Interleukin-10 Prevents Pathological Microglia Hyperactivation following Peripheral Endotoxin Challenge.

Microglia, the resident macrophages of the brain parenchyma, are key players in central nervous system (CNS) development, homeostasis, and disorders. Distinct brain pathologies seem associated with discrete microglia activation modules. How microglia regain quiescence following challenges remains less understood. Here, we explored the role of the interleukin-10 (IL-10) axis in restoring murine microglia homeostasis following a peripheral endotoxin challenge. Specifically, we show that lipopolysaccharide (LPS)-challenged mice harboring IL-10 receptor-deficient microglia displayed neuronal impairment and succumbed to fatal sickness. Addition of a microglial tumor necrosis factor (TNF) deficiency rescued these animals, suggesting a microglia-based circuit driving pathology. Single cell transcriptome analysis revealed various IL-10 producing immune cells in the CNS, including most prominently Ly49D+ NK cells and neutrophils, but not microglia. Collectively, we define kinetics of the microglia response to peripheral endotoxin challenge, including their activation and robust silencing, and highlight the critical role of non-microglial IL-10 in preventing deleterious microglia hyperactivation.

Thymic mimetic cells function beyond self-tolerance.

Development of immunocompetent T cells in the thymus is required for effective defence against all types of pathogens, including viruses, bacteria and fungi. To this end, T cells undergo a very strict educational program in the thymus, during which both non-functional and self-reactive T cell clones are eliminated by means of positive and negative selection1.Thymic epithelial cells (TECs) have an indispensable role in these processes, and previous studies have shown the notable heterogeneity of these cells2-7. Here, using multiomic analysis, we provide further insights into the functional and developmental diversity of TECs in mice, and reveal a detailed atlas of the TEC compartment according to cell transcriptional states and chromatin landscapes. Our analysis highlights unconventional TEC subsets that are similar to functionally well-defined parenchymal populations, including endocrine cells, microfold cells and myocytes. By focusing on the endocrine and microfold TEC populations, we show that endocrine TECs require Insm1 for their development and are crucial to maintaining thymus cellularity in a ghrelin-dependent manner; by contrast, microfold TECs require Spib for their development and are essential for the generation of thymic IgA+ plasma cells. Collectively, our study reveals that medullary TECs have the potential to differentiate into various types of molecularly distinct and functionally defined cells, which not only contribute to the induction of central tolerance, but also regulate the homeostasis of other thymus-resident populations.

Targeting Spt5-Pol II by Small-Molecule Inhibitors Uncouples Distinct Activities and Reveals Additional Regulatory Roles.

Spt5 is a conserved and essential transcription elongation factor that promotes promoter-proximal pausing, promoter escape, elongation, and mRNA processing. Spt5 plays specific roles in the transcription of inflammation and stress-induced genes and tri-nucleotide expanded-repeat genes involved in inherited neurological pathologies. Here, we report the identification of Spt5-Pol II small-molecule inhibitors (SPIs). SPIs faithfully reproduced Spt5 knockdown effects on promoter-proximal pausing, NF-κB activation, and expanded-repeat huntingtin gene transcription. Using SPIs, we identified Spt5 target genes that responded with profoundly diverse kinetics. SPIs uncovered the regulatory role of Spt5 in metabolism via GDF15, a food intake- and body weight-inhibitory hormone. SPIs further unveiled a role for Spt5 in promoting the 3' end processing of histone genes. While several SPIs affect all Spt5 functions, a few inhibit a single one, implying uncoupling and selective targeting of Spt5 activities. SPIs expand the understanding of Spt5-Pol II functions and are potential drugs against metabolic and neurodegenerative diseases.

Dicer Deficiency Differentially Impacts Microglia of the Developing and Adult Brain.

Microglia seed the embryonic neuro-epithelium, expand and actively sculpt neuronal circuits in the developing central nervous system, but eventually adopt relative quiescence and ramified morphology in the adult. Here, we probed the impact of post-transcriptional control by microRNAs (miRNAs) on microglial performance during development and adulthood by generating mice lacking microglial Dicer expression at these distinct stages. Conditional Dicer ablation in adult microglia revealed that miRNAs were required to limit microglial responses to challenge. After peripheral endotoxin exposure, Dicer-deficient microglia expressed more pro-inflammatory cytokines than wild-type microglia and thereby compromised hippocampal neuronal functions. In contrast, prenatal Dicer ablation resulted in spontaneous microglia activation and revealed a role for Dicer in DNA repair and preservation of genome integrity. Accordingly, Dicer deficiency rendered otherwise radio-resistant microglia sensitive to gamma irradiation. Collectively, the differential impact of the Dicer ablation on microglia of the developing and adult brain highlights the changes these cells undergo with time.

Inhibitors of eIF4G1-eIF1 uncover its regulatory role of ER/UPR stress-response genes independent of eIF2α-phosphorylation.

During translation initiation, eIF4G1 dynamically interacts with eIF4E and eIF1. While the role of eIF4E-eIF4G1 is well established, the regulatory functions of eIF4G1-eIF1 are poorly understood. Here, we report the identification of the eIF4G1-eIF1 inhibitors i14G1-10 and i14G1-12. i14G1s directly bind eIF4G1 and inhibit translation in vitro and in the cell, and their effects on translation are dependent on eIF4G1 levels. Translatome analyses revealed that i14G1s mimic eIF1 and eIF4G1 perturbations on the stringency of start codon selection and the opposing roles of eIF1-eIF4G1 in scanning-dependent and scanning-independent short 5' untranslated region (UTR) translation. Remarkably, i14G1s activate ER/unfolded protein response (UPR) stress-response genes via enhanced ribosome loading, elevated 5'UTR translation at near-cognate AUGs, and unexpected concomitant up-regulation of coding-region translation. These effects are, at least in part, independent of eIF2α-phosphorylation. Interestingly, eIF4G1-eIF1 interaction itself is negatively regulated by ER stress and mTOR inhibition. Thus, i14G1s uncover an unknown mechanism of ER/UPR translational stress response and are valuable research tools and potential drugs against diseases exhibiting dysregulated translation.

Bone morphology is regulated modularly by global and regional genetic programs.

Bone protrusions provide stable anchoring sites for ligaments and tendons and define the unique morphology of each long bone. Despite their importance, the mechanism by which superstructures are patterned is unknown. Here, we identify components of the genetic program that control the patterning of Sox9+/Scx+ superstructure progenitors in mouse and show that this program includes both global and regional regulatory modules. Using light-sheet fluorescence microscopy combined with genetic lineage labeling, we mapped the broad contribution of the Sox9+/Scx+ progenitors to the formation of bone superstructures. Then, by combining literature-based evidence, comparative transcriptomic analysis and genetic mouse models, we identified Gli3 as a global regulator of superstructure patterning, whereas Pbx1, Pbx2, Hoxa11 and Hoxd11 act as proximal and distal regulators, respectively. Moreover, by demonstrating a dose-dependent pattern regulation in Gli3 and Pbx1 compound mutations, we show that the global and regional regulatory modules work in a coordinated manner. Collectively, our results provide strong evidence for genetic regulation of superstructure patterning, which further supports the notion that long bone development is a modular process.This article has an associated 'The people behind the papers' interview.

Pseudo-mutant P53 is a unique phenotype of DNMT3A-mutated pre-leukemia.

Pre-leukemic clones carrying DNMT3A mutations have a selective advantage and an inherent chemoresistance, however the basis for this phenotype has not been fully elucidated. Mutations affecting the gene TP53 occur in pre-leukemic hematopoietic stem/progenitor cells (preL-HSPC) and lead to chemoresistance. Many of these mutations cause a conformational change and some of them were shown to enhance self-renewal capacity of preL-HSPC. Intriguingly, a misfolded P53 was described in AML blasts that do not harbor mutations in TP53, emphasizing the dynamic equilibrium between wild-type (WT) and "pseudo-mutant" conformations of P53. By combining single cell analyses and P53 conformation-specific monoclonal antibodies we studied preL-HSPC from primary human DNMT3A-mutated AML samples. We found that while leukemic blasts express mainly the WT conformation, in preL-HSPC the pseudo-mutant conformation is the dominant. HSPC from non-leukemic samples expressed both conformations to a similar extent. In a mouse model we found a small subset of HSPC with a dominant pseudo-mutant P53. This subpopulation was significantly larger among DNMT3AR882H-mutated HSPC, suggesting that while a pre-leukemic mutation can predispose for P53 misfolding, additional factors are involved as well. Treatment with a short peptide that can shift the dynamic equilibrium favoring the WT conformation of P53, specifically eliminated preL-HSPC that had dysfunctional canonical P53 pathway activity as reflected by single cell RNA sequencing. Our observations shed light upon a possible targetable P53 dysfunction in human preL-HSPC carrying DNMT3A mutations. This opens new avenues for leukemia prevention.

Dual role of starvation signaling in promoting growth and recovery.

Growing cells are subject to cycles of nutrient depletion and repletion. A shortage of nutrients activates a starvation program that promotes growth in limiting conditions. To examine whether nutrient-deprived cells prepare also for their subsequent recovery, we followed the transcription program activated in budding yeast transferred to low-phosphate media and defined its contribution to cell growth during phosphate limitation and upon recovery. An initial transcription wave was induced by moderate phosphate depletion that did not affect cell growth. A second transcription wave followed when phosphate became growth limiting. The starvation program contributed to growth only in the second, growth-limiting phase. Notably, the early response, activated at moderate depletion, promoted recovery from starvation by increasing phosphate influx upon transfer to rich medium. Our results suggest that cells subject to nutrient depletion prepare not only for growth in the limiting conditions but also for their predicted recovery once nutrients are replenished.

Cerebellar Learning Properties Are Modulated by the CRF Receptor.

Corticotropin-releasing factor (CRF) and its type 1 receptor (CRFR1) play an important role in the responses to stressful challenges. Despite the well established expression of CRFR1 in granular cells (GrCs), its role in procedural motor performance and memory formation remains elusive. To investigate the role of CRFR1 expression in cerebellar GrCs, we used a mouse model depleted of CRFR1 in these cells. We detected changes in the cellular learning mechanisms in GrCs depleted of CRFR1 in that they showed changes in intrinsic excitability and long-term synaptic plasticity. Analysis of cerebella transcriptome obtained from KO and control mice detected prominent alterations in the expression of calcium signaling pathways components. Moreover, male mice depleted of CRFR1 specifically in GrCs showed accelerated Pavlovian associative eye-blink conditioning, but no differences in baseline motor performance, locomotion, or fear and anxiety-related behaviors. Our findings shed light on the interplay between stress-related central mechanisms and cerebellar motor conditioning, highlighting the role of the CRF system in regulating particular forms of cerebellar learning.SIGNIFICANCE STATEMENT Although it is known that the corticotropin-releasing factor type 1 receptor (CRFR1) is highly expressed in the cerebellum, little attention has been given to its role in cerebellar functions in the behaving animal. Moreover, most of the attention was directed at the effect of CRF on Purkinje cells at the cellular level and, to this date, almost no data exist on the role of this stress-related receptor in other cerebellar structures. Here, we explored the behavioral and cellular effect of granular cell-specific ablation of CRFR1 We found a profound effect on learning both at the cellular and behavioral levels without an effect on baseline motor skills.

UTAP: User-friendly Transcriptome Analysis Pipeline.

BACKGROUND: RNA-Seq technology is routinely used to characterize the transcriptome, and to detect gene expression differences among cell types, genotypes and conditions. Advances in short-read sequencing instruments such as Illumina Next-Seq have yielded easy-to-operate machines, with high throughput, at a lower price per base. However, processing this data requires bioinformatics expertise to tailor and execute specific solutions for each type of library preparation. RESULTS: In order to enable fast and user-friendly data analysis, we developed an intuitive and scalable transcriptome pipeline that executes the full process, starting from cDNA sequences derived by RNA-Seq [Nat Rev Genet 10:57-63, 2009] and bulk MARS-Seq [Science 343:776-779, 2014] and ending with sets of differentially expressed genes. Output files are placed in structured folders, and results summaries are provided in rich and comprehensive reports, containing dozens of plots, tables and links. CONCLUSION: Our User-friendly Transcriptome Analysis Pipeline (UTAP) is an open source, web-based intuitive platform available to the biomedical research community, enabling researchers to efficiently and accurately analyse transcriptome sequence data.

Induction of the type I interferon response in neurological forms of Gaucher disease.

BACKGROUND: Neuroinflammation is a key phenomenon in the pathogenesis of many neurodegenerative diseases. Understanding the mechanisms by which brain inflammation is engaged and delineating the key players in the immune response and their contribution to brain pathology is of great importance for the identification of novel therapeutic targets for these devastating diseases. Gaucher disease, the most common lysosomal storage disease, is caused by mutations in the GBA1 gene and is a significant risk factor for Parkinson's disease; in some forms of Gaucher disease, neuroinflammation is observed. METHODS: An unbiased gene profile analysis was performed on a severely affected brain area of a neurological form of a Gaucher disease mouse at a pre-symptomatic stage; the mouse used for this study, the Gba (flox/flox); nestin-Cre mouse, was engineered such that GBA1 deficiency is restricted to cells of neuronal lineage, i.e., neurons and macroglia. RESULTS: The 10 most up-regulated genes in the ventral posteromedial/posterolateral region of the thalamus were inflammatory genes, with the gene expression signature significantly enriched in interferon signaling genes. Interferon ß levels were elevated in neurons, and interferon-stimulated genes were elevated mainly in microglia. Interferon signaling pathways were elevated to a small extent in the brain of another lysosomal storage disease mouse model, Krabbe disease, but not in Niemann-Pick C or Sandhoff mouse brain. Ablation of the type I interferon receptor attenuated neuroinflammation but had no effect on GD mouse viability. CONCLUSIONS: Our results imply that the type I interferon response is involved in the development of nGD pathology, and possibly in other lysosomal storage diseases in which simple glycosphingolipids accumulate, and support the notion that interferon signaling pathways play a vital role in the sterile inflammation that often occurs during chronic neurodegenerative diseases in which neuroinflammation is present.

Differences in microRNA detection levels are technology and sequence dependent.

Identification and quantification of small RNAs are challenging because of their short length, high sequence similarities within microRNA (miRNA) families, and the existence of miRNA isoforms and O-methyl 3' modifications. In this study, the detection performance of three high-throughput commercial platforms, Agilent and Affymetrix microarrays and Illumina next-generation sequencing, was systematically and comprehensively compared. The ability to detect miRNAs was shown to depend strongly on the platform and on miRNA modifications and sequence. Using synthetic transcripts, including mature, precursor, and O-methyl-modified miRNAs spiked into human RNA, a large intensity variation in all spiked-in miRNAs and a reduced capacity in detecting O-methyl-modified miRNAs were observed between the tested platforms. In addition, endogenous human miRNA expression levels were assessed across the platforms. Detected miRNA expression levels were not consistent between platforms. Although biases in miRNA detection were previously described, here the end-point result, i.e., detection intensity, of these biases was investigated on multiple platforms in a controlled fashion. A detailed exploration of a large number of attributes, including base composition, sequence structure, and isoform miRNA attributes, suggests their impact on miRNA expression detection level. This study provides a basis for understanding the attributes that should be considered to adjust platform-dependent detection biases.

Cell isolation induces fate changes of bone marrow mesenchymal cells leading to loss or alternatively to acquisition of new differentiation potentials.

Mesenchymal stromal cell populations include a fraction, termed mesenchymal stem cells, exhibiting multipotency. Other cells within this population possess a lesser differentiation range. This was assumed to be due to a mesenchymal cellular cascade topped by a multipotent cell, which gives rise to progeny with diminishing differentiation potentials. Here, we show that mesenchymal cells, a priori exhibiting a limited differentiation potential, may gain new capacities and become multipotent following single-cell isolation. These fate changes were accompanied by upregulation of differentiation promoting genes, many of which also became H4K20me1 methylated. Early events in the process included TGFß and Wnt modulation, and downregulation of hypoxia signaling. Indeed, hypoxic conditions inhibited the observed cell changes. Overall, cell isolation from neighboring partners caused major molecular changes and particularly, a newly established epigenetic state, ultimately leading to the acquisition of new differentiation potentials and an altered cell fate.

Induction of Superior Systemic and Mucosal Protective Immunity to SARS-CoV-2 by Nasal Administration of a VSV-ΔG-Spike Vaccine.

The emergence of rapidly spreading variants of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) poses a major challenge to vaccines' protective efficacy. Intramuscular (IM) vaccine administration induces short-lived immunity but does not prevent infection and transmission. New vaccination strategies are needed to extend the longevity of vaccine protection, induce mucosal and systemic immunity and prevent viral transmission. The intranasal (IN) administration of the VSV-ΔG-spike vaccine candidate directly to mucosal surfaces yielded superior mucosal and systemic immunity at lower vaccine doses. Compared to IM vaccination in the K18-hACE2 model, IN vaccination preferentially induced mucosal IgA and T-cells, reduced the viral load at the site of infection, and ameliorated disease-associated brain gene expression. IN vaccination was protective even one year after administration. As most of the world population has been vaccinated by IM injection, we demonstrate the potential of a heterologous IM + IN vaccination regimen to induce mucosal immunity while maintaining systemic immunity. Furthermore, the IM + IN regimen prevented virus transmission in a golden Syrian hamster co-caging model. Taken together, we show that IN vaccination with VSV-ΔG-spike, either as a homologous IN + IN regimen or as a boost following IM vaccination, has a favorable potential over IM vaccination in inducing efficient mucosal immunity, long-term protection and preventing virus transmission.

Tuft cells and fibroblasts promote thymus regeneration through ILC2-mediated type 2 immune response.

The thymus is a primary lymphoid organ that is essential for the establishment of adaptive immunity through generation of immunocompetent T cells. In response to various stress signals, the thymus undergoes acute but reversible involution. However, the mechanisms governing its recovery are incompletely understood. Here, we used a dexamethasone-induced acute thymic involution mouse model to investigate how thymic hematopoietic cells (excluding T cells) contribute to thymic regeneration. scRNA-seq analysis revealed marked transcriptional and cellular changes in various thymic populations and highlighted thymus-resident innate lymphoid cells type 2 (ILC2) as a key cell type involved in the response to damage. We identified that ILC2 are activated by the alarmins IL-25 and IL-33 produced in response to tissue damage by thymic tuft cells and fibroblasts, respectively. Moreover, using mouse models deficient in either tuft cells and/or IL-33, we found that these alarmins are required for effective thymus regeneration after dexamethasone-induced damage. We also demonstrate that upon their damage-dependent activation, thymic ILC2 produce several effector molecules linked to tissue regeneration, such as amphiregulin and IL-13, which in turn promote thymic epithelial cell differentiation. Collectively, our study elucidates a previously undescribed role for thymic tuft cells and fibroblasts in thymus regeneration through activation of the type 2 immune response.

The tomato SlSHINE3 transcription factor regulates fruit cuticle formation and epidermal patterning.

Fleshy tomato fruit typically lacks stomata; therefore, a proper cuticle is particularly vital for fruit development and interaction with the surroundings. Here, we characterized the tomato SlSHINE3 (SlSHN3) transcription factor to extend our limited knowledge regarding the regulation of cuticle formation in fleshy fruits. We created SlSHN3 overexpressing and silenced plants, and used them for detailed analysis of cuticular lipid compositions, phenotypic characterization, and the study on the mode of SlSHN3 action. Heterologous expression of SlSHN3 in Arabidopsis phenocopied overexpression of the Arabidopsis SHNs. Silencing of SlSHN3 results in profound morphological alterations of the fruit epidermis and significant reduction in cuticular lipids. We demonstrated that SlSHN3 activity is mediated by control of genes associated with cutin metabolism and epidermal cell patterning. As with SlSHN3 RNAi lines, mutation in the SlSHN3 target gene, SlCYP86A69, resulted in severe cutin deficiency and altered fruit surface architecture. In vitro activity assays demonstrated that SlCYP86A69 possesses NADPH-dependent ω-hydroxylation activity, particularly of C18:1 fatty acid to the 18-hydroxyoleic acid cutin monomer. This study provided insights into transcriptional mechanisms mediating fleshy fruit cuticle formation and highlighted the link between cutin metabolism and the process of fruit epidermal cell patterning.

Neural plate progenitors give rise to both anterior and posterior pituitary cells.

The pituitary is the master neuroendocrine gland, which regulates body homeostasis. It consists of the anterior pituitary/adenohypophysis harboring hormones producing cells and the posterior pituitary/neurohypophysis, which relays the passage of hormones from the brain to the periphery. It is accepted that the adenohypophysis originates from the oral ectoderm (Rathke's pouch), whereas the neural ectoderm contributes to the neurohypophysis. Single-cell transcriptomics of the zebrafish pituitary showed that cyp26b1-positive astroglial pituicytes of the neurohypophysis and prop1-positive adenohypophyseal progenitors expressed common markers implying lineage relatedness. Genetic tracing identifies that, in contrast to the prevailing dogma, neural plate precursors of zebrafish (her4.3+) and mouse (Sox1+) contribute to both neurohypophyseal and a subset of adenohypophyseal cells. Pituicyte-derived retinoic-acid-degrading enzyme Cyp26b1 fine-tunes differentiation of prop1+ progenitors into hormone-producing cells. These results challenge the notion that adenohypophyseal cells are exclusively derived from non-neural ectoderm and demonstrate that crosstalk between neuro- and adeno-hypophyseal cells affects differentiation of pituitary cells.

Molecular characterization of the intact mouse muscle spindle using a multi-omics approach.

The proprioceptive system is essential for the control of coordinated movement, posture, and skeletal integrity. The sense of proprioception is produced in the brain using peripheral sensory input from receptors such as the muscle spindle, which detects changes in the length of skeletal muscles. Despite its importance, the molecular composition of the muscle spindle is largely unknown. In this study, we generated comprehensive transcriptomic and proteomic datasets of the entire muscle spindle isolated from the murine deep masseter muscle. We then associated differentially expressed genes with the various tissues composing the spindle using bioinformatic analysis. Immunostaining verified these predictions, thus establishing new markers for the different spindle tissues. Utilizing these markers, we identified the differentiation stages the spindle capsule cells undergo during development. Together, these findings provide comprehensive molecular characterization of the intact spindle as well as new tools to study its development and function in health and disease.

Genome sequence of the pattern-forming social bacterium Paenibacillus dendritiformis C454 chiral morphotype.

Paenibacillus dendritiformis is a Gram-positive, soil-dwelling, spore-forming social microorganism. An intriguing collective faculty of this strain is manifested by its ability to switch between different morphotypes, such as the branching (T) and the chiral (C) morphotypes. Here we report the 6.3-Mb draft genome sequence of the P. dendritiformis C454 chiral morphotype.