Dictionary of immune responses to cytokines at single-cell resolution.

Cytokines mediate cell-cell communication in the immune system and represent important therapeutic targets1-3. A myriad of studies have highlighted their central role in immune function4-13, yet we lack a global view of the cellular responses of each immune cell type to each cytokine. To address this gap, we created the Immune Dictionary, a compendium of single-cell transcriptomic profiles of more than 17 immune cell types in response to each of 86 cytokines (>1,400 cytokine-cell type combinations) in mouse lymph nodes in vivo. A cytokine-centric view of the dictionary revealed that most cytokines induce highly cell-type-specific responses. For example, the inflammatory cytokine interleukin-1ß induces distinct gene programmes in almost every cell type. A cell-type-centric view of the dictionary identified more than 66 cytokine-driven cellular polarization states across immune cell types, including previously uncharacterized states such as an interleukin-18-induced polyfunctional natural killer cell state. Based on this dictionary, we developed companion software, Immune Response Enrichment Analysis, for assessing cytokine activities and immune cell polarization from gene expression data, and applied it to reveal cytokine networks in tumours following immune checkpoint blockade therapy. Our dictionary generates new hypotheses for cytokine functions, illuminates pleiotropic effects of cytokines, expands our knowledge of activation states of each immune cell type, and provides a framework to deduce the roles of specific cytokines and cell-cell communication networks in any immune response.

Single-cell atlas of the liver myeloid compartment before and after cure of chronic viral hepatitis.

BACKGROUND & AIMS: Chronic viral infections present serious public health challenges; however, direct-acting antivirals (DAAs) are now able to cure nearly all patients infected with hepatitis C virus (HCV), representing the only cure of a human chronic viral infection to date. DAAs provide a valuable opportunity to study immune pathways in the reversal of chronic immune failures in an in vivo human system. METHODS: To leverage this opportunity, we used plate-based single-cell RNA-seq to deeply profile myeloid cells from liver fine needle aspirates in patients with HCV before and after DAA treatment. We comprehensively characterised liver neutrophils, eosinophils, mast cells, conventional dendritic cells, plasmacytoid dendritic cells, classical monocytes, non-classical monocytes, and macrophages, and defined fine-grained subpopulations of several cell types. RESULTS: We discovered cell type-specific changes post-cure, including an increase in MCM7+STMN1+ proliferating CD1C+ conventional dendritic cells, which may support restoration from chronic exhaustion. We observed an expected downregulation of interferon-stimulated genes (ISGs) post-cure as well as an unexpected inverse relationship between pre-treatment viral load and post-cure ISG expression in each cell type, revealing a link between viral loads and sustained modifications of the host's immune system. We found an upregulation of PD-L1/L2 gene expression in ISG-high neutrophils and IDO1 expression in eosinophils, pinpointing cell subpopulations crucial for immune regulation. We identified three recurring gene programmes shared by multiple cell types, distilling core functions of the myeloid compartment. CONCLUSIONS: This comprehensive single-cell RNA-seq atlas of human liver myeloid cells in response to cure of chronic viral infections reveals principles of liver immunity and provides immunotherapeutic insights. CLINICAL TRIAL REGISTRATION: This study is registered at ClinicalTrials.gov (NCT02476617). IMPACT AND IMPLICATIONS: Chronic viral liver infections continue to be a major public health problem. Single-cell characterisation of liver immune cells during hepatitis C and post-cure provides unique insights into the architecture of liver immunity contributing to the resolution of the first curable chronic viral infection of humans. Multiple layers of innate immune regulation during chronic infections and persistent immune modifications after cure are revealed. Researchers and clinicians may leverage these findings to develop methods to optimise the post-cure environment for HCV and develop novel therapeutic approaches for other chronic viral infections.

Investigations of methylmercury-induced alterations in neurogenesis.

Methylmercury (MeHg) has been an environmental concern to public health and regulatory agencies for over 50 years because of its toxicity to the human nervous system. Its association with nervous system toxicity in adults and infants near Minamata Bay, Japan, in the 1950s initiated environmental health research inquiries that continue to this day. Observations of greater neurotoxicity with gestational compared with adult exposure suggest a unique susceptibility of the developing nervous system to MeHg. Despite extensive research conducted over the last half century, determination of definitive molecular mechanisms underlying the observed neurotoxic effects of MeHg have not been identified. This paper summarizes results of a series of experiments conducted to examine the effects of MeHg on neuroepithelial cell proliferation, a hypothesized mode of action for its selective effects on neurogenesis. Observed effects of MeHg on cell cycle entry and progression were associated with alterations in a variety of cell cycle regulatory molecules, including p21 signaling pathways. We place these studies in the context of other cellular responses involved in signal transduction, including oxidative stress, altered protein phosphorylation, and altered intracellular calcium homeostasis. Although existing information suggests that no single mechanism underlies the diverse array of effects associated with MeHg-induced developmental neurotoxicity, we demonstrate characteristic effects of MeHg on cell signaling that contribute to observed effects on cell proliferation. Experimentally derived cell cycle kinetic and cytotoxicity data allowed development of a biologically based dose-response model of MeHg-induced alterations in neurodevelopment, which can form the basis for information synthesis and hypothesis testing and for use in assessing risks from environmental exposures.

p21(WAF1/CIP1) inhibits cell cycle progression but not G2/M-phase transition following methylmercury exposure.

Methylmercury (MeHg) is an environmentally prevalent organometal that is particularly toxic to the developing central nervous system (CNS). Prenatal MeHg exposure is associated with reduced brain size and weight and a reduced number of neurons, which have been associated with impaired cell proliferation. We evaluate the role of p21, a cell cycle protein involved in the G1- and G2-phase checkpoint control, in the cell cycle inhibition induced by MeHg. Primary mouse embryonic fibroblasts (MEFs) of different p21 genotypes (wild-type, heterozygous, and null) were isolated at day 14 of gestation and treated at passages 4-6 with either 0, 2, 4, or 6 microM MeHg or 50 nM colchicine for 24 h. Changes in cell cycle distribution after continuous toxicant treatment were analyzed by DNA content-based flow cytometry using DAPI. MeHg induced an increase in the proportion of cells in G2/M at 2 and 4 microM MeHg (p < or = 0.05) irrespective of p21 genotype. Effects of MeHg on cell cycle progression were subsequently evaluated using BrdU-Hoechst flow cytometric analysis. Inhibition of cell cycle progression was observed in all p21 genotypes after continuous exposure to MeHg for 24 and 48 h. p21 null (-/-) cells reached the second-round G1 at a higher fraction compared to the wild type (+/+) and heterozygous (+/-) cells (p < or = 0.05). These data support previous observations that MeHg inhibits cell cycle progression through delayed G2/M transition. Whereas the G2/M accumulation induced by MeHg was independent of p21 status, a greater proportion of p21(-/-) cells were able to complete one round of cell division in the presence of MeHg compared to p21(+/-) or p21(+/+) cells. These data suggest a role for p21 in retarding cell cycle progression, but not mitotic inhibition, following exposure to MeHg.