Interferon treatment reduced adherence, invasiveness and intracellular multiplication of Shigella flexneri in coxsackie B1 virus-infected cells.

The effect of interferon treatment on interaction of Shigella flexneri with in vitro cultured cells was investigated. Pretreatment of HEp-2 cells with human interferons had no effect on the susceptibility of cells to S. flexneri, measured by invasiveness and adhesiveness. Human leukocyte interferon and human recombinant interferon-alpha-A reduced adhesiveness, intracellular multiplication and invasiveness of S. flexneri in HEp-2 cells preinfected with coxsackie B1 virus. Also non-receptor mediated-phagocytosis was reduced by interferon treatment in virus infected cells. The interferon effects were dependent on continuous protein synthesis, because they were not expressed when cycloheximide or abrin was added to the virus infected cell cultures. No effect of interferon was detected on intracellular content of Na+ or K+, Na(+)-K+ activated ATPase activity or cytoplasma membrane polarity, in virus infected or control cell cultures. The interferon effect on bacterial invasiveness seems to be dependent on an interferon receptor interaction on cytoplasma membrane level because directly microinjected interferon showed no effect.     

Inhibition of dengue virus entry and multiplication into monocytes using RNA interference

Background

Dengue infection ranks as one of the most significant viral diseases of the globe. Currently, there is no specific vaccine or antiviral therapy for prevention or treatment. Monocytes/macrophages are the principal target cells for dengue virus and are responsible for disseminating the virus after its transmission. Dengue virus enters target cells via receptor-mediated endocytosis after the viral envelope protein E attaches to the cell surface receptor. This study aimed to investigate the effect of silencing the CD-14 associated molecule and clathrin-mediated endocytosis using siRNA on dengue virus entry into monocytes.

Methodology/Principal Findings

Gene expression analysis showed a significant down-regulation of the target genes (82.7%, 84.9 and 76.3% for CD-14 associated molecule, CLTC and DNM2 respectively) in transfected monocytes. The effect of silencing of target genes on dengue virus entry into monocytes was investigated by infecting silenced and non-silenced monocytes with DENV-2. Results showed a significant reduction of infected cells (85.2%), intracellular viral RNA load (73.0%), and extracellular viral RNA load (63.0%) in silenced monocytes as compared to non-silenced monocytes.

Conclusions/Significance

Silencing the cell surface receptor and clathrin mediated endocytosis using RNA interference resulted in inhibition of the dengue virus entry and subsequently multiplication of the virus in the monocytes. This might serve as a novel promising therapeutic target to attenuate dengue infection and thus reduce transmission as well as progression to severe dengue hemorrhagic fever.     

RNA sensors enable human mast cell anti-viral chemokine production and IFN-mediated protection in response to antibody-enhanced dengue virus infection

Dengue hemorrhagic fever and/or dengue shock syndrome represent the most serious pathophysiological manifestations of human dengue virus infection. Despite intensive research, the mechanisms and important cellular players that contribute to dengue disease are unclear. Mast cells are tissue-resident innate immune cells that play a sentinel cell role in host protection against infectious agents via pathogen-recognition receptors by producing potent mediators that modulate inflammation, cell recruitment and normal vascular homeostasis. Most importantly, mast cells are susceptible to antibody-enhanced dengue virus infection and respond with selective cytokine and chemokine responses. In order to obtain a global view of dengue virus-induced gene regulation in mast cells, primary human cord blood-derived mast cells (CBMCs) and the KU812 and HMC-1 mast cell lines were infected with dengue virus in the presence of dengue-immune sera and their responses were evaluated at the mRNA and protein levels. Mast cells responded to antibody-enhanced dengue virus infection or polyinosiniċpolycytidylic acid treatment with the production of type I interferons and the rapid and potent production of chemokines including CCL4, CCL5 and CXCL10. Multiple interferon-stimulated genes were also upregulated as well as mRNA and protein for the RNA sensors PKR, RIG-I and MDA5. Dengue virus-induced chemokine production by KU812 cells was significantly modulated by siRNA knockdown of RIG-I and PKR, in a negative and positive manner, respectively. Pretreatment of fresh KU812 cells with supernatants from dengue virus-infected mast cells provided protection from subsequent infection with dengue virus in a type I interferon-dependent manner. These findings support a role for tissue-resident mast cells in the early detection of antibody-enhanced dengue virus infection via RNA sensors, the protection of neighbouring cells through interferon production and the potential recruitment of leukocytes via chemokine production.     

Gamma interferon augments Fc gamma receptor-mediated dengue virus infection of human monocytic cells.

It has been reported that anti-dengue antibodies at subneutralizing concentrations augment dengue virus infection of monocytic cells. This is due to the increased uptake of dengue virus in the form of virus-antibody complexes by cells via Fc gamma receptors. We analyzed the effects of recombinant human gamma interferon (rIFN-gamma) on dengue virus infection of human monocytic cells. U937 cells, a human monocytic cell line, were infected with dengue virus in the form of virus-antibody complexes after rIFN-gamma treatment. Pretreatment of U937 cells with rIFN-gamma resulted in a significant increase in the number of dengue virus-infected cells and in the yield of infectious virus. rIFN-gamma did not augment dengue virus infection when cells were infected with virus in the absence of anti-dengue antibodies. Gamma interferon (IFN-gamma) produced by peripheral blood lymphocytes from dengue-immune donors after in vitro stimulation with dengue antigens also augmented dengue virus infection of U937 cells. IFN-gamma did not augment dengue virus infections when cells were infected with virus in the presence of F(ab')2 prepared from anti-dengue immunoglobulin G. Human immunoglobulin inhibited IFN-gamma-induced augmentation. IFN-gamma increased the number of Fc gamma receptors on U937 cells. The increase in the percentage of dengue antigen-positive cells correlated with the increase in the number of Fc gamma receptors after rIFN-gamma treatment. These results indicate that IFN-gamma-induced augmentation of dengue virus infection is Fc gamma receptor mediated. Based on these results we conclude that IFN-gamma increases the number of Fc gamma receptors and that this leads to an augmented uptake of dengue virus in the form of dengue virus-antibody complexes, which results in augmented dengue virus infection.     

Influence of interferon on human monocyte to macrophage development

The effect of interferon-α (Wellferon) on human monocyte to macrophage maturation in vitro has been investigated. Cell volume and three markers, acid phosphatase, leucine aminopeptidase, and phagocytosis, which increase with maturation, have been studied employing recently developed flow cytofluorometric techniques. The increase in cell volume and in the expression of all three markers was inhibited in a dose-dependent manner in monocyte cultures given 50–300 U/ml of interferon within 2 hr of culture initiation. An initial dose of 300 U/ml of interferon, removed from the cultures after 24 hr, was as effective in inhibiting the development of each of the markers as three 100 U pulses on three consecutive days, and as effective as 300 U interferon left in throughout the culture period. Histogram analysis of marker expression indicated that all monocytes, and not a subpopulation, were affected by the interferon. Cytotoxic activity of freshly isolated monocytes rapidly decayed when the cells were cultured under standard maturation conditions. The addition of interferon to the cultures prevented the loss of this activity while also preventing the development of more mature cells. It appears that maintenance of the cytotoxic state is one influence of interferons; however, it may be that these cells have also been directed toward alternate pathways of macrophage differentiation.     

Susceptibility and response of human blood monocyte subsets to primary dengue virus infection

Human blood monocytes play a central role in dengue infections and form the majority of virus infected cells in the blood. Human blood monocytes are heterogeneous and divided into CD16(-) and CD16(+) subsets. Monocyte subsets play distinct roles during disease, but it is not currently known if monocyte subsets differentially contribute to dengue protection and pathogenesis. Here, we compared the susceptibility and response of the human CD16(-) and CD16(+) blood monocyte subsets to primary dengue virus in vitro. We found that both monocyte subsets were equally susceptible to dengue virus (DENV2 NGC), and capable of supporting the initial production of new infective virus particles. Both monocyte subsets produced anti-viral factors, including IFN-α, CXCL10 and TRAIL. However, CD16(+) monocytes were the major producers of inflammatory cytokines and chemokines in response to dengue virus, including IL-1β, TNF-α, IL-6, CCL2, 3 and 4. The susceptibility of both monocyte subsets to infection was increased after IL-4 treatment, but this increase was more profound for the CD16(+) monocyte subset, particularly at early time points after virus exposure. These findings reveal the differential role that monocyte subsets might play during dengue disease.     

Antiviral and protein-inducing activities of recombinant human leukocyte interferons and their hybrids.

The antiviral activities of recombinant human leukocyte interferons IFN-alpha A and IFN-alpha D as well as five hybrids of these interferons against retroviruses, vesicular stomatitis virus, and encephalomyocarditis virus were studied in feline, human, and murine cells. Although these interferon species had widely different potencies, their activities against these viruses were, in general, proportional. The IFN-alpha A/D (Bgl) hybrid was the most potent species, and the IFN-alpha D/A (Bgl) hybrid was the least potent. However, the latter species did not interfere with the action of the former species. Like natural human leukocyte interferon, each of the seven species of recombinant interferons induced the synthesis of at least five proteins in human fibroblasts, whereas induction of only one such protein was readily detected in a feline fibroblast line in which these interferon species inhibited the replication of all three viruses.     

New simple dye-uptake assay for interferon

Using the spectrophotometer that the authors developed, the amounts of human leukocyte and mouse L cell interferons on FL cells and L929 cells were measured and values were compared with those measured by the cytopathogenic effect (CPE) reduction method (CPE method). The spectrophotometric method, which was simpler than the original dye-uptake method, was found to be more sensitive than the latter. When Sindbis virus was used instead of vesicular stomatitis virus (VSV), there were no significant differences in the sensitivities of the two methods or the interferon titers estimated. When FL cells or L929 cells were treated with interferon at the time of their dispersion, their interferon titers were almost the same as those of cells treated with interferon 2 days after dispersion. It is concluded that this new dye-uptake method is useful for assay of human and mouse interferons.     

Reversible inhibition by interferon of the maturation of human peripheral blood monocytes to macrophages

We demonstrated that interferon delays the maturation of human monocytes to macrophages in vitro as assessed by morphologic, histochemical, and biochemical parameters. After exposure to interferon, monocytes were slightly smaller, less stretched out, and had a delayed loss of granules with peroxidase positive reactivity, as compared with control, noninterferon-treated cells. Also, interferon prevented the increase of the specific activity of three lysosomal enzymes (β-galactosidase, β-glucuronidase, and β-N-acetylglucosaminidase) in the monocytes, and enzyme activities were 30–40% of that observed in untreated cells. Both human leukocyte and human fibroblast interferons were effective in delaying the maturation process. The effects of the interferon were species specific and reversible and were neutralized by antiinterferon serum. These studies describe a new nonantiviral effect of interferon, unique in that it involves the delay of maturation of cells in a system which is not associated with cell proliferation. Thus interferon could potentially effect host defense mechanisms and aspects of the immune response which are dependent on the maturation of monocytes to macrophages.     

The monocyte-macrophage-mast cell axis in dengue pathogenesis

Dengue virus, the causative agent of dengue disease which may have hemorrhagic complications, poses a global health threat. Among the numerous target cells for dengue virus in humans are monocytes, macrophages and mast cells which are important regulators of vascular integrity and which undergo dramatic cellular responses after infection by dengue virus. The strategic locations of these three cell types, inside blood vessels (monocytes) or outside blood vessels (macrophages and mast cells) allow them to respond to dengue virus infection with the production of both intracellular and secretory factors which affect virus replication, vascular permeability and/or leukocyte extravasation. Moreover, the expression of Fc receptors on the surface of monocytes, macrophages and mast cells makes them important target cells for antibody-enhanced dengue virus infection which is a major risk factor for severe dengue disease, involving hemorrhage. Collectively, these features of monocytes, macrophages and mast cells contribute to both beneficial and harmful responses of importance to understanding and controlling dengue infection and disease.     

Comparison of the antiviral action of different human interferons against DNA and RNA viruses

Abstract Five different interferon preparations were compared for their antiviral activity against Herpes simplex virus type 1 (HSV-1) and several RNA viruses. The interferons used were: interferon α from human buffy coats, interferon β from human fibroblasts, interferon γ from human lymphocytes after stimulation with phytohemagglutinin (PHA), lymphoblastoid interferon from Namalva cells IFN-α (Ly) and cloned α ₂ interferon produced by Escherichia coli containing the human gene for interferon α ₂. All preparations were able to protect monolayers of HeLa cells against HSV-1 infection when low multiplicities were used. The five IFN preparations were also tested against encephalomyocarditis (EMC) virus, poliovirus and vesicular stomatitis virus (VSV).     

Tumor Necrosis Factor Alpha Exerts Powerful Anti-Influenza Virus Effects in Lung Epithelial Cells

Previous studies have associated influenza virus-induced expression of inflammatory cytokines, including tumor necrosis factor alpha (TNF-alpha), with influenza pathogenesis in the human respiratory tract and have suggested that alpha and beta interferons are the first cytokines recruited to counteract such infection. However, we report here that TNF-alpha has powerful anti-influenza virus activity. When infected with influenza virus, cultured porcine lung epithelial cells expressed TNF-alpha in a dose-dependent manner. Expression of TNF-alpha was induced only by replicating virus. TNF-alpha showed strong antiviral activity against avian, swine, and human influenza viruses, and the antiviral effect of TNF-alpha was greater than that of gamma or alpha interferon. These findings suggest that TNF-alpha serves as the first line of defense against influenza virus infection in the natural host.     

Effect of interferon on the number of cytogenetic disorders occurring in human cultured lymphocytes treated with thio-TEPA and fotrin

The paper deals with the modifying effects of natural (leukocytic) and synthesized (recombinant) interferons on the number of cytogenetic injuries in the cultured lymphocytes of human peripheric blood after exposure to alkylating chemicals--thio-TEPA and fotrin. The analysis of chromosomal aberration levels is suggestive of a significant protective effect exerted by interferons. The addition of the recombinant interferon increased the number of sister chromatid exchanged frequency in mutagen treated variants. The application of the test system that involves two types of interferon made it possible to reveal differences in their cytogenetic effect during the protector-sensitive period of cultivation.     

Heat shock protein 90 and heat shock protein 70 are components of dengue virus receptor complex in human cells

Dengue virus requires the presence of an unidentified cellular receptor on the surface of the host cell. By using a recently published affinity chromatography approach, an 84-kDa molecule, identified as heat shock protein 90 (HSP90) by matrix-assisted laser desorption ionization-time of flight mass spectrometry, was isolated from neuroblastoma and U937 cells. Based on the ability of HSP90 (84 kDa) to interact with HSP70 (74 kDa) on the surface of monocytes during lipopolysaccharide (LPS) signaling and evidence that LPS inhibits dengue virus infection, the presence of HSP70 was demonstrated in affinity chromatography eluates and by pull-down experiments. Infection inhibition assays support the conclusion that HSP90 and HSP70 participate in dengue virus entry as a receptor complex in human cell lines as well as in monocytes/macrophages. Additionally, our results indicate that both HSPs are associated with membrane microdomains (lipid rafts) in response to dengue virus infection. Moreover, methyl-beta-cyclodextrin, a raft-disrupting drug, inhibits dengue virus infection, supporting the idea that cholesterol-rich membrane fractions are important in dengue virus entry.     

Activation of endothelial cells via antibody-enhanced dengue virus infection of peripheral blood monocytes.

Although endothelial cells have been speculated to be a target in the pathogenesis of dengue hemorrhagic fever (DHF), there has been little evidence linking dengue virus infection to any alteration in endothelial cell function. In this study, we show that human umbilical vein endothelial cells become activated when exposed to culture fluids from dengue virus-infected peripheral blood monocytes. Maximum activation was achieved with culture fluids from monocytes in which virus infection was enhanced by the addition of dengue virus-immune serum, thus correlating with epidemiological evidence that prior immunity to dengue virus is a major risk factor for DHF. Activation was strongest for endothelial cell expression of VCAM-1 and ICAM-1. In contrast, activation of endothelial cell E-selectin expression appeared to be more transient, as indicated by its detection at 3 h, but not at 16 h, of treatment. Treatment of monocyte culture fluids with anti-tumor necrosis factor alpha (TNF-alpha) antibody largely abolished the activation effect (as measured by endothelial cell expression of ICAM-1), whereas treatment with IL-1beta receptor antagonist had a much smaller inhibitory effect on activation. Endothelial cells inoculated directly with dengue virus or with virus-antibody combinations were poorly infectable (compared to Vero cells or peripheral blood monocytes), and virus-inoculated endothelial cells showed no increased expression of VCAM-1, ICAM-1, or E-selectin. Taken together, the results strongly indicate that dengue virus can modulate endothelial cell function by an indirect route, in which a key intermediary is TNF-alpha released from virus-infected monocytes.     

Presence of human chromosome 21 alone is sufficient for hybrid cell sensitivity to human interferon.

Human/mouse somatic cell hybrids with chromosome 21 as the only detectable human genetic material were sensitive to both human leukocyte and fibroblast interferons. The presence of additional human chromosomes decreased the amount of interferon needed to attain a given level of virus resistance. Decreased cytopathic effects, decreased virus yields, and the appearance of a specific phosphorylated protein associated with interferon treatment were all observed in hybrids maintaining only human chromosome 21. The phosphorylated protein found in extracts of these human interferon-treated hybrid cells was of mouse origin.     

Comparison of dengue infection in human mononuclear leukocytes with mosquito C6/36 and mammalian Vero cells using flow cytometry to detect virus antigen

Fluorescent activated cell sorter (FACS) analysis is useful for the detection of cellular surface antigens and intracellular proteins. We used this methodology in order to detect and quantify dengue antigens in highly susceptible cells such as clone C6/36 (Aedes albopictus) and Vero cells (green monkey kidney). Additionally, we analyzed the infection in vitro of human peripheral blood mononuclear leukocytes (PBML). FACS analysis turned out to be a reliable technique to quantify virus growth in traditional cell cultures of C6/36 as well as Vero cells. High rates of infection were achieved with a good statistical correlation between the virus amount used in infection and the percentage of dengue antigen containing cells detected in infected cultures. We also showed that human monocytes (CD14+) are preferred target cells for in vitro dengue infection among PBML. Monocytes were much less susceptible to virus infection than cell lines but they displayed dengue antigens detected by FACS five days after infection. In contrast, lymphocytes showed no differences in their profile for dengue specific immunofluorescence. Without an animal model to reproduce dengue disease, alternative assays have been sought to correlate viral virulence with clinical manifestations and disease severity. Study of in vitro interaction of virus and host cells may highlight this relationship.     

Interferon-γ: A historical perspective

This article reviews the main lines of thinking and exploration that have led to our current conception of the role of IFN-γ in immune defense and autoimmunity. In 1965 the first report appeared describing production of an interferon-like virus inhibitor in cultured human leukocytes following exposure to the mitogen phytohemagglutinin. In the early 1970s the active principle became recognized as being distinct from classical virus-induced interferons, leading to its designation as immune interferon or Type II interferon, and eventually IFN-γ. Up to that point interest in the factor had come almost exclusively from virologists, in particular those among them who were believers in interferon. Evidence first coming forward in the 1980s that IFN-γ is indistinguishable from macrophage-activating factor (MAF), then a prototype lymphokine, was the signal for immunologists at large to become interested. Today IFN-γ ranks among the most important endogenous regulators of immune responses.     

Differential Expression of Toll-like Receptors in Dendritic Cells of Patients with Dengue during Early and Late Acute Phases of the Disease

Background

Dengue hemorrhagic fever (DHF) is observed in individuals that have pre-existing heterotypic dengue antibodies and is associated with increased viral load and high levels of pro-inflammatory cytokines early in infection. Interestingly, a recent study showed that dengue virus infection in the presence of antibodies resulted in poor stimulation of Toll-like receptors (TLRs), thereby facilitating virus particle production, and also suggesting that TLRs may contribute to disease pathogenesis.

Methodology/Principal Findings

We evaluated the expression levels of TLR2, 3, 4 and 9 and the co-stimulatory molecules CD80 and CD86 by flow cytometry. This was evaluated in monocytes, in myeloid and plasmacytoid dendritic cells (mDCs and pDCs) from 30 dengue patients with different clinical outcomes and in 20 healthy controls. Increased expression of TLR3 and TLR9 in DCs of patients with dengue fever (DF) early in infection was detected. In DCs from patients with severe manifestations, poor stimulation of TLR3 and TLR9 was observed. In addition, we found a lower expression of TLR2 in patients with DF compared to DHF. Expression levels of TLR4 were not affected. Furthermore, the expression of CD80 and CD86 was altered in mDCs and CD86 in pDCs of severe dengue cases. We show that interferon alpha production decreased in the presence of dengue virus after stimulation of PBMCs with the TLR9 agonist (CpG A). This suggests that the virus can affect the interferon response through this signaling pathway.

Conclusions/Significance

These results show that during dengue disease progression, the expression profile of TLRs changes depending on the severity of the disease. Changes in TLRs expression could play a central role in DC activation, thereby influencing the innate immune response.     

Selectivity of interferon action in simian virus 40-transformed cells superinfected with simian virus 40.

Treatment of African green monkey kidney CV-1 cells with human alpha interferons before infection with simian virus 40 (SV40) inhibited the accumulation of SV40 mRNAs and SV40 T-antigen (Tag). This inhibition persisted as long as the interferons were present in the medium. SV40-transformed human SV80 cells and mouse SV3T3-38 cells express Tag, and interferon treatment of these cells did not affect this expression. SV80 and SV3T3-38 cells which had been exposed to interferons were infected with a viable SV40 deletion mutant (SV40 dl1263) that codes for a truncated Tag. Exposure to interferons inhibited the accumulation of the truncated Tag (specified by the infecting virus) but had no significant effect on the accumulation of the endogenous Tag (specified by the SV40 DNA integrated into the cellular genome). The level of Tag in SV40-transformed mouse SV101 cells was not significantly decreased by interferon treatment. SV40 was rescued from SV101 cells and used to infect interferon-treated and control African green monkey kidney Vero cells. Tag accumulation was inhibited in the cells which had been treated with interferons before infection. Our data demonstrate that even within the same cell the interferon system can discriminate between expression of a gene in the SV40 viral genome and expression of the same gene integrated into a host chromosome.