Rapid unleashing of macrophage efferocytic capacity via transcriptional pause release.

During development, inflammation or tissue injury, macrophages may successively engulf and process multiple apoptotic corpses via efferocytosis to achieve tissue homeostasis1. How macrophages may rapidly adapt their transcription to achieve continuous corpse uptake is incompletely understood. Transcriptional pause/release is an evolutionarily conserved mechanism, in which RNA polymerase (Pol) II initiates transcription for 20-60 nucleotides, is paused for minutes to hours and is then released to make full-length mRNA2. Here we show that macrophages, within minutes of corpse encounter, use transcriptional pause/release to unleash a rapid transcriptional response. For human and mouse macrophages, the Pol II pause/release was required for continuous efferocytosis in vitro and in vivo. Interestingly, blocking Pol II pause/release did not impede Fc receptor-mediated phagocytosis, yeast uptake or bacterial phagocytosis. Integration of data from three genomic approaches-precision nuclear run-on sequencing, RNA sequencing, and assay for transposase-accessible chromatin using sequencing (ATAC-seq)-on efferocytic macrophages at different time points revealed that Pol II pause/release controls expression of select transcription factors and downstream target genes. Mechanistic studies on transcription factor EGR3, prominently regulated by pause/release, uncovered EGR3-related reprogramming of other macrophage genes involved in cytoskeleton and corpse processing. Using lysosomal probes and a new genetic fluorescent reporter, we identify a role for pause/release in phagosome acidification during efferocytosis. Furthermore, microglia from egr3-deficient zebrafish embryos displayed reduced phagocytosis of apoptotic neurons and fewer maturing phagosomes, supporting defective corpse processing. Collectively, these data indicate that macrophages use Pol II pause/release as a mechanism to rapidly alter their transcriptional programs for efficient processing of the ingested apoptotic corpses and for successive efferocytosis.

Enhanced epigenetic profiling of classical human monocytes reveals a specific signature of healthy aging in the DNA methylome.

The impact of healthy aging on molecular programming of immune cells is poorly understood. Here, we report comprehensive characterization of healthy aging in human classical monocytes, with a focus on epigenomic, transcriptomic, and proteomic alterations, as well as the corresponding proteomic and metabolomic data for plasma, using healthy cohorts of 20 young and 20 older males (~27 and ~64 years old on average). For each individual, we performed eRRBS-based DNA methylation profiling, which allowed us to identify a set of age-associated differentially methylated regions (DMRs) - a novel, cell-type specific signature of aging in DNA methylome. Hypermethylation events were associated with H3K27me3 in the CpG islands near promoters of lowly-expressed genes, while hypomethylated DMRs were enriched in H3K4me1 marked regions and associated with age-related increase of expression of the corresponding genes, providing a link between DNA methylation and age-associated transcriptional changes in primary human cells.

Semi-supervised peak calling with SPAN and JBR genome browser.

The widespread application of ChIP-seq led to a growing need for consistent analysis of multiple epigenetics profiles, for instance, in human studies where multiple replicates are a common element of design. Such multi-samples experimental designs introduced analytical and computational challenges. For example, when peak calling is done independently for each sample, small differences in signal strength/quality lead to a very different number of peaks for individual samples, making group-level analysis difficult. On the other side, when samples are pooled together for joint analysis, individual-level statistical differences are averaged out. Recently, we have demonstrated that a semi-supervised peak calling approach (SPAN) allows for robust analysis of multiple epigenetic profiles while preserving individual sample statistics. Here, we present this approach's implementation, centered around the JBR genome browser, a stand-alone tool that allows for accessible and streamlined annotation, analysis and visualization. Specifically, JBR supports graphical interactive manual region selection and annotation, thereby addressing supervised learning's key procedural challenge. Furthermore, JBR includes the capability for peak optimization, i.e. calibration of sample-specific peak calling parameters by leveraging manual annotation. This procedure can be applied to a broad range of ChIP-seq datasets of different quality and chromatin accessibility ATAC-seq, including single-cell experiments. JBR was designed for efficient data processing, resulting in fast viewing and analysis of multiple replicates, up to thousands of tracks. Accelerated execution and integrated semi-supervised peak calling make JBR and SPAN next-generation visualization and analysis tools for multi-sample epigenetic data. AVAILABILITY AND IMPLEMENTATION: SPAN and JBR run on Linux, Mac OS and Windows, and is freely available at https://research.jetbrains.org/groups/biolabs/tools/span-peak-analyzer and https://research.jetbrains.org/groups/biolabs/tools/jbr-genome-browser. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Comprehensive Profiling of an Aging Immune System Reveals Clonal GZMK+ CD8+ T Cells as Conserved Hallmark of Inflammaging.

Systematic understanding of immune aging on a whole-body scale is currently lacking. We characterized age-associated alterations in immune cells across multiple mouse organs using single-cell RNA and antigen receptor sequencing and flow cytometry-based validation. We defined organ-specific and common immune alterations and identified a subpopulation of age-associated granzyme K (GZMK)-expressing CD8+ T (Taa) cells that are distinct from T effector memory (Tem) cells. Taa cells were highly clonal, had specific epigenetic and transcriptional signatures, developed in response to an aged host environment, and expressed markers of exhaustion and tissue homing. Activated Taa cells were the primary source of GZMK, which enhanced inflammatory functions of non-immune cells. In humans, proportions of the circulating GZMK+CD8+ T cell population that shares transcriptional and epigenetic signatures with mouse Taa cells increased during healthy aging. These results identify GZMK+ Taa cells as a potential target to address age-associated dysfunctions of the immune system.

Bhlhe40 is an essential repressor of IL-10 during Mycobacterium tuberculosis infection.

The cytokine IL-10 antagonizes pathways that control Mycobacterium tuberculosis (Mtb) infection. Nevertheless, the impact of IL-10 during Mtb infection has been difficult to decipher because loss-of-function studies in animal models have yielded only mild phenotypes. We have discovered that the transcription factor basic helix-loop-helix family member e40 (Bhlhe40) is required to repress Il10 expression during Mtb infection. Loss of Bhlhe40 in mice results in higher Il10 expression, higher bacterial burden, and early susceptibility similar to that observed in mice lacking IFN-γ. Deletion of Il10 in Bhlhe40-/- mice reverses these phenotypes. Bhlhe40 deletion in T cells or CD11c+ cells is sufficient to cause susceptibility to Mtb Bhlhe40 represents the first transcription factor found to be essential during Mtb infection to specifically regulate Il10 expression, revealing the importance of strict control of IL-10 production by innate and adaptive immune cells during infection. Our findings uncover a previously elusive but significant role for IL-10 in Mtb pathogenesis.