scholarly journals FLAMs: A self-replicating ex vivo model of alveolar macrophages for functional genetic studies

2021 ◽  
Author(s):  
Sean Thomas ◽  
Kathryn Wierenga ◽  
James Pestka ◽  
Andrew Olive
Keyword(s):  
Alveolar Macrophages ◽  
Ex Vivo ◽  
Fetal Liver ◽  
Expression Profiles ◽  
Surface Expression ◽  
Specific Gene ◽  
Ex Vivo Model ◽  
A Genome ◽  
Underlying Mechanisms ◽  
And Function

Alveolar macrophages (AMs) are tissue resident cells in the lungs derived from the fetal liver that maintain lung homeostasis and respond to inhaled stimuli. While the importance of AMs is undisputed, they remain refractory to standard experimental approaches and high-throughput functional genetics as they are challenging to isolate and rapidly lose AM properties in standard culture. This limitation hinders our understanding of key regulatory mechanisms that control AM maintenance and function. Here, we describe the development of a new model, fetal liver-derived alveolar-like macrophages (FLAMs), which maintains cellular morphologies, expression profiles, and functional mechanisms similar to murine AMs. FLAMs combine treatment with two key cytokines for AM maintenance, GM-CSF and TGFβ. We leveraged the long-term stability of FLAMs to develop functional genetic tools using CRISPR-Cas9-mediated gene editing. Targeted editing confirmed the role of AM-specific gene Marco and the IL-1 receptor Il1r1 in modulating the AM response to crystalline silica. Furthermore, a genome-wide knockout library using FLAMs identified novel genes required for surface expression of the AM marker Siglec-F, most notably those related to the peroxisome. Taken together, our results suggest that FLAMs are a stable, self-replicating model of AM function that enables previously impossible global genetic approaches to define the underlying mechanisms of AM maintenance and function.

P-Selectin Expression and Platelet Function Are Developmentally Regulated

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1439-1439 ◽  
Author(s):  
Massiel Chavez Stolla ◽  
Kathryn Leyens ◽  
Seana C Catherman ◽  
Kathleen E McGrath ◽  
James Palis
Keyword(s):  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Fetal Liver ◽  
Surface Expression ◽  
Developmentally Regulated ◽  
Primary Mouse ◽  
Significant Difference ◽  
Platelet Aggregates ◽  
Granule Secretion ◽  
And Function

Abstract Platelets have numerous well-defined roles during adult hemostasis, however little is known about whether these functions are developmentally regulated. In the adult, P-selectin is constitutively expressed in megakaryocytes and localized to the membrane of alpha granules. Activation of platelets results in granule secretion and P-selectin surface exposure allowing for recruitment of leukocytes to sites of vascular injury and the formation of leukocyte-platelet aggregates. Here, we examined the expression and function of P-selectin in platelets during murine embryogenesis. Platelets were isolated from timed pregnant outbred-ICR mice at embryonic day 12.5 (E12.5), E15.5, postnatal day 1 (PN1), PN7, PN21, and were assayed for thrombin induced integrin activation and P-selectin translocation. While no significant differences were seen in the ability of embryonic platelets to be activated by thrombin, P-selectin surface expression after thrombin stimulation was significantly reduced in embryonic compared to adult platelets (E15.5 p=.0006, E12.5 p=.0004, PN1 p=.01, n=3, unpaired t-test). Surprisingly, P-selectin expression began to appear on the surface of activated platelets only after birth, approaching 50% and 75% of maternal expression at PN7 and PN21, respectively. No significant difference was seen in VEGF secretion after thrombin activation of E12.5, E15.5 and adult platelets (n=5), suggesting that the lack of P-selectin surface expression was not due to a loss of alpha granule secretion. To further characterize expression of P-selectin, real-time qPCR was performed on mRNA harvested from platelets at E12.5, E15.5, PN1 and adult stages. P-selectin transcript levels were significantly lower in embryonic and PN1 platelets, suggesting that P-selectin expression is transcriptionally regulated. Consistent with this concept, Imagestream analysis indicates that P-selectin protein is absent in megakaryocytes derived from the fetal liver. P-selectin expression on platelets is an important mediator of platelet-leukocyte interactions; therefore, we assayed the ability of platelets to bind to leukocytes in embryonic whole blood. Preliminary data suggest that embryonic platelets do not form leukocyte-platelet aggregates upon ex vivo stimulation with AYPGKF, a Par4 agonist. Taken together, our data indicate that P-selectin expression is developmentally regulated in primary mouse platelets. The absence of P-selectin expression in embryonic platelets correlates with an impaired ability to interact with leukocytes. Disclosures No relevant conflicts of interest to declare.

scholarly journals Discovery of novel mechanosensitive genes in vivo using mouse carotid artery endothelium exposed to disturbed flow

Blood ◽  
2010 ◽  
Vol 116 (15) ◽  
pp. e66-e73 ◽  
Author(s):  
Chih-Wen Ni ◽  
Haiwei Qiu ◽  
Amir Rezvan ◽  
Kihwan Kwon ◽  
Douglas Nam ◽  
...  
Keyword(s):  
Carotid Artery ◽  
Ex Vivo ◽  
Expression Profiles ◽  
Quantitative Polymerase Chain Reaction ◽  
Gene Expression Profiles ◽  
Disturbed Flow ◽  
A Genome ◽  
Pattern Comparison

Abstract Recently, we showed that disturbed flow caused by a partial ligation of mouse carotid artery rapidly induces atherosclerosis. Here, we identified mechanosensitive genes in vivo through a genome-wide microarray study using mouse endothelial RNAs isolated from the flow-disturbed left and the undisturbed right common carotid artery. We found 62 and 523 genes that changed significantly by 12 hours and 48 hours after ligation, respectively. The results were validated by quantitative polymerase chain reaction for 44 of 46 tested genes. This array study discovered numerous novel mechanosensitive genes, including Lmo4, klk10, and dhh, while confirming well-known ones, such as Klf2, eNOS, and BMP4. Four genes were further validated for protein, including LMO4, which showed higher expression in mouse aortic arch and in human coronary endothelium in an asymmetric pattern. Comparison of in vivo, ex vivo, and in vitro endothelial gene expression profiles indicates that numerous in vivo mechanosensitive genes appear to be lost or dysregulated during culture. Gene ontology analyses show that disturbed flow regulates genes involved in cell proliferation and morphology by 12 hours, followed by inflammatory and immune responses by 48 hours. Determining the functional importance of these novel mechanosensitive genes may provide important insights into understanding vascular biology and atherosclerosis.

scholarly journals BHLHE40 Regulates IL-10 and IFN-γ Production in T Cells but Does Not Interfere With Human Type 1 Regulatory T Cell Differentiation

2021 ◽  
Vol 12 ◽  
Author(s):  
Molly Javier Uyeda ◽  
Robert A. Freeborn ◽  
Brandon Cieniewicz ◽  
Rosa Romano ◽  
Ping (Pauline) Chen ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Differentiation ◽  
Ex Vivo ◽  
Surface Expression ◽  
Surface Molecules ◽  
Ifn Γ ◽  
And Function ◽  
Tr1 Cells

Type 1 regulatory T (Tr1) cells are subset of peripherally induced antigen-specific regulatory T cells. IL-10 signaling has been shown to be indispensable for polarization and function of Tr1 cells. However, the transcriptional machinery underlying human Tr1 cell differentiation and function is not yet elucidated. To this end, we performed RNA sequencing on ex vivo human CD49b+LAG3+ Tr1 cells. We identified the transcription factor, BHLHE40, to be highly expressed in Tr1 cells. Even though Tr1 cells characteristically produce high levels of IL-10, we found that BHLHE40 represses IL-10 and increases IFN-γ secretion in naïve CD4+ T cells. Through CRISPR/Cas9-mediated knockout, we determined that IL10 significantly increased in the sgBHLHE40-edited cells and BHLHE40 is dispensable for naïve CD4+ T cells to differentiate into Tr1 cells in vitro. Interestingly, BHLHE40 overexpression induces the surface expression of CD49b and LAG3, co-expressed surface molecules attributed to Tr1 cells, but promotes IFN-γ production. Our findings uncover a novel mechanism whereby BHLHE40 acts as a regulator of IL-10 and IFN-γ in human CD4+ T cells.

scholarly journals Development of a CRISPR-SaCas9 system for projection- and function-specific gene editing in the rat brain

2020 ◽  
Vol 6 (12) ◽  
pp. eaay6687 ◽  
Author(s):  
Haojie Sun ◽  
Su Fu ◽  
Shuang Cui ◽  
Xiangsha Yin ◽  
Xiaoyan Sun ◽  
...  
Keyword(s):  
Rat Brain ◽  
Gene Editing ◽  
High Efficiency ◽  
Protein A ◽  
Cell Types ◽  
Cell Labeling ◽  
Specific Gene ◽  
Creb Binding Protein ◽  
A Genome ◽  
And Function

A genome editing technique based on the clustered regularly interspaced short palindromic repeats (CRISPR)–associated endonuclease Cas9 enables efficient modification of genes in various cell types, including neurons. However, neuronal ensembles even in the same brain region are not anatomically or functionally uniform but divide into distinct subpopulations. Such heterogeneity requires gene editing in specific neuronal populations. We developed a CRISPR-SaCas9 system–based technique, and its combined application with anterograde/retrograde AAV vectors and activity-dependent cell-labeling techniques achieved projection- and function-specific gene editing in the rat brain. As a proof-of-principle application, we knocked down the cbp (CREB-binding protein), a sample target gene, in specific neuronal subpopulations in the medial prefrontal cortex, and demonstrated the significance of the projection- and function-specific CRISPR-SaCas9 system in revealing neuronal and circuit basis of memory. The high efficiency and specificity of our projection- and function-specific CRISPR-SaCas9 system could be widely applied in neural circuitry studies.

scholarly journals Citrullinated vimentin mediates development and progression of lung fibrosis

2021 ◽  
Vol 13 (585) ◽  
pp. eaba2927
Author(s):  
Fu Jun Li ◽  
Ranu Surolia ◽  
Huashi Li ◽  
Zheng Wang ◽  
Gang Liu ◽  
...  
Keyword(s):  
Lung Tissue ◽  
Lung Fibrosis ◽  
Ex Vivo ◽  
Lavage Fluid ◽  
Surface Expression ◽  
Lung Fibroblasts ◽  
Dependent Manner ◽  
Ex Vivo Model ◽  
Molecular Pattern ◽  
Citrullinated Vimentin

The mechanisms by which environmental exposures contribute to the pathogenesis of lung fibrosis are unclear. Here, we demonstrate an increase in cadmium (Cd) and carbon black (CB), common components of cigarette smoke (CS) and environmental particulate matter (PM), in lung tissue from subjects with idiopathic pulmonary fibrosis (IPF). Cd concentrations were directly proportional to citrullinated vimentin (Cit-Vim) amounts in lung tissue of subjects with IPF. Cit-Vim amounts were higher in subjects with IPF, especially smokers, which correlated with lung function and were associated with disease manifestations. Cd/CB induced the secretion of Cit-Vim in an Akt1- and peptidylarginine deiminase 2 (PAD2)–dependent manner. Cit-Vim mediated fibroblast invasion in a 3D ex vivo model of human pulmospheres that resulted in higher expression of CD26, collagen, and α-SMA. Cit-Vim activated NF-κB in a TLR4-dependent fashion and induced the production of active TGF-β1, CTGF, and IL-8 along with higher surface expression of TLR4 in lung fibroblasts. To corroborate ex vivo findings, mice treated with Cit-Vim, but not Vim, independently developed a similar pattern of fibrotic tissue remodeling, which was TLR4 dependent. Moreover, wild-type mice, but not PAD2−/− and TLR4 mutant (MUT) mice, exposed to Cd/CB generated high amounts of Cit-Vim, in both plasma and bronchoalveolar lavage fluid, and developed lung fibrosis in a stereotypic manner. Together, these studies support a role for Cit-Vim as a damage-associated molecular pattern molecule (DAMP) that is generated by lung macrophages in response to environmental Cd/CB exposure. Furthermore, PAD2 might represent a promising target to attenuate Cd/CB-induced fibrosis.

scholarly journals Imaging transcriptomics of brain disorders

2021 ◽  
Author(s):  
Aurina Arnatkeviciute ◽  
Ben Fulcher ◽  
Mark Bellgrove ◽  
Alex Fornito
Keyword(s):  
Gene Expression ◽  
Brain Structure ◽  
Ex Vivo ◽  
Specific Gene ◽  
Brain Disorders ◽  
Spatiotemporal Resolution ◽  
Indirect Measures ◽  
Regional Vulnerability ◽  
Brain Structure And Function ◽  
And Function

Non-invasive neuroimaging is a powerful tool for quantifying diverse aspects of brain structure and function invivo and has been used extensively to map the neural changes associated with different brain disorders. However,most neuroimaging techniques have limited spatiotemporal resolution and offer only indirect measures ofunderlying pathological mechanisms. The recent development of anatomically comprehensive gene-expressionatlases has opened new opportunities for studying the transcriptional correlates of non-invasively measured neuralphenotypes, offering a rich framework for evaluating pathophysiological hypotheses and putative mechanisms.Here, we overview some fundamental methods in imaging transcriptomics and outline their application tounderstanding brain disorders of neurodevelopment, adulthood, and neurodegeneration. Converging evidenceindicates that spatial variations in gene expression are linked to normative changes in brain structure during agerelatedmaturation and neurodegeneration that are in part associated with cell-specific gene expression markersof gene expression. Transcriptional correlates of disorder-related neuroimaging phenotypes are also linked totranscriptionally dysregulated genes identified in ex vivo analyses of patient brains. Modeling studies demonstratethat spatial patterns of gene expression are involved in regional vulnerability to neurodegeneration and the spreadof disease across the brain. This growing body of work supports the utility of transcriptional atlases in testinghypotheses about the molecular mechanism driving disease-related changes in macroscopic neuroimagingphenotypes.

scholarly journals Critical Role of cRel Subunit of NF-κB in Sepsis Survival

2011 ◽  
Vol 79 (5) ◽  
pp. 1848-1854 ◽  
Author(s):  
Emilie Courtine ◽  
Frédéric Pène ◽  
Nicolas Cagnard ◽  
Julie Toubiana ◽  
Catherine Fitting ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression Profiles ◽  
Critical Role ◽  
Cecal Ligation ◽  
Gene Expression Profiles ◽  
Severe Infection ◽  
A Genome ◽  
Underlying Mechanisms ◽  
Pathogen Clearance

ABSTRACTNF-κB is a critical regulator of gene expression during severe infections. NF-κB comprises homo- and heterodimers of proteins from the Rel family. Among them, p50 and p65 have been clearly implicated in the pathophysiology of sepsis. In contrast, the role of cRel in sepsis is still controversial and has been poorly studied in single-pathogen infections. We aimed to investigate the consequences of cRel deficiency in a cecal ligation and puncture (CLP) model of sepsis. We have approached the underlying mechanisms of host defense by analyzing bacterial clearance, systemic inflammation, and the distribution of spleen dendritic cell subsets. Moreover, by using a genome-wide technology, we have also analyzed the CLP-induced modifications in gene expression profiles both in wild-type (wt) and inrel−/−mice. The absence of cRel enhances mortality due to polymicrobial sepsis. Despite normal pathogen clearance, cRel deficiency leads to an altered systemic inflammatory response associated with a sustained loss of the spleen lymphoid dendritic cells. Furthermore, a whole-blood microarray study reveals that the differential outcome between wt andrel−/−mice during sepsis is preceded by remarkable changes in the expression of hundreds of genes involved in aspects of host-pathogen interaction, such as host survival and lipid metabolism. In conclusion, cRel is a key NF-κB member required for host antimicrobial defenses and a regulatory transcription subunit that controls the inflammatory and immune responses in severe infection.

scholarly journals A Computational Method for Classifying Different Human Tissues with Quantitatively Tissue-Specific Expressed Genes

Genes ◽  
2018 ◽  
Vol 9 (9) ◽  
pp. 449 ◽  
Author(s):  
JiaRui Li ◽  
Lei Chen ◽  
Yu-Hang Zhang ◽  
XiangYin Kong ◽  
Tao Huang ◽  
...  
Keyword(s):  
Expression Profiles ◽  
Expression Patterns ◽  
Machine Learning Algorithms ◽  
Computational Method ◽  
Support Vector ◽  
Specific Gene ◽  
Human Tissues ◽  
Tissue Development ◽  
Tissue Specific ◽  
And Function

Tissue-specific gene expression has long been recognized as a crucial key for understanding tissue development and function. Efforts have been made in the past decade to identify tissue-specific expression profiles, such as the Human Proteome Atlas and FANTOM5. However, these studies mainly focused on “qualitatively tissue-specific expressed genes” which are highly enriched in one or a group of tissues but paid less attention to “quantitatively tissue-specific expressed genes”, which are expressed in all or most tissues but with differential expression levels. In this study, we applied machine learning algorithms to build a computational method for identifying “quantitatively tissue-specific expressed genes” capable of distinguishing 25 human tissues from their expression patterns. Our results uncovered the expression of 432 genes as optimal features for tissue classification, which were obtained with a Matthews Correlation Coefficient (MCC) of more than 0.99 yielded by a support vector machine (SVM). This constructed model was superior to the SVM model using tissue enriched genes and yielded MCC of 0.985 on an independent test dataset, indicating its good generalization ability. These 432 genes were proven to be widely expressed in multiple tissues and a literature review of the top 23 genes found that most of them support their discriminating powers. As a complement to previous studies, our discovery of these quantitatively tissue-specific genes provides insights into the detailed understanding of tissue development and function.

scholarly journals The genome of the Hi5 germ cell line from Trichoplusia ni, an agricultural pest and novel model for small RNA biology

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Yu Fu ◽  
Yujing Yang ◽  
Han Zhang ◽  
Gwen Farley ◽  
Junling Wang ◽  
...  
Keyword(s):  
Cell Line ◽  
Small Rna ◽  
Trichoplusia Ni ◽  
Ex Vivo ◽  
Expression Profiles ◽  
Insect Pest ◽  
Gene Families ◽  
Specific Gene ◽  
Germ Cell Line ◽  
Lepidopteran Insect

We report a draft assembly of the genome of Hi5 cells from the lepidopteran insect pest, Trichoplusia ni, assigning 90.6% of bases to one of 28 chromosomes and predicting 14,037 protein-coding genes. Chemoreception and detoxification gene families reveal T. ni-specific gene expansions that may explain its widespread distribution and rapid adaptation to insecticides. Transcriptome and small RNA data from thorax, ovary, testis, and the germline-derived Hi5 cell line show distinct expression profiles for 295 microRNA- and >393 piRNA-producing loci, as well as 39 genes encoding small RNA pathway proteins. Nearly all of the W chromosome is devoted to piRNA production, and T. ni siRNAs are not 2´-O-methylated. To enable use of Hi5 cells as a model system, we have established genome editing and single-cell cloning protocols. The T. ni genome provides insights into pest control and allows Hi5 cells to become a new tool for studying small RNAs ex vivo.

R123: Gene Expression Profiles in Ex Vivo Model of Nasal Polyp

2006 ◽  
Vol 135 (2_suppl) ◽  
pp. P147-P147
Author(s):  
Kyung-Wook Heo ◽  
Seong-Kook Park
Keyword(s):  
Gene Expression ◽  
Nasal Polyp ◽  
Ex Vivo ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Ex Vivo Model
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