scholarly journals Mosquito cellular immunity at single-cell resolution

2020 ◽  
Author(s):  
Gianmarco Raddi ◽  
Ana Beatriz F Barletta ◽  
Mirjana Efremova ◽  
Jose Luis Ramirez ◽  
Rafael Cantera ◽  
...  
Keyword(s):  
Single Cell ◽  
Transmembrane Protein ◽  
Blood Feeding ◽  
Human Pathogens ◽  
Protein Marker ◽  
Immune Priming ◽  
Cellular Events ◽  
Invertebrate Immunology ◽  
Immune Challenges ◽  
Rna In Situ Hybridization

AbstractInsect hemocytes are the functional equivalents of leukocytes and limit the capacity of mosquitoes to transmit human pathogens through phagocytosis, encapsulation, secretion of immune factors and immune priming (1, 2). Here we profile the transcriptomes of 8506 hemocytes of Anopheles gambiae and Aedes aegypti, two important mosquito vectors. Blood feeding, infection with malaria parasites and other immune challenges reveal a previously unknown functional diversity of hemocytes, with different types of granulocytes expressing distinct and evolutionarily conserved subsets of effector genes. A new cell type, which we term megacyte, is defined in Anopheles by a unique transmembrane protein marker (TM7318) and high expression of LPS-Induced TNF-alpha transcription factor 3 (LL3). Knock-down experiments indicate that LL3 mediates hemocyte differentiation during immune priming. We identify two main hemocyte lineages and find evidence of proliferating granulocyte populations. We validate our analysis with RNA in-situ hybridization and highlight the mobilization of sessile hemocytes into circulation upon infection. Our data (https://hemocytes.cellgeni.sanger.ac.uk/) provide the first atlas of medically relevant invertebrate immune cells at single cell resolution. It provides an important resource for invertebrate immunology by identifying cellular events that underpin mosquito immunity to malaria infection.

scholarly journals Mosquito cellular immunity at single-cell resolution

Science ◽  
2020 ◽  
Vol 369 (6507) ◽  
pp. 1128-1132 ◽  
Author(s):  
Gianmarco Raddi ◽  
Ana Beatriz F. Barletta ◽  
Mirjana Efremova ◽  
Jose Luis Ramirez ◽  
Rafael Cantera ◽  
...  
Keyword(s):  
Single Cell ◽  
Transmembrane Protein ◽  
Human Pathogens ◽  
Protein Marker ◽  
Immune Priming ◽  
Cellular Events ◽  
Effector Genes ◽  
Evolutionarily Conserved ◽  
Factor Α ◽  
Necrosis Factor

Hemocytes limit the capacity of mosquitoes to transmit human pathogens. Here we profile the transcriptomes of 8506 hemocytes of Anopheles gambiae and Aedes aegypti mosquito vectors. Our data reveal the functional diversity of hemocytes, with different subtypes of granulocytes expressing distinct and evolutionarily conserved subsets of effector genes. A previously unidentified cell type in An. gambiae, which we term “megacyte,” is defined by a specific transmembrane protein marker (TM7318) and high expression of lipopolysaccharide-induced tumor necrosis factor–α transcription factor 3 (LL3). Knockdown experiments indicate that LL3 mediates hemocyte differentiation during immune priming. We identify and validate two main hemocyte lineages and find evidence of proliferating granulocyte populations. This atlas of medically relevant invertebrate immune cells at single-cell resolution identifies cellular events that underpin mosquito immunity to malaria infection.

scholarly journals Single-cell analysis of mosquito hemocytes identifies signatures of immune cell sub-types and cell differentiation

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Hyeogsun Kwon ◽  
Mubasher Mohammed ◽  
Oscar Franzén ◽  
Johan Ankarklev ◽  
Ryan Smith
Keyword(s):  
Single Cell ◽  
Cell Fate ◽  
Immune Cells ◽  
Cell Biology ◽  
Immune Cell ◽  
Single Cell Analysis ◽  
Immune Priming ◽  
Rna Fish ◽  
Invertebrate Immunology ◽  
Humoral Responses

Mosquito immune cells, known as hemocytes, are integral to cellular and humoral responses that limit pathogen survival and mediate immune priming. However, without reliable cell markers and genetic tools, studies of mosquito immune cells have been limited to morphological observations, leaving several aspects of their biology uncharacterized. Here, we use single-cell RNA sequencing (scRNA-seq) to characterize mosquito immune cells, demonstrating an increased complexity to previously defined prohemocyte, oenocytoid, and granulocyte subtypes. Through functional assays relying on phagocytosis, phagocyte depletion, and RNA-FISH experiments, we define markers to accurately distinguish immune cell subtypes and provide evidence for immune cell maturation and differentiation. In addition, gene-silencing experiments demonstrate the importance of lozenge in defining the mosquito oenocytoid cell fate. Together, our scRNA-seq analysis provides an important foundation for future studies of mosquito immune cell biology and a valuable resource for comparative invertebrate immunology.

scholarly journals Single-cell analysis of mosquito hemocytes identifies signatures of immune cell subtypes and cell differentiation

2020 ◽  
Author(s):  
Hyeogsun Kwon ◽  
Mubasher Mohammed ◽  
Oscar Franzén ◽  
Johan Ankarklev ◽  
Ryan C. Smith
Keyword(s):  
Single Cell ◽  
Cell Fate ◽  
Immune Cells ◽  
Cell Biology ◽  
Immune Cell ◽  
Single Cell Analysis ◽  
Immune Priming ◽  
Rna Fish ◽  
Invertebrate Immunology ◽  
Humoral Responses

AbstractMosquito immune cells, known as hemocytes, are integral to cellular and humoral responses that limit pathogen survival and mediate immune priming. However, without reliable cell markers and genetic tools, studies of mosquito immune cells have been limited to morphological observations, leaving several aspects of their biology uncharacterized. Here, we use single-cell RNA sequencing (scRNA-seq) to characterize mosquito immune cells, demonstrating an increased complexity to previously defined prohemocyte, oenocytoid, and granulocyte subtypes. Through functional assays relying on phagocytosis, phagocyte depletion, and RNA-FISH experiments, we define markers to accurately distinguish immune cell subtypes and provide evidence for immune cell maturation and differentiation. In addition, gene-silencing experiments demonstrate the importance of lozenge in defining the mosquito oenocytoid cell fate. Together, our scRNA-seq analysis provides an important foundation for studies of mosquito immune cell biology and a valuable resource for comparative invertebrate immunology.

scholarly journals Quantitative Single-Cell Transcript Assessment of Biomarkers Supports Cellular Heterogeneity in the Bovine IVD

2019 ◽  
Vol 6 (2) ◽  
pp. 42 ◽  
Author(s):  
Kangning Li ◽  
Devin Kapper ◽  
Sumona Mondal ◽  
Thomas Lufkin ◽  
Petra Kraus
Keyword(s):  
In Situ Hybridization ◽  
Single Cell ◽  
Gaussian Mixture ◽  
Cellular Heterogeneity ◽  
Rna In Situ Hybridization ◽  
Ivd Degeneration

Severe and chronic low back pain is often associated with intervertebral disc (IVD) degeneration. While imposing a considerable socio-economic burden worldwide, IVD degeneration is also severely impacting on the quality of life of affected individuals. Cell-based regenerative medicine approaches have moved into clinical trials, yet IVD cell identities in the mature disc remain to be fully elucidated and tissue heterogeneity exists, requiring a better characterization of IVD cells. The bovine coccygeal IVD is an accepted research model to study IVD mechano-biology and disc homeostasis. Recently, we identified novel IVD biomarkers in the outer annulus fibrosus (AF) and nucleus pulposus (NP) of the mature bovine coccygeal IVD through RNA in situ hybridization (AP-RISH) and z-proportion test. Here we follow up on Lam1, Thy1, Gli1, Gli3, Noto, Ptprc, Scx, Sox2 and Zscan10 with fluorescent RNA in situ hybridization (FL-RISH) and confocal microscopy. This permits sub-cellular transcript localization and the addition of quantitative single-cell derived values of mRNA expression levels to our previous analysis. Lastly, we used a Gaussian mixture modeling approach for the exploratory analysis of IVD cells. This work complements our earlier cell population proportion-based study, confirms the previously proposed biomarkers and indicates even further heterogeneity of cells in the outer AF and NP of a mature IVD.

scholarly journals Single-cell transcriptomics defines heterogeneity of epicardial cells and fibroblasts within the infarcted murine heart

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Julia Hesse ◽  
Christoph Owenier ◽  
Tobias Lautwein ◽  
Ria Zalfen ◽  
Jonas F Weber ◽  
...  
Keyword(s):  
Single Cell ◽  
Stromal Cells ◽  
Wilms Tumor ◽  
Hypoxia Inducible Factor ◽  
Lineage Tracing ◽  
Proliferating Cells ◽  
Paracrine Factors ◽  
Epicardial Cells ◽  
Cardiac Specification ◽  
Rna In Situ Hybridization

In the adult heart, the epicardium becomes activated after injury, contributing to cardiac healing by secretion of paracrine factors. Here, we analyzed by single-cell RNA sequencing combined with RNA in situ hybridization and lineage tracing of Wilms tumor protein 1-positive (WT1+) cells, the cellular composition, location, and hierarchy of epicardial stromal cells (EpiSC) in comparison to activated myocardial fibroblasts/stromal cells in infarcted mouse hearts. We identified 11 transcriptionally distinct EpiSC populations, which can be classified into three groups, each containing a cluster of proliferating cells. Two groups expressed cardiac specification markers and sarcomeric proteins suggestive of cardiomyogenic potential. Transcripts of hypoxia-inducible factor (HIF)-1α and HIF-responsive genes were enriched in EpiSC consistent with an epicardial hypoxic niche. Expression of paracrine factors was not limited to WT1+ cells but was a general feature of activated cardiac stromal cells. Our findings provide the cellular framework by which myocardial ischemia may trigger in EpiSC the formation of cardioprotective/regenerative responses.

scholarly journals Linear-time cluster ensembles of large-scale single-cell RNA-seq and multimodal data

2020 ◽  
Author(s):  
Van Hoan Do ◽  
Francisca Rojas Ringeling ◽  
Stefan Canzar
Keyword(s):  
Single Cell ◽  
Large Scale ◽  
Linear Time ◽  
Cell Types ◽  
Substantial Improvement ◽  
Rna Seq ◽  
Sampling Step ◽  
Protein Marker ◽  
Cluster Ensembles ◽  
Sequencing Technologies

AbstractA fundamental task in single-cell RNA-seq (scRNA-seq) analysis is the identification of transcriptionally distinct groups of cells. Numerous methods have been proposed for this problem, with a recent focus on methods for the cluster analysis of ultra-large scRNA-seq data sets produced by droplet-based sequencing technologies. Most existing methods rely on a sampling step to bridge the gap between algorithm scalability and volume of the data. Ignoring large parts of the data, however, often yields inaccurate groupings of cells and risks overlooking rare cell types. We propose method Specter that adopts and extends recent algorithmic advances in (fast) spectral clustering. In contrast to methods that cluster a (random) subsample of the data, we adopt the idea of landmarks that are used to create a sparse representation of the full data from which a spectral embedding can then be computed in linear time. We exploit Specter’s speed in a cluster ensemble scheme that achieves a substantial improvement in accuracy over existing methods and that is sensitive to rare cell types. Its linear time complexity allows Specter to scale to millions of cells and leads to fast computation times in practice. Furthermore, on CITE-seq data that simultaneously measures gene and protein marker expression we demonstrate that Specter is able to utilize multimodal omics measurements to resolve subtle transcriptomic differences between subpopulations of cells. Specter is open source and available at https://github.com/canzarlab/Specter.

scholarly journals Single-cell transcriptomics defines heterogeneity of epicardial cells and fibroblasts within the infarcted heart

2021 ◽  
Author(s):  
Julia Hesse ◽  
Christoph Owenier ◽  
Tobias Lautwein ◽  
Ria Zalfen ◽  
Jonas F. Weber ◽  
...  
Keyword(s):  
Single Cell ◽  
Stromal Cells ◽  
Lineage Tracing ◽  
Sarcomeric Proteins ◽  
Proliferating Cells ◽  
Paracrine Factors ◽  
Epicardial Cells ◽  
Cardiac Specification ◽  
Rna In Situ Hybridization

AbstractIn the adult heart, the epicardium becomes activated after injury, contributing to cardiac healing by secretion of paracrine factors. Here we analyzed by single-cell RNA sequencing combined with RNA in situ hybridization and lineage tracing of WT1+ cells the cellular composition, location, and hierarchy of epicardial stromal cells (EpiSC) in comparison to activated myocardial fibroblasts/stromal cells in infarcted mouse hearts. We identified 11 transcriptionally distinct EpiSC populations, that can be classified in three groups each containing a cluster of proliferating cells. Two groups expressed cardiac specification makers and sarcomeric proteins suggestive of cardiomyogenic potential. Transcripts of HIF-1α and HIF-responsive genes were enriched in EpiSC consistent with an epicardial hypoxic niche. Expression of paracrine factors was not limited to WT1+ cells but was a general feature of activated cardiac stromal cells. Our findings provide the cellular framework by which myocardial ischemia may trigger in EpiSC the formation of cardioprotective/regenerative responses.

scholarly journals Gregarine single-cell transcriptomics reveals differential mitochondrial remodeling and adaptation in apicomplexans

BMC Biology ◽  
2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Eric D. Salomaki ◽  
Kristina X. Terpis ◽  
Sonja Rueckert ◽  
Michael Kotyk ◽  
Zuzana Kotyková Varadínová ◽  
...  
Keyword(s):  
Single Cell ◽  
Evolutionary Relationship ◽  
Mitochondrial Metabolism ◽  
Human Pathogens ◽  
Cryptosporidium Hominis ◽  
Microbial Eukaryotes ◽  
Transport Chain ◽  
Phylogenomic Analyses ◽  
Mitochondrial Remodeling ◽  
Genome Scale

Abstract Background Apicomplexa is a diverse phylum comprising unicellular endobiotic animal parasites and contains some of the most well-studied microbial eukaryotes including the devastating human pathogens Plasmodium falciparum and Cryptosporidium hominis. In contrast, data on the invertebrate-infecting gregarines remains sparse and their evolutionary relationship to other apicomplexans remains obscure. Most apicomplexans retain a highly modified plastid, while their mitochondria remain metabolically conserved. Cryptosporidium spp. inhabit an anaerobic host-gut environment and represent the known exception, having completely lost their plastid while retaining an extremely reduced mitochondrion that has lost its genome. Recent advances in single-cell sequencing have enabled the first broad genome-scale explorations of gregarines, providing evidence of differential plastid retention throughout the group. However, little is known about the retention and metabolic capacity of gregarine mitochondria. Results Here, we sequenced transcriptomes from five species of gregarines isolated from cockroaches. We combined these data with those from other apicomplexans, performed detailed phylogenomic analyses, and characterized their mitochondrial metabolism. Our results support the placement of Cryptosporidium as the earliest diverging lineage of apicomplexans, which impacts our interpretation of evolutionary events within the phylum. By mapping in silico predictions of core mitochondrial pathways onto our phylogeny, we identified convergently reduced mitochondria. These data show that the electron transport chain has been independently lost three times across the phylum, twice within gregarines. Conclusions Apicomplexan lineages show variable functional restructuring of mitochondrial metabolism that appears to have been driven by adaptations to parasitism and anaerobiosis. Our findings indicate that apicomplexans are rife with convergent adaptations, with shared features including morphology, energy metabolism, and intracellularity.

scholarly journals Quantitative single-cell analysis of immunofluorescence protein multiplex images illustrates biomarker spatial heterogeneity within breast cancer subtypes

2021 ◽  
Vol 23 (1) ◽  
Author(s):  
Alison Min-Yan Cheung ◽  
Dan Wang ◽  
Kela Liu ◽  
Tyna Hope ◽  
Mayan Murray ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Single Cell ◽  
Diversity Index ◽  
Breast Cancer Subtype ◽  
Cellular Heterogeneity ◽  
Luminal A ◽  
Proliferating Cells ◽  
Protein Marker ◽  
Long Term Outcome ◽  
Luminal B

Abstract Background The extent of cellular heterogeneity in breast cancer could have potential impact on diagnosis and long-term outcome. However, pathology evaluation is limited to biomarker immunohistochemical staining and morphology of the bulk cancer. Inter-cellular heterogeneity of biomarkers is not usually assessed. As an initial evaluation of the extent of breast cancer cellular heterogeneity, we conducted quantitative and spatial imaging of Estrogen Receptor (ER), Progesterone Receptor (PR), Epidermal Growth Factor Receptor-2 (HER2), Ki67, TP53, CDKN1A (P21/WAF1), CDKN2A (P16INK4A), CD8 and CD20 of a tissue microarray (TMA) representing subtypes defined by St. Gallen surrogate classification. Methods Quantitative, single cell-based imaging was conducted using an Immunofluorescence protein multiplexing platform (MxIF) to study protein co-expression signatures and their spatial localization patterns. The range of MxIF intensity values of each protein marker was compared to the respective IHC score for the TMA core. Extent of heterogeneity in spatial neighborhoods was analyzed using co-occurrence matrix and Diversity Index measures. Results On the 101 cores from 59 cases studied, diverse expression levels and distributions were observed in MxIF measures of ER and PR among the hormonal receptor-positive tumor cores. As expected, Luminal A-like cancers exhibit higher proportions of cell groups that co-express ER and PR, while Luminal B-like (HER2-negative) cancers were composed of ER+, PR- groups. Proliferating cells defined by Ki67 positivity were mainly found in groups with PR-negative cells. Triple-Negative Breast Cancer (TNBC) exhibited the highest proliferative fraction and incidence of abnormal P53 and P16 expression. Among the tumors exhibiting P53 overexpression by immunohistochemistry, a group of TNBC was found with much higher MxIF-measured P53 signal intensity compared to HER2+, Luminal B-like and other TNBC cases. Densities of CD8 and CD20 cells were highest in HER2+ cancers. Spatial analysis demonstrated variability in heterogeneity in cellular neighborhoods in the cancer and the tumor microenvironment. Conclusions Protein marker multiplexing and quantitative image analysis demonstrated marked heterogeneity in protein co-expression signatures and cellular arrangement within each breast cancer subtype. These refined descriptors of biomarker expressions and spatial patterns could be valuable in the development of more informative tools to guide diagnosis and treatment.

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