Mitochondrial phenotypes in purified human immune cell subtypes and cell mixtures

Using a high-throughput mitochondrial phenotyping platform to quantify multiple mitochondrial features among molecularly-defined immune cell subtypes, we quantify the natural variation in citrate synthase, mitochondrial DNA copy number (mtDNAcn), and respiratory chain enzymatic activities in human neutrophils, monocytes, B cells, and naïve and memory T lymphocyte subtypes. In mixed peripheral blood mononuclear cells (PBMCs) from the same individuals, we show to what extent mitochondrial measures are confounded by both cell type distributions and contaminating platelets. Cell subtype-specific measures among women and men spanning 4 decades of life indicate potential age- and sex-related differences, including an age-related elevation in mtDNAcn, which are masked or blunted in mixed PBMCs. Finally, a proof-of-concept, repeated-measures study in a single individual validates cell type differences and also reveals week-to-week changes in mitochondrial activities. Larger studies are required to validate and mechanistically extend these findings. These mitochondrial phenotyping data build upon established immunometabolic differences among leukocyte sub-populations, and provide foundational quantitative knowledge to develop interpretable blood-based assays of mitochondrial health.

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Mitochondrial profiling reveals dynamic, sex- and age-specific mitochondrial phenotypes in human immune cell subtypes

10.1101/2020.10.16.342923 ◽

2020 ◽

Author(s):

Shannon Rausser ◽

Caroline Trumpff ◽

Marlon A McGill ◽

Alex Junker ◽

Wei Wang ◽

...

Keyword(s):

Respiratory Chain ◽

Repeated Measures ◽

Mononuclear Cells ◽

Immune Cell ◽

Cell Type ◽

Mitochondrial Content ◽

Peripheral Blood Mononuclear ◽

Mitochondrial Dna Copy Number ◽

Age Related ◽

Cell Type Specific

AbstractHow mitochondria functionally differ between immune cell subtypes, between the sexes, and with aging has not been defined in humans. We deploy a high-throughput mitochondrial phenotyping platform to define cell-type specific mitochondrial features in human circulating immune cell subtypes. In women and men spanning four decades of life, we find that mitochondrial content, mitochondrial DNA copy number (mtDNAcn), and respiratory chain enzymatic activities vary by up to 3.5-fold between neutrophils, monocytes, B and T lymphocyte subtypes. Mitochondria exhibit specific age- and sex-related differences among individual cell subtypes, including an age-related increase in mtDNAcn. In an intensive repeated-measures study, we also identify remarkable weekly variation in mitochondrial content and respiratory chain function, which is partially correlated with changes in circulating biomarkers. Our results also define multivariate mitochondrial phenotypes – mitotypes – that distinguish lymphoid from myeloid cell types, naïve-to-memory lymphocyte states, and moderately differ between women and men. Finally, a comparison of mitochondrial features in purified cell subtypes and in peripheral blood mononuclear cells (PBMCs) invites caution in using cell type mixtures to infer person-level mitochondrial function. Together, these findings identify dynamic cell-type specific variation in mitochondrial biology among circulating leukocytes and provide foundational knowledge to develop interpretable blood-based assays of mitochondrial health.

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Single cell resolution landscape of equine peripheral blood mononuclear cells reveals diverse immune cell subtypes including T-bet+ B cells

10.1101/2020.05.05.077362 ◽

2020 ◽

Cited By ~ 1

Author(s):

Roosheel S. Patel ◽

Joy E. Tomlinson ◽

Thomas J. Divers ◽

Gerlinde R. Van de Walle ◽

Brad R. Rosenberg

Keyword(s):

B Cells ◽

Immune Cells ◽

Peripheral Blood ◽

Mononuclear Cells ◽

Immune Cell ◽

Model Organisms ◽

Traditional Model ◽

Cell Type ◽

Peripheral Blood Mononuclear ◽

Blood Mononuclear Cells

ABSTRACTTraditional laboratory model organisms represent a small fraction of the diversity of multicellular life, and findings in any given experimental model often do not translate to other species. Immunology research in non-traditional model organisms can be advantageous or even necessary (e.g. for host-pathogen interaction studies), but presents multiple challenges, many stemming from an incomplete understanding of potentially species-specific immune cell types, frequencies and phenotypes. Identifying and characterizing immune cells in such organisms is frequently limited by the availability of species-reactive immunophenotyping reagents for flow cytometry, and insufficient prior knowledge of cell type-defining markers. Here, we demonstrate the utility of single cell RNA sequencing (scRNA-Seq) to characterize immune cells for which traditional experimental tools are limited. Specifically, we used scRNA-Seq to comprehensively define the cellular diversity of equine peripheral blood mononuclear cells (PBMCs) from healthy horses across different breeds, ages, and sexes. We identified 30 cell type clusters partitioned into five major populations: Monocytes/Dendritic Cells, B cells, CD3+PRF1+ lymphocytes, CD3+PRF1- lymphocytes, and Basophils. Comparative analyses revealed many cell populations analogous to human PBMC, including transcriptionally heterogeneous monocytes and distinct dendritic cell subsets (cDC1, cDC2, plasmacytoid DC). Unexpectedly, we found that a majority of the equine peripheral B cell compartment is comprised of T-bet+ B cells; an immune cell subpopulation typically associated with chronic infection and inflammation in human and mouse. Taken together, our results demonstrate the potential of scRNA-Seq for cellular analyses in non-traditional model organisms, and form the basis for an immune cell atlas of horse peripheral blood.

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Sexual-dimorphism in human immune system aging

10.1101/755702 ◽

2019 ◽

Author(s):

Eladio J. Márquez ◽

Cheng-han Chung ◽

Radu Marches ◽

Robert J. Rossi ◽

Djamel Nehar-Belaid ◽

...

Keyword(s):

Immune System ◽

Mononuclear Cells ◽

Immune Cell ◽

Rna Seq ◽

Human Immune System ◽

Peripheral Blood Mononuclear ◽

Cell Functions ◽

Age Related ◽

Male Specific

AbstractDifferences in immune function and responses contribute to health- and life-span disparities between sexes. However, the role of sex in immune system aging is not well understood. Here, we characterize peripheral blood mononuclear cells from 172 healthy adults 22-93 years of age using ATAC-seq, RNA-seq, and flow-cytometry. These data reveal a shared epigenomic signature of aging including declining naïve T cell and increasing monocyte/cytotoxic cell functions. These changes were greater in magnitude in men and accompanied by a male-specific genomic decline in B-cell specific loci. Age-related epigenomic changes first spike around late-thirties with similar timing and magnitude between sexes, whereas the second spike is earlier and stronger in men. Unexpectedly, genomic differences between sexes increase after age 65, with men having higher innate and pro-inflammatory activity and lower adaptive activity. Impact of age and sex on immune cell genomes can be visualized at https://immune-aging.jax.org to provide insights into future studies.

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Binding of Macrophage Receptor MARCO, LDL, and LDLR to Disease-Associated Crystalline Structures

Frontiers in Immunology ◽

10.3389/fimmu.2020.596103 ◽

2020 ◽

Vol 11 ◽

Author(s):

Anika Alberts ◽

Annika Klingberg ◽

Leonie Hoffmeister ◽

Anne Kathrin Wessig ◽

Korbinian Brand ◽

...

Keyword(s):

Cell Activation ◽

Mononuclear Cells ◽

Immune Cell ◽

Serum Proteins ◽

Sterile Inflammation ◽

Human Neutrophils ◽

Ros Production ◽

Peripheral Blood Mononuclear ◽

Cholesterol Crystals ◽

Crystalline Structures

Endogenous and exogenous crystalline structures are involved in various pathologies and diseases in humans by inducing sterile inflammation, mechanical stress, or obstruction of excretory organs. The best studied of these diseases is gout, in which crystallization of uric acid in the form of monosodium urate (MSU) mainly in synovial fluid of the joints leads to sterile inflammation. Though some of these diseases have been described for centuries, little is known about if and how the immune system recognizes the associated crystals. Thus, in this study we aimed at identifying possible recognition molecules of MSU using liquid chromatography-mass spectrometry (LC-MS) analysis of MSU-binding serum proteins. Among the strongest binding proteins, we unexpectedly found two transmembrane receptors, namely macrophage receptor with collagenous structure (MARCO) and low-density lipoprotein (LDL) receptor (LDLR). We show that recombinant versions of both human and mouse MARCO directly bind to unopsonized MSU and several other disease-associated crystals. Recombinant LDLR binds many types of crystals mainly when opsonized with serum proteins. We show that this interaction is predominantly mediated by LDL, which we found to bind to all crystalline structures tested except for cholesterol crystals. However, murine macrophages lacking LDLR expression do neither show altered phagocytosis nor interleukin-1β (IL-1β) production in response to opsonized crystals. Binding of LDL to MSU has previously been shown to inhibit the production of reactive oxygen species (ROS) by human neutrophils. We extend these findings and show that LDL inhibits neutrophil ROS production in response to most crystals tested, even cholesterol crystals. The inhibition of neutrophil ROS production only partly correlated with the inhibition of IL-1β production by peripheral blood mononuclear cells (PBMCs): LDL inhibited IL-1β production in response to large MSU crystals, but not small MSU or silica crystals. This may suggest distinct upstream signals for IL-1β production depending on the size or the shape of the crystals. Together, we identify MARCO and LDLR as potential crystal recognition receptors, and show that LDL binding to diverse disease-associated crystalline structures has variable effects on crystal-induced innate immune cell activation.

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Single-cell resolution landscape of equine peripheral blood mononuclear cells reveals diverse cell types including T-bet+ B cells

BMC Biology ◽

10.1186/s12915-020-00947-5 ◽

2021 ◽

Vol 19 (1) ◽

Author(s):

Roosheel S. Patel ◽

Joy E. Tomlinson ◽

Thomas J. Divers ◽

Gerlinde R. Van de Walle ◽

Brad R. Rosenberg

Keyword(s):

B Cells ◽

Immune Cells ◽

Peripheral Blood ◽

Mononuclear Cells ◽

Immune Cell ◽

Cell Types ◽

Model Organisms ◽

Traditional Model ◽

Cell Type ◽

Peripheral Blood Mononuclear

Abstract Background Traditional laboratory model organisms represent a small fraction of the diversity of multicellular life, and findings in any given experimental model often do not translate to other species. Immunology research in non-traditional model organisms can be advantageous or even necessary, such as when studying host-pathogen interactions. However, such research presents multiple challenges, many stemming from an incomplete understanding of potentially species-specific immune cell types, frequencies, and phenotypes. Identifying and characterizing immune cells in such organisms is frequently limited by the availability of species-reactive immunophenotyping reagents for flow cytometry, and insufficient prior knowledge of cell type-defining markers. Results Here, we demonstrate the utility of single-cell RNA sequencing (scRNA-Seq) to characterize immune cells for which traditional experimental tools are limited. Specifically, we used scRNA-Seq to comprehensively define the cellular diversity of equine peripheral blood mononuclear cells (PBMC) from healthy horses across different breeds, ages, and sexes. We identified 30 cell type clusters partitioned into five major populations: monocytes/dendritic cells, B cells, CD3+PRF1+ lymphocytes, CD3+PRF1− lymphocytes, and basophils. Comparative analyses revealed many cell populations analogous to human PBMC, including transcriptionally heterogeneous monocytes and distinct dendritic cell subsets (cDC1, cDC2, plasmacytoid DC). Remarkably, we found that a majority of the equine peripheral B cell compartment is comprised of T-bet+ B cells, an immune cell subpopulation typically associated with chronic infection and inflammation in human and mouse. Conclusions Taken together, our results demonstrate the potential of scRNA-Seq for cellular analyses in non-traditional model organisms and form the basis for an immune cell atlas of horse peripheral blood.

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Profiles of Peripheral Immune Cells of Uncomplicated COVID-19 Cases with Distinct Viral RNA Shedding Periods

Viruses ◽

10.3390/v13030514 ◽

2021 ◽

Vol 13 (3) ◽

pp. 514

Author(s):

Denise Utami Putri ◽

Cheng-Hui Wang ◽

Po-Chun Tseng ◽

Wen-Sen Lee ◽

Fu-Lun Chen ◽

...

Keyword(s):

Immune Response ◽

Immune Cells ◽

Mononuclear Cells ◽

Immune Cell ◽

Severe Disease ◽

Viral Rna ◽

Disease Course ◽

Peripheral Blood Mononuclear ◽

Peripheral Immune Cells ◽

Cell Profile

The heterogeneity of immune response to COVID-19 has been reported to correlate with disease severity and prognosis. While so, how the immune response progress along the period of viral RNA-shedding (VRS), which determines the infectiousness of disease, is yet to be elucidated. We aim to exhaustively evaluate the peripheral immune cells to expose the interplay of the immune system in uncomplicated COVID-19 cases with different VRS periods and dynamic changes of the immune cell profile in the prolonged cases. We prospectively recruited four uncomplicated COVID-19 patients and four healthy controls (HCs) and evaluated the immune cell profile throughout the disease course. Peripheral blood mononuclear cells (PBMCs) were collected and submitted to a multi-panel flowcytometric assay. CD19+-B cells were upregulated, while CD4, CD8, and NK cells were downregulated in prolonged VRS patients. Additionally, the pro-inflammatory-Th1 population showed downregulation, followed by improvement along the disease course, while the immunoregulatory cells showed upregulation with subsequent decline. COVID-19 patients with longer VRS expressed an immune profile comparable to those with severe disease, although they remained clinically stable. Further studies of immune signature in a larger cohort are warranted.

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Endogenous retrovirus-encoded Syncytin-2 contributes to exosome-mediated immunosuppression of T cells†

Biology of Reproduction ◽

10.1093/biolre/ioz124 ◽

2019 ◽

Cited By ~ 5

Author(s):

Adjimon G Lokossou ◽

Caroline Toudic ◽

Phuong Trang Nguyen ◽

Xavier Elisseeff ◽

Amandine Vargas ◽

...

Keyword(s):

T Cells ◽

T Cell Activation ◽

Map Kinases ◽

Cell Activation ◽

Mononuclear Cells ◽

Immune Cell ◽

Endogenous Retrovirus ◽

Jurkat Cells ◽

Peripheral Blood Mononuclear ◽

Interfering Rna

Abstract Modulation of the activation status of immune cell populations during pregnancy depends on placental villous cytotrophoblast (VCT) cells and the syncytiotrophoblast (STB). Failure in the establishment of this immunoregulatory function leads to pregnancy complications. Our laboratory has been studying Syncytin-2 (Syn-2), an endogenous retroviral protein expressed in placenta and on the surface of placental exosomes. This protein plays an important role not only in STB formation through its fusogenic properties, but also through its immunosuppressive domain (ISD). Considering that Syn-2 expression is importantly reduced in preeclamptic placentas, we were interested in addressing its possible immunoregulatory effects on T cells. Activated Jurkat T cells and peripheral blood mononuclear cells (PBMCs) were treated with monomeric or dimerized version of a control or a Syn-2 ISD peptide. Change in phosphorylation levels of ERK1/2 MAP kinases was selectively noted in Jurkat cells treated with the dimerized ISD peptide. Upon incubation with the dimerized Syn-2 ISD peptide, significant reduction in Th1 cytokine production was further demonstrated by ELISA and Human Th1/Th2 Panel Multi-Analyte Flow Assay. To determine if exosome-associated Syn-2 could also be immunosuppressive placental exosomes were incubated with activated Jurkat and PBMCs. Quantification of Th1 cytokines in the supernatants revealed severe reduction in T cell activation. Interestingly, exosomes from Syn-2-silenced VCT incubated with PBMCs were less suppressive when compared with exosome derived from VCT transfected with control small interfering RNA (siRNA). Our results suggest that Syn-2 is an important immune regulator both locally and systemically, via its association with placental exosomes.

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Anticancer immunity induced by a synthetic tumor-targeted CD137 agonist

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-001762 ◽

2021 ◽

Vol 9 (1) ◽

pp. e001762

Author(s):

Punit Upadhyaya ◽

Johanna Lahdenranta ◽

Kristen Hurov ◽

Sailaja Battula ◽

Rachel Dods ◽

...

Keyword(s):

Mononuclear Cells ◽

Tumor Antigens ◽

Immune Cell ◽

Checkpoint Inhibitors ◽

Human Peripheral Blood ◽

Interleukin 2 ◽

Cancer Therapeutics ◽

Peripheral Blood Mononuclear ◽

Tnf Superfamily

BackgroundIn contrast to immune checkpoint inhibitors, the use of antibodies as agonists of immune costimulatory receptors as cancer therapeutics has largely failed. We sought to address this problem using a new class of modular synthetic drugs, termed tumor-targeted immune cell agonists (TICAs), based on constrained bicyclic peptides (Bicycles).MethodsPhage libraries displaying Bicycles were panned for binders against tumor necrosis factor (TNF) superfamily receptors CD137 and OX40, and tumor antigens EphA2, Nectin-4 and programmed death ligand 1. The CD137 and OX40 Bicycles were chemically conjugated to tumor antigen Bicycles with different linkers and stoichiometric ratios of binders to obtain a library of low molecular weight TICAs (MW <8 kDa). The TICAs were evaluated in a suite of in vitro and in vivo assays to characterize their pharmacology and mechanism of action.ResultsLinking Bicycles against costimulatory receptors (e.g., CD137) to Bicycles against tumor antigens (e.g., EphA2) created potent agonists that activated the receptors selectively in the presence of tumor cells expressing these antigens. An EphA2/CD137 TICA (BCY12491) efficiently costimulated human peripheral blood mononuclear cells in vitro in the presence of EphA2 expressing tumor cell lines as measured by the increased secretion of interferon γ and interleukin-2. Treatment of C57/Bl6 mice transgenic for the human CD137 extracellular domain (huCD137) bearing EphA2-expressing MC38 tumors with BCY12491 resulted in the infiltration of CD8+ T cells, elimination of tumors and generation of immunological memory. BCY12491 was cleared quickly from the circulation (plasma t1/2 in mice of 1–2 hr), yet intermittent dosing proved effective.ConclusionTumor target-dependent CD137 agonism using a novel chemical approach (TICAs) afforded elimination of tumors with only intermittent dosing suggesting potential for a wide therapeutic index in humans. This work unlocks a new path to effective cancer immunotherapy via agonism of TNF superfamily receptors.

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The Effect of Sulforaphane on Glyoxalase I Expression and Activity in Peripheral Blood Mononuclear Cells

Nutrients ◽

10.3390/nu10111773 ◽

2018 ◽

Vol 10 (11) ◽

pp. 1773 ◽

Cited By ~ 6

Author(s):

Michela Alfarano ◽

Donato Pastore ◽

Vincenzo Fogliano ◽

Casper Schalkwijk ◽

Teresa Oliviero

Keyword(s):

Gene Expression ◽

Peripheral Blood Mononuclear Cells ◽

Peripheral Blood ◽

Mononuclear Cells ◽

Limit Of Detection ◽

Peripheral Blood Mononuclear ◽

Potential Health ◽

Age Related ◽

Blood Mononuclear Cells ◽

Expression Activity

Studies demonstrate that the potential health-beneficial effect of sulforaphane (SR), a compound formed in broccoli, is the result of a number of mechanisms including upregulation of phase two detoxification enzymes. Recent studies suggest that SR increases expression/activity of glyoxalase 1 (Glo1), an enzyme involved in the degradation of methylglyoxal, is major precursor of advanced glycation end products. Those compounds are associated with diabetes complications and other age-related diseases. In this study, the effect of SR on the expression/activity of Glo1 in peripheral blood mononuclear cells (PBMCs) from 8 healthy volunteers was investigated. PBMCs were isolated and incubated with SR (2.5 μM-concentration achievable by consuming a broccoli portion) for 24 h and 48 h. Glo1 activity/expression, reduced glutathione (GSH), and glutathione-S-transferase gene expression were measured. Glo1 activity was not affected while after 48 h a slight but significant increase of its gene expression (1.03-fold) was observed. GSTP1 expression slightly increased after 24 h incubation (1.08-fold) while the expressions of isoform GSTT2 and GSTM2 were below the limit of detection. GSH sharply decreased, suggesting the formation of GSH-SR adducts that may have an impact SR availability. Those results suggest that a regular exposure to SR by broccoli consumption or SR supplements may enhance Glo1.

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Effects of 30 min of aerobic exercise on gene expression in human neutrophils

Journal of Applied Physiology ◽

10.1152/japplphysiol.00872.2007 ◽

2008 ◽

Vol 104 (1) ◽

pp. 236-243 ◽

Cited By ~ 71

Author(s):

Shlomit Radom-Aizik ◽

Frank Zaldivar ◽

Szu-Yun Leu ◽

Pietro Galassetti ◽

Dan M. Cooper

Keyword(s):

Gene Expression ◽

Mononuclear Cells ◽

Cycle Ergometer ◽

Human Neutrophils ◽

Peripheral Blood Mononuclear ◽

False Discovery ◽

Ergometer Exercise ◽

Before And After ◽

Ease Score ◽

Repair Genes

Relatively brief bouts of exercise alter gene expression in peripheral blood mononuclear cells (PBMCs), but whether exercise changes gene expression in circulating neutrophils (whose numbers, like PBMCs, increase) is not known. We hypothesized that exercise would activate neutrophil genes involved in apoptosis, inflammation, and cell growth and repair, since these functions in leukocytes are known to be influenced by exercise. Blood was sampled before and immediately after 30 min of constant, heavy (∼80% peak O2uptake) cycle ergometer exercise in 12 healthy men (19–29 yr old) of average fitness. Neutrophils were isolated using density gradients; RNA was hybridized to Affymetrix U133+2 Genechip arrays. With false discovery rate (FDR) <0.05 with 95% confidence, a total of 526 genes were differentially expressed between before and after exercise. Three hundred and sixteen genes had higher expression after exercise. The Jak/STAT pathway, known to inhibit apoptosis, was significantly activated (EASE score, P < 0.005), but 14 genes were altered in a way likely to accelerate apoptosis as well. Similarly, both proinflammatory (e.g., IL-32, TNFSF8, and CCR5) and anti-inflammatory (e.g., ANXA1) were affected. Growth and repair genes like AREG and FGF2 receptor genes (involved in angiogenesis) were also activated. Finally, a number of neutrophil genes known to be involved in pathological conditions like asthma and arthritis were altered by exercise, suggesting novel links between physical activity and disease or its prevention. In summary, brief heavy exercise leads to a previously unknown substantial and significant alteration in neutrophil gene expression.

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