Search

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., In Brief Kambara et al. find that GSDMD deficiency augments host responses to extracellular Escherichia coli, mainly by delaying neutrophil death, establishing GSDMD as a negative regulator of innate immunity. GSDMD cleavage and activation in neutrophils is mediated by ELANE, released from cytoplasmic granules into the cytosol in aging neutrophils.

Gasdermin D (GSDMD) is considered a pro-inflammatory factor that mediates lytic pyroptotic cell death in macrophages to protect hosts from intracellular bacteria (He et al., 2015; Kayagaki et al., 2015; Shi et al., 2015a). However, GSDMD’s role in clearing extracellular pathogens has not been directly examined. Here we reveal that GSDMD deficiency unexpectedly and paradoxically augmented host responses to extracellular Escherichia coli, mainly by delaying neutrophil death, for the first time establishing GSDMD as a negative regulator of innate immunity. The levels of pro-inflammatory cytokines in the peritoneal cavity were significantly elevated in GSDMD-deficient mice, suggesting that the production of these cytokines was not mainly mediated by macrophage pyroptosis. This further confirmed that in E.coli-induced peritonitis GSDMD acted as an anti-inflammatory factor. 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 and mediated by a neutrophil-specific serine protease, neutrophil elastase (ELANE), released from cytoplasmic granules into the cytosol during neutrophil death. ELANE-mediated GSDMD cleavage was upstream of the caspase cleavage site and produced a fully active ELANE-derived Nterminal fragment (GSDMD-eNT) that induced lytic cell death as efficiently as GSDMD-cNT. This is the first description of GSDMD’s role in neutrophil death and the negative regulation of neutrophil-mediated innate immunity. Thus, GSDMD is pleiotropic, exerting both pro- and anti-inflammatory effects that make it a unique target for novel anti-bacterial and anti-inflammatory therapies.

Japanese encephalitis virus (JEV) is a mosquito-borne RNA virus that belongs to the Flaviviridae family. In this study, we have examined the effect of cyclosporin A (CsA) on the propagation of JEV. CsA exhibited potent anti-JEV activity in various mammalian cell lines through the inhibition of CypB. The propagation of JEV was impaired in the CypB-knockdown cells and this reduction was cancelled by the expression of wild-type but not of peptidylprolyl cis-trans isomerase (PPIase)-deficient CypB, indicating that PPIase activity of CypB is critical for JEV propagation. Infection of pseudotype viruses bearing JEV envelope proteins was not impaired by the knockdown of CypB, suggesting that CypB participates in the replication but not in the entry of JEV. CypB was colocalized and immunoprecipitated with JEV NS4A in infected cells. These results suggest that CypB plays a crucial role in the replication of JEV through an interaction with NS4A.

Novel 2-deoxo-2-phenyl-5-deazaflavins and 2-deoxo-2-phenylflavin-5-oxides were prepared as a new class of antitumor agents and showed significant antitumor activities against NCI-H 460, HCT 116, A 431, CCRF-HSB-2, andKB cell lines. In vivo investigation, 2-deoxo-10-methyl-2-phenyl-5-deazaflavin exhibited the effective antitumor activity against A 431 human adenocarcinoma cells transplanted subcutaneously into nude mouse. Furthermore, AutoDock study has been done by binding of the flavin analogs into PTK pp60(c-src), where a good correlation between their IC(50) and AutoDock binding free energy was exhibited. In particular, 2-deoxo-2-phenylflavin-5-oxides exhibited the highest potential binding affinity within the binding pocket of PTK.