The vascular interface of the brain, known as the blood-brain barrier (BBB), is understood to maintain brain function in part via its low transcellular permeability
. Yet, recent studies have ...demonstrated that brain ageing is sensitive to circulatory proteins
. Thus, it is unclear whether permeability to individually injected exogenous tracers-as is standard in BBB studies-fully represents blood-to-brain transport. Here we label hundreds of proteins constituting the mouse blood plasma proteome, and upon their systemic administration, study the BBB with its physiological ligand. We find that plasma proteins readily permeate the healthy brain parenchyma, with transport maintained by BBB-specific transcriptional programmes. Unlike IgG antibody, plasma protein uptake diminishes in the aged brain, driven by an age-related shift in transport from ligand-specific receptor-mediated to non-specific caveolar transcytosis. This age-related shift occurs alongside a specific loss of pericyte coverage. Pharmacological inhibition of the age-upregulated phosphatase ALPL, a predicted negative regulator of transport, enhances brain uptake of therapeutically relevant transferrin, transferrin receptor antibody and plasma. These findings reveal the extent of physiological protein transcytosis to the healthy brain, a mechanism of widespread BBB dysfunction with age and a strategy for enhanced drug delivery.
Early engagement of the lymphatic system by solid tumors in peripheral, nonlymphoid tissues is a clinical hallmark of cancer and often forecasts poor prognosis. The significance of lymph node ...metastasis for distant spread, however, has been questioned by large-scale lymph node dissection trials and the likely prevalence of direct hematogenous metastasis. Still, an emerging appreciation for the immunological role of the tumor-draining lymph node has renewed interest in its basic biology, role in metastatic progression, antitumor immunity, and patient outcomes. In this review, we discuss our current understanding of the early mechanisms through which tumors engage lymphatic transport and condition tumor-draining lymph nodes, the significance of these changes for both metastasis and immunity, and potential implications of the tumor-draining lymph node for immunotherapy.
Bioorthogonal tools enable cell-type-specific proteomics, a prerequisite to understanding biological processes in multicellular organisms. Here we report two engineered aminoacyl-tRNA synthetases for ...mammalian bioorthogonal labeling: a tyrosyl (ScTyrY43G) and a phenylalanyl (MmPheT413G) tRNA synthetase that incorporate azide-bearing noncanonical amino acids specifically into the nascent proteomes of host cells. Azide-labeled proteins are chemoselectively tagged via azide–alkyne cycloadditions with fluorophores for imaging or affinity resins for mass spectrometric characterization. Both mutant synthetases label human, hamster, and mouse cell line proteins and selectively activate their azido-bearing amino acids over 10-fold above the canonical. ScTyrY43G and MmPheT413G label overlapping but distinct proteomes in human cell lines, with broader proteome coverage upon their coexpression. In mice, ScTyrY43G and MmPheT413G label the melanoma tumor proteome and plasma secretome. This work furnishes new tools for mammalian residue-specific bioorthogonal chemistry, and enables more robust and comprehensive cell-type-specific proteomics in live mammals.
Lymphatic vessels are often considered passive conduits that flush antigenic material, pathogens, and cells to draining lymph nodes. Recent evidence, however, suggests that lymphatic vessels actively ...regulate diverse processes from antigen transport to leukocyte trafficking and dietary lipid absorption. Here we tested the hypothesis that infection-induced changes in lymphatic transport actively contribute to innate host defense. We demonstrate that cutaneous vaccinia virus infection by scarification activates dermal lymphatic capillary junction tightening (zippering) and lymph node lymphangiogenesis, which are associated with reduced fluid transport and cutaneous viral sequestration. Lymphatic-specific deletion of VEGFR2 prevented infection-induced lymphatic capillary zippering, increased fluid flux out of tissue, and allowed lymphatic dissemination of virus. Further, a reduction in dendritic cell migration to lymph nodes in the absence of lymphatic VEGFR2 associated with reduced antiviral CD8+ T cell expansion. These data indicate that VEGFR2-driven lymphatic remodeling is a context-dependent, active mechanism of innate host defense that limits viral dissemination and facilitates protective, antiviral CD8+ T cell responses.
Plasmid vectors that express the full‐length human dystrophin coding sequence in human cells were developed. Dystrophin, the protein mutated in Duchenne muscular dystrophy, is extraordinarily large, ...providing challenges for cloning and plasmid production in Escherichia coli. The authors expressed dystrophin from the strong, widely expressed CAG promoter, along with co‐transcribed luciferase and mCherry marker genes useful for tracking plasmid expression. Introns were added at the 3' and 5' ends of the dystrophin sequence to prevent translation in E. coli, resulting in improved plasmid yield. Stability and yield were further improved by employing a lower‐copy number plasmid origin of replication. The dystrophin plasmids also carried an attB site recognized by phage phiC31 integrase, enabling the plasmids to be integrated into the human genome at preferred locations by phiC31 integrase. The authors demonstrated single‐copy integration of plasmid DNA into the genome and production of human dystrophin in the human 293 cell line, as well as in induced pluripotent stem cells derived from a patient with Duchenne muscular dystrophy. Plasmid‐mediated dystrophin expression was also demonstrated in mouse muscle. The dystrophin expression plasmids described here will be useful in cell and gene therapy studies aimed at ameliorating Duchenne muscular dystrophy.
Gene therapy approaches that seek to utilize full‐length dystrophin (DMD), the gene mutated in Duchenne muscular dystrophy, are challenging, primarily due to the large size of DMD cDNA. To create better options, the authors constructed plasmids that robustly co‐express human DMD (hDMD) with several marker genes on a polycistron and validated these vectors in a DMD patient induced pluripotent cell line. Our vectors are well suited for large‐scale production in E. coli, can be stably integrated into the genome with phiC31 integrase, and can be used to optimize delivery and expression of full‐length hDMD in muscle cells and tissue.
Here we present a compendium of single-cell transcriptomic data from the model organism Mus musculus that comprises more than 100,000 cells from 20 organs and tissues. These data represent a new ...resource for cell biology, reveal gene expression in poorly characterized cell populations and enable the direct and controlled comparison of gene expression in cell types that are shared between tissues, such as T lymphocytes and endothelial cells from different anatomical locations. Two distinct technical approaches were used for most organs: one approach, microfluidic droplet-based 3'-end counting, enabled the survey of thousands of cells at relatively low coverage, whereas the other, full-length transcript analysis based on fluorescence-activated cell sorting, enabled the characterization of cell types with high sensitivity and coverage. The cumulative data provide the foundation for an atlas of transcriptomic cell biology.
Ageing is characterized by a progressive loss of physiological integrity, leading to impaired function and increased vulnerability to death
. Despite rapid advances over recent years, many of the ...molecular and cellular processes that underlie the progressive loss of healthy physiology are poorly understood
. To gain a better insight into these processes, here we generate a single-cell transcriptomic atlas across the lifespan of Mus musculus that includes data from 23 tissues and organs. We found cell-specific changes occurring across multiple cell types and organs, as well as age-related changes in the cellular composition of different organs. Using single-cell transcriptomic data, we assessed cell-type-specific manifestations of different hallmarks of ageing-such as senescence
, genomic instability
and changes in the immune system
. This transcriptomic atlas-which we denote Tabula Muris Senis, or 'Mouse Ageing Cell Atlas'-provides molecular information about how the most important hallmarks of ageing are reflected in a broad range of tissues and cell types.
Mutations in the gene for dysferlin cause a degenerative disorder of skeletal muscle known as limb girdle muscular dystrophy 2B. To achieve gene delivery of plasmids encoding dysferlin to hind limb ...muscles of dysferlin knockout mice, we used a vascular injection method that perfused naked plasmid DNA into all major muscle groups of the hind limb. We monitored delivery by luciferase live imaging and western blot, confirming strong dysferlin expression that persisted over the 3-month time course of the experiment. Co-delivery of the follistatin gene, which may promote muscle growth, was monitored by ELISA. Immunohistochemistry documented the presence of dysferlin in muscle fibers in treated limbs, and PCR confirmed the presence of plasmid DNA. Because dysferlin is involved in repair of the sarcolemmal membrane, dysferlin loss leads to fragile sarcolemmal membranes that can be detected by permeability to Evan's blue dye. We showed that after gene therapy with a plasmid encoding both dysferlin and follistatin, statistically significant reduction in Evan's blue dye permeability was present in hamstring muscles. These results suggest that vascular delivery of plasmids carrying these therapeutic genes may lead to simple and effective approaches for improving the clinical condition of limb girdle muscular dystrophy 2B.
Ageing is the single greatest cause of disease and death worldwide, and understanding the associated processes could vastly improve quality of life. Although major categories of ageing damage have ...been identified-such as altered intercellular communication, loss of proteostasis and eroded mitochondrial function
-these deleterious processes interact with extraordinary complexity within and between organs, and a comprehensive, whole-organism analysis of ageing dynamics has been lacking. Here we performed bulk RNA sequencing of 17 organs and plasma proteomics at 10 ages across the lifespan of Mus musculus, and integrated these findings with data from the accompanying Tabula Muris Senis
-or 'Mouse Ageing Cell Atlas'-which follows on from the original Tabula Muris
. We reveal linear and nonlinear shifts in gene expression during ageing, with the associated genes clustered in consistent trajectory groups with coherent biological functions-including extracellular matrix regulation, unfolded protein binding, mitochondrial function, and inflammatory and immune response. Notably, these gene sets show similar expression across tissues, differing only in the amplitude and the age of onset of expression. Widespread activation of immune cells is especially pronounced, and is first detectable in white adipose depots during middle age. Single-cell RNA sequencing confirms the accumulation of T cells and B cells in adipose tissue-including plasma cells that express immunoglobulin J-which also accrue concurrently across diverse organs. Finally, we show how gene expression shifts in distinct tissues are highly correlated with corresponding protein levels in plasma, thus potentially contributing to the ageing of the systemic circulation. Together, these data demonstrate a similar yet asynchronous inter- and intra-organ progression of ageing, providing a foundation from which to track systemic sources of declining health at old age.
Antigen-specific CD8
T cell accumulation in tumors is a prerequisite for effective immunotherapy, and yet the mechanisms of lymphocyte transit are not well defined. Here we show that tumor-associated ...lymphatic vessels control T cell exit from tumors via the chemokine CXCL12, and intratumoral antigen encounter tunes CXCR4 expression by effector CD8
T cells. Only high-affinity antigen downregulates CXCR4 and upregulates the CXCL12 decoy receptor, ACKR3, thereby reducing CXCL12 sensitivity and promoting T cell retention. A diverse repertoire of functional tumor-specific CD8
T cells, therefore, exit the tumor, which limits the pool of CD8
T cells available to exert tumor control. CXCR4 inhibition or loss of lymphatic-specific CXCL12 boosts T cell retention and enhances tumor control. These data indicate that strategies to limit T cell egress might be an approach to boost the quantity and quality of intratumoral T cells and thereby response to immunotherapy.
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