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Cytometry. Part a : the Journal of the... Nov 2019We developed this comprehensive 28-color flow cytometry panel with the aim to measure a variety of T cell effector functions in combination with T cell differentiation...
We developed this comprehensive 28-color flow cytometry panel with the aim to measure a variety of T cell effector functions in combination with T cell differentiation markers (CCR7, CD27, CD28, CD45RO, CD95) in γδ T cells and CD4 and CD8 αβ T cells (Table 1). The effector functions measured in this panel include activation and co-stimulatory molecules (CD69, CD137, and CD154), cytokines (IL-2, IL-13, IL-17A, IL-21, IL-22, TNF, and IFNγ), the chemokine IL-8, cytotoxic molecules (perforin and granzyme B), and the degranulation marker CD107a. In addition, Ki67 enables the identification and analysis of recently activated T cells. To characterize regulatory T cells (T ), we included CD25, CD39, and the canonical T transcription factor FoxP3. We developed and optimized this panel for cryopreserved human peripheral blood mononuclear cells (PBMC) and stimulation with phorbol 12-myristate 13-acetate (PMA) and ionomycin. However, we successfully tested other types of stimulation such as staphylococcus enterotoxin B (SEB) or a mix of immunodominant peptides (CEF peptide pool) from cytomegalovirus (CMV), Epstein-Barr virus (EBV) and influenza. Published 2019. This article is a U.S. Government work and is in the public domain in the USA.
Topics: Antigens, CD; Antigens, Differentiation, T-Lymphocyte; CD28 Antigens; CD40 Ligand; Cytokines; Flow Cytometry; Forkhead Transcription Factors; Granzymes; Humans; Immunophenotyping; Interleukin-8; Ki-67 Antigen; Lectins, C-Type; Leukocyte Common Antigens; Leukocytes, Mononuclear; Lymphocyte Activation; Lysosomal-Associated Membrane Protein 1; Perforin; Receptors, CCR7; T-Lymphocytes; T-Lymphocytes, Regulatory; Tumor Necrosis Factor Receptor Superfamily, Member 7; Tumor Necrosis Factor Receptor Superfamily, Member 9; fas Receptor
PubMed: 31334913
DOI: 10.1002/cyto.a.23853 -
Journal of Hepatology Jan 2022Patients with acute-on-chronic liver failure (ACLF) present a systemic hyperinflammatory response associated with increased circulating levels of small-molecule...
BACKGROUND & AIMS
Patients with acute-on-chronic liver failure (ACLF) present a systemic hyperinflammatory response associated with increased circulating levels of small-molecule metabolites. To investigate whether these alterations reflect inadequate cell energy output, we assessed mitochondrial morphology and central metabolic pathways with emphasis on the tricarboxylic acid (TCA) cycle in peripheral leukocytes from patients with acutely decompensated (AD) cirrhosis, with and without ACLF.
METHODS
The study included samples from patients with AD cirrhosis (108 without and 128 with ACLF) and 41 healthy individuals. Leukocyte mitochondrial ultrastructure was visualized by transmission electron microscopy and cytosolic and mitochondrial metabolic fluxes were determined by assessing NADH/FADH production from various substrates. Plasma GDF15 and FGF21 were determined by Luminex and acylcarnitines by LC-MS/MS. Gene expression was analyzed by RNA-sequencing and PCR-based glucose metabolism profiler array.
RESULTS
Mitochondrial ultrastructure in patients with advanced cirrhosis was distinguished by cristae rarefication and swelling. The number of mitochondria per leukocyte was higher in patients, accompanied by a reduction in their size. Increased FGF21 and C6:0- and C8:0-carnitine predicted mortality whereas GDF15 strongly correlated with a gene set signature related to leukocyte activation. Metabolic flux analyses revealed increased energy production in mononuclear leukocytes from patients with preferential involvement of extra-mitochondrial pathways, supported by upregulated expression of genes encoding enzymes of the glycolytic and pentose phosphate pathways. In patients with ACLF, mitochondrial function analysis uncovered break-points in the TCA cycle at the isocitrate dehydrogenase and succinate dehydrogenase level, which were bridged by anaplerotic reactions involving glutaminolysis and nucleoside metabolism.
CONCLUSIONS
Our findings provide evidence at the cellular, organelle and biochemical levels that severe mitochondrial dysfunction governs immunometabolism in leukocytes from patients with AD cirrhosis and ACLF.
LAY SUMMARY
Patients at advanced stages of liver disease have dismal prognosis due to vital organ failures and the lack of treatment options. In this study, we report that the functioning of mitochondria, which are known as the cell powerhouse, is severely impaired in leukocytes of these patients, probably as a consequence of intense inflammation. Mitochondrial dysfunction is therefore a hallmark of advanced liver disease.
Topics: Acute-On-Chronic Liver Failure; Humans; Immunologic Factors; Leukocytes; Leukocytes, Mononuclear; Mitochondrial Diseases; Tandem Mass Spectrometry
PubMed: 34450236
DOI: 10.1016/j.jhep.2021.08.009 -
Nature Genetics Apr 2018Genome-wide association studies have identified thousands of genetic variants that are associated with disease . Most of these variants have small effect sizes, but...
Genome-wide association studies have identified thousands of genetic variants that are associated with disease . Most of these variants have small effect sizes, but their downstream expression effects, so-called expression quantitative trait loci (eQTLs), are often large and celltype-specific. To identify these celltype-specific eQTLs using an unbiased approach, we used single-cell RNA sequencing to generate expression profiles of ~25,000 peripheral blood mononuclear cells from 45 donors. We identified previously reported cis-eQTLs, but also identified new celltype-specific cis-eQTLs. Finally, we generated personalized co-expression networks and identified genetic variants that significantly alter co-expression relationships (which we termed 'co-expression QTLs'). Single-cell eQTL analysis thus allows for the identification of genetic variants that impact regulatory networks.
Topics: Epistasis, Genetic; Gene Regulatory Networks; Genetic Variation; Genome-Wide Association Study; Humans; Leukocytes, Mononuclear; Quantitative Trait Loci; Sequence Analysis, RNA; Single-Cell Analysis; Transcriptome
PubMed: 29610479
DOI: 10.1038/s41588-018-0089-9 -
Nature Medicine Feb 2020Drug-induced hypersensitivity syndrome/drug reaction with eosinophilia and systemic symptoms (DiHS/DRESS) is a potentially fatal multiorgan inflammatory disease...
Drug-induced hypersensitivity syndrome/drug reaction with eosinophilia and systemic symptoms (DiHS/DRESS) is a potentially fatal multiorgan inflammatory disease associated with herpesvirus reactivation and subsequent onset of autoimmune diseases. Pathophysiology remains elusive and therapeutic options are limited. Cases refractory to corticosteroid therapy pose a clinical challenge and approximately 30% of patients with DiHS/DRESS develop complications, including infections and inflammatory and autoimmune diseases. Progress in single-cell RNA sequencing (scRNA-seq) provides an opportunity to dissect human disease pathophysiology at unprecedented resolutions, particularly in diseases lacking animal models, such as DiHS/DRESS. We performed scRNA-seq on skin and blood from a patient with refractory DiHS/DRESS, identifying the JAK-STAT signaling pathway as a potential target. We further showed that central memory CD4 T cells were enriched with DNA from human herpesvirus 6b. Intervention via tofacitinib enabled disease control and tapering of other immunosuppressive agents. Tofacitinib, as well as antiviral agents, suppressed culprit-induced T cell proliferation in vitro, further supporting the roles of the JAK-STAT pathway and herpesviruses in mediating the adverse drug reaction. Thus, scRNA-seq analyses guided successful therapeutic intervention in the patient with refractory DiHS/DRESS. scRNA-seq may improve our understanding of complicated human disease pathophysiology and provide an alternative approach in personalized medicine.
Topics: Adrenal Cortex Hormones; Adult; Antiviral Agents; Autoimmune Diseases; CD4-Positive T-Lymphocytes; Cell Proliferation; Cell Separation; Drug Hypersensitivity Syndrome; Flow Cytometry; Herpesvirus 6, Human; Humans; Immunosuppressive Agents; Leukocytes, Mononuclear; Lymphocytes; Male; Piperidines; Pyrimidines; Pyrroles; RNA-Seq; Signal Transduction; Single-Cell Analysis; T-Lymphocytes, Regulatory; Transcriptome; VDJ Recombinases
PubMed: 31959990
DOI: 10.1038/s41591-019-0733-7 -
Arthritis Research & Therapy Aug 2019The in vitro pharmacology of baricitinib, upadacitinib, and tofacitinib was evaluated to understand differences among these JAK inhibitors (JAKis) at the cellular level. (Comparative Study)
Comparative Study
BACKGROUND
The in vitro pharmacology of baricitinib, upadacitinib, and tofacitinib was evaluated to understand differences among these JAK inhibitors (JAKis) at the cellular level.
METHODS
Peripheral blood mononuclear cells from healthy donors were incubated with different JAKis, levels of phosphorylated signal transducer and activator of transcription (pSTAT) were measured following cytokine stimulation, and half maximum inhibitory concentration (IC) values were calculated in phenotypically gated leukocyte subpopulations. Therapeutic dose relevance of the in vitro analysis was assessed using calculated mean concentration-time profiles over 24 h obtained from JAKi-treated subjects. Time above IC and average daily percent inhibition of pSTAT formation were calculated for each JAKi, cytokine, and cell type.
RESULTS
Distinct JAKis displayed different in vitro pharmacologic profiles. For example, tofacitinib and upadacitinib were the most potent inhibitors of the JAK1/3-dependent cytokines tested (interleukin [IL]-2, IL-4, IL-15, and IL-21) with lower IC values and increased time above IC translating to a greater overall inhibition of STAT signaling during the dosing interval. All JAKis tested inhibited JAK1/2-dependent cytokines (e.g., IL-6 and interferon [IFN]-γ), the JAK1/tyrosine kinase 2 (TYK2)-dependent cytokines IL-10 and IFN-α, the JAK2/2-dependent cytokines IL-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF), and the JAK2/TYK2-dependent cytokine granulocyte colony-stimulating factor (G-CSF), but often to significantly differing degrees.
CONCLUSIONS
Different JAKis modulated distinct cytokine pathways to varying degrees, and no agent potently or continuously inhibited an individual cytokine signaling pathway throughout the dosing interval. Notably, baricitinib inhibited JAK1/3 signaling to a lesser extent than upadacitinib and tofacitinib, while upadacitinib, baricitinib, and tofacitinib inhibited the signaling of JAK2/2-dependent cytokines, including GM-CSF and IL-3, as well as the signaling of the JAK2/TYK2-dependent cytokine G-CSF.
Topics: Arthritis, Rheumatoid; Azetidines; Biomarkers; Cytokines; Flow Cytometry; Heterocyclic Compounds, 3-Ring; Humans; Janus Kinase Inhibitors; Leukocytes, Mononuclear; Piperidines; Protein Kinase Inhibitors; Purines; Pyrazoles; Pyrimidines; Pyrroles; Signal Transduction; Sulfonamides
PubMed: 31375130
DOI: 10.1186/s13075-019-1964-1 -
Chemical & Pharmaceutical Bulletin 2020Over the past decade, a number of new 1,4-naphthoquinones have been isolated from natural sources and new 1,4-naphthoquinones with diverse structural features have been... (Review)
Review
Over the past decade, a number of new 1,4-naphthoquinones have been isolated from natural sources and new 1,4-naphthoquinones with diverse structural features have been synthesized. Cardioprotective, anti-ischemic, hepatoprotective, neuroprotective and some other new properties were found for these compounds; their role in protecting against neurodegenerative diseases has been established. Their anti-inflammatory, antimicrobial and antitumor activities have been studied in more detail; new, previously unknown intracellular molecular targets and mechanisms of action have been discovered. Some compounds of this class are already being used as a medicinal drugs and some substances can be used as biochemical tools and probes for non-invasive detection of pathological areas in cells and tissues in myocardial infarction and neurodegenerative diseases using modern molecular imaging techniques.
Topics: Anti-Infective Agents; Anti-Inflammatory Agents; Bacteria; Cytokines; Humans; Leukocytes, Mononuclear; Naphthoquinones; Oxidative Stress; Protective Agents; Trypanosoma
PubMed: 31902901
DOI: 10.1248/cpb.c19-00911 -
STAR Protocols Mar 2021A growing number of studies show that innate immune cells can undergo functional reprogramming, facilitating a faster and enhanced response to heterologous secondary...
A growing number of studies show that innate immune cells can undergo functional reprogramming, facilitating a faster and enhanced response to heterologous secondary stimuli. This concept has been termed "trained immunity." We outline here a protocol to recapitulate this using adherent monocytes from consecutive isolation of peripheral blood mononuclear cells. The induction of trained immunity and the associated functional reprogramming of monocytes is described in detail using β-glucan (from ) and Bacillus Calmette-Guérin as examples. For complete details on the use and execution of this protocol, please refer to Repnik et al. (2003) and Bekkering et al. (2016).
Topics: Cellular Reprogramming; Cellular Reprogramming Techniques; Cytokines; Humans; Immunity, Innate; Leukocytes, Mononuclear; Monocytes; Mycobacterium bovis; beta-Glucans
PubMed: 33718890
DOI: 10.1016/j.xpro.2021.100365 -
Redox Biology Dec 2022Type 1 diabetes (T1D) involves critical metabolic disturbances that contribute to an increased cardiovascular risk. Leukocytes are key players in the onset of...
Type 1 diabetes (T1D) involves critical metabolic disturbances that contribute to an increased cardiovascular risk. Leukocytes are key players in the onset of atherosclerosis due to their interaction with the endothelium. However, whether mitochondrial redox impairment, altered bioenergetics and abnormal autophagy in leukocytes contribute to T1D physiopathology is unclear. In this study we aimed to evaluate the bioenergetic and redox state of peripheral blood mononuclear cells (PBMCs) from T1D patients in comparison to those from healthy subjects, and to assess autophagy induction and leukocyte-endothelial interactions. T1D patients presented lower levels of fast-acting and total antioxidants in their blood, and their leukocytes produced higher amounts of total reactive oxygen species (ROS) and superoxide radical with respect to controls. Basal and ATP-linked respiration were similar in PBMCs from T1D and controls, but T1D PBMCs exhibited reduced spare respiratory capacity and a tendency toward decreased maximal respiration and reduced non-mitochondrial respiration, compared to controls. The autophagy markers P-AMPK, Beclin-1 and LC3-II/LC3-I were increased, while P62 and NBR1 were decreased in T1D PBMCs versus those from controls. Leukocytes from T1D patients displayed lower rolling velocity, higher rolling flux and more adhesion to the endothelium versus controls. Our findings show that T1D impairs mitochondrial function and promotes oxidative stress and autophagy in leukocytes, and suggest that these mechanisms contribute to an increased risk of atherosclerosis by augmenting leukocyte-endothelial interactions.
Topics: Humans; Leukocytes, Mononuclear; Diabetes Mellitus, Type 1; Diabetes Mellitus, Type 2; Leukocytes; Mitochondria; Autophagy; Oxidation-Reduction; Atherosclerosis
PubMed: 36455476
DOI: 10.1016/j.redox.2022.102551 -
Hypertension (Dallas, Tex. : 1979) Jul 2020Endothelial inflammation and mitochondrial dysfunction have been implicated in cardiovascular diseases, yet, a unifying mechanism tying them together remains limited....
Endothelial inflammation and mitochondrial dysfunction have been implicated in cardiovascular diseases, yet, a unifying mechanism tying them together remains limited. Mitochondrial dysfunction is frequently associated with mitochondrial fission/fragmentation mediated by the GTPase Drp1 (dynamin-related protein 1). Nuclear factor (NF)-κB, a master regulator of inflammation, is implicated in endothelial dysfunction and resultant complications. Here, we explore a causal relationship between mitochondrial fission and NF-κB activation in endothelial inflammatory responses. In cultured endothelial cells, TNF-α (tumor necrosis factor-α) or lipopolysaccharide induces mitochondrial fragmentation. Inhibition of Drp1 activity or expression suppresses mitochondrial fission, NF-κB activation, vascular cell adhesion molecule-1 induction, and leukocyte adhesion induced by these proinflammatory factors. Moreover, attenuations of inflammatory leukocyte adhesion were observed in Drp1 heterodeficient mice as well as endothelial Drp1 silenced mice. Intriguingly, inhibition of the canonical NF-κB signaling suppresses endothelial mitochondrial fission. Mechanistically, NF-κB p65/RelA seems to mediate inflammatory mitochondrial fission in endothelial cells. In addition, the classical anti-inflammatory drug, salicylate, seems to maintain mitochondrial fission/fusion balance against TNF-α via inhibition of NF-κB. In conclusion, our results suggest a previously unknown mechanism whereby the canonical NF-κB cascade and a mitochondrial fission pathway interdependently regulate endothelial inflammation.
Topics: 3T3 Cells; Animals; Aorta; Cell Adhesion; Cells, Cultured; Dynamins; Endothelial Cells; Endothelium, Vascular; Leukocytes, Mononuclear; Membrane Proteins; Mice; Mitochondrial Dynamics; Mitochondrial Proteins; Mutation, Missense; NF-kappa B; Phosphorylation; Phosphoserine; Protein Processing, Post-Translational; Proteome; RNA Interference; RNA, Small Interfering; Rats; Sodium Salicylate; Tumor Necrosis Factor-alpha; Vascular Cell Adhesion Molecule-1; Vasculitis
PubMed: 32389075
DOI: 10.1161/HYPERTENSIONAHA.120.14686 -
Frontiers in Immunology 2018Antibody-mediated rejection (AMR) of solid organ transplants is characterized by the activation and injury of the allograft endothelium. Histological and transcriptomic... (Review)
Review
Antibody-mediated rejection (AMR) of solid organ transplants is characterized by the activation and injury of the allograft endothelium. Histological and transcriptomic studies have associated microvascular inflammation and endothelial lesions with the severity of rejection and poor graft outcomes. The allograft endothelium forms the physical barrier between the donor organ and the recipient; this position directly exposes the endothelium to alloimmune responses. However, endothelial cells are not just victims and can actively participate in the pathogenesis of rejection. In healthy tissues, the endothelium plays a major role in vascular and immune homeostasis. Organ transplantation, however, subjects the endothelium to an environment of inflammation, alloreactive lymphocytes, donor-specific antibodies, and potentially complement activation. As a result, endothelial cells become activated and have modified interactions with the cellular effectors of allograft damage: lymphocytes, natural killer, and myeloid cells. Activated endothelial cells participate in leukocyte adhesion and recruitment, lymphocyte activation and differentiation, as well as the secretion of cytokines and chemokines. Ultimately, highly activated endothelial cells promote pro-inflammatory alloresponses and become accomplices to AMR.
Topics: Animals; Antibodies; Endothelial Cells; Endothelium, Vascular; Graft Rejection; Humans; Leukocytes, Mononuclear
PubMed: 29434607
DOI: 10.3389/fimmu.2018.00106