Single-cell transcriptomics reveals bimodality in expression and splicing in immune cells

Recent molecular studies have shown that, even when derived from a seemingly homogenous population, individual cells can exhibit substantial differences in gene expression, protein levels and phenotypic output1, 2, 3, 4, 5, with important functional consequences4, 5. Existing studies of cellular heterogeneity, however, have typically measured only a few pre-selected RNAs1, 2 or proteins5, 6 simultaneously, because genomic profiling methods3 could not be applied to single cells until very recently7, 8, 9, 10. Here we use single-cell RNA sequencing to investigate heterogeneity in the response of mouse bone-marrow-derived dendritic cells (BMDCs) to lipopolysaccharide. We find extensive, and previously unobserved, bimodal variation in messenger RNA abundance and splicing patterns, which we validate by RNA-fluorescence in situ hybridization for select transcripts. In particular, hundreds of key immune genes are bimodally expressed across cells, surprisingly even for genes that are very highly expressed at the population average. Moreover, splicing patterns demonstrate previously unobserved levels of heterogeneity between cells. Some of the observed bimodality can be attributed to closely related, yet distinct, known maturity states of BMDCs; other portions reflect differences in the usage of key regulatory circuits. For example, we identify a module of 137 highly variable, yet co-regulated, antiviral response genes. Using cells from knockout mice, we show that variability in this module may be propagated through an interferon feedback circuit, involving the transcriptional regulators Stat2 and Irf7. Our study demonstrates the power and promise of single-cell genomics in uncovering functional diversity between cells and in deciphering cell states and circuits.National Institutes of Health (U.S.) (NIH Postdoctoral Fellowship (1F32HD075541-01))Charles H. Hood Foundation (Postdoctoral Fellowship)National Institutes of Health (U.S.) (NIH grant U54 AI057159)National Institutes of Health (U.S.) (NIH New Innovator Award (DP2 OD002230))National Institutes of Health (U.S.) (NIH CEGS Award (1P50HG006193-01))National Institutes of Health (U.S.) (NIH Pioneer Awards (5DP1OD003893-03))National Institutes of Health (U.S.) (NIH Pioneer Awards (DP1OD003958-01))Broad Institute of MIT and HarvardBroad Institute of MIT and Harvard (Klarman Cell Observatory

Single-cell RNA-seq reveals dynamic paracrine control of cellular variation

High-throughput single-cell transcriptomics offers an unbiased approach for understanding the extent, basis and function of gene expression variation between seemingly identical cells. Here we sequence single-cell RNA-seq libraries prepared from over 1,700 primary mouse bone-marrow-derived dendritic cells spanning several experimental conditions. We find substantial variation between identically stimulated dendritic cells, in both the fraction of cells detectably expressing a given messenger RNA and the transcript’s level within expressing cells. Distinct gene modules are characterized by different temporal heterogeneity profiles. In particular, a ‘core’ module of antiviral genes is expressed very early by a few ‘precocious’ cells in response to uniform stimulation with a pathogenic component, but is later activated in all cells. By stimulating cells individually in sealed microfluidic chambers, analysing dendritic cells from knockout mice, and modulating secretion and extracellular signalling, we show that this response is coordinated by interferon-mediated paracrine signalling from these precocious cells. Notably, preventing cell-to-cell communication also substantially reduces variability between cells in the expression of an early-induced ‘peaked’ inflammatory module, suggesting that paracrine signalling additionally represses part of the inflammatory program. Our study highlights the importance of cell-to-cell communication in controlling cellular heterogeneity and reveals general strategies that multicellular populations can use to establish complex dynamic responses.National Human Genome Research Institute (U.S.). Centers of Excellence in Genomic Science (1P50HG006193-01)National Institutes of Health (U.S.). Pioneer Award (DP1OD003958-01)Howard Hughes Medical InstituteBroad Institute of MIT and Harvard. Klarman Cell Observator

Single cell RNA Seq reveals dynamic paracrine control of cellular variation

High-throughput single-cell transcriptomics offers an unbiased approach for understanding the extent, basis, and function of gene expression variation between seemingly identical cells. Here, we sequence single-cell RNA-Seq libraries prepared from over 1,700 primary mouse bone marrow derived dendritic cells (DCs) spanning several experimental conditions. We find substantial variation between identically stimulated DCs, in both the fraction of cells detectably expressing a given mRNA and the transcript’s level within expressing cells. Distinct gene modules are characterized by different temporal heterogeneity profiles. In particular, a “core” module of antiviral genes is expressed very early by a few “precocious” cells, but is later activated in all cells. By stimulating cells individually in sealed microfluidic chambers, analyzing DCs from knockout mice, and modulating secretion and extracellular signaling, we show that this response is coordinated via interferon-mediated paracrine signaling. Surprisingly, preventing cell-to-cell communication also substantially reduces variability in the expression of an early-induced “peaked” inflammatory module, suggesting that paracrine signaling additionally represses part of the inflammatory program. Our study highlights the importance of cell-to-cell communication in controlling cellular heterogeneity and reveals general strategies that multicellular populations use to establish complex dynamic responses

Whole exome sequencing of circulating tumor cells provides a window into metastatic prostate cancer

Comprehensive analyses of cancer genomes promise to inform prognoses and precise cancer treatments. A major barrier, however, is inaccessibility of metastatic tissue. A potential solution is to characterize circulating tumor cells (CTCs), but this requires overcoming the challenges of isolating rare cells and sequencing low-input material. Here we report an integrated process to isolate, qualify and sequence whole exomes of CTCs with high fidelity, using a census-based sequencing strategy. Power calculations suggest that mapping of >99.995% of the standard exome is possible in CTCs. We validated our process in two prostate cancer patients including one for whom we sequenced CTCs, a lymph node metastasis and nine cores of the primary tumor. Fifty-one of 73 CTC mutations (70%) were observed in matched tissue. Moreover, we identified 10 early-trunk and 56 metastatic-trunk mutations in the non-CTC tumor samples and found 90% and 73% of these, respectively, in CTC exomes. This study establishes a foundation for CTC genomics in the clinic

The neuropeptide NMU amplifies ILC2-driven allergic lung inflammation

Type 2 innate lymphoid cells (ILC2s) both contribute to mucosal homeostasis and initiate pathologic inflammation in allergic asthma. However, the signals that direct ILC2s to promote homeostasis versus inflammation are unclear. To identify such molecular cues, we profiled mouse lung-resident ILCs using single-cell RNA sequencing at steady state and after in vivo stimulation with the alarmin cytokines IL-25 and IL-33. ILC2s were transcriptionally heterogeneous after activation, with subpopulations distinguished by expression of proliferative, homeostatic and effector genes. The neuropeptide receptor Nmur1 was preferentially expressed by ILC2s at steady state and after IL-25 stimulation. Neuromedin U (NMU), the ligand of NMUR1, activated ILC2s in vitro, and in vivo co-administration of NMU with IL-25 strongly amplified allergic inflammation. Loss of NMU-NMUR1 signalling reduced ILC2 frequency and effector function, and altered transcriptional programs following allergen challenge in vivo. Thus, NMUR1 signalling promotes inflammatory ILC2 responses, highlighting the importance of neuro-immune crosstalk in allergic inflammation at mucosal surfaces

Calcineurin Interacts with PERK and Dephosphorylates Calnexin to Relieve ER Stress in Mammals and Frogs

Background: The accumulation of misfolded proteins within the endoplasmic reticulum (ER) triggers a cellular process known as the Unfolded Protein Response (UPR). One of the earliest responses is the attenuation of protein translation. Little is known about the role that Ca 2+ mobilization plays in the early UPR. Work from our group has shown that cytosolic phosphorylation of calnexin (CLNX) controls Ca 2+ uptake into the ER via the sarco-endoplasmic reticulum Ca 2+-ATPase (SERCA) 2b. Methodology/Principal Findings: Here, we demonstrate that calcineurin (CN), a Ca 2+ dependent phosphatase, associates with the (PKR)-like ER kinase (PERK), and promotes PERK auto-phosphorylation. This association, in turn, increases the phosphorylation level of eukaryotic initiation factor-2 a (eIF2-a) and attenuates protein translation. Data supporting these conclusions were obtained from co-immunoprecipitations, pull-down assays, in-vitro kinase assays, siRNA treatments and [ 35 S]-methionine incorporation measurements. The interaction of CN with PERK was facilitated at elevated cytosolic Ca 2+ concentrations and involved the cytosolic domain of PERK. CN levels were rapidly increased by ER stressors, which could be blocked by siRNA treatments for CN-Aa in cultured astrocytes. Downregulation of CN blocked subsequent ER-stress-induced increases in phosphorylated elF2-a. CN knockdown in Xenopus oocytes predisposed them to induction of apoptosis. We also found that CLNX was dephosphorylated by CN when Ca 2+ increased. These data were obtained from [c 32 P]-CLN

Rethinking energy, climate and security: a critical analysis of energy security in the US

Understanding the complicated relationship between energy, climate and security is vital both to the study of international relations and to ensure the continued survival of a world increasingly threatened by environmental change. Climate change is largely caused by burning fossil fuels for energy, but while discussions on the climate consider the role of energy, energy security debates largely overlook climate concerns. This article traces the separation between energy and climate through an analysis of US energy security discourse and policy. It shows that energy security is continually constructed as national security, which enables very particular policy choices and prioritises it above climate concerns. Thus, in many cases, policies undertaken in the name of energy security contribute directly to climate insecurity. The article argues that the failure to consider securing the climate as inherently linked to energy security is not just problematic, but, given global warming, potentially harmful. Consequently, any approach to dealing with climate change has to begin by rethinking energy security and security more broadly, as national (energy) security politics no longer provides security in any meaningful sense

The IDENTIFY study: the investigation and detection of urological neoplasia in patients referred with suspected urinary tract cancer - a multicentre observational study

Objective To evaluate the contemporary prevalence of urinary tract cancer (bladder cancer, upper tract urothelial cancer [UTUC] and renal cancer) in patients referred to secondary care with haematuria, adjusted for established patient risk markers and geographical variation. Patients and Methods This was an international multicentre prospective observational study. We included patients aged ≥16 years, referred to secondary care with suspected urinary tract cancer. Patients with a known or previous urological malignancy were excluded. We estimated the prevalence of bladder cancer, UTUC, renal cancer and prostate cancer; stratified by age, type of haematuria, sex, and smoking. We used a multivariable mixed-effects logistic regression to adjust cancer prevalence for age, type of haematuria, sex, smoking, hospitals, and countries. Results Of the 11 059 patients assessed for eligibility, 10 896 were included from 110 hospitals across 26 countries. The overall adjusted cancer prevalence (n = 2257) was 28.2% (95% confidence interval [CI] 22.3–34.1), bladder cancer (n = 1951) 24.7% (95% CI 19.1–30.2), UTUC (n = 128) 1.14% (95% CI 0.77–1.52), renal cancer (n = 107) 1.05% (95% CI 0.80–1.29), and prostate cancer (n = 124) 1.75% (95% CI 1.32–2.18). The odds ratios for patient risk markers in the model for all cancers were: age 1.04 (95% CI 1.03–1.05; P < 0.001), visible haematuria 3.47 (95% CI 2.90–4.15; P < 0.001), male sex 1.30 (95% CI 1.14–1.50; P < 0.001), and smoking 2.70 (95% CI 2.30–3.18; P < 0.001). Conclusions A better understanding of cancer prevalence across an international population is required to inform clinical guidelines. We are the first to report urinary tract cancer prevalence across an international population in patients referred to secondary care, adjusted for patient risk markers and geographical variation. Bladder cancer was the most prevalent disease. Visible haematuria was the strongest predictor for urinary tract cancer

Centrality evolution of the charged-particle pseudorapidity density over a broad pseudorapidity range in Pb-Pb collisions at root s(NN)=2.76TeV

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