Single cell resolution landscape of equine peripheral blood mononuclear cells reveals diverse immune cell subtypes including T-bet+ B 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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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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Statin-induced microRNAome alterations modulating inflammation pathways of peripheral blood mononuclear cells in patients with hypercholesterolemia

Bioscience Reports ◽

10.1042/bsr20201885 ◽

2020 ◽

Vol 40 (9) ◽

Author(s):

Hung-Ju Lin ◽

Sung-Liang Yu ◽

Ta-Chen Su ◽

Hsiu-Ching Hsu ◽

Ming-Fong Chen ◽

...

Keyword(s):

Peripheral Blood Mononuclear Cells ◽

Immune Cells ◽

Peripheral Blood ◽

Mononuclear Cells ◽

Transforming Growth Factor ◽

Quantitative Polymerase Chain Reaction ◽

Density Lipoprotein ◽

Cardiovascular Risks ◽

Peripheral Blood Mononuclear ◽

Blood Mononuclear Cells

Abstract Statins inhibit cholesterol biogenesis and modulate atheroma inflammation to reduce cardiovascular risks. Promoted by immune and non-immune cells, serum C-reactive protein (CRP) might be a biomarker suboptimal to assess inflammation status. Although it has been reported that statins modulated inflammation via microRNAs (miRNAs), evidence remains lacking on comprehensive profiling of statin-induced miRNAome alterations in immune cells. We recruited 19 hypercholesterolemic patients receiving 2 mg/day pitavastatin and 15 ones receiving 10 mg/day atorvastatin treatment for 12 weeks, and performed microarray-based profiling of 1733 human mature miRNAs in peripheral blood mononuclear cells (PBMCs) before and after statin treatment. Differentially expressed miRNAs were determined if their fold changes were >1.50 or <0.67, after validated using quantitative polymerase chain reaction (qPCR). The miRSystem and miTALOS platforms were utilized for pathway analysis. Of the 34 patients aged 63.7 ± 6.2 years, 27 were male and 19 were with coronary artery disease. We discovered that statins induced differential expressions of miR-483-5p, miR-4667-5p, miR-1244, and miR-3609, with qPCR-validated fold changes of 1.74 (95% confidence interval, 1.33–2.15), 1.61 (1.25–1.98), 1.61 (1.01–2.21), and 1.68 (1.19–2.17), respectively. The fold changes of the four miRNAs were not correlated with changes of low-density-lipoprotein cholesterol or CRP, after sex, age, and statin type were adjusted. We also revealed that RhoA and transforming growth factor-β signaling pathways might be regulated by the four miRNAs. Given our findings, miRNAs might be involved in statin-induced inflammation modulation in PBMCs, providing likelihood to assess and reduce inflammation in patients with atherosclerotic cardiovascular diseases.

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MicroRNA expression profiling of peripheral blood mononuclear cells associated with syphilis

10.21203/rs.2.9964/v2 ◽

2019 ◽

Author(s):

Tao Huang ◽

Jun Zhang ◽

Wujian Ke ◽

Xiaohui Zhang ◽

Wentao Chen ◽

...

Keyword(s):

Peripheral Blood Mononuclear Cells ◽

Peripheral Blood ◽

Mononuclear Cells ◽

Cell Function ◽

Immune Cell ◽

Treponema Pallidum ◽

Target Sequence ◽

Healthy Individuals ◽

Peripheral Blood Mononuclear ◽

Blood Mononuclear Cells

Abstract Background Treponema pallidum ( T. pallidum ) infection evokes significant immune responses, resulting in tissue damage. The immune mechanism underlying T. pallidum infection is still unclear, although microRNAs (miRNAs) have been shown to influence immune cell function and, consequently, the generation of antibody responses during other microbe infections. However, these mechanisms are unknown for T. pallidum . Methods In this study, we performed a comprehensive analysis of differentially expressed miRNAs in healthy individuals, untreated patients with syphilis, patients in the serofast state, and serologically cured patients. miRNAs were profiled from the peripheral blood of patients obtained at the time of serological diagnosis. Then, both the target sequence analysis of these different miRNAs and pathway analysis were performed to identify important immune and cell signaling pathways. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) was performed for microRNA analysis. Results A total of 89 differentially regulated miRNAs were identified. Following RT-qPCR confirmation, three miRNAs (hsa-miR-195-5p, hsa-miR-223-3p, hsa-miR-589-3p) showed significant differences in the serofast and serologically cured states ( P <0.05). One miRNA (hsa-miR-195-5p) showed significant differences between untreated patients and healthy individuals. Conclusions This is the first study of miRNA expression differences in peripheral blood mononuclear cells (PBMCs) in different stages of T. pallium infection. Our study suggests that the combination of three miRNAs has great potential to serve as a non-invasive biomarker of T. pallium infections, which will facilitate better diagnosis and treatment of T. pallium infections.

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Norepinephrine Inhibits Energy Metabolism of Human Peripheral Blood Mononuclear Cells via Adrenergic Receptors

Bioscience Reports ◽

10.1023/a:1014768909442 ◽

2001 ◽

Vol 21 (5) ◽

pp. 627-635 ◽

Cited By ~ 22

Author(s):

Jan D. Lünemann ◽

Frank Buttgereit ◽

Robert Tripmacher ◽

Christoph G. O. Baerwald ◽

Gerd-Rüdiger Burmester ◽

...

Keyword(s):

Energy Metabolism ◽

Peripheral Blood Mononuclear Cells ◽

Immune Cells ◽

Peripheral Blood ◽

Adrenergic Receptors ◽

Mononuclear Cells ◽

Human Peripheral Blood ◽

Peripheral Blood Mononuclear ◽

Immune Effector ◽

Blood Mononuclear Cells

Previous studies demonstrated that the adaptive response to stressors and inflammatory signals involves the activation of the automotic nervous system. Catecholamines have been shown to modulate the activity of various immune effector cells directly via membrane adrenergic receptors. Here, we investigated immediate effects of norepinephrine on energy metabolism of immune cells. Norepinephrine inhibits oxygen consumption of human peripheral blood mononuclear cells at concentrations that are relevant to its physiological range. The ?-adrenoreceptor antagonist propranolol, but not the ?-adrenoreceptor antagonist phentolamine reversed the norepinephrine induced inhibition in quiescent cells. Conversely, phentolamine but not propranolol is capable of blocking norepinephrine mediated effects in mitogen activated human peripheral blood mononuclear cells. Our data indicate that the sensitization of ?- and ?-adrenoreceptors on immune cells is differentially regulated, and that these processes depend on the activation state of these cells. These findings have important implications for the understanding of stress-induced suppression of immune function and may contribute to the elucidation of the pathogenesis of immunologically mediated diseases.

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Distribution of integrins on human peripheral blood mononuclear cells

Blood ◽

10.1182/blood.v74.4.1348.1348 ◽

1989 ◽

Vol 74 (4) ◽

pp. 1348-1354 ◽

Cited By ~ 27

Author(s):

HG Klingemann ◽

S Dedhar

Keyword(s):

T Cells ◽

B Cells ◽

Peripheral Blood Mononuclear Cells ◽

Peripheral Blood ◽

Mononuclear Cells ◽

Human Peripheral Blood ◽

Peptide Sequence ◽

Peripheral Blood Mononuclear ◽

Blood Mononuclear Cells ◽

Beta 1 Integrin

Abstract The receptors for fibronectin (FN-R) and vitronectin (VN-R) belong to a family of integral membrane glycoproteins known to be involved in cell- extracellular matrix and cell-cell interactions named integrins (FN-R = beta 1 integrin and VN-R = beta 3 integrin). Adhesion studies using FN- coated plastic dishes and highly purified subpopulations of peripheral blood mononuclear cells (PBMCs) showed a strong binding of monocytes and T lymphocytes to FN but virtually no binding of B cells to FN. Binding of monocytes and T cells to FN could be partially inhibited by a hexapeptide (GRGDSP) containing the adhesive peptide sequence Arg-Gly- Asp (RGD) as well as by an anti-FN-R antibody. The distribution of beta 1 and beta 3 integrin complexes on PBMCs was characterized by immunoprecipitation of detergent extracts of 125I-labeled cells using polyclonal antibodies against these two receptors. Two surface polypeptides corresponding to the alpha and beta chains of FN-R and VN- R were found on all three cell types. To characterize these receptors further, monoclonal antibodies (MoAbs) against the very late antigens (VLAs) 1, 3, and 5 were used for immunoprecipitation studies. Monocytes and T cells reacted with VLA 5 that was previously identified as the human FN receptor, whereas no labeling with anti-VLA 5 could be shown for B cells. When cell populations were cultured in 10% human serum for 24 hours, an increase in beta 1-integrin+ monocytes and T cells was observed. The number of beta 3-integrin+ cells remained essentially unchanged. The presence of beta 1 and beta 3 integrins on monocytes as well as on T and B lymphocytes may be of significance in the ability of these cells to interact with each other and participate in hematopoiesis and certain immune reactions.

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Oropouche Virus Infects, Persists and Induces IFN Response in Human Peripheral Blood Mononuclear Cells as Identified by RNA PrimeFlow™ and qRT-PCR Assays

Viruses ◽

10.3390/v12070785 ◽

2020 ◽

Vol 12 (7) ◽

pp. 785

Author(s):

Mariene Ribeiro Amorim ◽

Marjorie Cornejo Pontelli ◽

Gabriela Fabiano de Souza ◽

Stéfanie Primon Muraro ◽

Daniel A. Toledo-Teixeira ◽

...

Keyword(s):

T Cells ◽

B Cells ◽

Peripheral Blood Mononuclear Cells ◽

Peripheral Blood ◽

Mononuclear Cells ◽

Peripheral Blood Mononuclear ◽

Human Leukocytes ◽

Qrt Pcr ◽

Viral Antigens ◽

Blood Mononuclear Cells

Oropouche orthobunyavirus (OROV) is an emerging arbovirus with a high potential of dissemination in America. Little is known about the role of peripheral blood mononuclear cells (PBMC) response during OROV infection in humans. Thus, to evaluate human leukocytes susceptibility, permissiveness and immune response during OROV infection, we applied RNA hybridization, qRT-PCR and cell-based assays to quantify viral antigens, genome, antigenome and gene expression in different cells. First, we observed OROV replication in human leukocytes lineages as THP-1 monocytes, Jeko-1 B cells and Jurkat T cells. Interestingly, cell viability and viral particle detection are maintained in these cells, even after successive passages. PBMCs from healthy donors were susceptible but the infection was not productive, since neither antigenome nor infectious particle was found in the supernatant of infected PBMCs. In fact, only viral antigens and small quantities of OROV genome were detected at 24 hpi in lymphocytes, monocytes and CD11c+ cells. Finally, activation of the Interferon (IFN) response was essential to restrict OROV replication in human PBMCs. Increased expression of type I/III IFNs, ISGs and inflammatory cytokines was detected in the first 24 hpi and viral replication was re-established after blocking IFNAR or treating cells with glucocorticoid. Thus, in short, our results show OROV is able to infect and remain in low titers in human T cells, monocytes, DCs and B cells as a consequence of an effective IFN response after infection, indicating the possibility of leukocytes serving as a trojan horse in specific microenvironments during immunosuppression.

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Dynamic changes in human single cell transcriptional signatures during fatal sepsis

10.1101/2021.03.01.21252411 ◽

2021 ◽

Author(s):

Xinru Qiu ◽

Jiang Li ◽

Jeff Bonenfant ◽

Lukasz Jaroszewski ◽

Walter Klein ◽

...

Keyword(s):

Single Cell ◽

Peripheral Blood Mononuclear Cells ◽

Peripheral Blood ◽

Metabolic Pathways ◽

Mononuclear Cells ◽

Immune Cell ◽

Peripheral Blood Mononuclear ◽

Repeated Measures Design ◽

Blood Mononuclear Cells ◽

Fatal Sepsis

AbstractSystemic infections, especially in patients with chronic diseases, result in sepsis: an explosive, uncoordinated immune response that can lead to multisystem organ failure with a high mortality rate. Sepsis survivors and non-survivors oftentimes have similar clinical phenotypes or sepsis biomarker expression upon diagnosis, suggesting that the dynamics of sepsis in the critical early stage may have an impact on these opposite outcomes. To investigate this, we designed a within-subject study of patients with systemic gram-negative bacterial sepsis with surviving and fatal outcomes and performed single-cell transcriptomic analyses of peripheral blood mononuclear cells (PBMC) collected during the critical period between sepsis recognition and 6 hours. We observed that the largest sepsis-induced expression changes over time in surviving versus fatal sepsis were in CD14+ monocytes, including gene signatures previously reported for sepsis outcomes. We further identify changes in the metabolic pathways of both monocytes and platelets, the emergence of erythroid precursors, and T cell exhaustion signatures, with the most extreme differences occurring between the non-sepsis control and the sepsis non-survivor. Our single-cell observations are consistent with trends from public datasets but also reveal specific effects in individual immune cell populations, which change within hours. In conclusion, this pilot study provides the first single-cell results with a repeated measures design in sepsis to analyze the temporal changes in the immune cell population behavior in surviving or fatal sepsis. These findings indicate that tracking temporal expression changes in specific cell-types could lead to more accurate predictions of sepsis outcomes. We also identify molecular pathways that could be therapeutically controlled to improve the sepsis trajectory toward better outcomes.Summary sentenceSingle cell transcriptomics of peripheral blood mononuclear cells in surviving and fatal sepsis reveal inflammatory and metabolic pathways that change within hours of sepsis recognition.

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Function of ex vivo stimulated Natural killer T cells from patients with chronic HIV-1 infection

10.1101/831834 ◽

2019 ◽

Author(s):

Prakash Thapa ◽

Pramod Nehete ◽

Hong He ◽

Bharti Nehete ◽

Stephanie J. Buchl ◽

...

Keyword(s):

Natural Killer ◽

Peripheral Blood Mononuclear Cells ◽

Immune Cells ◽

Peripheral Blood ◽

Mononuclear Cells ◽

Ex Vivo ◽

Nkt Cells ◽

Peripheral Blood Mononuclear ◽

Blood Mononuclear Cells ◽

Hiv 1

AbstractNatural killer T (NKT) cells are innate immune cells that are responsible for the first line of antiviral defense, through crosstalk with downstream antigen-presenting cells, natural killer cells, and adaptive immune cells. Previous studies have indicated that NKT cell function is severely impaired in patients with chronic HIV-1 infection. It was reported that alpha-galactosylceramide, a potent agonist antigen for NKT cells, failed to trigger the expansion of NKT cells, or the production of anti-viral cytokines by NKT cells from HIV-1 infected patients in an in vitro assay, in which peripheral blood mononuclear cells (PBMCs) were cultured in the presence of alpha-galactosylceramide. In this study, we stimulated banked peripheral blood mononuclear cells from HIV-1-infected patients with dendritic cells (DC) generated ex vivo and loaded with alpha-galactosylceramide. The results showed that NKT cells were expanded in HIV infected subjects except in patients with advanced AIDS. Expanded NKT cells were capable of producing antiviral cytokines. Our results indicate that NKT cells in HIV infected individuals are potential targets for therapeutic intervention.

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