Biology subjects

Daharsh, L.

Publications and source records attributed to Daharsh, L..

4 recordsLinked to original sources

bioRxiv2024

Definition of regulatory elements and transcription factors controlling immune cell gene expression at single cell resolution using single nucleus ATAC-seq

The transcriptome of porcine peripheral blood mononuclear cells (PBMC) at single cell (sc) resolution is well described, but little is understood about the cis-regulatory mechanism behind scPBMC gene expression. Here, we profiled the open chromatin landscape of porcine PBMC using single nucleus ATAC sequencing (snATAC-seq). Approximately 22% of the identified peaks overlapped with annotated transcription start sites (TSS). Using clustering based on open chromatin pattern similarity, we demonstrate that cell type annotations using snATAC-seq are highly concordant to that reported for sc RNA sequencing (scRNA-seq). The differentially accessible peaks (DAPs) for each cell type were characterized and the pattern of accessibility of the DAPs near cell type markers across cell types was similar to that of the average gene expression level of corresponding marker genes. Additionally, we found that peaks identified in snATAC-seq have the potential power to predict the cell type specific transcription starting site (TSS). We identified both transcription factors (TFs) whose binding motif were enriched in cell type DAPs of multiple cell types and cell type specific TFs by conducting transcription factor binding motif (TFBM) analysis. Furthermore, we identified the putative enhancer or promoter regions bound by TFs for each differentially expressed gene (DEG) having a DAP that overlapped with its TSS by generating cis-co-accessibility networks (CCAN). To predict the regulators of such DEGs, TFBM analysis was performed for each CCAN. The regulator TF-target DEG pair predicted in this way was largely consistent with the results reported in the ENCODE Transcription Factor Targets Dataset (TFTD). This snATAC-seq approach provides insights into the chromatin accessibility landscape of porcine PBMCs and enables discovery of TFs predicted to control DEG through binding regulatory elements whose chromatin accessibility correlates with the DEG promoter region.

bioinformatics↗

bioRxiv2021

Reference transcriptomes of porcine peripheral immune cells created through bulk and single-cell RNA sequencing

Pigs are a valuable human biomedical model and an important protein source supporting global food security. The transcriptomes of peripheral blood immune cells in pigs were defined at the bulk cell-type and single cell levels. First, eight cell types were isolated in bulk from peripheral blood mononuclear cells (PBMCs) by cell sorting, representing Myeloid, NK cells and specific populations of T and B cells. Transcriptomes for each bulk population of cells were generated by RNA-seq with 10,974 expressed genes detected. Pairwise comparisons between cell types revealed specific expression, while enrichment analysis identified 1,885 to 3,591 significantly enriched genes across all 8 cell types. Gene Ontology analysis for the top 25% of significantly enriched genes (SEG) showed high enrichment of biological processes related to the nature of each cell type. Comparison of gene expression indicated highly significant correlations between pig cells and corresponding human PBMC bulk RNA-seq data available in Haemopedia. Second, higher resolution of distinct cell populations was obtained by single-cell RNA-sequencing (scRNA-seq) of PBMC. Seven PBMC samples were partitioned and sequenced that produced 28,810 single cell transcriptomes distributed across 36 clusters and classified into 13 general cell types including plasmacytoid dendritic cells (DC), conventional DCs, monocytes, B cell, conventional CD4 and CD8 {beta} T cells, NK cells, and {gamma}{delta} T cells. Signature gene sets from the human Haemopedia data were assessed for relative enrichment in genes expressed in pig cells and integration of pig scRNA-seq with a public human scRNA-seq dataset provided further validation for similarity between human and pig data. The sorted porcine bulk RNAseq dataset informed classification of scRNA-seq PBMC populations; specifically, an integration of the datasets showed that the pig bulk RNAseq data helped define the CD4CD8 double-positive T cell populations in the scRNA-seq data. Overall, the data provides deep and well-validated transcriptomic data from sorted PBMC populations and the first single-cell transcriptomic data for porcine PBMCs. This resource will be invaluable for annotation of pig genes controlling immunogenetic traits as part of the porcine Functional Annotation of Animal Genomes (FAANG) project, as well as further study of, and development of new reagents for, porcine immunology.

immunology↗

bioRxiv2020

Alterations in the gut microbiome by HIV-1 infection or a high-fat diet associates with systemic immune activation and inflammation in double humanized-BLT mice

BackgroundWhile the translatability of gut microbiome studies utilizing animal models to humans has proven difficult, studying the gut microbiome directly in humans is also challenging due to the existence of many confounding variables. Therefore, we utilized double humanized mice, which have both an engrafted stable human-like gut microbiome and functional human immune system. With this model, we were able to determine the in vivo impact of HIV-1 infection or a high-fat diet (HFD) on gut human microbiome composition, and its relationship with human immune cell activation and systemic inflammation. ResultsSurgery was performed on NSG mice to create humanized bone-marrow, liver, thymus mice (hu-mice). In order to create double hu-mice, the hu-mice were treated with broad spectrum antibiotics to deplete murine gut bacteria and subsequently transplanted with human fecal material from healthy human donors. We characterized 262 fecal samples from hu-mice, double hu-mice, and human fecal donors to determine the impact of HIV-1 infection or HFD on the gut microbiome and systemic immune activation and inflammation. We found that HIV-1 infection altered the human-like gut microbiome of double hu-mice, which was associated with decreased human CD4 T cells and increased systemic inflammation and immune activation. Further, using a HFD we induced gut microbial dysbiosis in double hu-mice which corresponded with increased systemic immune activation and inflammation. ConclusionsHere, we describe the changes in the human gut microbiome and human immune system due to HIV-1 infection or HFD using our double hu-mice model. HIV-1 infection led to changes in the composition of the human-like gut microbiome that was associated with human CD4 T cell loss and high levels of inflammation and immune activation. The HFD quickly changed the composition of the gut microbiome and led to systemic immune activation and inflammation. We further identified a subset of gut bacteria in HIV-1 infected and HFD fed double hu-mice that was closely associated with systemic inflammation and immune activation. This study demonstrated how double humanized mice can be used to study the complex in vivo interactions of the gut microbiome and human immune system in the context of both disease and diet.

microbiology↗

bioRxiv2019

Stable Engraftment of a Human Gut Bacterial Microbiome in Double Humanized BLT-mice

BackgroundHumanized mice featuring a functional human immune system are an important pre-clinical model for examining immune responses to human-specific pathogens. This model has been widely utilized to study human diseases that are otherwise impossible or difficult to investigate in humans or with other animal models. However, one limitation of using humanized mice is their native murine gut microbiome, which significantly differs from the one found in humans. These differences may be even greater for mice housed and bred in specific pathogen free conditions. Given the importance of the gut microbiome to human health and disease, these differences may profoundly impact the ability to translate the results from humanized mice studies to human disease. Further, there is a critical need for improved pre-clinical models to study the complex in vivo relationships of the gut microbiome, immune system, and human disease. We therefore created double humanized mice with both a functional human immune system and stable human-like gut microbiome.\n\nResultsSurgery was performed on NOD.Cg-PrkdcscidII2rgtm1Wjl/SzJ (NSG) mice to create bone-marrow, liver, thymus (BLT) humanized mice. After immune reconstitution, mice were treated with broad spectrum antibiotics to deplete murine gut bacteria and then transplanted with fecal material from healthy human donors. Characterization of 173 fecal samples obtained from 45 humanized mice revealed that double humanized mice had unique 16S rRNA gene profiles consistent with those of the individual human donor samples. Importantly, transplanted human-like gut microbiomes were stable in mice for the duration of the study, up to 14.5 weeks post-transplant. Microbiomes of double humanized mice also harbored predicted functional capacities that more closely resembled those of the human donors compared to humanized mice.\n\nConclusionsHere, we describe successful engraftment of a stable human microbiome in BLT humanized mice to further improve this preclinical humanized mouse model. These double humanized mice represent a unique and tractable new model to study the complex relationships between the human gut microbiome, human immune system, and human disease in vivo.

microbiology↗