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Single-cell RNA sequencing (scRNA-seq) has revolutionized the study of the cellular landscape of organs. Most single-cell protocols require fresh material, which limits sample size per experiment, and consequently, introduces batch effects. This is especially true for samples acquired through complex medical procedures, such as intestinal mucosal biopsies. Moreover, the tissue dissociation procedure required for obtaining single cells is a major source of noise; different dissociation procedures applied to different compartments of the tissue induce artificial gene expression differences between cell subsets. To overcome these challenges, we have developed a one-step dissociation protocol and demonstrated its use on cryopreserved gut mucosal biopsies. Using flow cytometry and scRNA-seq analysis, we compared this one-step dissociation protocol with the current gold standard, two-step collagenase digestion, and an adaptation of a recently published alternative, three-step cold-active Bacillus licheniformus protease digestion. Both cell viability and cell type composition were comparable between the one-step and two-step collagenase dissociation, with the former being more time-efficient. The cold protease digestion resulted in equal cell viability, but better preserves the epithelial cell types. Consequently, to analyze the rarer cell types, such as glial cells, larger total biopsy cell numbers are required as input material. The multi-step protocols affected cell types spanning multiple compartments differently. In summary, we show that cryopreserved gut mucosal biopsies can be used to overcome the logistical challenges and batch effects in large scRNA-seq studies. Furthermore, we demonstrate that using cryopreserved biopsies digested using a one-step collagenase protocol enables large-scale scRNA-seq, FACS, organoid generation and intraepithelial lymphocyte expansion. [ABSTRACT FROM AUTHOR]

Single-cell RNA sequencing (scRNA-seq) has revolutionized the study of the cellular landscape of organs. Most single-cell protocols require fresh material, which limits sample size per experiment, and consequently, introduces batch effects. This is especially true for samples acquired through complex medical procedures, such as intestinal mucosal biopsies. Moreover, the tissue dissociation procedure required for obtaining single cells is a major source of noise; different dissociation procedures applied to different compartments of the tissue induce artificial gene expression differences between cell subsets. To overcome these challenges, we have developed a one-step dissociation protocol and demonstrated its use on cryopreserved gut mucosal biopsies. Using flow cytometry and scRNA-seq analysis, we compared this one-step dissociation protocol with the current gold standard, two-step collagenase digestion, and an adaptation of a recently published alternative, three-step cold-active Bacillus licheniformus protease digestion. Both cell viability and cell type composition were comparable between the one-step and two-step collagenase dissociation, with the former being more time-efficient. The cold protease digestion resulted in equal cell viability, but better preserves the epithelial cell types. Consequently, to analyze the rarer cell types, such as glial cells, larger total biopsy cell numbers are required as input material. The multi-step protocols affected cell types spanning multiple compartments differently. In summary, we show that cryopreserved gut mucosal biopsies can be used to overcome the logistical challenges and batch effects in large scRNA-seq studies. Furthermore, we demonstrate that using cryopreserved biopsies digested using a one-step collagenase protocol enables large-scale scRNA-seq, FACS, organoid generation and intraepithelial lymphocyte expansion. [ABSTRACT FROM AUTHOR]

Celiac disease is an auto-immune disease in which an immune response to dietary gluten leads to inflammation and subsequent atrophy of small intestinal villi, causing severe bowel discomfort and malabsorption of nutrients. The major instigating factor for the immune response in celiac disease is the activation of gluten-specific CD4+ T cells expressing T cell receptors that recognize gluten peptides presented in the context of HLA-DQ2 and DQ8. Here we provide an in-depth characterization of 28 gluten-specific T cell clones. We assess their transcriptional and epigenetic response to T cell receptor stimulation and link this to genetic factors associated with celiac disease. Gluten-specific T cells have a distinct transcriptional profile that mostly resembles that of Th1 cells but also express cytokines characteristic of other types of T-helper cells. This transcriptional response appears not to be regulated by changes in chromatin state, but rather by early upregulation of transcription factors and non-coding RNAs that likely orchestrate the subsequent activation of genes that play a role in immune pathways. Finally, integration of chromatin and transcription factor binding profiles suggest that genes activated by T cell receptor stimulation of gluten‑specific T cells may be impacted by genetic variation at several genetic loci associated with celiac disease. [ABSTRACT FROM AUTHOR]

Background: Expression quantitative trait loci (eQTL) studies are used to interpret the function of disease-associated genetic risk factors. To date, most eQTL analyses have been conducted in bulk tissues, such as whole blood and tissue biopsies, which are likely to mask the cell type-context of the eQTL regulatory effects. Although this context can be investigated by generating transcriptional profiles from purified cell subpopulations, current methods to do this are labor-intensive and expensive. We introduce a new method, Decon2, as a framework for estimating cell proportions using expression profiles from bulk blood samples (Decon-cell) followed by deconvolution of cell type eQTLs (Decon-eQTL). Results: The estimated cell proportions from Decon-cell agree with experimental measurements across cohorts (R ≥ 0.77). Using Decon-cell, we could predict the proportions of 34 circulating cell types for 3194 samples from a population-based cohort. Next, we identified 16,362 whole-blood eQTLs and deconvoluted cell type interaction (CTi) eQTLs using the predicted cell proportions from Decon-cell. CTi eQTLs show excellent allelic directional concordance with eQTL (≥ 96–100%) and chromatin mark QTL (≥87–92%) studies that used either purified cell subpopulations or single-cell RNA-seq, outperforming the conventional interaction effect. Conclusions: Decon2 provides a method to detect cell type interaction effects from bulk blood eQTLs that is useful for pinpointing the most relevant cell type for a given complex disease. Decon2 is available as an R package and Java application (https://github.com/molgenis/systemsgenetics/tree/master/Decon2) and as a web tool (www.molgenis.org/deconvolution). [ABSTRACT FROM AUTHOR]

Phage-displayed immunoprecipitation sequencing (PhIP-seq) has enabled high-throughput profiling of human antibody repertoires. However, a comprehensive overview of environmental and genetic determinants shaping human adaptive immunity is lacking. In this study, we investigated the effects of genetic, environmental, and intrinsic factors on the variation in human antibody repertoires. We characterized serological antibody repertoires against 344,000 peptides using PhIP-seq libraries from a wide range of microbial and environmental antigens in 1,443 participants from a population cohort. We detected individual-specificity, temporal consistency, and co-housing similarities in antibody repertoires. Genetic analyses showed the involvement of the HLA , IGHV , and FUT2 gene regions in antibody-bound peptide reactivity. Furthermore, we uncovered associations between phenotypic factors (including age, cell counts, sex, smoking behavior, and allergies, among others) and particular antibody-bound peptides. Our results indicate that human antibody epitope repertoires are shaped by both genetics and environmental exposures and highlight specific signatures of distinct phenotypes and genotypes. [Display omitted] • PhIP-seq libraries from microbial and environmental antigens in a population cohort • Antibody-bound peptide co-reactivity highlights bacterial mimicry • Common genetic variants in HLA, IGHV, and FUT2 determinate antibody reactivity • Antibody reactivity is associated with widespread phenotypical factors In this study, Andreu-Sánchez et al. utilize phage-displayed immunoprecipitation sequencing to investigate the environmental and genetic determinants shaping human adaptive immunity. The results suggest that both genetics and environmental exposures shape human antibody epitope repertoires, with specific signatures of distinct phenotypes and genotypes. Furthermore, co-occurring antibody responses suggest a link between bacterial immunity and the development of allergies or autoimmunity. [ABSTRACT FROM AUTHOR]

Candidaemia is a bloodstream infection caused by Candida species that primarily affects specific groups of at-risk patients. Because only small candidaemia patient cohorts are available, classical genome wide association cannot be used to identify Candida susceptibility genes. Therefore, we have applied an integrative genomics approach to identify novel susceptibility genes and pathways for candidaemia. Candida-induced transcriptome changes in human primary leukocytes were assessed by RNA sequencing. Genetic susceptibility to candidaemia was assessed using the Illumina immunochip platform for genotyping of a cohort of 217 patients. We then integrated genetics data with gene-expression profiles, Candida-induced cytokine production capacity, and circulating concentrations of cytokines. Based on the intersection of transcriptome pathways and genomic data, we prioritized 31 candidate genes for candidaemia susceptibility. This group of genes was enriched with genes involved in inflammation, innate immunity, complement, and hemostasis. We then validated the role of MAP3K8 in cytokine regulation in response to Candida stimulation. Here, we present a new framework for the identification of susceptibility genes for infectious diseases that uses an unbiased, hypothesis-free, systems genetics approach. By applying this approach to candidaemia, we identified novel susceptibility genes and pathways for candidaemia, and future studies should assess their potential as therapeutic targets. [ABSTRACT FROM AUTHOR]

Background Although genome-wide association studies (GWAS) have identified hundreds of variants associated with a risk for autoimmune and immune-related disorders (AID), our understanding of the disease mechanisms is still limited. In particular, more than 90% of the risk variants lie in non-coding regions, and almost 10% of these map to long non-coding RNA transcripts (lncRNAs). LncRNAs are known to show more cell-type specificity than protein-coding genes. Methods We aimed to characterize lncRNAs and protein-coding genes located in loci associated with nine AID which have been well-defined by Immunochip analysis and by transcriptome analysis across seven populations of peripheral blood leukocytes (granulocytes, monocytes, NK cells, B-cells, memory-T cells, naive CD3+ and naive CD8+ T-cells) and four populations of cord blood-derived T-helper cells (precursor, primary, and polarized (Th1, Th2) T-helper cells). Results We show that lncRNAs mapping to loci shared between AID are significantly enriched in immune cell types compared to lncRNAs from the whole genome (α <0.005). We were not able to prioritize single cell types relevant for specific diseases, but we observed five different cell types enriched (α <0.005) in five AID (NK cells for inflammatory bowel disease, juvenile idiopathic arthritis, primary biliary cirrhosis, and psoriasis; memory-T and CD8+ T-cells in juvenile idiopathic arthritis, primary biliary cirrhosis, psoriasis, and rheumatoid arthritis; Th0 and Th2 cells for inflammatory bowel disease, juvenile idiopathic arthritis, primary biliary cirrhosis, psoriasis, and rheumatoid arthritis). Furthermore, we show that co-expression analyses of lncRNAs and protein-coding genes can predict the signaling pathways in which these AID-associated lncRNAs are involved. [ABSTRACT FROM AUTHOR]

It has been found that the majority of disease-associated genetic variants identified by genome-wide association studies are located outside of protein-coding regions, where they seem to affect regions that control transcription (promoters, enhancers) and non-coding RNAs that also can influence gene expression. In this review, we focus on two classes of non-coding RNAs that are currently a major focus of interest: micro-RNAs and long non-coding RNAs. We describe their biogenesis, suggested mechanism of action, and discuss how these non-coding RNAs might be affected by disease-associated genetic alterations. The discovery of these alterations has already contributed to a better understanding of the etiopathology of human diseases and yielded insight into the function of these non-coding RNAs. We also provide an overview of available databases, bioinformatics tools, and high-throughput techniques that can be used to study the mechanism of action of individual non-coding RNAs. This article is part of a Special Issue entitled: From Genome to Function. [ABSTRACT FROM AUTHOR]

Celiac disease (CeD) is triggered by gluten and results in inflammation and villous atrophy of the small intestine. We aimed to explore the role of miRNA-mediated deregulation of the transcriptome in CeD. Duodenal biopsies of CeD patients (n = 33) and control subjects (n = 10) were available for miRNA-sequencing, with RNA-sequencing also available for controls (n = 5) and CeD (n = 6). Differential expression analysis was performed to select CeD-associated miRNAs and genes. MiRNA‒target transcript pairs selected from public databases that also displayed a strong negative expression correlation in the current dataset (R < −0.7) were used to construct a CeD miRNA‒target transcript interaction network. The network includes 2030 miRNA‒target transcript interactions, including 423 experimentally validated pairs. Pathway analysis found that interactions are involved in immune-related pathways (e.g., interferon signaling) and metabolic pathways (e.g., lipid metabolism). The network includes 13 genes previously prioritized to be causally deregulated by CeD-associated genomic variants, including STAT1. CeD-associated miRNAs might play a role in promoting inflammation and decreasing lipid metabolism in the small intestine, thereby contributing unbalanced cell turnover in the intestinal crypt. Some CeD-associated miRNAs deregulate genes that are also affected by genomic CeD-risk variants, adding an additional layer of complexity to the deregulated transcriptome in CeD. [ABSTRACT FROM AUTHOR]

Recently it has become clear that only a small percentage (7%) of disease-associated single nucleotide polymorphisms (SNPs) are located in protein-coding regions, while the remaining 93% are located in gene regulatory regions or in intergenic regions. Thus, the understanding of how genetic variations control the expression of non-coding RNAs (in a tissue-dependent manner) has far-reaching implications. We tested the association of SNPs with expression levels (eQTLs) of large intergenic non-coding RNAs (lincRNAs), using genome-wide gene expression and genotype data from five different tissues. We identified 112 cis-regulated lincRNAs, of which 45% could be replicated in an independent dataset. We observed that 75% of the SNPs affecting lincRNA expression (lincRNA cis-eQTLs) were specific to lincRNA alone and did not affect the expression of neighboring protein-coding genes. We show that this specific genotype-lincRNA expression correlation is tissue-dependent and that many of these lincRNA cis-eQTL SNPs are also associated with complex traits and diseases. [ABSTRACT FROM AUTHOR]

Non-alcoholic fatty liver disease (NAFLD) is the most prevalent chronic liver disease and is characterized by different stages varying from benign fat accumulation to non-alcoholic steatohepatitis (NASH) that may progress to cirrhosis and liver cancer. In recent years, a regulatory role of long non-coding RNAs (lncRNAs) in NAFLD has emerged. Therefore, we aimed to characterize the still poorly understood lncRNA contribution to disease progression. Transcriptome analysis in 60 human liver samples with various degrees of NAFLD/NASH was combined with a functional genomics experiment in an in vitro model where we exposed HepG2 cells to free fatty acids (FFA) to induce steatosis, then stimulated them with tumor necrosis factor alpha (TNFα) to mimic inflammation. Bioinformatics analyses provided a functional prediction of novel lncRNAs. We further functionally characterized the involvement of one novel lncRNA in the nuclear-factor-kappa B (NF-κB) signaling pathway by its silencing in Hepatoma G2 (HepG2) cells. We identified 730 protein-coding genes and 18 lncRNAs that responded to FFA/TNFα and associated with human NASH phenotypes with consistent effect direction, with most being linked to inflammation. One novel intergenic lncRNA, designated lncTNF, was 20-fold up-regulated upon TNFα stimulation in HepG2 cells and positively correlated with lobular inflammation in human liver samples. Silencing lncTNF in HepG2 cells reduced NF-κB activity and suppressed expression of the NF-κB target genes A20 and NFKBIA. The lncTNF we identified in the NF-κB signaling pathway may represent a novel target for controlling liver inflammation. [ABSTRACT FROM AUTHOR]

Phagocytosis and autophagy are two ancient, highly conserved processes involved, respectively, in the removal of extracellular organisms and the destruction of organisms in the cytosol. Autophagy, for either metabolic regulation or defence, involves the formation of a double membrane called the autophagosome, which then fuses with lysosomes to degrade the contents, a process that has similarities with phagosome maturation. Toll-like-receptor (TLR) engagement activates a variety of defence mechanisms within phagocytes, including facilitation of phagosome maturation, and also engages autophagy. Therefore we speculated that TLR signalling might link these processes to enhance the function of conventional phagosomes. Here we show that a particle that engages TLRs on a murine macrophage while it is phagocytosed triggers the autophagosome marker LC3 to be rapidly recruited to the phagosome in a manner that depends on the autophagy pathway proteins ATG5 and ATG7; this process is preceded by recruitment of beclin 1 and phosphoinositide-3-OH kinase activity. Translocation of beclin 1 and LC3 to the phagosome was not associated with observable double-membrane structures characteristic of conventional autophagosomes, but was associated with phagosome fusion with lysosomes, leading to rapid acidification and enhanced killing of the ingested organism. [ABSTRACT FROM AUTHOR]

Celiac disease (CeD) is a complex immune-mediated disorder that is triggered by dietary gluten in genetically predisposed individuals. CeD is characterized by inflammation and villous atrophy of the small intestine, which can lead to gastrointestinal complaints, malnutrition, and malignancies. Currently, diagnosis of CeD relies on serology (antibodies against transglutaminase and endomysium) and small-intestinal biopsies. Since small-intestinal biopsies require invasive upper-endoscopy, and serology cannot predict CeD in an early stage or be used for monitoring disease after initiation of a gluten-free diet, the search for non-invasive biomarkers is ongoing. Here, we summarize current and up-and-coming non-invasive biomarkers that may be able to predict, diagnose, and monitor the progression of CeD. We further discuss how current and emerging techniques, such as (single-cell) transcriptomics and genomics, can be used to uncover the pathophysiology of CeD and identify non-invasive biomarkers. [ABSTRACT FROM AUTHOR]

Summary As part of the Human Functional Genomics Project, which aims to understand the factors that determine the variability of immune responses, we investigated genetic variants affecting cytokine production in response to ex vivo stimulation in two independent cohorts of 500 and 200 healthy individuals. We demonstrate a strong impact of genetic heritability on cytokine production capacity after challenge with bacterial, fungal, viral, and non-microbial stimuli. In addition to 17 novel genome-wide significant cytokine QTLs (cQTLs), our study provides a comprehensive picture of the genetic variants that influence six different cytokines in whole blood, blood mononuclear cells, and macrophages. Important biological pathways that contain cytokine QTLs map to pattern recognition receptors ( TLR1-6-10 cluster), cytokine and complement inhibitors, and the kallikrein system. The cytokine QTLs show enrichment for monocyte-specific enhancers, are more often located in regions under positive selection, and are significantly enriched among SNPs associated with infections and immune-mediated diseases. PaperClip [ABSTRACT FROM AUTHOR]