Cytosolic flagellin receptor NLRC4 protects mice against mucosal and systemic challenges.

Bacterial flagellin is a dominant innate immune activator of the intestine. Therefore, we examined the role of the intracellular flagellin receptor, NLRC4, in protecting the gut and/or driving inflammation. In accordance with NLRC4 acting through transcription-independent pathways, loss of NLRC4 did not reduce the rapid robust changes in intestinal gene expression induced by flagellin administration. Loss of NLRC4 did not alter basal intestinal homeostasis nor predispose mice to development of colitis upon administration of an anti-interleukin (IL)-10R monoclonal antibody. However, epithelial injury induced by dextran sulfate sodium in mice lacking NLRC4 resulted in a more severe disease, indicating a role for NLRC4 in protecting the gut. Moreover, loss of NLRC4 resulted in increased mortality in response to flagellate, but not aflagellate Salmonella infection. Thus, despite not being involved in rapid intestinal gene remodeling upon detection of flagellin, NLRC4-mediated inflammasome activation results in production of IL-1ß and IL-18, two cytokines that protect mice from mucosal and systemic challenges.

TLR5 activation induces secretory interleukin-1 receptor antagonist (sIL-1Ra) and reduces inflammasome-associated tissue damage.

Toll-like receptor-5 (TLR5)-mediated detection of flagellin induces nuclear factor (NF)-κB-mediated transcription of host defense gene expression, whereas recognition of intracellular flagellin by interleukin (IL)-1-converting enzyme protease-activation factor (IPAF) results in maturation/secretion of the inflammasome cytokine IL-1ß. The potent effects of IL-1ß are counter-regulated by secretory IL-1 receptor antagonist (sIL-1Ra). We studied the roles of flagellin receptors in regulating the expression of IL-1ß and sIL-1Ra and their subsequent roles in inflammation. Flagellin induced sIL-1Ra in intestinal epithelia and macrophages in a dose- and time-dependent manner, whereas IL-1ß was only induced in macrophages. In vivo, flagellin-induced sIL-1Ra, but not IL-1ß, was absolutely dependent upon TLR5 expressed on non-hemopioetic cells. Thus, loss of TLR5 increased the IL-1ß/sIL-1Ra ratio on flagellin treatment, which correlated with increased inflammatory pathology in response to this product. Furthermore, the flagellin/TLR5 interaction was important for the induction of sIL-1Ra and limiting inflammatory pathology on Salmonella infection. Finally, reduced sIL-1Ra levels in TLR5KO mice correlated with spontaneous colitis. Taken together, we demonstrate that intestinal epithelia, despite not expressing IL-1ß, secrete sIL-1Ra in a TLR5-dependent manner suggesting that loss of TLR5 may promote inflammation by increasing IL-1ß activity. Thus, optimizing the balance between inflammasome cytokines and their endogenous inhibitors might prove a useful strategy to treat inflammatory disorders.

The role of the gene 71 product in the life cycle of equine herpesvirus 1.

Equine herpesvirus type 1 (EHV-1) gene 71 encodes a heavily O-glycosylated 192 kDa protein with no identified herpesvirus homologue. Isolation of a deletion mutant in gene 71 (ED71) demonstrated that its protein product is not essential in vitro. To investigate the role of the gene 71 protein in the virus life cycle, ED71 has been characterized in vitro in terms of cellular adsorption, penetration, egress and transmission compared to wild-type and revertant virus. ED71 virions adsorbed to cells less efficiently than wild-type and revertant virus with a consequential effect on virus penetration; virus egress was significantly impaired and the timing of release was also delayed. The percentage of both full and empty capsids accumulating in the nuclei of ED71-infected cells was significantly higher than in wild-type virus-infected cells but the most notable differences were the low number of particles and the low ratio of enveloped to unenveloped capsids in the cytoplasm. The primary mode of transmission of the mutant virus is by direct cell-to-cell spread and the fact that a neutralizing antiserum did not reduce ED71 plaque size, supported the conclusion that deletion of gene 71 impairs the ability of virus to spread via release and readsorption to uninfected cells. Thus, deletion of EHV-1 gene 71 results in a defect in virus maturation and capsid envelopment. Progeny virus is consequently impaired in adsorption/penetration presumably due to the particles lacking the glycoprotein spikes predicted to be encoded by this gene and hence spreads by direct cell-to-cell contact.

ICP34.5 influences herpes simplex virus type 1 maturation and egress from infected cells in vitro.

We have previously demonstrated that efficient replication of mutant herpes simplex virus which fails to synthesize the polypeptide ICP34.5 is cell type and cell state dependent. ICP34.5 negative viruses do not grow in stationary state mouse embryo fibroblast 3T6 cells whereas the growth kinetics in BHK cells are indistinguishable from those of wild-type. We now demonstrate that this defect is not due to an inability of mutant virus to adsorb to 3T6 cells but rather to an inability to spread from the initially infected cells. Electron microscopic studies with wild-type HSV in both BHK and 3T6 cells revealed virus particles equally distributed between nucleus and cytoplasm, and additionally in the extracellular matrix. In BHK cells infected with the ICP34.5 negative mutant 1716, virus is likewise distributed between nucleus and cytoplasm but in 50% of the infected cells there is marked delamination and swelling of the nuclear membrane. In addition there is evidence of a significant number of particles trapped between the nuclear lamellae. When 1716 is used to infect 3T6 cells, over 90% of the virus particles are confined to the nuclei and the number of infected cells remains constant between 24 and 48 h with no increase in the proportion of extracellular virus. Failure to express ICP34.5 appears therefore to result in a defect in virus maturation and egress from the nuclei of infected cells. Egress of HSV from the nuclei to the extracellular space is thought to occur via two pathways. We postulate that lack of expression of ICP34.5 results in one of these pathways being blocked. In BHK cells this leads to overloading of the alternative pathway with a buildup of particles in the nuclear lamellae and associated endoplasmic reticulum. In stationary state 3T6 cells, it appears that there is no functional alternative pathway. We conclude that ICP34.5 exerts an effect on HSV maturation by controlling the passage of virus through infected cells.

The effect of triterpenoid compounds on uninfected and herpes simplex virus-infected cells in culture. III. Ultrastructural study of virion maturation.

Electron microscope studies been made of mock-infected or herpes simplex virus (HSV) type 1 (strain 17)- or HSV-2(strain HG52)-infected Flow 2002 cells grown in the absence or presence of various concentrations of cicloxolone sodium (CCX). Fifty non-serial, thin sections of mock-infected or HSV-infected cells, which contained a portion of the nucleus, were examined by transmission electron microscopy. With increasing drug concentration, counts of capsid structures both in the nucleus and cytoplasm showed a decrease in the number of virions per cell section, an increase in the ratio of nuclear to cytoplasmic virus, a relative reduction in the percentage of cytoplasmic enveloped virus particles, but no effect on the ratio of empty to core-containing capsid structures. The high particle to p.f.u. ratio induced by CCX treatment is thus not explained through failure to assemble morphologically mature core-containing capsids. In part it can be explained by non-envelopment, but in addition, specific effects on other virion proteins (tegument and envelope) must be involved. The extracellular virus particle yield was unaffected, indicating that CCX treatment enhances the egress of HSV. In the presence of CCX the encapsidation of HSV DNA into DNase-resistant structures was unaffected.