under which NGFR+ MNCs are involved in arterial remodelling. Methods Adult C57BL/6J male NGFR-wild-type (WT) or -bone marrow (BM)-specific (KO) mice were subjected to unilateral carotid artery ligation. The ligated and the opposite-sided, sham-operated arteries were assessed by immunohistology and gene expression analysis using a PCR on day 28. Also, human NGFR+ MNCs from a healthy volunteer were sorted using fluorescence-activated cell sorting and characterised using RNA sequencing. The cell apoptosis (AnnexinV+7AAD-) and chemotaxis in response to the ligands, NGF and its precursor proNGF, were assessed using flow cytometry and a Transwell migration. Results In WT mice, BM-derived NGFR+ cells accumulated in the neointima after ligation. The neointimal area was significantly greater in the BM-specific depletion of NGFR than in WT mice. NGFR+ cells in the neointima exhibited apoptosis (TUNEL+ and cleaved caspase-3+) accompanied by promoted F4/80+ macrophage and anti-inflammatory IL-10. By contrast, in the BM-specific NGFR-KO model, non-BM-derived SMCs increased in the neointima with augmented proliferation, and the accumulation of BM-derived cells and the expression of IL-10 were decreased. The expressions of proNGF/NGF and inflammatory cytokines, including IL-6, were not changed in a ligated artery, irrespective of NGFR+ depletion. Human NGFR+ MNCs expressed TrkB, TrkC, and sortilin co-receptors but not macrophage markers. Human NGFR+ MNCs, but not NGFR- MNCs, co-cultured with artery SMCs were susceptible to apoptosis in response to proNGF. Additionally, PDGF stimulated proNGF/NGF expression in SMCs, and proNGF/NGF did not affect the proliferation of SMCs in vitro. Furthermore, NGFR+ MNCs, but not NGFR- MNCs, increased migration toward NGF and did not show chemotaxis toward proNGF. Conclusions These data imply that local NGF might draw NGFR+ MNCs to the injured artery, in which proNGF induces NGFR+ MNCs apoptosis. Apoptotic NGFR+ cells promoted macrophage chemotaxis to resolve inflammation and decrease neointimal formation. Thus, we propose the proNGF/NGF-NGFR axis as potentially contributing to the suppression of arterial remodelling.

The myocardium of human dilated nonischemic cardiomyopathy harbors unique tissue-resident lymphocyte subsets

Abstract Purpose The etiology of dilated nonischemic cardiomyopathy (NICM) is often unclear, but thought to be multifactorial, involving an interplay between genetic factors and direct myocardial damage caused by infection, autoimmunity, or toxins. Tissue-resident lymphocytes are non-circulating immune cells which are retained within various tissues and play integral roles in maintaining homeostasis, responding to infection, regulating inflammation, and mediating tissue repair. Despite their likely involvement in various cardiac pathologies, the presence of tissue-resident lymphocytes in the human heart has not yet been well-characterized, neither in health nor in disease. Methods Human left ventricular tissue and paired peripheral blood samples were obtained from six adult organ donors with normal heart function who died of noncardiac causes and from the explants of six adult patients with end-stage nonischemic cardiomyopathy. Heart tissue was processed using mechanical and enzymatic digestion. Mononuclear cells were isolated from both heart and paired blood by density centrifugation. Isolated mononuclear cells were surface stained with an antibody cocktail and interrogated using multidimensional flow cytometry. Results The adult human heart contains a diverse immune cell population (Fig 1A). Both healthy and NICM cardiac lymphocytes express distinct phenotypic profiles which resemble those of resident lymphocytes found across various tissues, distinct from those isolated from paired blood. Frequencies of effector memory T (Tem) cells (CD45RA- CCR7-) were higher in heart than in blood among both CD4+ and CD8+ T-cells. In heart compared to blood, significantly more Tem expressed CD69, a canonical marker of tissue-resident memory T (Trm) cells (Fig 1B). Cardiac Trm, especially CD8+ Trm, expressed classical Trm signature markers including integrins CD103 (epithelial binding) and CD49a (collagen binding). NICM Trm expressed more CD49a than healthy heart Trm (Fig 1C). NK-cells also demonstrated higher CD69 expression (trNK) in heart than in blood, with associated expression of CD103, CD49a, and CXCR6 (Fig 1D). Compared to healthy hearts, NICM trNK were predominantly CD56bright/CD16- (Fig 1E and 1F) and expressed less CD57 (Fig 1D), overall indicating a less mature state with lower cytotoxic potential. Confocal imaging of cardiac tissue revealed CD69-expressing tissue-resident lymphocytes within the cardiac interstitium. Conclusions The adult human heart contains diverse populations of lymphocytes which express canonical markers of tissue residency, similar to lymphocytes resident in other tissues. Compared to healthy hearts, NICM hearts have more CD49a+ Trm, perhaps related to greater fibrosis, and also harbor a distinct subset of immature tissue-resident NK cells which may shed light on pathophysiology. Future work is focused on better exploring the functional phenotypes of these resident cells and better localizing them within the myocardium.FA - Figure 1

Expression of cardiomyopathy-related genes in mononuclear blood cells predicts all-cause mortality in patients presenting with acute versus chronic coronary syndrome requiring revascularisation

Abstract Objective Despite advances in the revascularisation techniques their net clinical benefit differs substantially between patients presenting with myocardial infarction (ACS) and chronic coronary syndrome (CCS) due to obstructive coronary atherosclerosis. Considering that leukocytes represents a key pathophysiological component of myocardial ischaemia, exploration of mononuclear blood cell (PBMC) gene expression profile in CCS and ACS setting could reveal clinically relevant transcriptomic signals. The aim of the present study was to identify differences in PBMC transcriptome between ACS and CCS patients which are linked to mortality after revascularisation. Methods and Results We analysed PBMC transcriptomes from719 ACS and 486 CCS patients creating discovery cohort. The results of transcriptomic analysis were verified in replication cohort (ACS: N=682 for CCS: N=489) generated based on propensity score matching. Median time from revascularisation to blood collection (IQR) was 15.68 (-4.52, 23.23) hrs. Patients with ACS showed consistent differential expression of 8173 genes (4544 upregulated, 3629 downregulated) when compared with CCS patients. The subsequent pathway analysis also showed significant enrichment of several canonical pathways involved in major cardiovascular pathologies i.e. atherosclerosis (p = 1.22-06, enrichment = 3.39), vascular endothelial growth factor production (p = 1.16-06, enrichment = 16.73), positive regulation of vascular development (p = 2.78-06, enrichment = 5.30) and heterotopic cell adhesion including gene desmocollin 2 (DSC2, p = 3.64-04, enrichment = 6.30). In der multivariate survival analysis with differentially expressed genes, increased expression of DCS2, lamin A/C (LMNA), and UGGT2, and decreased expression of SNRNP70 predicted poor survival over the median follow-up 11.35 yrs. Consistently, controlling the false discovery rate at ≤ 5% these genes enriched pathways involved in cardiomyopathies such as arrhythmogenic right-ventricular cardiomyopathy (p = 4.19x10-4, enrichment = 58.9) and collagen disease (p = 2.29x10-3, enrichment = 23.8). Conclusion We found altered PBMC expression of multiple genes in patients requiring revascularisation for ACS versus CCS. Several differentially expressed genes included in cardiomyopathy pathways predicted long-term all-cause mortality in these patients.

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.

Peripheral immune landscape of pediatric fulminant myocarditis revealed by single-cell sequencing

Abstract Fulminant myocarditis (FM) is one of the most feared diseases in children because of its fulminant nature and high mortality rate. However, the precise pathological immune subsets and molecular changes in FM are unknown. Here, we present the first comprehensive cellular and transcriptomic profiling of the peripheral blood mononuclear cells of children in FM acute and recovery phases using Single-cell RNA sequencing. 21 cell clusters are identified among 79542 cells. The proportion of T cells and NK cells increase, while myeloid cells and B cells reduce in acute phase, which were consistent with our flow cytometry data of 35 FM patients. Several unique cell types in peripheral blood are identified, including regulatory B cells, MAIT cells, adaptive NK cells and CD8+Tpex cells. The transcriptomic analysis exhibited elevated expression levels of chemokine receptor CXCR4 and S100A family genes almost in all cell types in FM acute phase, as well as MHC-II molecules in antigen-presenting cells. TCR and BCR exhibit remarkable amplification and obvious skewed usages of V genes in FM. Ligand receptor analysis show myeloid cells maintained active communication with other immune cells. Considering that CXCR4 may play an important role in myocarditis, we designed experiments to explore its function in mouse model. Vital myocarditis (VMC) mouse model was constructed using Coxsackie virus type3（CVB3）.We found that myocardial inflammation and myocardial injury of VMC mice were alleviated when treated with CXCR4 blocker. CXCR4 may be a potential therapeutic target for myocarditis. Our study will provide useful insights into key immune cell subsets and transcriptomic profiles implicated in pediatric fulminant myocarditis acute phase and recovery phase, thus providing several potential diagnostic and therapeutic targets for this disease.

Integrated multi-omics predictive analysis of atherosclerosis: a sub-study from the Mineralocorticoid Receptor Antagonism in Diabetic Atherosclerosis (MAGMA) trial

Abstract Background/Introduction Mineralocorticoid receptor (MR) antagonists (MRA) are beneficial in cardiorenal outcomes in randomized controlled trials but the mechanisms are unclear. MAGMA was an NHLBI sponsored randomized, double-blind, placebo controlled, 12-month trial comparing Spironolactone (n = 37) vs. placebo (n = 42) in Type 2 diabetics with CKD stages 3-4 on maximal renin-angiotensin system (RAS) blockade and a prior atherosclerotic event and/or left ventricular (LV) hypertrophy. The primary outcome of percent (%) change in total aortic wall volume (TWV) at 12 months, measured by magnetic resonance imaging (MRI), was significantly reduced by Spironolactone. Purpose/Originality The purpose of this analysis was to understand mechanistic pathways of MRAs using a multi-omics approach that could help tease out molecular mechanisms of benefit. Revealing for the first time which of these integrated pathways are predictive of the primary outcome will help design treatments for targeted interventions. Methods Plasma and peripheral blood mononuclear cells from patients randomized to Spironolactone or placebo at baseline and 3-months were measured for aptamer-based proteomic biomarkers (7,596 proteins) and 10-X platform-based single-cell RNA-sequencing (scRNAseq), respectively. Pre-selected candidate predictors were used as inputs of a predictive model of changes of TWV. We fit a Mixed-Multivariate Random Forest (RF) model, a variation of the RF supervised tree-based machine learning method to take the experimental design into account in the regression formulation. The two sources of "omics predictors" were integrated into a supervised multi-omics model to jointly explain the outcome using an extension of Sparse Generalized Canonical Correlation Analysis (SGCCA). Integrated multiome functional analyses with graphical visualizations were carried out by statistical enrichment analysis using Over Representation Analysis (ORA) and Gene Set Enrichment Analysis (GSEA) against databases of gene ontologies, biological pathways, putative regulatory motifs, proteins, or disease annotations. Results The plasma proteome in response to Spironolactone revealed downregulation of MR targets including fibrosis, immune activation/inflammation, leukocyte activation, proliferation and pathways involved in cytokine stimulation. scRNAseq pathways revealed negative regulation of cytokines production such as IL-2 and redistribution of multiple cell types. Predictors of plaque progression involved cytokine-receptor, complement-coagulation, cell adhesion and axonal guidance targets. Multiome integrated functional analyses results of predictive pathways will be presented. Conclusions The changes in plasma proteomic profile with Spironolactone were consistent with the phenotype of reduced atherosclerosis and downregulation of multiple inflammatory, immune response and profibrotic pathways.

Sex differences in the expression of metabolic and pro-inflammatory markers in the initial post-COVID-19 phase

Abstract Cardiovascular complications are a common manifestation of post-acute COVID-19 infection. Post-COVID-19 syndrome is often associated with the female sex and exacerbated inflammation; however, the underlying molecular mechanisms of this sex difference remain poorly understood. In the present study, we aimed to investigate several metabolic, mitochondrial, autophagy, oxidative stress, and inflammatory markers in peripheral blood mononuclear cells (PBMCs) and plasma isolated from older (>75) male and female patients at the initial phase (1-2 weeks) after COVID-19 pneumonia. Age-matched non-infected women and men were used as a control group. We found a significantly higher plasma TNF-α in post-COVID-19 women, but not in men, compared to the control group. Similarly, a significant elevation of IL-1β and IL-18 mRNA in PBMCs from post-COVID-19 women, but not men, was observed. These effects were associated with marked activation of AMPK (defined as pAMPK/AMPK ratio), suggesting potential metabolic stress in female post-COVID-19 PBMCs. Further analysis revealed increased expression of mitochondrial genes, which may be due to AMPK activation as a compensatory reaction to metabolic stress. Analysis of autophagy or oxidative stress (serum 8-OHdG) markers did not reveal any differences between the groups investigated. We conclude that women in the initial phase of post-COVID-19 have elevated expression of inflammatory markers in plasma and in immune cells, which is associated with metabolic stress. This may contribute to the cardiovascular complications in female post-COVID-19 patients.

Successful eculizumab treatment as an adjunctive therapy to desensitization in ABO-incompatible living donor kidney transplantation and its molecular phenotypes

IntroductionABO-incompatible (ABOi) kidney transplantation (KT) has become an important option to overcome organ shortage. Plasmapheresis/rituximab-based desensitization therapy has successfully reduced anti-ABO antibody levels and suppressed antibody-mediated rejection (AMR) in ABOi KT. However, high titers of anti-ABO antibodies in some patients are refractory to standard desensitization, leading to loss of KT opportunities or AMR.MethodsEculizumab treatment was used an adjunctive therapy to rescue high-titer ABOi KT patients refractory to plasmapheresis/rituximab-based desensitization. Molecular phenotypes of allograft biopsies and cellular phenotypes of peripheral blood mononuclear cells of eculizumab group were compared with those of control groups using the Banff Human Organ Transplant gene panel and flow-cytometric analysis, respectively.ResultsThe initial titers of anti-ABO antibodies in the two patients were 1:512 and >1:1024; the final pre-transplant titers after desensitization were 1:128 and 1:64. Both patients received eculizumab from KT day to two or four weeks post-KT and maintained stable renal function up to one-year post-transplantation without overt infection, despite early episodes of probable AMR or borderline T cell-mediated rejection. Molecular phenotype analysis revealed that gene expression patterns in the ABOi KT with eculizumab group overlapped with those in the ABOi KT with AMR group more than in the ABOi KT without AMR group, except for complement pathway-related gene expression. Anti-ABO antibody titers decreased to low levels 1–3 months post-transplant in the eculizumab group in parallel with decreasing anti-B-specific B cells.ConclusionsShort-term eculizumab therapy is promising for rescuing ABOi KT recipients with high anti-ABO antibody titers refractory to plasmapheresis-based desensitization therapy.

Changes in blood metabolomes as potential biomarkers for severity and prognosis in doxorubicin-induced heart failure: a study in HER2-positive and HER2-negative breast cancer patients

Abstract Background The use of doxorubicin for breast cancer treatment is often limited due to cardiotoxicity. Doxorubicin was shown to cause the alterations of cardiac metabolome levels in doxorubicin-treated rats. Interestingly, these changes were robustly associated with the development of doxorubicin-induced heart failure. Unfortunately, measurements of cardiac metabolome levels are rarely possible in humans. Hence, non-invasive biomarkers as representatives of doxorubicin-induced heart failure are required in clinical research and practice. Human epidermal growth factor receptor 2 (HER2) is a point of interest in breast cancer treatment. HER2 is associated with tumor aggressiveness. However, it was observed that HER2 protected against systemic oxidative stress and dilated cardiomyopathy. Therefore, we hypothesized that the alterations of blood metabolome levels and cardiac functions following doxorubicin treatment were different between HER2-positive and HER2-negative breast cancer patients. Purpose We determined the potential roles of changes in blood metabolomes as severity and prognostic markers of doxorubicin-induced heart failure. In addition, the changes in blood metabolomes and cardiac parameters following doxorubicin treatment in HER2-positive and HER2-negative breast cancer patients were compared. Methods HER2-positive (n=37) and HER2-negative (n=37) breast cancer patients were enrolled. Echocardiography, heart rate variability, and blood collection were performed at baseline and 2 weeks after completion of doxorubicin treatment in all patients, as well as at 3 months after completion of doxorubicin treatment in HER2-negative breast cancer patients. Blood obtained at all 3 time points was processed for measuring cardiac injury markers. Blood obtained at baseline and 2 weeks after completion of doxorubicin treatment was also processed for measuring oxidative stress in peripheral blood mononuclear cells using flow cytometry and 85 metabolome levels in plasma using mass spectrometry-based targeted metabolomics. Results Cardiac injury and systolic dysfunction at 2 weeks after completion of doxorubicin treatment were comparable between HER2-positive and HER2-negative breast cancer patients. However, only HER2-negative breast cancer patients exhibited increased systemic oxidative stress and cardiac autonomic dysfunction at this time point. Moreover, 33 and 29 blood metabolomes were altered at 2 weeks after completion of doxorubicin treatment in HER2-positive and HER2-negative breast cancer patients, respectively (Figure). Interestingly, the changes in most of these metabolomes were correlated with the changes in cardiac parameters, both at 2 weeks and 3 months after completion of doxorubicin treatment (Figure). Conclusions The changes in blood metabolomes following doxorubicin treatment were dependent on HER2 status, which might be served as severity and prognostic markers of doxorubicin-induced heart failure.

Fluconazole worsened lung inflammation, partly through lung microbiome dysbiosis in mice with ovalbumin-induced asthma

Innate immunity in asthma may be influenced by alterations in lung microbiota, potentially affecting disease severity. This study investigates the differences in lung inflammation and microbiome between asthma-ovalbumin (OVA) administered with and without fluconazole treatment in C57BL/6 mice. Additionally, the role of inflammation was examined in an in vitro study using a pulmonary cell line. At 30 days post-OVA administration, allergic asthma mice exhibited increased levels of IgE and IL-4 in serum and lung tissue, higher pathological scores, and elevated eosinophils in bronchoalveolar lavage fluid (BALF) compared to control mice. Asthma inflammation was characterized by elevated serum IL-6, increased lung cytokines (TNF-α, IL-6, IL-10), and higher fungal abundance confirmed by polymerase chain reaction (PCR). Fluconazole-treated asthma mice displayed higher levels of cytokines in serum and lung tissue (TNF-α and IL-6), increased pathological scores, and a higher number of mononuclear cells in BALF, with undetectable fungal levels compared to untreated mice. Lung microbiome analysis revealed similarities between control and asthma mice; however, fluconazole-treated asthma mice exhibited higher Bacteroidota levels, lower Firmicutes, and reduced bacterial abundance. Pro-inflammatory cytokine production was increased in supernatants of the pulmonary cell line (NCI-H292) after co-stimulation with LPS and beta-glucan (BG) compared to LPS alone. Fluconazole treatment in OVA-induced asthma mice exacerbated inflammation, partially due to fungi and Gram-negative bacteria, as demonstrated by LPS+BG-activated pulmonary cells. Therefore, fluconazole should be reserved for treating fungal asthma rather than asthma caused by other etiologies.

Selenopolysaccharide Isolated from Lentinula edodes Mycelium Affects Human T-Cell Function

Lentinula edodes polysaccharides are natural immunomodulators. SeLe30, analyzed in this study, is a new mixture of selenium-enriched linear 1,4-α-glucans and 1,3-β- and 1,6-β-glucans isolated from L. edodes mycelium. In the present study, we evaluated its immunomodulatory properties in human T cells. Peripheral blood mononuclear cells (PBMCs) and T cells were isolated from healthy donors’ buffy coats. The effects of SeLe30 on CD25, CD366, and CD279 expression, the subsets of CD8+ T cells, and IFN-γ, IL-6, and TNF-α production were analyzed. SeLe30 downregulated CD25, CD279, and CD366 expression on T cells stimulated by the anti-CD3 antibody (Ab) and upregulated in unstimulated and anti-CD3/CD28-Abs-stimulated T cells. It increased the percentage of central memory CD8+ T cells in unstimulated PBMCs and naïve and central memory T cells in anti-CD3-Ab-stimulated PBMCs. SeLe30 decreased the number of central memory and naïve CD8+ T cells in anti-CD3/CD28-stimulated T cells, whereas, in PBMCs, it reduced the percentage of effector memory CD8+ T cells. Moreover, SeLe30 upregulated cytokine production. SeLe30 exhibits context-dependent effects on T cells. It acts on unstimulated T cells, affecting their activation while increasing the expression of immune checkpoints, which sensitizes them to inhibitory signals that can silence this activation. In the case of a lack of costimulation, SeLe30 exhibits an inhibitory effect, reducing T-cell activation. In cells stimulated by dual signals, its effect is further enhanced, again increasing the “safety brake” of CD366 and CD279. However, the final SeLe30 effect is mediated by its indirect impacts by altering interactions with other immune cells.

SNORD3A: a new possible circulating biomarker of human heart failure

Abstract Background Heart failure (HF) is a complex clinical syndrome and the leading cause of mortality, morbidity and hospitalization in industrialized countries. Human cardiac biopsies are invaluable resources for identifying cardiac signaling pathways, however blood fractions and peripheral blood mononuclear cells (PBMCs) could be used as a source of surrogate biomarkers of signaling pathway activation in human heart failure. Purpose In the present study we aimed to identify novel circulating biomarkers mirroring human myocardial signaling in HF and to study the role played by SNORD3A in cardiomyocyte survival and death. Methods Cardiac left ventricle samples and PBLs from 8 HF patients undergoing heart transplantation and 2 control patients (CTRL) were analyzed by RNA sequencing (RNASeq) to identify transcripts that were regulated in both hearts and PBLs. Five identified transcripts, KCNQOT1, MIAT, SCRN1, MALAT1 and SNORD3A, were then validated by quantitative real-time PCR in PBMCs and in LVs. Next, we analyzed the expression of the Small Nucleolar RNA (snoRNA) SNORD3A in LVs and PBMCs from 8-week-old wild type C57BL/6 mice with pressure overload-induced HF by transverse aortic constriction (TAC). Sham-operated mice (sham) were used as controls. To further investigate the role of SNORD3A in cardiomyocyte function and survival, SNORD3A antisense oligonucleotides (SNOaso) and scramble control sequences (GFPaso) were synthesized and tested in cardiomyoblasts H9C2 cells under normoxic or hypoxic conditions to evaluate SNORD3A transcription levels, cell death and protein synthesis. Results SNORD3A expression was significantly increased in both LVs and PBMCs from HF patients compared to control subjects. Consistent with these results, SNORD3A levels were increased both in PBMCs and LVs from TAC mice compared to sham. In vitro hypoxia significantly increased SNORD3A expression levels in H9C2 cells, compared to normoxia. After SNOaso transfection, SNORD3A transcripts were downregulated, and they were further reduced following 3-hour-hypoxia. Depletion of SNORD3A levels increased cardiomyocyte death and reduced protein synthesis in H9C2 cells. Conclusions Our data suggest that SNORD3A might be involved in the regulation of cardiomyocyte survival in HF and could be useful for diagnostic and prognostic applications in HF.

Drug eluting stents trigger the secretion of inflammatory proteins in an experimental in vitro study

Abstract Introduction Ischemic cardiovascular diseases commonly appear following the occlusion of one or more of the coronary arteries. Drug eluting stents (DES) constitute the gold standard to help to keep open the arteries and maintain the blood flow. After their placement into the coronary arteries, they stay in direct contact with the blood stream, disturbing the physiological conditions and interacting with several cells and proteins within the surrounding blood and vessel wall. Besides the beneficial effects of DES, their clinical application is also associated with complications, such as (late) stent thromboses. Previous studies have shown that thrombosis and inflammation are tightly linked with each other by activating inflammatory cells and secreting cytokines and chemokines to further promote an inflammatory surrounding. However, the influence of DES and their components on the inflammatory cascade and their corresponding players was not investigated yet. Methods To investigate the inflammatory secretome in response to DES, we performed an in vitro study with isolated human peripheral blood mononuclear cells (PBMCs). Therefore, PBMCs were isolated from the whole blood of four healthy volunteers. In total, 63 stents were used in the study, including Medtronic Resolute Onyx (n=37), Abbott XIENCE Sierra (n=5), Boston Scientific SYNERGY (n=4), Terumo Ultimaster (n=3), and Biotronik Orsiro (n=14). We measured the secretion of cytokines and chemokines, including IL-1β, IL-6, TNF-α, IL-8, and IL-12, as well as ICAM-1, VCAM-1, and MCP-1 by commercially available enzyme-linked immunosorbent assay (ELISA). Results Our results showed that DES significantly increase the secretion of the cytokines IL-1β (p=0.0005), and IL-6 (p=0.0002), as well as the chemokine IL-8 (p=0.0010). Additionally, we also detected an elevation of the reaction product ICAM-1 (p=0.0003). Further analyses resulted in a reduced secretion of IL-1β, IL-6, TNF-α, IL-8, and ICAM-1 into the cells’ supernatants in response to Resolute Onyx stents, in comparison to the other stents. Moreover, multiple regression analysis revealed a high dependency of the inflammatory reaction on the elution drug, the polymer type and the stent’s length. Additionally, the material of the stent platform, as well as the polymer thickness further have impact on the cytokine and chemokine response of PBMCs. Conclusion Our study indicate that DES by themselves trigger an inflammatory reaction of blood cells by the secretion of cytokines and chemokines. Furthermore, we provided first insights into the composition of the inflammosome in response to DES. A triggered inflammation may increase the vulnerability for complications and the failure of the DES. As these complications were already observed in clinical studies, our results would potentially provide predictions about the application of certain DES designs and may help to choose the appropriate stents in the clinical setting.

Human coronary blood single-cell sequencing combined with exosome proteomic profiling reveals the molecular mechanism of recurrent in-stent restenosis (R-ISR)

Abstract Background Percutaneous coronary intervention (PCI) is recognized as the core treatment for symptomatic myocardial ischemia and acute coronary syndrome (ACS). However, the complex pathogenesis of recurrent in-stent restenosis (R-ISR) after stent implantation is still insufficiently understood. Purpose This study reports the first single-cell RNA sequencing and proteomic profiling to characterize pathogenesis of R-ISR in humans. Methods To reveal the pathophysiology and molecular mechanism of R-ISR, we performed single-cell RNA sequencing to identify RISR-induced changes in transcriptional landscape in coronary blood mononuclear cell composition. Besides, proteomic profiling of human coronary plasma-derived exosomes was used to further screen different proteins. Transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA) and western blot analysis were performed to identify exosomes. In vitro cell biology experiments were used to validate the discoveries of bioinformatics analysis. Results Compared with normal subjects, the percentage of FCGR3B high monocytes, memory T cells and plasmacytoid dendritic cells (DCs) was increased in R-ISR. Furthermore, we observed a marked increase in the expression of JUN, FOS, CXCL8, S100A8 enriched in the IL-17 signaling pathway that is known to play a crucial role in activating inflammation, mediating immune response, and even maintaining inflammation to chronicity. According to in vitro experiments, the co-cultured system demonstrated that FCGR3B high monocytes isolated from RISR patients activated the expression of inflammatory factors in endothelial cells (ECs), such as IL-1β, IL-6 and TNF-α. Inflammatory activated endothelial cells could release a large amount of exosomes, proteomic profiling of exosomes isolated from ECs observed a significant upregulation of MYLK expression, which could promote proliferation and migration of vascular smooth muscle cells (VSMCs) by activating VEGFR-2 signaling pathway. Conclusions Our study reveals a novel subset of FCGR3B high monocytes in R-ISR patients that could activate ECs inflammation via IL-17 signaling pathway. Activated ECs release exosomes rich in MYLK, which promote proliferation and migration of VSMCs, ultimately leading to repeated restenosis.

Gut microbiota predict retinopathy in patients with diabetes: A longitudinal cohort study

The gut microbiota has emerged as an independent risk factor for diabetes and its complications. This research aimed to delve into the intricate relationship between the gut microbiome and diabetic retinopathy (DR) through a dual approach of cross-sectional and prospective cohort studies. In our cross-sectional study cross-sectional investigation involving ninety-nine individuals with diabetes, distinct microbial signatures associated with DR were identified. Specifically, gut microbiome profiling revealed decreased levels of Butyricicoccus and Ruminococcus torques group, alongside upregulated methanogenesis pathways among DR patients. These individuals concurrently exhibited lower concentrations of short-chain fatty acids in their plasma. Leveraging machine learning models, including random forest classifiers, we constructed a panel of microbial genera and genes that robustly differentiated DR cases. Importantly, these genera also demonstrated significant correlations with dietary patterns and the molecular profiles of peripheral blood mononuclear cells. Building upon these findings, our prospective cohort study followed 62 diabetes patients over a 2-year period to assess the predictive value of these microbial markers. The results underlined the panel’s efficacy in predicting DR incidence. By stratifying patients based on the predictive genera and metabolites identified in the cross-sectional phase, we established significant associations between reduced levels of Butyricicoccus, plasma acetate, and increased susceptibility to DR. This investigation not only deepens our understanding of how gut microbiota influences DR but also underscores the potential of microbial markers as early indicators of disease risk. These insights hold promise for developing targeted interventions aimed at mitigating the impact of diabetic complications.Key points• Microbial signatures are differed in diabetic patients with and without retinopathy• DR-related taxa are linked to dietary habits and transcriptomic profiles• Lower abundances of Butyricicoccus and acetate were prospectively associated with DR

Immunomodulatory effects of cysteamine and its potential use as a host-directed therapy for tuberculosis

ObjectiveCysteamine, a drug approved to treat cystinosis, has been proposed as a host-directed therapy for M. tuberculosis (Mtb) and SARS-CoV-2. The impact of cysteamine on the immune responses has not been fully investigated. We aimed to in vitro evaluate the immunomodulatory effects of cysteamine on peripheral blood mononuclear cells (PBMCs) using the purified protein derivative (PPD) as a recall antigen, and an unspecific stimulus as staphylococcal enterotoxin B (SEB).MethodsPBMCs isolated from subjects with tuberculosis infection (TBI), those with tuberculosis disease (TB), and healthy controls (HC) were in vitro stimulated with PPD or SEB and treated or not with cysteamine at different concentrations (50 µM–400 µM) for 6 hours (h) and 24 h. We evaluated the T helper1 (Th1) and T cytotoxic1 (Tc1) cell cytokine production by flow cytometry and immune-enzymatic assays. In HC, we also evaluated apoptosis and/or necrosis by flow cytometry.ResultsWe observed an immunomodulatory effect of cysteamine at 400 µM in PBMCs from TB and TBI subjects. It significantly reduced PPD-specific Th1 responses at 24 h and at 6 h (p=0.0004 and p=0.0009, respectively), and a similar non-significant trend was observed with cysteamine at 200 µM (p=0.06 at 24 h and p=0.14 at 6 h). Moreover, cysteamine at both 400 µM (p<0.0001 and p=0.0187 at 24 h, respectively, and p<0.0001 at 6 h for both) and 200 µM (p=0.0119 and p=0.0028 at 24 h and p=0.0028 and p=0.0003 at 6 h, respectively) significantly reduced SEB-induced Th1 and Tc1 responses. Furthermore, we found that cysteamine induced morphological lymphocyte changes and significantly reduced the lymphocyte percentage in a dose- and time-dependent manner. Cysteamine at 400 µM induced 8% late apoptosis and 1.6% necrosis (p<0.05) at 24 h. In contrast, despite significant differences from untreated conditions (p<0.05), cysteamine at 400 µM for 6 h induced approximately 1% late apoptosis and 0.1% necrosis in the cells.ConclusionsHigh doses of cysteamine in vitro reduce the percentages of PPD- and SEB-induced Th1 and Tc1 cells and induce late apoptosis and necrosis. Differently, cysteamine at lower doses retains the immunomodulatory effect without affecting cell viability. These findings suggest cysteamine as a potential adjunct to antimicrobial regimens as in the TB or COVID-19 field, for its ability to reduce the inflammatory status.

OMIP-109: 45-color full spectrum flow cytometry panel for deep immunophenotyping of the major lineages present in human peripheral blood mononuclear cells with emphasis on the T cell memory compartment.

The need for more in-depth exploration of the human immune system has moved the flow cytometry field forward with advances in instrumentation, reagent development and availability, and user-friendly implementation of data analysis methods. We developed a high-quality human 45-color panel, for comprehensive characterization of major cell lineages present in circulation including T cells, γδ T cells, NKT-like cells, B cells, NK cells, monocytes, basophils, dendritic cells, and ILCs. Assay optimization steps are described in detail to ensure that each marker in the panel was optimally resolved. In addition, we highlight the outstanding discernment of cell activation, exhaustion, memory, and differentiation states of CD4+ and CD8+ T cells using this 45-color panel. The panel enabled an in-depth description of very distinct phenotypes associated with the complexity of the T cell memory response. Furthermore, we present how this panel can be effectively used for cell sorting on instruments with a similar optical layout to achieve the same level of resolution. Functional evaluation of sorted specific rare cell subsets demonstrated significantly different patterns of immunological responses to stimulation, supporting functional and phenotypic differences within the T cell memory subsets. In summary, the combination of full spectrum profiling technology and careful assay design and optimization results in a high resolution multiparametric 45-color assay. This panel offers the opportunity to fully characterize immunological profiles present in peripheral blood in the context of infectious diseases, autoimmunity, neurodegeneration, immunotherapy, and biomarker discovery.

HIV DNA Levels in Persons Who Acquired HIV in the Setting of Long-Acting Cabotegravir for HIV Prevention: Analysis of Cases from HPTN 083 and 084.

We evaluated HIV DNA levels in individuals who received long-acting cabotegravir (CAB-LA) or tenofovir disoproxil fumarate/emtricitabine (TDF/FTC) pre-exposure prophylaxis in the HPTN 083 and 084 trials and had HIV DNA testing performed to help determine HIV status. HIV DNA testing was performed using peripheral blood mononuclear cell (PBMC) samples collected after a reactive HIV test was obtained at a study site. DNA was quantified using droplet digital PCR (lower limit of detection [LLOD]: 4.09 copies/million PBMCs). Final HIV status and the timing of the first HIV-positive visit were determined by an independent adjudication committee based on HIV test results from real-time site testing and retrospective testing at a centralized laboratory. HIV DNA testing was performed for 133 participants [21 HIV-positive (7 CAB-LA arm, 14 TDF/FTC arm) and 112 HIV-negative; 1-6 tests/person]. For persons with HIV, the time between the first HIV-positive visit and collection of the first sample for DNA testing was a median of 81 days for those receiving CAB-LA (range 41-246) and 11 days for those receiving TDF/FTC (range 3-127). Four (57.1%) of the seven CAB-LA cases with infection had a low initial DNA result [three detected <LLOD; one near the LLOD (4.2 copies/106 PBMCs); in 2/4 cases, the DNA level was still <10 copies/106 PBMCs ≥40 weeks after the first HIV-positive visit. In contrast, only 3/14 (21.4%) of the TDF/FTC cases had a low or negative initial DNA test result (one not detected; two <10 copies/106 PBMCs). In this study, the time between the first HIV-positive visit and the first DNA test was longer in the CAB-LA cases than the TDF/FTC cases. Despite this difference, low or undetectable DNA levels were more frequently observed in the CAB-LA cases. This suggests that CAB-LA exposure may limit seeding of the HIV reservoir in early infection.

Deciphering the Multifaceted Immune Landscape of Unresectable Primary Liver Cancer to Predict Immunotherapy Response.

Immunotherapies employing PD-1/PD-L1 immune checkpoint inhibitors (ICIs) are vital for primary liver cancer (PLC), but response rates remain unsatisfying. Accurate differentiation of responders from non-responders to immunotherapy is imperative. Here, single-cell-scaled mass cytometry analysis on sequential peripheral blood mononuclear cells (PBMCs) from ICI-treated PLC patients is conducted, and tissue residence of immune subpopulations is assessed via multiplex immunohistochemistry. In the discovery cohort (n = 24), responders have lower baseline B cell and HLA-DR+CD8+T cell, and higher CD14+CD16- classical monocyte (CM) proportions. CMs decrease more in responders PBMCs, while HLA-DR+CD8+T cells conformably amplify after ICI-exposure. Responsive individuals display upregulated exhaustion and activation markers in peripheral immune lineages. In the expanded cohort of 77 patients, the augment of the B cells in non-responders is re-confirmed. Responders demonstrate much higher enrichment of B cells or tertiary lymphoid structures in tumor compared to non-responders. A prospective model that excelled in early discrimination of responders is developed using generalized linear model and achieves a satisfactory AUC over 0.9 in all three independent cohorts. Integratedly, the study unveils dynamic immune landscapes in PLC patients undergoing ICI-based therapy, aiding in PLC patient stratification for ICI-based treatment and fostering new response monitoring strategies.