Spatially resolved single-cell RNA sequencing (scRNAseq) is a powerful approach for inferring connections between a cell's identity and its position in a tissue. We recently combined scRNAseq with spatially mapped landmark genes to infer the expression zonation of hepatocytes. However, determining zonation of small cells with low mRNA content, or without highly expressed landmark genes, remains challenging. Here we used paired-cell sequencing, in which mRNA from pairs of attached mouse cells were sequenced and gene expression from one cell type was used to infer the pairs' tissue coordinates. We applied this method to pairs of hepatocytes and liver endothelial cells (LECs). Using the spatial information from hepatocytes, we reconstructed LEC zonation and extracted a landmark gene panel that we used to spatially map LEC scRNAseq data. Our approach revealed the expression of both Wnt ligands and the Dkk3 Wnt antagonist in distinct pericentral LEC sub-populations. This approach can be used to reconstruct spatial expression maps of non-parenchymal cells in other tissues.

SummarySenescent cells are present in premalignant lesions and sites of tissue damage and accumulate in tissues with age. In vivo identification, quantification and characterization of senescent cells are challenging tasks that limit our understanding of the role of senescent cells in diseases and aging. Here, we present a new way to precisely quantify and identify senescent cells in tissues on a single‐cell basis. The method combines a senescence‐associated beta‐galactosidase assay with staining of molecular markers for cellular senescence and of cellular identity. By utilizing technology that combines flow cytometry with high‐content image analysis, we were able to quantify senescent cells in tumors, fibrotic tissues, and tissues of aged mice. Our approach also yielded the finding that senescent cells in tissues of aged mice are larger than nonsenescent cells. Thus, this method provides a basis for quantitative assessment of senescent cells and it offers proof of principle for combination of different markers of senescence. It paves the way for screening of senescent cells for identification of new senescence biomarkers, genes that bypass senescence or senolytic compounds that eliminate senescent cells, thus enabling a deeper understanding of the senescent state in vivo.

STING is an innate immune cytosolic adaptor for DNA sensors that engage malaria parasite (Plasmodium falciparum) or other pathogen DNA. As P. falciparum infects red blood cells and not leukocytes, how parasite DNA reaches such host cytosolic DNA sensors in immune cells is unclear. Here we show that malaria parasites inside red blood cells can engage host cytosolic innate immune cell receptors from a distance by secreting extracellular vesicles (EV) containing parasitic small RNA and genomic DNA. Upon internalization of DNA-harboring EVs by human monocytes, P. falciparum DNA is released within the host cell cytosol, leading to STING-dependent DNA sensing. STING subsequently activates the kinase TBK1, which phosphorylates the transcription factor IRF3, causing IRF3 to translocate to the nucleus and induce STING-dependent gene expression. This DNA-sensing pathway may be an important decoy mechanism to promote P. falciparum virulence and thereby may affect future strategies to treat malaria.

We recently reported that the posttraumatic spread of degeneration in the damaged optic nerve can be attenuated by the adoptive transfer of autoimmune T cells specific to myelin basic protein. However, it would be desirable to obtain immune neuroprotection free of any possible autoimmune disease. In an attempt to obtain disease-free immune neuroprotection, we used the synthetic fouramino acid polymer copolymer 1 (Cop-1), which is known not to be encephalitogenic despite its cross-reactivity with myelin basic protein. We show here that active immunization with Cop-1 administered in adjuvant, as well as adoptive transfer of T cells reactive to Cop-1, can inhibit the progression of secondary degeneration after crush injury of the rat optic nerve. These results have implications for the treatment of optic neuropathies.

Retention of long-term repopulating hematopoietic stem cells (LT-HSCs) in the bone marrow is essential for hematopoiesis and for protection from myelotoxic injury. We report that signaling cascades that are traditionally viewed as coagulation-related also control retention of EPCR+ LT-HSCs in the bone marrow and their recruitment to the blood via two different protease activated receptor 1 (PAR1)-mediated pathways. Thrombin-PAR1 signaling induces nitric oxide (NO) production, leading to TACE-mediated EPCR shedding, enhanced CXCL12-CXCR4-induced motility, and rapid stem and progenitor cell mobilization. Conversely, bone marrow blood vessels provide a microenvironment enriched with protein C that retain EPCR+ LT-HSCs by limiting NO generation, reducing Cdc42 activity and enhancing VLA4 affinity and adhesion. Inhibition of NO production by activated protein C (aPC)-EPCR-PAR1 signaling reduces progenitor cell egress, increases NOlow bone marrow EPCR+ LT-HSCs retention and protects mice from chemotherapy-induced hematological failure and death. Our study reveals new roles for PAR1 and EPCR that control NO production to balance maintenance and recruitment of bone marrow EPCR+ LT-HSCs with clinical relevance.