Characterizing intergenic transcription at RNA polymerase II binding sites in normal and cancer tissues.

Intergenic transcription in normal and cancerous tissues is pervasive but incompletely understood. To investigate this, we constructed an atlas of over 180,000 consensus RNA polymerase II (RNAPII)-bound intergenic regions from 900 RNAPII chromatin immunoprecipitation sequencing (ChIP-seq) experiments in normal and cancer samples. Through unsupervised analysis, we identified 51 RNAPII consensus clusters, many of which mapped to specific biotypes and revealed tissue-specific regulatory signatures. We developed a meta-clustering methodology to integrate our RNAPII atlas with active transcription across 28,797 RNA sequencing (RNA-seq) samples from The Cancer Genome Atlas (TCGA), Genotype-Tissue Expression (GTEx), and Encyclopedia of DNA Elements (ENCODE). This analysis revealed strong tissue- and disease-specific interconnections between RNAPII occupancy and transcriptional activity. We demonstrate that intergenic transcription at RNAPII-bound regions is a novel per-cancer and pan-cancer biomarker. This biomarker displays genomic and clinically relevant characteristics, distinguishing cancer subtypes and linking to overall survival. Our results demonstrate the effectiveness of coherent data integration to uncover intergenic transcriptional activity in normal and cancer tissues.

Peyer's patch phagocytes acquire specific transcriptional programs that influence their maturation and activation profiles.

Peyer's patches (PPs) are secondary lymphoid organs in contact with the external environment via the intestinal lumen, thus combining antigen sampling and immune response initiation sites. Therefore, they provide a unique opportunity to study the entire process of phagocyte differentiation and activation in vivo. Here, we deciphered the transcriptional and spatial landscape of PP phagocyte populations from their emergence in the tissue to their final maturation state at homeostasis and under stimulation. Activation of monocyte-derived Lysozyme-expressing dendritic cells (LysoDCs) differs from that of macrophages by their upregulation of conventional DC (cDC) signature genes such as Ccr7 and downregulation of typical monocyte-derived cell genes such as Cx3cr1. We identified gene sets that distinguish PP cDCs from the villus ones and from LysoDCs. We also identified key immature, early, intermediate, and late maturation markers of PP phagocytes. Finally, exploiting the ability of the PP interfollicular region to host both villous and subepithelial dome emigrated cDCs, we showed that the type of stimulus, the subset, but also the initial location of cDCs shape their activation profile and thus direct the immune response. Our study highlights the importance of targeting the right phagocyte subset at the right place and time to manipulate the immune response.

Meningeal macrophages protect against viral neuroinfection.

The surface of the central nervous system (CNS) is protected by the meninges, which contain a dense network of meningeal macrophages (MMs). Here, we examined the role of tissue-resident MM in viral infection. MHC-II- MM were abundant neonatally, whereas MHC-II+ MM appeared over time. These barrier macrophages differentially responded to in vivo peripheral challenges such as LPS, SARS-CoV-2, and lymphocytic choriomeningitis virus (LCMV). Peripheral LCMV infection, which was asymptomatic, led to a transient infection and activation of the meninges. Mice lacking macrophages but conserving brain microglia, or mice bearing macrophage-specific deletion of Stat1 or Ifnar, exhibited extensive viral spread into the CNS. Transcranial pharmacological depletion strategies targeting MM locally resulted in several areas of the meninges becoming infected and fatal meningitis. Low numbers of MHC-II+ MM, which is seen upon LPS challenge or in neonates, corelated with higher viral load upon infection. Thus, MMs protect against viral infection and may present targets for therapeutic manipulation.

Blood DNA methylation marks discriminate Chagas cardiomyopathy disease clinical forms.

Chagas disease is a parasitic disease from South America, affecting around 7 million people worldwide. Decades after the infection, 30% of people develop chronic forms, including Chronic Chagas Cardiomyopathy (CCC), for which no treatment exists. Two stages characterized this form: the moderate form, characterized by a heart ejection fraction (EF) ≥ 0.4, and the severe form, associated to an EF < 0.4. We propose two sets of DNA methylation biomarkers which can predict in blood CCC occurrence, and CCC stage. This analysis, based on machine learning algorithms, makes predictions with more than 95% accuracy in a test cohort. Beyond their predictive capacity, these CpGs are located near genes involved in the immune response, the nervous system, ion transport or ATP synthesis, pathways known to be deregulated in CCCs. Among these genes, some are also differentially expressed in heart tissues. Interestingly, the CpGs of interest are tagged to genes mainly involved in nervous and ionic processes. Given the close link between methylation and gene expression, these lists of CpGs promise to be not only good biomarkers, but also good indicators of key elements in the development of this pathology.

Type 1 Innate Lymphoid Cells Limit the Antitumoral Immune Response.

Natural killer (NK) cells are known to be able to kill established tumor cell lines, but important caveats remain regarding their roles in the detection and elimination of developing primary tumors. Using a genetic model of selective ILC1 and NK cell deficiency, we showed that these cells were dispensable for tumor immunosurveillance and immunoediting in the MCA-induced carcinogenesis model. However, we were able to generate primary cell lines derived from MCA-induced tumors with graded sensitivity to NK1.1+ cells (including NK cells and ILC1). This differential sensitivity was associated neither with a modulation of intratumoral NK cell frequency, nor the capacity of tumor cells to activate NK cells. Instead, ILC1 infiltration into the tumor was found to be a critical determinant of NK1.1+ cell-dependent tumor growth. Finally, bulk tumor RNAseq analysis identified a gene expression signature associated with tumor sensitivity to NK1.1+ cells. ILC1 therefore appear to play an active role in inhibiting the antitumoral immune response, prompting to evaluate the differential tumor infiltration of ILC1 and NK cells in patients to optimize the harnessing of immunity in cancer therapies.

Distinct Waves from the Hemogenic Endothelium Give Rise to Layered Lymphoid Tissue Inducer Cell Ontogeny.

During embryogenesis, lymphoid tissue inducer (LTi) cells are essential for lymph node organogenesis. These cells are part of the innate lymphoid cell (ILC) family. Although their earliest embryonic hematopoietic origin is unclear, other innate immune cells have been shown to be derived from early hemogenic endothelium in the yolk sac as well as the aorta-gonad-mesonephros. A proper model to discriminate between these locations was unavailable. In this study, using a Cxcr4-CreERT2 lineage tracing model, we identify a major contribution from embryonic hemogenic endothelium, but not the yolk sac, toward LTi progenitors. Conversely, embryonic LTi cells are replaced by hematopoietic stem cell-derived cells in adults. We further show that, in the fetal liver, common lymphoid progenitors differentiate into highly dynamic alpha-lymphoid precursor cells that, at this embryonic stage, preferentially mature into LTi precursors and establish their functional LTi cell identity only after reaching the periphery.

Reticular Fibroblasts Expressing the Transcription Factor WT1 Define a Stromal Niche that Maintains and Replenishes Splenic Red Pulp Macrophages.

Located within red pulp cords, splenic red pulp macrophages (RPMs) are constantly exposed to the blood flow, clearing senescent red blood cells (RBCs) and recycling iron from hemoglobin. Here, we studied the mechanisms underlying RPM homeostasis, focusing on the involvement of stromal cells as these cells perform anchoring and nurturing macrophage niche functions in lymph nodes and liver. Microscopy revealed that RPMs are embedded in a reticular meshwork of red pulp fibroblasts characterized by the expression of the transcription factor Wilms' Tumor 1 (WT1) and colony stimulating factor 1 (CSF1). Conditional deletion of Csf1 in WT1+ red pulp fibroblasts, but not white pulp fibroblasts, drastically altered the RPM network without altering circulating CSF1 levels. Upon RPM depletion, red pulp fibroblasts transiently produced the monocyte chemoattractants CCL2 and CCL7, thereby contributing to the replenishment of the RPM network. Thus, red pulp fibroblasts anchor and nurture RPM, a function likely conserved in humans.

Differentiation Paths of Peyer's Patch LysoDCs Are Linked to Sampling Site Positioning, Migration, and T Cell Priming.

The monocyte-derived phagocytes termed LysoDCs are hallmarks of Peyer's patches, where their main function is to sample intestinal microorganisms. Here, we study their differentiation pathways in relation with their sampling, migratory, and T cell-priming abilities. Among four identified LysoDC differentiation stages displaying similar phagocytic activity, one is located in follicles, and the others reside in subepithelial domes (SED), where they proliferate and mature as they get closer to the epithelium. Mature LysoDCs but not macrophages express a gene set in common with conventional dendritic cells and prime naive helper T cells in vitro. At steady state, they do not migrate into naive T cell-enriched interfollicular regions (IFRs), but upon stimulation, they express the chemokine receptor CCR7 and migrate from SED to the IFR periphery, where they strongly interact with proliferative immune cells. Finally, we show that LysoDCs populate human Peyer's patches, strengthening their interest as targets for modulating intestinal immunity.

High-Dimensional Single-Cell Analysis Identifies Organ-Specific Signatures and Conserved NK Cell Subsets in Humans and Mice.

Natural killer (NK) cells are innate lymphoid cells (ILCs) involved in antimicrobial and antitumoral responses. Several NK cell subsets have been reported in humans and mice, but their heterogeneity across organs and species remains poorly characterized. We assessed the diversity of human and mouse NK cells by single-cell RNA sequencing on thousands of individual cells isolated from spleen and blood. Unbiased transcriptional clustering revealed two distinct signatures differentiating between splenic and blood NK cells. This analysis at single-cell resolution identified three subpopulations in mouse spleen and four in human spleen, and two subsets each in mouse and human blood. A comparison of transcriptomic profiles within and between species highlighted the similarity of the two major subsets, NK1 and NK2, across organs and species. This unbiased approach provides insight into the biology of NK cells and establishes a rationale for the translation of mouse studies to human physiology and disease.

Human germinal center transcriptional programs are de-synchronized in B cell lymphoma.

Most adult B cell lymphomas originate from germinal center (GC) B cells, but it is unclear to what extent B cells in overt lymphoma retain the functional dynamics of GC B cells or are blocked at a particular stage of the GC reaction. Here we used integrative single-cell analysis of phenotype, gene expression and variable-region sequence of the immunoglobulin heavy-chain locus to track the characteristic human GC B cell program in follicular lymphoma B cells. By modeling the cyclic continuum of GC B cell transitional states, we identified characteristic patterns of synchronously expressed gene clusters. GC-specific gene-expression synchrony was lost in single lymphoma B cells. However, distinct follicular lymphoma-specific cell states co-existed within single patient biopsies. Our data show that lymphoma B cells are not blocked in a GC B cell state but might adopt new dynamic modes of functional diversity, which opens the possibility of novel definitions of lymphoma identity.

Perturbed human sub-networks by Fusobacterium nucleatum candidate virulence proteins.

BACKGROUND: Fusobacterium nucleatum is a gram-negative anaerobic species residing in the oral cavity and implicated in several inflammatory processes in the human body. Although F. nucleatum abundance is increased in inflammatory bowel disease subjects and is prevalent in colorectal cancer patients, the causal role of the bacterium in gastrointestinal disorders and the mechanistic details of host cell functions subversion are not fully understood. RESULTS: We devised a computational strategy to identify putative secreted F. nucleatum proteins (FusoSecretome) and to infer their interactions with human proteins based on the presence of host molecular mimicry elements. FusoSecretome proteins share similar features with known bacterial virulence factors thereby highlighting their pathogenic potential. We show that they interact with human proteins that participate in infection-related cellular processes and localize in established cellular districts of the host-pathogen interface. Our network-based analysis identified 31 functional modules in the human interactome preferentially targeted by 138 FusoSecretome proteins, among which we selected 26 as main candidate virulence proteins, representing both putative and known virulence proteins. Finally, six of the preferentially targeted functional modules are implicated in the onset and progression of inflammatory bowel diseases and colorectal cancer. CONCLUSIONS: Overall, our computational analysis identified candidate virulence proteins potentially involved in the F. nucleatum-human cross-talk in the context of gastrointestinal diseases.

Guanabenz Prevents d-Galactosamine/Lipopolysaccharide-Induced Liver Damage and Mortality.

Multi-organ failure in response to uncontrolled microbial infection is characterized by low blood pressure accompanied by a systemic over-inflammation state, caused by massive pro-inflammatory cytokines release and liver damage. Recently, the integrated stress response (ISR), characterized by eukaryotic translation initiation factor 2α (eIF2α) phosphorylation, was involved with controlling apoptosis in stressed hepatocytes and associated with poor survival to endotoxin challenge. Lipopolysaccharide (LPS) alone is able to induce the ISR in hepatocytes and can trigger massive liver damage along with tumor necrosis factor-alpha (TNF-α) expression. Consequently, drugs interfering with eIF2α phosphorylation may represent potential candidates for the treatment of such pathologies. We, therefore, used Guanabenz (GBZ), a small compound with enhancing eIF2α phosphorylation activity to evaluate its effect on bacterial LPS sensing and endotoxemia. GBZ is confirmed here to have an anti-inflammatory activity by increasing in vitro interleukin-10 (IL-10) production by LPS-stimulated dendritic cells. We further show that in the d-galactosamine (d-galN)/LPS-dependent lethality model, intraperitoneal injection of GBZ promoted mice survival, prevented liver damage, increased IL-10 levels, and inhibited TNF-α production. GBZ and its derivatives could therefore represent an interesting pharmacological solution to control systemic inflammation and associated acute liver failure.

Tyrosine phosphorylation of RNA polymerase II CTD is associated with antisense promoter transcription and active enhancers in mammalian cells.

In mammals, the carboxy-terminal domain (CTD) of RNA polymerase (Pol) II consists of 52 conserved heptapeptide repeats containing the consensus sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7. Post-translational modifications of the CTD coordinate the transcription cycle and various steps of mRNA maturation. Here we describe Tyr1 phosphorylation (Tyr1P) as a hallmark of promoter (5' associated) Pol II in mammalian cells, in contrast to what was described in yeast. Tyr1P is predominantly found in antisense orientation at promoters but is also specifically enriched at active enhancers. Mutation of Tyr1 to phenylalanine (Y1F) prevents the formation of the hyper-phosphorylated Pol IIO form, induces degradation of Pol II to the truncated Pol IIB form, and results in a lethal phenotype. Our results suggest that Tyr1P has evolved specialized and essential functions in higher eukaryotes associated with antisense promoter and enhancer transcription, and Pol II stability.DOI: http://dx.doi.org/10.7554/eLife.02105.001.