The full neutrophil heterogeneity and differentiation landscape remains incompletely characterized. Here we profiled >25,000 differentiating and mature mouse neutrophils using single-cell RNA sequencing to provide a comprehensive transcriptional landscape of neutrophil maturation, function, and fate decision in their steady state and during bacterial infection. Eight neutrophil populations were defined by distinct molecular signatures. The three mature peripheral blood neutrophil subsets arise from distinct maturing bone marrow neutrophil subsets. Driven by both known and uncharacterized transcription factors, neutrophils gradually acquire microbicidal capability as they traverse the transcriptional landscape, representing an evolved mechanism for fine-tuned regulation of an effective but balanced neutrophil response. Bacterial infection reprograms the genetic architecture of neutrophil populations, alters dynamic transition between each subpopulation, and primes neutrophils for augmented functionality without affecting overall heterogeneity. In summary, these data establish a reference model and general framework for studying neutrophil-related disease mechanisms, biomarkers, and therapeutic targets at single-cell resolution.

Long non-coding RNAs (lncRNAs) play critical roles in diverse cellular processes; however, their involvement in many critical aspects of the immune response including the interferon (IFN) response remains poorly understood. To address this gap, we compared the global gene expression pattern of primary human hepatocytes before and at three time points after treatment with IFN-α. Among ∼200 IFN-induced lncRNAs, one transcript showed ∼100-fold induction. This RNA, which we named lncRNA-CMPK2, was a spliced, polyadenylated nuclear transcript that was induced by IFN in diverse cell types from human and mouse. Similar to protein-coding IFN-stimulated genes (ISGs), its induction was dependent on JAK-STAT signaling. Intriguingly, knockdown of lncRNA-CMPK2 resulted in a marked reduction in HCV replication in IFN-stimulated hepatocytes, suggesting that it could affect the antiviral role of IFN. We could show that lncRNA-CMPK2 knockdown resulted in upregulation of several protein-coding antiviral ISGs. The observed upregulation was caused by an increase in both basal and IFN-stimulated transcription, consistent with loss of transcriptional inhibition in knockdown cells. These results indicate that the IFN response involves a lncRNA-mediated negative regulatory mechanism. lncRNA-CMPK2 was strongly upregulated in a subset of HCV-infected human livers, suggesting a role in modulation of the IFN response in vivo.

SUMMARY Gasdermin D (GSDMD) is considered a proinflammatory factor that mediates pyroptosis in macrophages to protect hosts from intracellular bacteria. Here, we reveal that GSDMD deficiency paradoxically augmented host responses to extracellular Escherichia coli, mainly by delaying neutrophil death, which established GSDMD as a negative regulator of innate immunity. In contrast to its activation in macrophages, in which activated inflammatory caspases cleave GSDMD to produce an N-terminal fragment (GSDMD-cNT) to trigger pyroptosis, GSDMD cleavage and activation in neutrophils was caspase independent. It was mediated by a neutrophil-specific serine protease, neutrophil elastase (ELANE), released from cytoplasmic granules into the cytosol in aging neutrophils. ELANE-mediated GSDMD cleavage was upstream of the caspase cleavage site and produced a fully active ELANE-derived NT fragment (GSDMD-eNT) that induced lytic cell death as efficiently as GSDMD-cNT. Thus, GSDMD is pleiotropic, exerting both pro- and anti-inflammatory effects that make it a potential target for antibacterial and anti-inflammatory therapies.

Inflammasomes promote interleukin (IL)-1β secretion and pyroptosis. Kanneganti et al. now show that the pyroptosis effector gasdermin D (GSDMD) is required for systemic IL-1β secretion and autoinflammatory pathology in a mouse model of Familial Mediterranean Fever (FMF), suggesting GSDMD inhibitors as potential antiinflammatory treatments.

dStress granules (SGs) are cytoplasmic foci composed of stalled translation preinitiation complexes induced by environmental stress stimuli, including viral infection. Since viral propagation completely depends on the host translational machinery, many viruses have evolved to circumvent the induction of SGs or co-opt SG components. In this study, we found that expression of Japanese encephalitis virus (JEV) core protein inhibits SG formation. Caprin-1 was identified as a binding partner of the core protein by an affinity capture mass spectrometry analysis. Alanine scanning mutagenesis revealed that Lys 97 and Arg 98 in the ␣-helix of the JEV core protein play a crucial role in the interaction with Caprin-1. In cells infected with a mutant JEV in which Lys 97 and Arg 98 were replaced with alanines in the core protein, the inhibition of SG formation was abrogated, and viral propagation was impaired. Furthermore, the mutant JEV exhibited attenuated virulence in mice. These results suggest that the JEV core protein circumvents translational shutoff by inhibiting SG formation through an interaction with Caprin-1 and facilitates viral propagation in vitro and in vivo.

No abstract

Japanese encephalitis virus (JEV) is a small, enveloped virus belonging to the family Flaviviridae and the genus Flavivirus, which also includes Dengue virus (DENV), West Nile virus (WNV), Yellow fever virus, and Tick-borne encephalitis virus (11). JEV is the most common agent of viral encephalitis, causing approximately 50,000 cases annually, of which 15,000 will die, and up to 50% of survivors are left with severe residual neurological complications. JEV has a single-stranded positive-sense RNA genome of approximately 11 kb, encoding a single large polyprotein, which is cleaved by the host-and virus-encoded proteases into three structural proteins, capsid (C), premembrane (PrM), and envelope (E), and seven nonstructural proteins. The structural proteins are components of viral particles, and the E protein is suggested to interact with a cell surface receptor molecule(s). Although a number of cellular components, including heat shock cognate protein 70 (33), glycosaminoglycans, such as heparin or heparan sulfate (21, 41), and laminin (3), have been shown to participate in JEV infection, the precise mechanisms by which these receptor candidates participate in JEV infection remain largely unclear.In addition to the many studies identifying and characterizing receptor molecules in numerous viruses, data suggesting the involvement of membrane lipids, such as sphingolipids and cholesterol, in viral infection have also been accumulating. Lipid rafts consisting of sphingolipids and cholesterol and distributing to the outer leaflet of the cell membrane have been shown to be involved in the infection of not only many viruses but also several bacteria and parasites (24), in addition to playing roles in various functions such as lipid sorting, protein trafficking (26, 47), cell polarity, and signal transduction (38). With respect to cholesterol itself, various aspects of the life cycle of flaviviruses have been shown to involve this lipid, including the entry of DENV (34), hepatitis C virus (HCV) (16), and WNV (27), the membrane fusion of tick-borne encephalitis virus (40), and the replication of HCV (14, 17), WNV (23), and DENV (35). Recently Lee et al. (20) showed that treatment with cholesterol efficiently impairs both the entry and replication steps of JEV and DENV-2 but enhances infection with the Sindbis virus (22).On the other hand, sphingolipids, including sphingomyelins and glycosphingolipids, are ubiquitous components of eukaryotic cell membrane structures, providing integrity to cellular membranes. Ceramide is one of the intermediates of sphingolipids and plays roles in cell differentiation, regulation of apoptosis and protein secretion, induction of cellular senescence, and other processes (2). Ceramide is generated from the hydrolysis of sphingomyelin by sphingomyelinase (SMase) or from catalysis by serine-palmitoyl-coenzyme A (CoA) transferase and ceramide synthase. Ceramide spontaneously selfassociates to form ceramide-enriched microdomains and then to form larger ceramide-enriched membrane platforms which...

The robust cell culture systems for hepatitis C virus (HCV) are limited to those using cell culture-adapted clones (HCV in cell culture [HCVcc]) and cells derived from the human hepatoma cell line Huh7. However, accumulating data suggest that host factors, including innate immunity and gene polymorphisms, contribute to the variation in host response to HCV infection. Therefore, the existing in vitro systems for HCV propagation are not sufficient to elucidate the life cycle of HCV. A liver-specific microRNA, miR122, has been shown to participate in the efficient replication of HCV. In this study, we examined the possibility of establishing a new permissive cell line for HCV propagation by the expression of miR122. A high level of miR122 was expressed by a lentiviral vector placed into human liver cell lines at a level comparable to the endogenous level in Huh7 cells. Among the cell lines that we examined, Hep3B cells stably expressing miR122 (Hep3B/miR122) exhibited a significant enhancement of HCVcc propagation. Surprisingly, the levels of production of infectious particles in Hep3B/miR122 cells upon infection with HCVcc were comparable to those in Huh7 cells. Furthermore, a line of "cured" cells, established by elimination of HCV RNA from the Hep3B/miR122 replicon cells, exhibited an enhanced expression of miR122 and a continuous increase of infectious titers of HCVcc in every passage. The establishment of the new permissive cell line for HCVcc will have significant implications not only for basic HCV research but also for the development of new therapeutics.