Novel biomarkers of preterm brain injury from blood transcriptome in sheep model of intrauterine asphyxia.

BACKGROUND: Infants born preterm have a higher incidence of neurological deficits. A key step in finding effective treatments is to identify biomarkers that reliably predict outcome. METHODS: Following umbilical cord occlusion (UCO) in pregnant sheep, whole fetal blood RNA was sequenced pre- and post-UCO, brain injury outcome was determined by battery of neuropathology scoring and the transcriptome signature correlated to the degree of brain injury. Additionally, we developed a novel analytical procedure to deduce cell blood composition over time. RESULTS: Sixty-one genes were identified with significant altered expression after UCO. In pre-UCO blood, the level of three mRNAs (Trex2, Znf280b, novel miRNA) and in post-UCO, four mRNAs (Fam184a, Angptl2, novel lincRNA and an unknown protein-coding gene) were associated to brain injury (FDR < 0.01). Several of these mRNAs are related to inflammation and angiogenesis. Pathway analysis highlighted genes playing a role in perinatal death and growth failure. Results also indicate that several leukocyte populations undergo significant changes after UCO. CONCLUSION: We have used a whole transcriptomic approach to uncover novel biomarkers in fetal blood that correlate to neuropathology in the preterm sheep brain. The current data forms a basis for future studies to investigate mechanisms of these mRNAs in the injury progression. IMPACT: Trend analysis of genes following asphyxia reveal a group of genes associated with perinatal death and growth failure. Several pre-asphyxia transcripts were associated to brain injury severity suggesting genomic susceptibility to injury. Several post-asphyxia transcripts were correlated to brain injury severity, thus, serve as potential novel biomarkers of injury outcome. Successfully adaptation of cell profiling algorithms suggests significant changes in blood cell composition following asphyxia.

Analysis of archaic human haplotypes suggests that 5hmC acts as an epigenetic guide for NCO recombination.

BACKGROUND: Non-crossover (NCO) refers to a mechanism of homologous recombination in which short tracks of DNA are copied between homologue chromatids. The allelic changes are typically restricted to one or few SNPs, which potentially allow for the gradual adaptation and maturation of haplotypes. It is assumed to be a stochastic process but the analysis of archaic and modern human haplotypes revealed a striking variability in local NCO recombination rates. METHODS: NCO recombination rates of 1.9 million archaic SNPs shared with Denisovan hominids were defined by a linkage study and correlated with functional and genomic annotations as well as ChIP-Seq data from modern humans. RESULTS: We detected a strong correlation between NCO recombination rates and the function of the respective region: low NCO rates were evident in introns and quiescent intergenic regions but high rates in splice sites, exons, 5'- and 3'-UTRs, as well as CpG islands. Correlations with ChIP-Seq data from ENCODE and other public sources further identified epigenetic modifications that associated directly with these recombination events. A particularly strong association was observed for 5-hydroxymethylcytosine marks (5hmC), which were enriched in virtually all of the functional regions associated with elevated NCO rates, including CpG islands and 'poised' bivalent regions. CONCLUSION: Our results suggest that 5hmC marks may guide the NCO machinery specifically towards functionally relevant regions and, as an intermediate of oxidative demethylation, may open a pathway for environmental influence by specifically targeting recently opened gene loci.

Deep phenotyping of Tregs identifies an immune signature for idiopathic aplastic anemia and predicts response to treatment.

Idiopathic aplastic anemia (AA) is an immune-mediated and serious form of bone marrow failure. Akin to other autoimmune diseases, we have previously shown that in AA regulatory T cells (Tregs) are reduced in number and function. The aim of this study was to further characterize Treg subpopulations in AA and investigate the potential correlation between specific Treg subsets and response to immunosuppressive therapy (IST) as well as their in vitro expandability for potential clinical use. Using mass cytometry and an unbiased multidimensional analytical approach, we identified 2 specific human Treg subpopulations (Treg A and Treg B) with distinct phenotypes, gene expression, expandability, and function. Treg B predominates in IST responder patients, has a memory/activated phenotype (with higher expression of CD95, CCR4, and CD45RO within FOXP3(hi), CD127(lo) Tregs), expresses the interleukin-2 (IL-2)/STAT5 pathway and cell-cycle commitment genes. Furthermore, in vitro-expanded Tregs become functional and take on the characteristics of Treg B. Collectively, this study identifies human Treg subpopulations that can be used as predictive biomarkers for response to IST in AA and potentially other autoimmune diseases. We also show that Tregs from AA patients are IL-2-sensitive and expandable in vitro, suggesting novel therapeutic approaches such as low-dose IL-2 therapy and/or expanded autologous Tregs and meriting further exploration.

Cell Specific eQTL Analysis without Sorting Cells.

The functional consequences of trait associated SNPs are often investigated using expression quantitative trait locus (eQTL) mapping. While trait-associated variants may operate in a cell-type specific manner, eQTL datasets for such cell-types may not always be available. We performed a genome-environment interaction (GxE) meta-analysis on data from 5,683 samples to infer the cell type specificity of whole blood cis-eQTLs. We demonstrate that this method is able to predict neutrophil and lymphocyte specific cis-eQTLs and replicate these predictions in independent cell-type specific datasets. Finally, we show that SNPs associated with Crohn's disease preferentially affect gene expression within neutrophils, including the archetypal NOD2 locus.

Cluster stability in the analysis of mass cytometry data.

Manual gating has been traditionally applied to cytometry data sets to identify cells based on protein expression. The advent of mass cytometry allows for a higher number of proteins to be simultaneously measured on cells, therefore providing a means to define cell clusters in a high dimensional expression space. This enhancement, whilst opening unprecedented opportunities for single cell-level analyses, makes the incremental replacement of manual gating with automated clustering a compelling need. To this aim many methods have been implemented and their successful applications demonstrated in different settings. However, the reproducibility of automatically generated clusters is proving challenging and an analytical framework to distinguish spurious clusters from more stable entities, and presumably more biologically relevant ones, is still missing. One way to estimate cell clusters' stability is the evaluation of their consistent re-occurrence within- and between-algorithms, a metric that is commonly used to evaluate results from gene expression. Herein we report the usage and importance of cluster stability evaluations, when applied to results generated from three popular clustering algorithms - SPADE, FLOCK and PhenoGraph - run on four different data sets. These algorithms were shown to generate clusters with various degrees of statistical stability, many of them being unstable. By comparing the results of automated clustering with manually gated populations, we illustrate how information on cluster stability can assist towards a more rigorous and informed interpretation of clustering results. We also explore the relationships between statistical stability and other properties such as clusters' compactness and isolation, demonstrating that whilst cluster stability is linked to other properties it cannot be reliably predicted by any of them. Our study proposes the introduction of cluster stability as a necessary checkpoint for cluster interpretation and contributes to the construction of a more systematic and standardized analytical framework for the assessment of cytometry clustering results. © 2016 International Society for Advancement of Cytometry.

Phenotypic Complexity of the Human Regulatory T Cell Compartment Revealed by Mass Cytometry.

Regulatory T cells (Tregs) are an essential component of the cellular immune response, occupying a key role in maintaining immunological tolerance and present an attractive therapeutic target in a range of immunopathologies. Comprehensive analysis of the human Treg compartment has been restricted due to technical limitations. The advent of mass cytometry enables simultaneous assessment of vastly increased phenotypic parameters at single-cell resolution. In this study, we used mass cytometry to examine the complexity of human Tregs using an extensive panel of surface markers associated with Treg function and phenotype. We applied unsupervised clustering analysis, revealing 22 distinct subpopulations of Tregs, representing previously identified and novel subpopulations. Our data represent the most in-depth phenotypic description of the human Treg compartment at single-cell resolution and show a hitherto unrecognized degree of phenotypic complexity among cells of the regulatory lineage.

Genetic analysis of an allergic rhinitis cohort reveals an intercellular epistasis between FAM134B and CD39.

BACKGROUND: Extracellular ATP is a pro-inflammatory molecule released by damaged cells. Regulatory T cells (Treg) can suppress inflammation by hydrolysing this molecule via ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1), also termed as CD39. Multiple studies have reported differences in CD39+ Treg percentages in diseases such as multiple sclerosis, Hepatitis B and HIV-1. In addition, CD39 polymorphisms have been implicated in immune-phenotypes such as susceptibility to inflammatory bowel disease and AIDS progression. However none of the studies published so far has linked disease-associated variants with differences in CD39 Treg surface expression. This study aims at identifying variants affecting CD39 expression on Treg and at evaluating their association with allergic rhinitis, a disease characterized by a strong Treg involvement. METHODS: Cohorts consisting of individuals of different ethnicities were employed to identify any association of CD39 variants to surface expression. Significant variant(s) were tested for disease association in a published GWAS cohort by one-locus and two-locus genetic analyses based on logistic models. Further functional characterization was performed using existing microarray data and quantitative RT-PCR on sorted cells. RESULTS: Our study shows that rs7071836, a promoter SNP in the CD39 gene region, affects the cell surface expression on Treg cells but not on other CD39+ leukocyte subsets. Epistasis analysis revealed that, in conjunction with a SNP upstream of the FAM134B gene (rs257174), it increased the risk of allergic rhinitis (P = 1.98 × 10-6). As a promoter SNP, rs257174 controlled the expression of the gene in monocytes but, notably, not in Treg cells. Whole blood transcriptome data of three large cohorts indicated an inverse relation in the expression of the two proteins. While this observation was in line with the epistasis data, it also implied that a functional link must exist. Exposure of monocytes to extracellular ATP resulted in an up-regulation of FAM134B gene expression, suggesting that extracellular ATP released from damaged cells represents the connection for the biological interaction of CD39 on Treg cells with FAM134B on monocytes. CONCLUSIONS: The interplay between promoter SNPs of CD39 and FAM134B results in an intercellular epistasis which influences the risk of a complex inflammatory disease.

The cholesterol biosynthesis pathway regulates IL-10 expression in human Th1 cells.

The mechanisms controlling CD4+ T cell switching from an effector to an anti-inflammatory (IL-10+) phenotype play an important role in the persistence of chronic inflammatory diseases. Here, we identify the cholesterol biosynthesis pathway as a key regulator of this process. Pathway analysis of cultured cytokine-producing human T cells reveals a significant association between IL-10 and cholesterol metabolism gene expression. Inhibition of the cholesterol biosynthesis pathway with atorvastatin or 25-hydroxycholesterol during switching from IFNγ+ to IL-10+ shows a specific block in immune resolution, defined as a significant decrease in IL-10 expression. Mechanistically, the master transcriptional regulator of IL10 in T cells, c-Maf, is significantly decreased by physiological levels of 25-hydroxycholesterol. Strikingly, progression to rheumatoid arthritis is associated with altered expression of cholesterol biosynthesis genes in synovial biopsies of predisposed individuals. Our data reveal a link between sterol metabolism and the regulation of the anti-inflammatory response in human CD4+ T cells.

A functional SNP associated with atopic dermatitis controls cell type-specific methylation of the VSTM1 gene locus.

BACKGROUND: Expression quantitative trait loci (eQTL) databases represent a valuable resource to link disease-associated SNPs to specific candidate genes whose gene expression is significantly modulated by the SNP under investigation. We previously identified signal inhibitory receptor on leukocytes-1 (SIRL-1) as a powerful regulator of human innate immune cell function. While it is constitutively high expressed on neutrophils, on monocytes the SIRL-1 surface expression varies strongly between individuals. The underlying mechanism of regulation, its genetic control as well as potential clinical implications had not been explored yet. METHODS: Whole blood eQTL data of a Chinese cohort was used to identify SNPs regulating the expression of VSTM1, the gene encoding SIRL-1. The genotype effect was validated by flow cytometry (cell surface expression), correlated with electrophoretic mobility shift assay (EMSA), chromatin immunoprecipitation (ChIP) and bisulfite sequencing (C-methylation) and its functional impact studied the inhibition of reactive oxygen species (ROS). RESULTS: We found a significant association of a single CpG-SNP, rs612529T/C, located in the promoter of VSTM1. Through flow cytometry analysis we confirmed that primarily in the monocytes the protein level of SIRL-1 is strongly associated with genotype of this SNP. In monocytes, the T allele of this SNP facilitates binding of the transcription factors YY1 and PU.1, of which the latter has been recently shown to act as docking site for modifiers of DNA methylation. In line with this notion rs612529T associates with a complete demethylation of the VSTM1 promoter correlating with the allele-specific upregulation of SIRL-1 expression. In monocytes, this upregulation strongly impacts the IgA-induced production of ROS by these cells. Through targeted association analysis we found a significant Meta P value of 1.14 × 10-6 for rs612529 for association to atopic dermatitis (AD). CONCLUSION: Low expression of SIRL-1 on monocytes is associated with an increased risk for the manifestation of an inflammatory skin disease. It thus underlines the role of both the cell subset and this inhibitory immune receptor in maintaining immune homeostasis in the skin. Notably, the genetic regulation is achieved by a single CpG-SNP, which controls the overall methylation state of the promoter gene segment.

Loss of TLR3 aggravates CHIKV replication and pathology due to an altered virus-specific neutralizing antibody response.

RNA-sensing toll-like receptors (TLRs) mediate innate immunity and regulate anti-viral response. We show here that TLR3 regulates host immunity and the loss of TLR3 aggravates pathology in Chikungunya virus (CHIKV) infection. Susceptibility to CHIKV infection is markedly increased in human and mouse fibroblasts with defective TLR3 signaling. Up to 100-fold increase in CHIKV load was observed in Tlr3-/- mice, alongside increased virus dissemination and pro-inflammatory myeloid cells infiltration. Infection in bone marrow chimeric mice showed that TLR3-expressing hematopoietic cells are required for effective CHIKV clearance. CHIKV-specific antibodies from Tlr3-/- mice exhibited significantly lower in vitro neutralization capacity, due to altered virus-neutralizing epitope specificity. Finally, SNP genotyping analysis of CHIKF patients on TLR3 identified SNP rs6552950 to be associated with disease severity and CHIKV-specific neutralizing antibody response. These results demonstrate a key role for TLR3-mediated antibody response to CHIKV infection, virus replication and pathology, providing a basis for future development of immunotherapeutics in vaccine development.

Genome-wide analysis of the genetic regulation of gene expression in human neutrophils.

Neutrophils are an abundant immune cell type involved in both antimicrobial defence and autoimmunity. The regulation of their gene expression, however, is still largely unknown. Here we report an eQTL study on isolated neutrophils from 114 healthy individuals of Chinese ethnicity, identifying 21,210 eQTLs on 832 unique genes. Unsupervised clustering analysis of these eQTLs confirms their role in inflammatory responses and immunological diseases but also indicates strong involvement in dermatological pathologies. One of the strongest eQTL identified (rs2058660) is also the tagSNP of a linkage block reported to affect leprosy and Crohn's disease in opposite directions. In a functional study, we can link the C allele with low expression of the ß-chain of IL18-receptor (IL18RAP). In neutrophils, this results in a reduced responsiveness to IL-18, detected both on the RNA and protein level. Thus, the polymorphic regulation of human neutrophils can impact beneficial as well as pathological inflammatory responses.

ArchiLD: hierarchical visualization of linkage disequilibrium in human populations.

Linkage disequilibrium (LD) is an essential metric for selecting single-nucleotide polymorphisms (SNPs) to use in genetic studies and identifying causal variants from significant tag SNPs. The explosion in the number of polymorphisms that can now be genotyped by commercial arrays makes the interpretation of triangular correlation plots, commonly used for visualizing LD, extremely difficult in particular when large genomics regions need to be considered or when SNPs in perfect LD are not adjacent but scattered across a genomic region. We developed ArchiLD, a user-friendly graphical application for the hierarchical visualization of LD in human populations. The software provides a powerful framework for analyzing LD patterns with a particular focus on blocks of SNPs in perfect linkage as defined by r(2). Thanks to its integration with the UCSC Genome Browser, LD plots can be easily overlapped with additional data on regulation, conservation and expression. ArchiLD is an intuitive solution for the visualization of LD across large or highly polymorphic genomic regions. Its ease of use and its integration with the UCSC Genome Browser annotation potential facilitates the interpretation of association results and enables a more informed selection of tag SNPs for genetic studies.