Delayed administration of interleukin-12 is efficacious in promoting recovery from lethal viral encephalitis.

Vesicular stomatitis virus (VSV) applied intranasally to mice initially infects the olfactory receptor neurons, and then spreads quickly to the rest of the central nervous system (CNS). Previously, we have shown that the cytokine interleukin-12 (IL-12) has a significant survival and recovery promoting effect in mice infected with VSV when administered at the time of infection. The question of whether IL-12 is efficacious under the more clinically relevant condition of post-infection administration was explored. The data show that when IL-12 is administered post-infection, it is as effective as at the time of infection.     

Uncoupling of macrophage inflammation from self-renewal modulates host recovery from respiratory viral infection

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The herpes simplex virus type 1 immediate-early polypeptide ICP4 is required for expression of globin genes located in the viral genome.

We infected Vero cells with ICP4-deficient herpes simplex virus recombinants bearing the rabbit beta-globin and human alpha 2-globin genes under the control of their own promoters and found that globin gene expression occurred only when ICP4 was provided in trans. These results demonstrate that ICP4 is required for the activity of globin promoters located in the viral genome and support the hypothesis that these cellular promoters are functionally equivalent to HSV early regulatory regions.     

Downregulation of CD4 is required for maintenance of viral infectivity of HIV-1

Downregulation of virus receptors on the cell surface is considered to be important in preventing superinfection. HIV-1 encodes multiple gene products, Env, Vpu, and Nef, involved in downregulation of CD4, a major HIV-1 receptor. We found that simultaneous mutations in both vpu and nef severely impaired virus replication. We examined the involvement of CD4 downregulation mediated by Vpu and Nef in the modification of virus infectivity. The mutation in vpu increased CD4 incorporation into virions without affecting the Env content in it, inhibiting the attachment step of virions to the CD4-positive cell surface. Although a single mutation in nef suppresses virus infectivity via a CD4-independent mechanism, it could augment CD4 incorporation in virions in combination with a vpu mutation. These results indicated that CD4 downregulation was necessary for maintenance of Env function in the virion.     

Bovine herpesvirus 1 glycoprotein G is required for viral growth by cell-to-cell infection

The bovine herpesvirus 1 (BHV-1) US4 gene encodes glycoprotein G (gG), which is conserved in the majority of alphaherpesviruses. In order to identify the role of BHV-1 gG in the viral infection cycle, a gG minus BHV-1 mutant and its gG-positive revertant were constructed and their growth characteristics in Madin-Darby bovine kidney (MDBK) cells were compared. The gG minus mutant formed smaller plaques than the gG-positive BHV-1 in MDBK cells. When a monolayer culture of MDBK cells was infected with BHV-1 at a low multiplicity of infection and overlaid with semi-solid growth medium, under which adsorption of the mature virion released in the medium was inhibited, gG-positive BHV-1 multiplied, while the growth of the gG negative BHV-1 was severely inhibited. These data suggest that BHV-1 gG functions in direct cell-to-cell transmission mechanism of BHV-1 in tissue culture.     

Fission yeast Ku protein is required for recovery from DNA replication stress

The fundamental function of the conserved Ku70–Ku80 heterodimer is to promote the non-homologous end-joining (NHEJ) pathway in double-strand break repair. Although it is thought that Ku plays several roles other than NHEJ in maintaining chromosomal integrity including telomere protection, these precise functions remain unclear. In this study, we describe a novel role of fission yeast Ku proteins encoded by pku70 ⁺ and pku80 ⁺ genes in dealing with DNA replication stress. In the absence of Rqh1, the fission yeast RecQ helicase, the cells are sensitive to reagents inducing replication stress. pku Δ rqh1 Δ double mutant showed synergistic sensitivities to these reagents. However, this synthetic phenotype was not observed when rqh1 Δ mutant was coupled with the deletion of lig4 ⁺ that encodes a ligase essential for NHEJ, indicating that the role of Ku in replication stress is NHEJ independent. pku Δ rqh1 Δ double mutant also showed highly variable copy numbers of rDNA repeats even under unstressed condition. Furthermore, the double mutant exhibited inefficient replication resumption after transient replication stalling. These results suggest the possibility that Ku proteins play an important role in genome integrity recovering replication stress.     

Beta1 integrin expression on endothelial cells is required for angiogenesis but not for vasculogenesis.

Integrins are a family of cell adhesion receptors that are involved in cell-matrix and cell-cell communications. They facilitate cell proliferation, migration, and survival. Using the Cre-Lox system, we deleted beta1 integrin on Tie2-positive (Tie2-cre beta1 Int (fl/fl)) vascular endothelial cells. Deletion of beta1 integrin on vascular endothelial cells results in embryonic lethality. Blood vessel defects are encountered in the Tie2-Cre beta1 Int (fl/fl) embryos at embryonic age (E9.5), and embryos die before reaching E10.5. The embryos exhibit growth retardation and both histological evaluation and PECAM-1 staining of E9.5 embryos revealed defects in angiogenic sprouting and vascular branching morphogenesis. Large and medium-size vessel formation is not affected in these embryos. Angiogenic defects were observed in several regions of the embryo and yolk sacs. These results indicate that beta1 integrin expression on vascular endothelial cells is crucial for embryonic angiogenesis but dispensable for vasculogenesis.     

NOS 1 is required for allergen-induced expression of NOS 2 in mice

BACKGROUND: Although increased nitric oxide (NO) production in asthma is mediated largely by upregulation of the inducible form of nitric oxide synthase (iNOS, or NOS 2), some studies have suggested an important role for the usually constitutive neural NOS isoform (nNOS, or NOS 1). AIM: To investigate how NOS 1 may influence allergic inflammation, we used NOS 1 knockout mice and their wild-type (WT) controls. METHODS: Mice were sensitized and challenged with ovalbumin (OVA) using a protocol known to upregulate NOS 2 in the airways. RESULTS: In addition to expected increases in NOS 2 activity, OVA challenge led to increases in calcium-dependent NOS activity, which was accounted for by increased expression of NOS 1 at both mRNA (n = 5, p < 0.001) and protein levels (n = 5, p < 0.01). In NOS-1-deficient mice, OVA challenge induced less eosinophilia (n = 7, p < 0.05) and much less NO production (n = 10, p < 0.01) than in WT controls, reflecting not only the expected absence of NOS 1, but also lack of upregulation of NOS 2. This interaction appeared to be stimulus specific as NOS-1-deficient mice did upregulate NOS 2 following exposure to lipopolysaccharide (n = 5, p < 0.001). CONCLUSIONS: These findings underscore the importance of NOS 1 in allergic airway inflammation and suggest a mechanism by which NOS 1 may influence overall NO production in the airways.     

Dectin-1 is required for host defense against Pneumocystis carinii but not against Candida albicans

Dectin-1 is a C-type lectin involved in the recognition of beta-glucans found in the cell walls of fungi. We generated dectin-1-deficient mice to determine the importance of dectin-1 in the defense against pathogenic fungi. In vitro, beta-glucan-induced cytokine production from wild-type dendritic cells and macrophages was abolished in cells homozygous for dectin-1 deficiency ('dectin-1-knockout' cells). In vivo, dectin-1-knockout mice were more susceptible than wild-type mice to pneumocystis infection, even though their cytokine production was normal. However, pneumocystis-infected dectin-1-knockout macrophages did show defective production of reactive oxygen species. In contrast to those results, wild-type and dectin-1-knockout mice were equally susceptible to candida infection. Thus, dectin-1 is required for immune responses to some fungal infections, as protective immunity to pneumocystis, but not to candida, required dectin-1 for the production of antifungal reactive oxygen species.     

E1 protein of bovine papillomavirus 1 is not required for the maintenance of viral plasmid DNA replication

Derivatives of bovine papillomavirus 1 (BPV1) with temperature-sensitive and dominant-negative mutation the E1 gene were used to determine the requirement for E1 in the maintenance of viral plasmid DNA replication. The abilities of these mutant BPV1 genomes to replicate as nuclear plasmids were monitored at permissive (32 degrees C) and nonpermissive (37 degrees C) temperatures in mouse C127 cells. We found that the temperature-sensitive E1 mutant BPV1 genomes replicate as nuclear plasmids as efficiently as does wild-type BPV1 in C127 cells after shifting to the nonpermissive temperature. These findings indicate that BPV1 does not require E1 for the maintenance of viral plasmids.     

c-Jun N-terminal kinase 1 is required for Toll-like receptor 1 gene expression in macrophages

The regulation of innate immune responses to pathogens occurs through the interaction of Toll-like receptors (TLRs) with pathogen-associated molecular patterns and the activation of several signaling pathways whose contribution to the overall innate immune response to pathogens is poorly understood. We demonstrate a mechanism of control of murine macrophage responses mediated by TLR1/2 heterodimers through c-Jun N-terminal kinase 1 (JNK1) activity. JNK controls tumor necrosis factor alpha production and TLR-mediated macrophage responses to Borrelia burgdorferi, the causative agent of Lyme disease, and the TLR1/TLR2-specific agonist PAM(3)CSK(4). JNK1, but not JNK2, activity regulates the expression of the tlr1 gene in the macrophage cell line RAW264.7, as well as in primary CD11b(+) cells. We also show that the proximal promoter region of the human tlr1 gene contains an AP-1 binding site that is subjected to regulation by the kinase and binds two complexes that involve the JNK substrates c-Jun, JunD, and ATF-2. These results demonstrate that JNK1 regulates the response to TLR1/2 ligands and suggest a positive feedback loop that may serve to increase the innate immune response to the spirochete.     

Protein synthesis is required for expression of anthrax lethal toxin cytotoxicity.

Anthrax lethal toxin, which is composed of two proteins, i.e., protective antigen and lethal factor, is cytolytic to mouse peritoneal macrophages and the macrophage-like cell line J774A.1. After exposure of cells to lethal toxin, inhibition of protein synthesis occurred only slightly before the onset of cytolysis. Thus, cell death did not appear to be due to inhibition of protein synthesis. However, prior treatment of J774A.1 cells with cycloheximide or puromycin, which inhibited protein synthesis, protected them completely against lethal toxin-induced cytolysis, which suggested that continuous protein synthesis is required for the expression of lethal toxin activity. Inhibition of protein synthesis had no appreciable effect on the binding of protective antigen to the cell surface receptor or on proteolytic cleavage of surface-bound protective antigen. Furthermore, inhibition of protein synthesis did not alter the uptake of toxin, which suggested that protein synthesis is required at a later stage of the intoxication process. The protection provided by inhibition of protein synthesis was effective, even up to 1 h after exposure to anthrax lethal toxin. The increased uptake of calcium observed in cells exposed to lethal toxin did not occur when they were protected by blocking protein synthesis. Identifying the protein(s) synthesized during the intoxication process may help to understand the mechanism of cell death produced by anthrax lethal toxin.     

Interleukin (IL)-12 receptor beta1 or IL-12 receptor beta 2 deficiency in mice indicates that IL-12 and IL-23 are not essential for host recovery from viral encephalitis

Vesicular stomatitis virus (VSV), a negative-sense, single-stranded RNA rhabdovirus, causes acute viral encephalitis when administered intranasally to mice. Interleukin-12 (IL-12) is a key pro-inflammatory cytokine that is produced largely by the antigen presenting cells (APC) and that bridges the innate and acquired immune responses. IL-12 is efficacious in enhancing recovery from VSV infection of the murine central nervous system. This effect is mediated by nitric oxide (NO) produced by the neuronal isoform of nitric oxide synthase (NOS-1), and is independent of the pro-inflammatory cytokines interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha). These data implied a link between IL-12 and NOS-1. Here we investigate the role of the IL-12R during VSV pathogenesis, using IL-12R beta2 and IL-12R beta1-deficient mice. We showed that a deficiency in either IL-12R beta2 or IL-12R beta1 had no effect on the outcome of VSV infection of the CNS or on the clearance of VSV from the CNS. Furthermore, these data indicate that IL-23 is not acting redundantly in the absence of IL-12 during VSV-induced encephalitis.     

Astrocytic Fas ligand expression is required to induce T‐cell apoptosis and recovery from experimental autoimmune encephalomyelitis

In T‐cell‐mediated autoimmune diseases of the CNS, apoptosis of Fas⁺ T cells by FasL contributes to resolution of disease. However, the apoptosis‐inducing cell population still remains to be identified. To address the role of astrocytic FasL in the regulation of T‐cell apoptosis in experimental autoimmune encephalomyelitis, we immunized C57BL/6 glial fibrillary acid protein (GFAP)‐Cre FasL^fl/fl mice selectively lacking FasL in astrocytes with MOG_35–55 peptide. GFAP‐Cre FasL^fl/fl mice were unable to resolve EAE and suffered from persisting demyelination and paralysis, while FasL^fl/fl control mice recovered. In contrast to FasL^fl/fl mice, GFAP‐Cre FasL^fl/fl mice failed to induce apoptosis of Fas⁺ activated CD4⁺ T cells and to increase numbers of Foxp3⁺ Treg cells beyond day 15 post immunization, the time point of maximal clinical disease in control mice. The persistence of activated and GM‐CSF‐producing CD4⁺ T cells in GFAP‐Cre FasL^fl/fl mice also resulted in an increased IL‐17, IFN‐γ, TNF, and GM‐CSF mRNA expression in the CNS. In vitro, FasL⁺ but not FasL^? astrocytes induced caspase‐3 expression and apoptosis of activated T cells. In conclusion, FasL expression of astrocytes plays an important role in the control and elimination of autoimmune T cells from the CNS, thereby determining recovery from EAE.     

Surface expression of ferripyochelin-binding protein is required for virulence of Pseudomonas aeruginosa.

Pseudomonas aeruginosa mutants which do not express ferripyochelin-binding protein (FBP) on the cell surface have previously been isolated. These mutants were used to assess the role of FBP in virulence in an acute and a systemic animal infection model. In a mouse corneal infection model, the pathology of eyes infected with the mutant strains was significantly less than that of eyes infected with the parent strain. The mutants were also cleared more rapidly from the eye. In a burn infection model, the mortality rate in mice infected with mutant FBP-28 was much less than that of mice infected with the parent strain at an inoculum of 10(2) CFU. At higher inocula (10(4) CFU), the mortality rate was not significantly different but the survival time was dramatically longer with the mutant strain. Quantitative bacteriology of blood and tissue homogenates revealed that P. aeruginosa PAO could multiply in the skin and could also be cultured from the blood, livers, and spleens of infected mice. FBP-28 could only be cultured from the skin. Therefore, this mutant could colonize the skin but could not disseminate. These data indicate that functional, exposed FBP is required for virulence of PAO.