Microglia-mediated demyelination protects against CD8+ T cell-driven axon degeneration in mice carrying PLP defects

Axon degeneration and functional decline in myelin diseases are often attributed to loss of myelin but their relation is not fully understood. Perturbed myelinating glia can instigate chronic neuroinflammation and contribute to demyelination and axonal damage. Here we study mice with distinct defects in the proteolipid protein 1 gene that develop axonal damage which is driven by cytotoxic T cells targeting myelinating oligodendrocytes. We show that persistent ensheathment with perturbed myelin poses a risk for axon degeneration, neuron loss, and behavioral decline. We demonstrate that CD8(+) T cell-driven axonal damage is less likely to progress towards degeneration when axons are efficiently demyelinated by activated microglia. Mechanistically, we show that cytotoxic T cell effector molecules induce cytoskeletal alterations within myelinating glia and aberrant actomyosin constriction of axons at paranodal domains. Our study identifies detrimental axon-glia-immune interactions which promote neurodegeneration and possible therapeutic targets for disorders associated with myelin defects and neuroinflammation. Demyelination is often suggested to cause axonal degeneration. Here, the authors study mice carrying distinct PLP defects and reveal how persistent ensheathment with perturbed myelin poses a risk for CD8+T cell-driven axon loss and behavioral decline

Time-resolved single-cell RNA-seq using metabolic RNA labelling

Single-cell RNA sequencing offers snapshots of whole transcriptomes but obscures the temporal RNA dynamics. Here we present single-cell metabolically labeled new RNA tagging sequencing (scNT-seq), a method for massively parallel analysis of newly transcribed and pre-existing mRNAs from the same cell. This droplet microfluidics-based method enables high-throughput chemical conversion on barcoded beads, efficiently marking newly transcribed mRNAs with T-to-C substitutions. Using scNT-seq, we jointly profiled new and old transcriptomes in ~55,000 single cells. These data revealed time-resolved transcription factor activities and cell-state trajectories at the single-cell level in response to neuronal activation. We further determined rates of RNA biogenesis and decay to uncover RNA regulatory strategies during stepwise conversion between pluripotent and rare totipotent two-cell embryo (2C)-like stem cell states. Finally, integrating scNT-seq with genetic perturbation identifies DNA methylcytosine dioxygenase as an epigenetic barrier into the 2C-like cell state. Time-resolved single-cell transcriptomic analysis thus opens new lines of inquiry regarding cell-type-specific RNA regulatory mechanisms

Interleukin-23 receptor expressing γδ T cells locally promote early atherosclerotic lesion formation and plaque necrosis in mice

Aims Atherosclerosis is a chronic inflammatory disease of the vessel wall controlled by local and systemic immune responses. The role of interleukin-23 receptor (IL-23R), expressed in adaptive immune cells (mainly T-helper 17 cells) and gamma delta T cells, in atherosclerosis is only incompletely understood. Here, we investigated the vascular cell types expressing IL-23R and addressed the function of IL-23R and gamma delta T cells in atherosclerosis. Methods and results IL-23R(+) cells were frequently found in the aortic root in contrast to the aorta in low-density lipoprotein receptor deficient IL-23R reporter mice (Ldlr(-/-)Il23r(gfp/+)), and mostly identified as gamma delta T cells that express IL-17 and GM-CSF. scRNA-seq confirmed gamma delta T cells as the main cell type expressing Il23r and Il17a in the aorta. Ldlr(-)(/)(-)Il23r(gfp/gfp) mice deficient in IL-23R showed a loss of IL-23R(+) cells in the vasculature, and had reduced atherosclerotic lesion formation in the aortic root compared to Ldlr(-/-) controls after 6 weeks of high-fat diet feeding. In contrast, Ldlr(-/-)Tcr delta(-/-) mice lacking all gamma delta T cells displayed unaltered early atherosclerotic lesion formation compared to Ldlr(-)(/-) mice. In both HFD-fed Ldlr(-/-)Il23r(gfp/gfp) and Ldlr(-/-)Tcr delta(-/-) mice a reduction in the plaque necrotic core area was noted as well as an expansion of splenic regulatory T cells. In vitro, exposure of bone marrow-derived macrophages to both IL-17A and GM-CSF induced cell necrosis, and necroptotic RIP3K and MLKL expression, as well as inflammatory mediators. Conclusions IL-23R(+) gamma delta T cells are predominantly found in the aortic root rather than the aorta and promote early atherosclerotic lesion formation, plaque necrosis, and inflammation at this site. Targeting IL-23R may thus be explored as a therapeutic approach to mitigate atherosclerotic lesion development

Genetic inhibition of CARD9 accelerates the development of atherosclerosis in mice through CD36 dependent-defective autophagy

Caspase recruitment-domain containing protein 9 (CARD9) is a key signaling pathway in macrophages but its role in atherosclerosis is still poorly understood. Global deletion of Card9 in Apoe -/- mice as well as hematopoietic deletion in Ldlr -/- mice increases atherosclerosis. The acceleration of atherosclerosis is also observed in Apoe -/- Rag2 -/- Card9 -/- mice, ruling out a role for the adaptive immune system in the vascular phenotype of Card9 deficient mice. Card9 deficiency alters macrophage phenotype through CD36 overexpression with increased IL-1β production, increased lipid uptake, higher cell death susceptibility and defective autophagy. Rapamycin or metformin, two autophagy inducers, abolish intracellular lipid overload, restore macrophage survival and autophagy flux in vitro and finally abolish the pro-atherogenic effects of Card9 deficiency in vivo. Transcriptomic analysis of human CARD9-deficient monocytes confirms the pathogenic signature identified in murine models. In summary, CARD9 is a key protective pathway in atherosclerosis, modulating macrophage CD36-dependent inflammatory responses, lipid uptake and autophagy

Laboratoires-sur-puces et billes magnétiques auto-organisées pour l'analyse de cellules et d'ADN

We present here two microfluidic lab-on-chip devices based on self-organization of magnetic beads for biological analyses. The first one is dedicated to Circulating Tumor Cells (CTC) sorting. Magnetic beads are self-organized on a magnetic ferrofluid pattern obtained by micro-contact printing. Magnetic beads, coated with an antibody directed against a cell membrane antigen, form a tridimensional array of columns. Experiences measure the yield and specificity of the cell separation. Also this system was used to culture lymphocytes. As clinical validation, this system was used to immunophenotype leukemia and lymphoma cells coupled with high resolution imaging. A second system has been used to infectious bacteria detection. A technological module for DNA preconcentration was demonstrated using closed packed magnetic beads. DNA was retained by electrostatic interaction at low pH and eluted at basic pH. The proof of principle of this system is established.Nous présentons deux dispositifs microfluidiques, aussi appelés Laboratoires-sur-Puces, fondés sur deux technologies : la microfluidique et les billes magnétiques. Un premier laboratoire-sur-puce est dédié à la recherche de cellules tumorales circulantes. Les billes magnétiques sont auto-organisées sur un dépôt magnétique de ferrofluide obtenu par tamponnage moléculaire et les cellules sont séparées sur la base de protéines de membranes à leur surface. Les expériences menées établissent les performances de rendement et de pureté de la séparation cellulaire. On démontre que les cellules capturées peuvent être remises en culture. Comme validation clinique, ce système a été utilisé pour le typage de cellules tumorales circulantes lymphoïdes issues de leucémies et de lymphomes à l'aide de microscopie confocale à haute résolution. Un deuxième laboratoire-sur-puce a été utilisé pour la recherche de bactéries infectieuses. Un module de concentration d'ADN a été mis en place à l'aide de billes magnétiques organisées en réseau très dense de billes. L'ADN est retenu par sa charge sur les billes à faible pH et élué par augmentation de pH. La preuve de concept du système est apportée

Single-cell RNA-seq: advances and future challenges

Phenotypically identical cells can dramatically vary with respect to behavior during their lifespan and this variation is reflected in their molecular composition such as the transcriptomic landscape. Singlecell transcriptomics using next-generation transcript sequencing (RNA-seq) is now emerging as a powerful tool to profile cell-to-cell variability on a genomic scale. Its application has already greatly impacted our conceptual understanding of diverse biological processes with broad implications for both basic and clinical research. Different single-cell RNAseq protocols have been introduced and are reviewed here – each one with its own strengths and current limitations. We further provide an overview of the biological questions single-cell RNA-seq has been used to address, the major findings obtained from such studies, and current challenges and expected future developments in this booming field

New RNA-seq approaches for the study of bacterial pathogens.

Understanding how bacteria cause disease requires knowledge of which genes are expressed and how they are regulated during infection. While RNA-seq is now a routine method for gene expression analysis in bacterial pathogens, the past years have also witnessed a surge of novel RNA-seq based approaches going beyond standard mRNA profiling. These include variations of the technique to capture post-transcriptional networks controlled by small RNAs and to discover associated RNA-binding proteins in the pathogen itself. Dual RNA-seq analyzing pathogen and host simultaneously has revealed roles of noncoding RNAs during infection and enabled the correlation of bacterial gene activity with specific host responses. Single-cell RNA-seq studies have addressed how heterogeneity among individual host cells may determine infection outcomes