Transmission of trained immunity and heterologous resistance to infections across generations.

Intergenerational inheritance of immune traits linked to epigenetic modifications has been demonstrated in plants and invertebrates. Here we provide evidence for transmission of trained immunity across generations to murine progeny that survived a sublethal systemic infection with Candida albicans or a zymosan challenge. The progeny of trained mice exhibited cellular, developmental, transcriptional and epigenetic changes associated with the bone marrow-resident myeloid effector and progenitor cell compartment. Moreover, the progeny of trained mice showed enhanced responsiveness to endotoxin challenge, alongside improved protection against systemic heterologous Escherichia coli and Listeria monocytogenes infections. Sperm DNA of parental male mice intravenously infected with the fungus C. albicans showed DNA methylation differences linked to immune gene loci. These results provide evidence for inheritance of trained immunity in mammals, enhancing protection against infections.

Peptide-specific recognition of human cytomegalovirus strains controls adaptive natural killer cells.

Natural killer (NK) cells are innate lymphocytes that lack antigen-specific rearranged receptors, a hallmark of adaptive lymphocytes. In some people infected with human cytomegalovirus (HCMV), an NK cell subset expressing the activating receptor NKG2C undergoes clonal-like expansion that partially resembles anti-viral adaptive responses. However, the viral ligand that drives the activation and differentiation of adaptive NKG2C+ NK cells has remained unclear. Here we found that adaptive NKG2C+ NK cells differentially recognized distinct HCMV strains encoding variable UL40 peptides that, in combination with pro-inflammatory signals, controlled the population expansion and differentiation of adaptive NKG2C+ NK cells. Thus, we propose that polymorphic HCMV peptides contribute to shaping of the heterogeneity of adaptive NKG2C+ NK cell populations among HCMV-seropositive people.

Epigenomic Profiling of Human CD4+ T Cells Supports a Linear Differentiation Model and Highlights Molecular Regulators of Memory Development.

The impact of epigenetics on the differentiation of memory T (Tmem) cells is poorly defined. We generated deep epigenomes comprising genome-wide profiles of DNA methylation, histone modifications, DNA accessibility, and coding and non-coding RNA expression in naive, central-, effector-, and terminally differentiated CD45RA+ CD4+ Tmem cells from blood and CD69+ Tmem cells from bone marrow (BM-Tmem). We observed a progressive and proliferation-associated global loss of DNA methylation in heterochromatic parts of the genome during Tmem cell differentiation. Furthermore, distinct gradually changing signatures in the epigenome and the transcriptome supported a linear model of memory development in circulating T cells, while tissue-resident BM-Tmem branched off with a unique epigenetic profile. Integrative analyses identified candidate master regulators of Tmem cell differentiation, including the transcription factor FOXP1. This study highlights the importance of epigenomic changes for Tmem cell biology and demonstrates the value of epigenetic data for the identification of lineage regulators.

Resident memory CD4+ T lymphocytes mobilize from bone marrow to contribute to a systemic secondary immune reaction.

Resident memory T lymphocytes (TRM ) of epithelial tissues and the Bm protect their host tissue. To what extent these cells are mobilized and contribute to systemic immune reactions is less clear. Here, we show that in secondary immune reactions to the measles-mumps-rubella (MMR) vaccine, CD4+ TRM are mobilized into the blood within 16 to 48 h after immunization in humans. This mobilization of TRM is cognate: TRM recognizing other antigens are not mobilized, unless they cross-react with the vaccine. We also demonstrate through methylome analyses that TRM are mobilized from the Bm. These mobilized cells make significant contribution to the systemic immune reaction, as evidenced by their T-cell receptor Vß clonotypes represented among the newly generated circulating memory T-cells, 14 days after vaccination. Thus, TRM of the Bm confer not only local, but also systemic immune memory.

Identification of an FXR-modulated liver-intestine hybrid state in iPSC-derived hepatocyte-like cells.

BACKGROUND & AIMS: Pluripotent stem cell (PSC)-derived hepatocyte-like cells (HLC) have enormous potential as a replacement for primary hepatocytes in drug screening, toxicology and cell replacement therapy, but their genome-wide expression patterns differ strongly from primary human hepatocytes (PHH). METHODS: We differentiated human induced pluripotent stem cells (hiPSC) via definitive endoderm to HLC and characterized the cells by single-cell and bulk RNA-seq, with complementary epigenetic analyses. We then compared HLC to PHH and publicly available data on human fetal hepatocytes (FH) ex vivo; we performed bioinformatics-guided interventions to improve HLC differentiation via lentiviral transduction of the nuclear receptor FXR and agonist exposure. RESULTS: Single-cell RNA-seq revealed that transcriptomes of individual HLC display a hybrid state, where hepatocyte-associated genes are expressed in concert with genes that are not expressed in PHH - mostly intestinal genes - within the same cell. Bulk-level overrepresentation analysis, as well as regulon analysis at the single-cell level, identified sets of regulatory factors discriminating HLC, FH, and PHH, hinting at a central role for the nuclear receptor FXR in the functional maturation of HLC. Combined FXR expression plus agonist exposure enhanced the expression of hepatocyte-associated genes and increased the ability of bile canalicular secretion as well as lipid droplet formation, thereby increasing HLCs' similarity to PHH. The undesired non-liver gene expression was reproducibly decreased, although only by a moderate degree. CONCLUSION: In contrast to physiological hepatocyte precursor cells and mature hepatocytes, HLC co-express liver and hybrid genes in the same cell. Targeted modification of the FXR gene regulatory network improves their differentiation by suppressing intestinal traits whilst inducing hepatocyte features. LAY SUMMARY: Generation of human hepatocytes from stem cells represents an active research field but its success is hampered by the fact that the stem cell-derived 'hepatocytes' still show major differences to hepatocytes obtained from a liver. Here, we identified an important reason for the difference, specifically that the stem cell-derived 'hepatocyte' represents a hybrid cell with features of hepatocytes and intestinal cells. We show that a specific protein (FXR) suppresses intestinal and induces liver features, thus bringing the stem cell-derived cells closer to hepatocytes derived from human livers.

Manganese-Induced Toxicity in C. elegans: What Can We Learn from the Transcriptome?

Manganese (Mn) is an essential ubiquitous transition metal and, when occupationally or environmentally overexposed, a well-known risk factor for several neurological pathologies. However, the molecular mechanisms underlying Mn-induced neurotoxicity are largely unknown. In this study, addressing RNA-Seq analysis, bioavailability and survival assays, key pathways of transcriptional responses to Mn overexposure were investigated in the model organism Caenorhabditis elegans (C. elegans), providing insights into the Mn-induced cellular stress and damage response. Comparative transcriptome analyses identified a large number of differentially expressed genes (DEGs) in nematodes exposed to MnCl2, and functional annotation suggested oxidative nucleotide damage, unfolded protein response and innate immunity as major damage response pathways. Additionally, a time-dependent increase in the transcriptional response after MnCl2 exposure was identified by means of increased numbers of DEGs, indicating a time-dependent response and activation of the stress responses in Mn neurotoxicity. The data provided here represent a powerful transcriptomic resource in the field of Mn toxicity, and therefore, this study provides a useful basis for further planning of targeted mechanistic studies of Mn-induced neurotoxicity that are urgently needed in the face of increasing industrially caused environmental pollution with Mn.

Epigenetic control of region-specific transcriptional programs in mouse cerebellar and cortical astrocytes.

Astrocytes from the cerebral cortex (CTX) and cerebellum (CB) share basic molecular programs, but also form distinct spatial and functional subtypes. The regulatory epigenetic layers controlling such regional diversity have not been comprehensively investigated so far. Here, we present an integrated epigenome analysis of methylomes, open chromatin, and transcriptomes of astroglia populations isolated from the cortex or cerebellum of young adult mice. Besides a basic overall similarity in their epigenomic programs, cortical astrocytes and cerebellar astrocytes exhibit substantial differences in their overall open chromatin structure and in gene-specific DNA methylation. Regional epigenetic differences are linked to differences in transcriptional programs encompassing genes of region-specific transcription factor networks centered around Lhx2/Foxg1 in CTX astrocytes and the Zic/Irx families in CB astrocytes. The distinct epigenetic signatures around these transcription factor networks point to a complex interconnected and combinatorial regulation of region-specific transcriptomes. These findings suggest that key transcription factors, previously linked to temporal, regional, and spatial control of neurogenesis, also form combinatorial networks important for astrocytes. Our study provides a valuable resource for the molecular basis of regional astrocyte identity and physiology.

Neuronal deficiency of p38α-MAPK ameliorates symptoms and pathology of APP or Tau-transgenic Alzheimer's mouse models.

Alzheimer's disease (AD) is the leading cause of dementia with very limited therapeutic options. Amyloid ß (Aß) and phosphorylated Tau (p-Tau) are key pathogenic molecules in AD. P38α-MAPK is specifically activated in AD lesion sites. However, its effects on AD pathogenesis, especially on p-Tau-associated brain pathology, and the underlying molecular mechanisms remain unclear. We mated human APP-transgenic mice and human P301S Tau-transgenic mice with mapk14-floxed and neuron-specific Cre-knock-in mice. We observed that deletion of p38α-MAPK specifically in neurons improves the cognitive function of both 9-month-old APP and Tau-transgenic AD mice, which is associated with decreased Aß and p-Tau load in the brain. We further used next-generation sequencing to analyze the gene transcription in brains of p38α-MAPK deficient and wild-type APP-transgenic mice, which indicated that deletion of p38α-MAPK regulates the transcription of calcium homeostasis-related genes, especially downregulates the expression of grin2a, a gene encoding NMDAR subunit NR2A. Cell culture experiments further verified that deletion of p38α-MAPK inhibits NMDA-triggered calcium influx and neuronal apoptosis. Our systemic studies of AD pathogenic mechanisms using both APP- and Tau-transgenic mice suggested that deletion of neuronal p38α-MAPK attenuates AD-associated brain pathology and protects neurons in AD pathogenesis. This study supports p38α-MAPK as a novel target for AD therapy.

Two Piwis with Ago-like functions silence somatic genes at the chromatin level.

Most sRNA biogenesis mechanisms involve either RNAse III cleavage or ping-pong amplification by different Piwi proteins harbouring slicer activity. Here, we follow the question why the mechanism of transgene-induced silencing in the ciliate Paramecium needs both Dicer activity and two Ptiwi proteins. This pathway involves primary siRNAs produced from non-translatable transgenes and secondary siRNAs from targeted endogenous loci. Our data does not indicate any signatures from ping-pong amplification but Dicer cleavage of long dsRNA. Ptiwi13 and 14 prefer different sub-cellular localizations and different preferences for primary and secondary siRNAs but do not load them mutually exclusive. Both Piwis enrich for antisense RNAs and show a general preference for uridine-rich sRNAs along the entire sRNA length. In addition, Ptiwi14-loaded siRNAs show a 5´-U signature. Our data indicates both Ptiwis and 2´-O-methylation contributing to strand selection of Dicer cleaved siRNAs. This unexpected function of the two distinct vegetative Piwis extends the increasing knowledge of the diversity of Piwi functions in diverse silencing pathways. We describe an unusual mode of action of Piwi proteins extending not only the great variety of Piwi-associated RNAi pathways but moreover raising the question whether this could have been the primordial one.

The sncRNA Zoo: a repository for circulating small noncoding RNAs in animals.

The repertoire of small noncoding RNAs (sncRNAs), particularly miRNAs, in animals is considered to be evolutionarily conserved. Studies on sncRNAs are often largely based on homology-based information, relying on genomic sequence similarity and excluding actual expression data. To obtain information on sncRNA expression (including miRNAs, snoRNAs, YRNAs and tRNAs), we performed low-input-volume next-generation sequencing of 500 pg of RNA from 21 animals at two German zoological gardens. Notably, none of the species under investigation were previously annotated in any miRNA reference database. Sequencing was performed on blood cells as they are amongst the most accessible, stable and abundant sources of the different sncRNA classes. We evaluated and compared the composition and nature of sncRNAs across the different species by computational approaches. While the distribution of sncRNAs in the different RNA classes varied significantly, general evolutionary patterns were maintained. In particular, miRNA sequences and expression were found to be even more conserved than previously assumed. To make the results available for other researchers, all data, including expression profiles at the species and family levels, and different tools for viewing, filtering and searching the data are freely available in the online resource ASRA (Animal sncRNA Atlas) at https://www.ccb.uni-saarland.de/asra/.

Unique and assay specific features of NOMe-, ATAC- and DNase I-seq data.

Chromatin accessibility maps are important for the functional interpretation of the genome. Here, we systematically analysed assay specific differences between DNase I-seq, ATAC-seq and NOMe-seq in a side by side experimental and bioinformatic setup. We observe that most prominent nucleosome depleted regions (NDRs, e.g. in promoters) are roboustly called by all three or at least two assays. However, we also find a high proportion of assay specific NDRs that are often 'called' by only one of the assays. We show evidence that these assay specific NDRs are indeed genuine open chromatin sites and contribute important information for accurate gene expression prediction. While technically ATAC-seq and DNase I-seq provide a superb high NDR calling rate for relatively low sequencing costs in comparison to NOMe-seq, NOMe-seq singles out for its genome-wide coverage allowing to not only detect NDRs but also endogenous DNA methylation and as we show here genome wide segmentation into heterochromatic B domains and local phasing of nucleosomes outside of NDRs. In summary, our comparisons strongly suggest to consider assay specific differences for the experimental design and for generalized and comparative functional interpretations.

Yeast Viral Killer Toxin K1 Induces Specific Host Cell Adaptions via Intrinsic Selection Pressure.

The killer phenomenon in yeast (Saccharomyces cerevisiae) not only provides the opportunity to study host-virus interactions in a eukaryotic model but also represents a powerful tool to analyze potential coadaptional events and the role of killer yeast in biological diversity. Although undoubtedly having a crucial impact on the abundance and expression of the killer phenotype in killer-yeast harboring communities, the influence of a particular toxin on its producing host cell has not been addressed sufficiently. In this study, we describe a model system of two K1 killer yeast strains with distinct phenotypical differences pointing to substantial selection pressure in response to the toxin secretion level. Transcriptome and lipidome analyses revealed specific and intrinsic host cell adaptions dependent on the amount of K1 toxin produced. High basal expression of genes coding for osmoprotectants and stress-responsive proteins in a killer yeast strain secreting larger amounts of active K1 toxin implies a generally increased stress tolerance. Moreover, the data suggest that immunity of the host cell against its own toxin is essential for the balanced virus-host interplay providing valuable hints to elucidate the molecular mechanisms underlying K1 immunity and implicating an evolutionarily conserved role for toxin immunity in natural yeast populations.IMPORTANCE The killer phenotype in Saccharomyces cerevisiae relies on the cytoplasmic persistence of two RNA viruses. In contrast to bacterial toxin producers, killer yeasts necessitate a specific immunity mechanism against their own toxin because they bear the same receptor populations as sensitive cells. Although the killer phenomenon is highly abundant and has a crucial impact on the structure of yeast communities, the influence of a particular toxin on its host cell has been barely addressed. In our study, we used two derivatives secreting different amount of the killer toxin K1 to analyze potential coadaptional events in this particular host/virus system. Our data underline the dependency of the host cell's ability to cope with extracellular toxin molecules and intracellular K1 molecules provided by the virus. Therefore, this research significantly advances the current understanding of the evolutionarily conserved role of this molecular machinery as an intrinsic selection pressure in yeast populations.

Comparative Analysis of Biochemical Biases by Ligation- and Template-Switch-Based Small RNA Library Preparation Protocols.

BACKGROUND: Small RNAs are key players in the regulation of gene expression and differentiation. However, many different classes of small RNAs (sRNAs) have been described with distinct biogenesis pathways and, as a result, with different biochemical properties. To analyze sRNAs by deep sequencing, complementary DNA synthesis requires manipulation of the RNA molecule itself. Thus, enzymatic activities during library preparation bias the library content owing to biochemical criteria. METHODS: We compared 4 different manipulations of RNA for library preparation: (a) a ligation-based procedure allowing only 5'-mono-phosphorylated RNA to enter the library, (b) a ligation-based procedure allowing additional 5'-triphosphates and Cap structures, (c) a ligation-independent, template-switch-based library preparation, and (d) a template-switch-based library preparation allowing 3'-phosphorylated RNAs to enter the library. RESULTS: Our data show large differences between ligation-dependent and ligation-independent libraries in terms of their preference for individual sRNA classes such as microRNAs (miRNAs), Piwi-interacting RNAs (piRNAs), and transfer RNA fragments. Moreover, the miRNA composition is different between both procedures, and more microRNA isoforms (isomiRs) can be identified after pyrophosphatase treatment. piRNAs are enriched in template-switch libraries, and this procedure apparently includes more different RNA species. CONCLUSIONS: Our data indicate that miRNAomics from both methods will hardly be comparable. Ligation-based libraries enrich for canonical miRNAs, which thus may be suitable methods for miRNAomics. Template-switch libraries contain increased numbers and different compositions of fragments and long RNAs. Following different interests for other small RNA species, ligation-independent libraries appear to show a more realistic sRNA landscape with lower bias against biochemical modifications.

Combining transcription factor binding affinities with open-chromatin data for accurate gene expression prediction.

The binding and contribution of transcription factors (TF) to cell specific gene expression is often deduced from open-chromatin measurements to avoid costly TF ChIP-seq assays. Thus, it is important to develop computational methods for accurate TF binding prediction in open-chromatin regions (OCRs). Here, we report a novel segmentation-based method, TEPIC, to predict TF binding by combining sets of OCRs with position weight matrices. TEPIC can be applied to various open-chromatin data, e.g. DNaseI-seq and NOMe-seq. Additionally, Histone-Marks (HMs) can be used to identify candidate TF binding sites. TEPIC computes TF affinities and uses open-chromatin/HM signal intensity as quantitative measures of TF binding strength. Using machine learning, we find low affinity binding sites to improve our ability to explain gene expression variability compared to the standard presence/absence classification of binding sites. Further, we show that both footprints and peaks capture essential TF binding events and lead to a good prediction performance. In our application, gene-based scores computed by TEPIC with one open-chromatin assay nearly reach the quality of several TF ChIP-seq data sets. Finally, these scores correctly predict known transcriptional regulators as illustrated by the application to novel DNaseI-seq and NOMe-seq data for primary human hepatocytes and CD4+ T-cells, respectively.

Next Generation Sequencing Analysis of Total Small Noncoding RNAs from Low Input RNA from Dried Blood Sampling.

Circulating miRNAs are favored for biomarker candidates as they can reflect tissue specific miRNA dysregulation in disease contexts. Moreover, they have the additional advantage that they can be monitored in a minimally invasive manner. Blood-borne miRNAs are therefore currently characterized to identify, describe, and validate their potential suitability as biomarkers; however, sampling and as well miRNA detection methods limit these studies in terms of sensitivity but also practicability in clinical, at-home, or low-resource sampling of high-quality circulating RNA samples. We describe here a novel and innovative method of circulating RNA microsampling from minimal volume dried blood samples with direct enrichment for small RNA fractions in combination with ligation free library preparation. We evaluated crucial parameters for efficient library preparation from low RNA inputs of 50 pg for efficient dissection not only of miRNAs but also isomiRs, piRNAs, and lincRNAs. We compared these data to classical microarrays and characterize the technical reproducibility and its sensitivity. We demonstrate and evaluate a method for easy low resource sampling and NGS analysis of circulating RNAs providing a powerful tool for massive cohort and remote patient monitoring.

Human cytomegalovirus drives epigenetic imprinting of the IFNG locus in NKG2Chi natural killer cells.

Memory type 1 T helper (T(H)1) cells are characterized by the stable expression of interferon (IFN)-γ as well as by the epigenetic imprinting of the IFNG locus. Among innate cells, NK cells play a crucial role in the defense against cytomegalovirus (CMV) and represent the main source of IFN-γ. Recently, it was shown that memory-like features can be observed in NK cell subsets after CMV infection. However, the molecular mechanisms underlying NK cell adaptive properties have not been completely defined. In the present study, we demonstrated that only NKG2Chi NK cells expanded in human CMV (HCMV) seropositive individuals underwent epigenetic remodeling of the IFNG conserved non-coding sequence (CNS) 1, similar to memory CD8(+) T cells or T(H)1 cells. The accessibility of the CNS1 was required to enhance IFN-γ transcriptional activity in response to NKG2C and 2B4 engagement, which led to consistent IFN-γ production in NKG2C(hi) NK cells. Thus, our data identify epigenetic imprinting of the IFNG locus as selective hallmark and crucial mechanism driving strong and stable IFN-γ expression in HCMV-specific NK cell expansions, providing a molecular basis for the regulation of adaptive features in innate cells.