p16High immune cell - controlled disease tolerance as a broad defense and healthspan extending strategy.

The ability of an organism to overcome infectious diseases has traditionally been linked to killing invading pathogens. Accumulating evidence, however, indicates that, apart from restricting pathogen loads, organismal survival is coupled to an additional yet poorly understood mechanism called disease tolerance. Here we report that p16High immune cells play a key role in establishing disease tolerance. We found that the FDA-approved BNT162b2 mRNA COVID-19 vaccine is a potent and rapid inducer of p16High immune subsets both in mice and humans. In turn, p16High immune cells were indispensable for counteracting different lethal conditions, including LPS-induced sepsis, acute SARS-CoV-2 infection and ionizing irradiation. Mechanistically, we propose that activation of TLR7 or a low physiological activity of STING is sufficient to induce p16High immune subset that, in turn, establishes a low adenosine environment and disease tolerance. Furthermore, containing these signals within a beneficial range by deleting MDA5 that appeared sufficient to maintain a low activity of STING, induces p16High immune cells and delays organ deterioration upon aging with improved healthspan. Our data highlight the beneficial role of p16High immune subsets in establishing a low adenosine environment and disease tolerance.

Assessment of a novel NRAS in-frame tandem duplication causing a myelodysplastic/myeloproliferative neoplasm.

Myelodysplastic/myeloproliferative diseases of childhood cause a relevant disease burden, and many of these diseases may have a fatal course. The use of next-generation sequencing (NGS) has led to the identification of novel genetic variants in patients with these diseases, advancing our understanding of the underlying pathophysiology. However, novel mutations can often only be interpreted as variants of unknown significance (VUS), hindering adequate diagnosis and the use of a targeted therapy. To improve variant interpretation and test targeted therapies in a preclinical setting, we are using a rapid zebrafish embryo model that allows functional evaluation of the novel variant and possible therapeutic approaches within days. Thereby, we accelerate the translation from genetic findings to treatment options. Here, we establish this workflow on a novel in-frame tandem duplication in NRAS (c.192_227dup; p.G75_E76insDS65_G75) identified by Sanger sequencing in a 2.5-year-old patient with an unclassifiable myelodysplastic/myeloproliferative neoplasm (MDS/MPN-U). We show that this variant results in a myeloproliferative phenotype in zebrafish embryos with expansion of immature myeloid cells in the caudal hematopoietic tissue, which can be reversed by MEK inhibition. Thus, we could reclassify the variant from likely pathogenic to pathogenic using the American College of Medical Genetics (ACMG) criteria.

Nucleic acid-induced inflammation on hematopoietic stem cells.

Hematopoiesis, the process of generating blood cells, starts during development with the primitive, pro-definitive, and definitive hematopoietic waves. The first two waves will generate erythrocytes and myeloid cells, although the definitive wave will give rise to hematopoietic stem cells (HSCs) that are multipotent and can produce most of the blood cells in an adult. Although HSCs are highly proliferative during development, during adulthood they remain quiescent in the bone marrow. Inflammatory signaling in the form of interferons, interleukins, tumor necrosis factors, and others is well-established to influence both developmental and adult hematopoiesis. Here we discuss the role of specific inflammatory pathways that are induced by sensing nucleic acids. We discuss the role of RNA-sensing members of the Toll-like, Rig-I-like, nucleotide-binding oligomerization domain (NOD)-like, and AIM2-like protein kinase receptors and the DNA-sensing receptors, DEAD-Box helicase 41 (DDX41) and cGAS. The main downstream pathways of these receptors are discussed, as well as their influence on developmental and adult hematopoiesis, including hematopoietic pathologies.

Transposable elements in normal and malignant hematopoiesis.

Transposable elements (TEs) are dispersed repetitive DNA sequences that can move within a genome. Even though hundreds of years of evolution have led to the accumulation of mutations that render most TEs unable to transpose, they still exert multiple important functions. They play a role in hematopoiesis, especially during periods of high cellular plasticity, such as development, regeneration and aging. This is because TEs can populate functional elements, such as enhancers. Furthermore, TE RNA can be sensed by innate immune sensors that play a role in inflammation and inflammaging. TEs also play an important role in different aspects of leukemia and lymphoma, leading to either beneficial or detrimental outcomes. Further studies into the function of TEs in healthy or diseased hematopoietic systems are necessary to manipulate them for therapeutic benefit.

TFEB induces mitochondrial itaconate synthesis to suppress bacterial growth in macrophages.

Successful elimination of bacteria in phagocytes occurs in the phago-lysosomal system, but also depends on mitochondrial pathways. Yet, how these two organelle systems communicate is largely unknown. Here we identify the lysosomal biogenesis factor transcription factor EB (TFEB) as regulator for phago-lysosome-mitochondria crosstalk in macrophages. By combining cellular imaging and metabolic profiling, we find that TFEB activation, in response to bacterial stimuli, promotes the transcription of aconitate decarboxylase (Acod1, Irg1) and synthesis of its product itaconate, a mitochondrial metabolite with antimicrobial activity. Activation of the TFEB-Irg1-itaconate signalling axis reduces the survival of the intravacuolar pathogen Salmonella enterica serovar Typhimurium. TFEB-driven itaconate is subsequently transferred via the Irg1-Rab32-BLOC3 system into the Salmonella-containing vacuole, thereby exposing the pathogen to elevated itaconate levels. By activating itaconate production, TFEB selectively restricts proliferating Salmonella, a bacterial subpopulation that normally escapes macrophage control, which contrasts TFEB's role in autophagy-mediated pathogen degradation. Together, our data define a TFEB-driven metabolic pathway between phago-lysosomes and mitochondria that restrains Salmonella Typhimurium burden in macrophages in vitro and in vivo.

Multilayer omics analysis reveals a non-classical retinoic acid signaling axis that regulates hematopoietic stem cell identity.

Hematopoietic stem cells (HSCs) rely on complex regulatory networks to preserve stemness. Due to the scarcity of HSCs, technical challenges have limited our insights into the interplay between metabolites, transcription, and the epigenome. In this study, we generated low-input metabolomics, transcriptomics, chromatin accessibility, and chromatin immunoprecipitation data, revealing distinct metabolic hubs that are enriched in HSCs and their downstream multipotent progenitors. Mechanistically, we uncover a non-classical retinoic acid (RA) signaling axis that regulates HSC function. We show that HSCs rely on Cyp26b1, an enzyme conventionally considered to limit RA effects in the cell. In contrast to the traditional view, we demonstrate that Cyp26b1 is indispensable for production of the active metabolite 4-oxo-RA. Further, RA receptor beta (Rarb) is required for complete transmission of 4-oxo-RA-mediated signaling to maintain stem cells. Our findings emphasize that a single metabolite controls stem cell fate by instructing epigenetic and transcriptional attributes.

Sensing Stemness.

PURPOSE OF REVIEW: Hematopoietic stem cells (HSCs) are formed embryonically during a dynamic developmental process and later reside in adult hematopoietic organs in a quiescent state. In response to their changing environment, HSCs have evolved diverse mechanisms to cope with intrinsic and extrinsic challenges. This review intends to discuss how HSCs and other stem cells co-opted DNA and RNA innate immune pathways to fine-tune developmental processes. RECENT FINDINGS: Innate immune receptors for nucleic acids like the RIG-I-like family receptors and members of DNA sensing pathways are expressed in HSCs and other stem cells. Even though the "classic" role of these receptors is recognition of foreign DNA or RNA from pathogens, it was recently shown that cellular transposable element (TE) RNA or R-loops activate such receptors, serving as endogenous triggers of inflammatory signaling that can shape HSC formation during development and regeneration. SUMMARY: Endogenous TEs and R-loops activate RNA and DNA sensors, which trigger distinct inflammatory signals to fine-tune stem cell decisions. This phenomenon could have broad implications for diverse somatic stem cells, for a variety of diseases and during aging.

Inflammation, Aging and Hematopoiesis: A Complex Relationship.

All vertebrate blood cells descend from multipotent hematopoietic stem cells (HSCs), whose activity and differentiation depend on a complex and incompletely understood relationship with inflammatory signals. Although homeostatic levels of inflammatory signaling play an intricate role in HSC maintenance, activation, proliferation, and differentiation, acute or chronic exposure to inflammation can have deleterious effects on HSC function and self-renewal capacity, and bias their differentiation program. Increased levels of inflammatory signaling are observed during aging, affecting HSCs either directly or indirectly via the bone marrow niche and contributing to their loss of self-renewal capacity, diminished overall functionality, and myeloid differentiation skewing. These changes can have significant pathological consequences. Here, we provide an overview of the current literature on the complex interplay between HSCs and inflammatory signaling, and how this relationship contributes to age-related phenotypes. Understanding the mechanisms and outcomes of this interaction during different life stages will have significant implications in the modulation and restoration of the hematopoietic system in human disease, recovery from cancer and chemotherapeutic treatments, stem cell transplantation, and aging.

Chemotherapy-induced transposable elements activate MDA5 to enhance haematopoietic regeneration.

Haematopoietic stem cells (HSCs) are normally quiescent, but have evolved mechanisms to respond to stress. Here, we evaluate haematopoietic regeneration induced by chemotherapy. We detect robust chromatin reorganization followed by increased transcription of transposable elements (TEs) during early recovery. TE transcripts bind to and activate the innate immune receptor melanoma differentiation-associated protein 5 (MDA5) that generates an inflammatory response that is necessary for HSCs to exit quiescence. HSCs that lack MDA5 exhibit an impaired inflammatory response after chemotherapy and retain their quiescence, with consequent better long-term repopulation capacity. We show that the overexpression of ERV and LINE superfamily TE copies in wild-type HSCs, but not in Mda5-/- HSCs, results in their cycling. By contrast, after knockdown of LINE1 family copies, HSCs retain their quiescence. Our results show that TE transcripts act as ligands that activate MDA5 during haematopoietic regeneration, thereby enabling HSCs to mount an inflammatory response necessary for their exit from quiescence.

ZFP451-mediated SUMOylation of SATB2 drives embryonic stem cell differentiation.

The establishment of cell fates involves alterations of transcription factor repertoires and repurposing of transcription factors by post-translational modifications. In embryonic stem cells (ESCs), the chromatin organizers SATB2 and SATB1 balance pluripotency and differentiation by activating and repressing pluripotency genes, respectively. Here, we show that conditional Satb2 gene inactivation weakens ESC pluripotency, and we identify SUMO2 modification of SATB2 by the E3 ligase ZFP451 as a potential driver of ESC differentiation. Mutations of two SUMO-acceptor lysines of Satb2 (Satb2K →R ) or knockout of Zfp451 impair the ability of ESCs to silence pluripotency genes and activate differentiation-associated genes in response to retinoic acid (RA) treatment. Notably, the forced expression of a SUMO2-SATB2 fusion protein in either Satb2K →R or Zfp451-/- ESCs rescues, in part, their impaired differentiation potential and enhances the down-regulation of Nanog The differentiation defect of Satb2K →R ESCs correlates with altered higher-order chromatin interactions relative to Satb2wt ESCs. Upon RA treatment of Satb2wt ESCs, SATB2 interacts with ZFP451 and the LSD1/CoREST complex and gains binding at differentiation genes, which is not observed in RA-treated Satb2K →R cells. Thus, SATB2 SUMOylation may contribute to the rewiring of transcriptional networks and the chromatin interactome of ESCs in the transition of pluripotency to differentiation.

New tools for 'ZEBRA-FISHING'.

Zebrafish has been established as a classical model for developmental studies, yet in the past years, with the explosion of novel technological methods, the use of zebrafish as a model has expanded. One of the prominent fields that took advantage of zebrafish as a model organism early on is hematopoiesis, the process of blood cell generation from hematopoietic stem and progenitor cells (HSPCs). In zebrafish, HSPCs are born early during development in the aorta-gonad-mesonephros region and then translocate to the caudal hematopoietic tissue, where they expand and finally take residence in the kidney marrow. This journey is tightly regulated at multiple levels from extracellular signals to chromatin. In order to delineate the mechanistic underpinnings of this process, next-generation sequencing techniques could be an important ally. Here, we describe genome-wide approaches that have been undertaken to delineate zebrafish hematopoiesis.

Dynamic Cardiolipin Synthesis Is Required for CD8+ T Cell Immunity.

Mitochondria constantly adapt to the metabolic needs of a cell. This mitochondrial plasticity is critical to T cells, which modulate metabolism depending on antigen-driven signals and environment. We show here that de novo synthesis of the mitochondrial membrane-specific lipid cardiolipin maintains CD8+ T cell function. T cells deficient for the cardiolipin-synthesizing enzyme PTPMT1 had reduced cardiolipin and responded poorly to antigen because basal cardiolipin levels were required for activation. However, neither de novo cardiolipin synthesis, nor its Tafazzin-dependent remodeling, was needed for T cell activation. In contrast, PTPMT1-dependent cardiolipin synthesis was vital when mitochondrial fitness was required, most notably during memory T cell differentiation or nutrient stress. We also found CD8+ T cell defects in a small cohort of patients with Barth syndrome, where TAFAZZIN is mutated, and in a Tafazzin-deficient mouse model. Thus, the dynamic regulation of a single mitochondrial lipid is crucial for CD8+ T cell immunity.

Common variants in signaling transcription-factor-binding sites drive phenotypic variability in red blood cell traits.

Genome-wide association studies identify genomic variants associated with human traits and diseases. Most trait-associated variants are located within cell-type-specific enhancers, but the molecular mechanisms governing phenotypic variation are less well understood. Here, we show that many enhancer variants associated with red blood cell (RBC) traits map to enhancers that are co-bound by lineage-specific master transcription factors (MTFs) and signaling transcription factors (STFs) responsive to extracellular signals. The majority of enhancer variants reside on STF and not MTF motifs, perturbing DNA binding by various STFs (BMP/TGF-ß-directed SMADs or WNT-induced TCFs) and affecting target gene expression. Analyses of engineered human blood cells and expression quantitative trait loci verify that disrupted STF binding leads to altered gene expression. Our results propose that the majority of the RBC-trait-associated variants that reside on transcription-factor-binding sequences fall in STF target sequences, suggesting that the phenotypic variation of RBC traits could stem from altered responsiveness to extracellular stimuli.

Repetitive Elements Trigger RIG-I-like Receptor Signaling that Regulates the Emergence of Hematopoietic Stem and Progenitor Cells.

Inflammatory signaling is required for hematopoietic stem and progenitor cell (HSPC) development. Here, we studied the involvement of RIG-I-like receptors (RLRs) in HSPC formation. Rig-I or Mda5 deficiency impaired, while Lgp2 deficiency enhanced, HSPC emergence in zebrafish embryos. Rig-I or Mda5 deficiency reduced HSPC numbers by inhibiting inflammatory signals that were in turn enhanced in Lgp2 deficient embryos. Simultaneous reduction of Lgp2 and either Rig-I or Mda5 rescued inflammatory signals and HSPC numbers. Modulating the expression of the signaling mediator Traf6 in RLR deficient embryos restored HSPC numbers. Repetitive element transcripts could be detected in hemogenic endothelial cells and HSPCs, suggesting a role as RLR ligands. Indeed, ectopic expression of repetitive elements enhanced HSPC formation in wild-type, but not in Rig-I or Mda5 deficient embryos. Manipulation of RLR expression in mouse fetal liver HSPCs indicated functional conservation among species. Thus, repetitive elements transcribed during development drive RLR-mediated inflammatory signals that regulate HSPC formation.

DOT1L-mediated murine neuronal differentiation associates with H3K79me2 accumulation and preserves SOX2-enhancer accessibility.

During neuronal differentiation, the transcriptional profile and the epigenetic context of neural committed cells is subject to significant rearrangements, but a systematic quantification of global histone modification changes is still missing. Here, we show that H3K79me2 increases and H3K27ac decreases globally during in-vitro neuronal differentiation of murine embryonic stem cells. DOT1L mediates all three degrees of methylation of H3K79 and its enzymatic activity is critical to modulate cellular differentiation and reprogramming. In this context, we find that inhibition of DOT1L in neural progenitor cells biases the transcriptional state towards neuronal differentiation, resulting in transcriptional upregulation of genes marked with H3K27me3 on the promoter region. We further show that DOT1L inhibition affects accessibility of SOX2-bound enhancers and impairs SOX2 binding in neural progenitors. Our work provides evidence that DOT1L activity gates differentiation of progenitors by allowing SOX2-dependent transcription of stemness programs.

CHD7 and Runx1 interaction provides a braking mechanism for hematopoietic differentiation.

Hematopoietic stem and progenitor cell (HSPC) formation and lineage differentiation involve gene expression programs orchestrated by transcription factors and epigenetic regulators. Genetic disruption of the chromatin remodeler chromodomain-helicase-DNA-binding protein 7 (CHD7) expanded phenotypic HSPCs, erythroid, and myeloid lineages in zebrafish and mouse embryos. CHD7 acts to suppress hematopoietic differentiation. Binding motifs for RUNX and other hematopoietic transcription factors are enriched at sites occupied by CHD7, and decreased RUNX1 occupancy correlated with loss of CHD7 localization. CHD7 physically interacts with RUNX1 and suppresses RUNX1-induced expansion of HSPCs during development through modulation of RUNX1 activity. Consequently, the RUNX1:CHD7 axis provides proper timing and function of HSPCs as they emerge during hematopoietic development or mature in adults, representing a distinct and evolutionarily conserved control mechanism to ensure accurate hematopoietic lineage differentiation.