Pathogenesis of Sendasi virus infection in mice. On the possible role of interferon on the development of disease

Intraperitoneally (i.p.) injected interferon prolonged the survival time of mice inoculated intranasally (i.n.) with Sendai virus and reduced the mortality in mice inoculated i.n. with Haemophilus influenzae. Moderate concentrations of interferon were demonstrated in homogenized lungs of Sendai virus infected mice as long as the virus was present. Similar concentrations could be produced by i.p. injection of Sendai virus or interferon. Alveolar macrophages from mice treated i.p. with interferon or Sendai virus phagocytized more actively than control macropages. From the present and earlier data it is concluded that interferon may have a direct effect on the Sendai virus infection. The total effect of virus pneumonia is a reduction of the lung macrophage antimicrobial activity, and therefore the phagocytosis-modifying effect of interferon produced in the lungs is probably of minor importance for the outcome of the disease.     

Influence of Statolon on Resistance of Mice to Influenza

Various interferon inducers are known to elicit protection against lethal or infecting doses of certain viral agents. Because of the relatively high morbidity rate of influenza and its seasonal occurrence, we wished to determine whether statolon-induced interferon might be effective in controlling this disease. Mice were treated intraperitoneally with statolon and challenged with influenza A(2) virus by the intranasal route. Although interferon was present in the serum at the time of virus administration, no change in mortality rate was observed. There was, however, a significant increase in the mean survival time of treated animals. Similar results were obtained when Newcastle disease virus was used as the interferon inducer. To determine the effect of the route of challenge, other mice were treated with statolon or Newcastle disease virus and inoculated with mengovirus by the intranasal or intraperitoneal route. The results demonstrated that the treated mice were protected to similar degree against challenge by either route. It is suggested that the relative ineffectiveness of interferon in protecting mice against influenza is due to an intrinsic characteristic of the virus itself rather than the type of interferon induced or the route of virus challenge.     

Enhancement of vaccinia virus based oncolysis with histone deacetylase inhibitors

Histone deacetylase inhibitors (HDI) dampen cellular innate immune response by decreasing interferon production and have been shown to increase the growth of vesicular stomatitis virus and HSV. As attenuated tumour-selective oncolytic vaccinia viruses (VV) are already undergoing clinical evaluation, the goal of this study is to determine whether HDI can also enhance the potency of these poxviruses in infection-resistant cancer cell lines. Multiple HDIs were tested and Trichostatin A (TSA) was found to potently enhance the spread and replication of a tumour selective vaccinia virus in several infection-resistant cancer cell lines. TSA significantly decreased the number of lung metastases in a syngeneic B16F10LacZ lung metastasis model yet did not increase the replication of vaccinia in normal tissues. The combination of TSA and VV increased survival of mice harbouring human HCT116 colon tumour xenografts as compared to mice treated with either agent alone. We conclude that TSA can selectively and effectively enhance the replication and spread of oncolytic vaccinia virus in cancer cells.     

Use of a Recombinant Vaccinia Virus Expressing Interferon Gamma for Post-Exposure Protection against Vaccinia and Ectromelia Viruses

Post-exposure vaccination with vaccinia virus (VACV) has been suggested to be effective in minimizing death if administered within four days of smallpox exposure. While there is anecdotal evidence for efficacy of post-exposure vaccination this has not been definitively studied in humans. In this study, we analyzed post-exposure prophylaxis using several attenuated recombinant VACV in a mouse model. A recombinant VACV expressing murine interferon gamma (IFN-γ) was most effective for post-exposure protection of mice infected with VACV and ectromelia virus (ECTV). Untreated animals infected with VACV exhibited severe weight loss and morbidity leading to 100% mortality by 8 to 10 days post-infection. Animals treated one day post-infection had milder symptoms, decreased weight loss and morbidity, and 100% survival. Treatment on days 2 or 3 post-infection resulted in 40% and 20% survival, respectively. Similar results were seen in ECTV-infected mice. Despite the differences in survival rates in the VACV model, the viral load was similar in both treated and untreated mice while treated mice displayed a high level of IFN-γ in the serum. These results suggest that protection provided by IFN-γ expressed by VACV may be mediated by its immunoregulatory activities rather than its antiviral effects. These results highlight the importance of IFN-γ as a modulator of the immune response for post-exposure prophylaxis and could be used potentially as another post-exposure prophylaxis tool to prevent morbidity following infection with smallpox and other orthopoxviruses.     

Essential role for the p40 subunit of interleukin-12 in neutrophil-mediated early host defense against pulmonary infection with Streptococcus pneumoniae: involvement of interferon-gamma

Interleukin (IL)-12 is a critical cytokine in the T helper (Th)1 response and host defense against intracellular microorganisms, while its role in host resistance to extracellular bacteria remains elusive. In the present study, we elucidated the role of IL-12 in the early-phase host defense against acute pulmonary infection with Streptococcus pneumoniae, a typical extracellular bacterium, using IL-12p40 gene-disrupted (IL-12p40KO) mice. IL-12p40KO mice were highly susceptible to S. pneumoniae infection, as indicated by the shortened survival time, which was completely restored by the replacement therapy with recombinant (r) IL-12, and increased bacterial counts in the lung. In these mice, recruitment of neutrophils in the lung was significantly attenuated when compared to that in wild-type (WT) mice, which correlated well with the reduced production of macrophage inflammatory protein (MIP-2) and tumor necrosis factor (TNF)-alpha in the infected tissues at the early phase of infection. In vitro synthesis of both cytokines by S. pneumoniae-stimulated lung leukocytes was significantly lower in IL-12p40KO mice than in WT mice, and addition of rIL-12 or interferon (IFN)-gamma restored the reduced production of MIP-2 and TNF-alpha in IL-12p40KO mice. Neutralizing anti-IFN-gamma monoclonal antibody (mAb) significantly decreased the effect of rIL-12. Anti-IFN-gamma mAb shortened the survival time of infected mice and reduced the recruitment of neutrophils and production of MIP-2 and TNF-alpha in the lungs. Our results indicated that IL-12p40 plays a critical role in the early-phase host defense against S. pneumoniae infection by promoting the recruitment of neutrophils to the infected tissues.     

Protection against avian influenza H9N2 virus challenge by immunization with hemagglutinin- or neuraminidase-expressing DNA in BALB/c mice

Avian influenza viruses of H9N2 subtype are widely spread in avian species. The viruses have recently been transmitted to mammalian species, including humans, accelerating the efforts to devise protective strategies against them. In this study, an avian influenza H9N2 virus strain (A/Chicken/Jiangsu/7/2002), isolated in Jiangsu Province, China, was used to infect BALB/c mice for adaptation. After five lung-to-lung passages, the virus was stably proliferated in a large quantity in the murine lung and caused the deaths of mice. In addition, we explored the protection induced by H9N2 virus hemagglutinin (HA)- and neuraminidase (NA)-expressing DNAs in BALB/c mice. Female BALB/c mice aged 6-8 weeks were immunized once or twice at a 3-week interval with HA-DNA and NA-DNA by electroporation, respectively, each at a dose of 3, 10 or 30microg. The mice were challenged with a lethal dose (40x LD(50)) of influenza H9N2 virus four weeks after immunization once or one week after immunization twice. The protections of DNA vaccines were evaluated by the serum antibody titers, residual lung virus titers, and survival rates of the mice. The result showed that immunization once with not less than 10microg or twice with 3microg HA-DNA or NA-DNA provided effective protection against homologous avian influenza H9N2 virus.     

Pulmonary compartmentalization of interferon and natural killer cell activity

Studies were performed to determine if natural killer (NK) activity in the mononuclear cells harvested from infected lungs was dependent on local or systemic factors. Mice were inoculated by intratracheal (it), intraperitoneal (ip), or intravenous (iv) routes with (a LD50 dose of) influenza virus A PR/8/34. At various days postinoculation cells from lungs, spleens, and peripheral blood were assayed for NK activity, and lung wash, lung homogenates, and serum were assayed for interferon. After it inoculation there was three- to fourfold increase of NK activity in the lung with little or no increase in NK activity in spleens or peripheral blood. The local augmentation of NK activity in the lung correlated with an increase in interferon (IFN) titer in the lung wash and lung homogenate of PR8 inoculated mice. The virus failed to induce IFN or augment NK activity when it was inoculated systemically. The observed local augmentation of NK activity and local induction of interferon production following it inoculation suggests that the NK population in the lung is capable of responding to locally derived regulatory factors.     

Interferon: a mediator of indoleamine 2,3-dioxygenase induction by lipopolysaccharide, poly(I) X poly(C), and pokeweed mitogen in mouse lung

When C3H/He mice were treated with lipopolysaccharide, poly(I) X poly(C), or pokeweed mitogen, the serum interferon titer increased almost instantaneously (100-2000 units/ml), and then the pulmonary indoleamine 2,3-dioxygenase was induced 50- to 140-fold. The peaks corresponding to interferon induction always preceded (approximately 24 h) those corresponding to dioxygenase induction. In C3H/HeJ (lipopolysaccharide-nonresponder) mice, however, lipopolysaccharide was totally inert in induction of both interferon and dioxygenase, although treatment with poly(I) X poly(C) and pokeweed mitogen led to a remarkable increase in the serum interferon titer and the enzyme activity. When lymphocytes of C3H/HeJ mice were inactivated by X irradiation and then reconstituted by the transfer of spleen cells from C3H/He mice, both enzyme and interferon from C3H/HeJ mice thus treated were induced almost normally after the lipopolysaccharide treatment. In addition, murine interferon alpha/beta, which was injected intravenously in C3H/He or C3H/HeJ mice, almost instantaneously and dose-dependently induced the pulmonary enzyme, and at a dose of 10(5) units per mouse the enzyme activity was enhanced 20- to 26-fold in these two strains of mice. These results suggest that interferon, which is generated by the interaction of lymphocytes with lipopolysaccharide, poly(I) X poly(C), or pokeweed mitogen, is a mediator of indoleamine 2,3-dioxygenase induction in the mouse lung by these agents.     

人H7N9禽流感病毒、高致病H5N1禽流感病毒及H1N1流感病毒感染小鼠特征分析

目的对比分析人高致病H5N1禽流感病毒、H7N9禽流感病毒及H1N1流感病毒分别感染BALB/c小鼠后的机体反应特征。方法分别以H7N9病毒、H5N1病毒和H1N1病毒滴鼻感染BALB/c小鼠,观察小鼠存活率、体征变化及感染后肺组织病理损伤差异,检测小鼠感染流感病毒后肺组织增殖细胞核抗原(PCNA)表达并观察小鼠感染后修复状况。结果 H7N9病毒、H5N1病毒和H1N1病毒均感染BALB/c小鼠,小鼠存活率依次为H7N9H1N1H5N1,肺组织病理损伤严重程度依次为H5N1H1N1H7N9,PCNA表达水平依次为H7N9H1N1H5N1。结论 H7N9病毒感染后宿主炎症反应较小,感染后小鼠肺组织自我修复能力较强;H5N1病毒感染BALB/c小鼠后的机体反应最为强烈,感染后恢复能力差,致死率高。     

Characterization of Cells Infiltrating the Lungs of X-Irradiated and Nude Mice after Influenza Virus Infection

After X-irradiated and nonirradiated mice (C3H/He) as well as athymic nude mice and haired littermates (BALB/c) were infected with influenza A virus (Kumamoto strain, H2N2), they were examined for survival period, the development of consolidation in the lungs and the characteristics of the cells infiltrating the lung tissues. In two different T-cell deficient groups, there was a definite delay in the development of consolidation compared with their respective controls and this was reflected in prolonged survival periods: 5 days longer for irradiated mice and 6 days longer for nude mice. In both T-cell deficient and normal groups, about 70% of the cells obtained from consolidated lung tissues after virus infection were found to be small lymphoid cells and there were no morphological differences between the T-cell deficient and normal groups. None of these small lymphoid cells from the peripheral blood or the spleens of T-cell deficient mice responded to concanavalin A. In the lungs of both X-irradiated mice and nude mice, however, a definite increase in cells having natural killer activity was found at the late stages of the influenza infection, suggesting their participation in the development of consolidation.     

Effect of Chlorite-Oxidized Oxyamylose on Influenza Virus Infection in Mice

Intraperitoneally administered chlorite-oxidized oxyamylose (COAM) provided protection of mice against intranasal infection with several influenza virus strains. Treated animals invariably showed a reduced consolidation of the lungs and, in the case of infection with lethal strains of virus, also a delay in mortality. With a small dose of influenza A/PR8 virus, an increase in final survival rate could be observed. The effect of COAM on influenza virus infection lasted for at least 4 to 8 days. Inhibition of lung consolidation was not paralleled by a decrease in virus multiplication in the lung. The significance of this finding in relation to the mechanism of the antiviral action of COAM is discussed.     

Importance of interferons in recovery from mousepox.

Gamma interferon is shown to be critical in recovery of C57BL/6 mice from mousepox. Anti-gamma interferon treatment of mice infected in the footpad with ectromelia virus resulted in enhanced spread to and efficient virus replication in the spleen, lungs, ovaries, and, especially, liver. All treated, infected mice died within a mean of 7 days, 2.5 days earlier than mice with severe combined immunodeficiency that were given a comparable infection. On the other hand, alpha interferon appeared not to have a major role in controlling virus replication in tissues examined, and beta interferon was important for virus clearance in the liver and ovaries but not the spleen. Either anti-alpha, beta interferon or anti-beta interferon antibody therapy resulted in only 25% mortality. Infected control mice survived but showed persistence of ectromelia virus at the site of infection (the footpad) and transient presence of the virus in the spleen, liver, lungs, and ovaries and in the fibroreticular but not lymphoid cells of the draining popliteal lymph node. Depletion of gamma interferon but not alpha and/or beta interferon resulted in a significant reduction in the numbers of splenic T (especially gamma delta-TCR+), B, and Mac-1+ cells, although the proportion of Mac-1+ cells in the spleen increased compared with control values. Depletion of alpha, beta, or gamma interferons did not severely affect the generation of virus-specific cytotoxic T-lymphocyte responses or natural killer cell cytolytic activity. This study, in which a natural virus disease model was used, underscores the crucial importance of gamma interferon in virus clearance at all stages of infection and in all tissues tested except the primary site of infection, where virus clearance appears to be delayed.     

Laninamivir Octanoate and Artificial Surfactant Combination Therapy Significantly Increases Survival of Mice Infected with Lethal Influenza H1N1 Virus

Background

Patients with influenza virus infection can develop severe pneumonia and acute respiratory distress syndrome (ARDS) which have a high mortality. Influenza virus infection is treated worldwide mainly by neuraminidase inhibitors (NAIs). However, monotherapy with NAIs is insufficient for severe pneumonia secondary to influenza virus infection. We previously demonstrated that mice infected with a lethal dose of influenza virus develop diffuse alveolar damage (DAD) with alveolar collapse similar to that seen in ARDS in humans. Additionally, pulmonary surfactant proteins were gradually increased in mouse serum, suggesting a decrease in pulmonary surfactant in the lung. Therefore, the present study examined whether combination therapy of NAI with exogenous artificial surfactant affects mortality of influenza virus-infected mice.

Methodology/Principal Findings

BALB/c mice were inoculated with several viral doses of influenza A/Puerto Rico/8/34 (PR8) virus (H1N1). The mice were additionally administered exogenous artificial surfactant in the presence or absence of a new NAI, laninamivir octanoate. Mouse survival, body weight and general condition were observed for up to 20 days after inoculation. Viral titer and cytokine/chemokine levels in the lungs, lung weight, pathological analysis, and blood O₂ and CO₂ pressures were evaluated. Infected mice treated with combination therapy of laninamivir octanoate with artificial surfactant showed a significantly higher survival rate compared with those that received laninamivir octanoate monotherapy (p = 0.003). However, virus titer, lung weight and cytokine/chemokine responses were not different between the groups. Histopathological examination, a hydrostatic lung test and blood gas analysis showed positive results in the combination therapy group.

Conclusions/Significance

Combination therapy of laninamivir octanoate with artificial surfactant reduces lethality in mice infected with influenza virus, and eventually suppresses DAD formation and preserves lung function. This combination could be effective for prevention of severe pneumonia secondary to influenza virus infection in humans, which is not improved by NAI monotherapy.     

Parasite-mediated upregulation of NK cell-derived gamma interferon protects against severe highly pathogenic H5N1 influenza virus infection

Outbreaks of influenza A viruses are associated with significant human morbidity worldwide. Given the increasing resistance to the available influenza drugs, new therapies for the treatment of influenza virus infection are needed. An alternative approach is to identify products that enhance a protective immune response. In these studies, we demonstrate that infecting mice with the Th1-inducing parasite Toxoplasma gondii prior to highly pathogenic avian H5N1 influenza virus infection led to decreased lung viral titers and enhanced survival. A noninfectious fraction of T. gondii soluble antigens (STAg) elicited an immune response similar to that elicited by live parasites, and administration of STAg 2 days after H5N1 influenza virus infection enhanced survival, lowered viral titers, and reduced clinical disease. STAg administration protected H5N1 virus-infected mice lacking lymphocytes, suggesting that while the adaptive immune response was not required for enhanced survival, it was necessary for STAg-mediated viral clearance. Mechanistically, we found that administration of STAg led to increased production of gamma interferon (IFN-γ) from natural killer (NK) cells, which were both necessary and sufficient for survival. Further, administration of exogenous IFN-γ alone enhanced survival from H5N1 influenza virus infection, although not to the same level as STAg treatment. These studies demonstrate that a noninfectious T. gondii extract enhances the protective immune response against severe H5N1 influenza virus infections even when a single dose is administered 2 days postinfection.     

In vivo blockade of gamma interferon affects the influenza virus-induced humoral and the local cellular immune response in lung tissue.

Influenza virus infection induces the local production of gamma interferon (IFN-gamma) by T cells and non-T cells in the respiratory tract. To elucidate the possible functions of this cytokine, the humoral and local cellular immune responses to influenza virus were studied in BALB/c mice with or without in vivo neutralization of IFN-gamma by using monoclonal antibodies. Neutralization of IFN-gamma led to a significant reduction in virus-specific titers of immunoglobulins G2a and G3 in serum but had little effect on other isotypes. Studies on cells isolated from the lung parenchyma itself revealed that at the height of the immune response the ability of these cells to produce cytokines after antigen or T-cell receptor/CD3 stimulation was not affected. Ex vivo cytolytic activity by lung parenchyma cells, which is induced by infection with this virus in normal mice, was also found to be undisturbed by this treatment, even though anti-IFN-gamma antibody activity was recovered from lung lavage samples and sera at all days studied. Surprisingly, in vivo neutralization of IFN-gamma led to a significant reduction in the magnitude of the cellular infiltrate in the lung tissue which followed infection, suggesting an involvement of IFN-gamma in the mechanisms that regulate increased leucocyte traffic in the inflamed lung parenchyma. This conclusion was supported by findings of differences between mock-treated and anti-IFN-gamma-treated mice in the number of CD8+ lung T cells expressing CD49d (alpha4-integrin) and CD62L at various times after influenza virus infection. This study therefore demonstrates that IFN-gamma affects the local cellular response in the respiratory tract as well as the systemic humoral response to influenza virus infection.     

Delivery of CFTR by adenoviral vector to cystic fibrosis mouse lung in a model of chronic Pseudomonas aeruginosa lung infection

In cystic fibrosis (CF) there is an excessive inflammatory response to lung infections with Pseudomonas aeruginosa, which causes significant morbidity and mortality. Mice deficient in the cystic fibrosis conductance transmembrane regulator homolog (Cftr) have exaggerated production of proinflammatory cytokines in epithelial lining fluid and increased mortality in response to chronic bronchopulmonary infection with mucoid P. aeruginosa, compared with infected wild-type littermates. Whether delivery of CFTR to CF airways by an adenoviral vector (Ad2/CFTR-16) decreases cytokine production and mortality in response to chronic bronchopulmonary infection with mucoid P. aeruginosa was tested. CF mice [stock Cftrtm1Unc-TgN(FABPCFTR)#Jaw] were anesthetized with isoflurane and inoculated intranasally with either Ad2/CFTR-16, diluent (sucrose), or empty vector (Ad2/EV). Two weeks later, mice were anesthetized with 2.5% Avertin and inoculated transtracheally with P. aeruginosa-laden agarose beads (PA M57-15). The cumulative 10-day survival of mice pretreated with Ad2/CFTR-16 was significantly higher compared with mice pretreated with sucrose but not significantly higher than mice pretreated with Ad2/EV. After adjusting for differences in experiment, we found weight loss at 3 days for mice treated with Ad2/CFTR-16 to be significantly less than for the sucrose- or Ad2/EV-treated groups. However, cytokine responses were similar in all groups 3 days after infection. In conclusion, the observed survival advantage of adenoviral delivery of CFTR to the CF lung may be due either to CFTR expression or possibly to proinflammatory effects of the adenoviral vector, or both.     

Production of Interferon in Mice: Effect of Altered Gaseous Environments

Effects of altered gaseous environments (parabarosis) on interferon production in mice were studied, with Newcastle disease virus (NDV) as the inducer. Increased levels of interferon in lung tissue were observed when mice were exposed to 11% O₂ in N₂ for 3 days before and after, or only after, injection of NDV. However, serum interferon levels remained unchanged. Exposure of mice to 77% O₂ for up to 7 days did not affect the response to interferon induction as assayed in lungs or sera. Interferon levels were significantly depressed in mice exposed to a simulated depth of 213 ft in seawater [with normal partial pressure of O₂ (pO₂) in N₂] for 2 or 4 weeks. Whereas definite depression of interferon was also observed in mice maintained at a simulated altitude of 37,000 ft (with normal pO₂) for 2 weeks, those maintained at the same condition for 4 weeks showed a normal level of interferon. The results obtained with hypoxia are compatible with other reports on the influence of O₂ tension on viral infection. The factors responsible for alterations observed in interferon level in mice kept in normal pO₂, but under altered pressure, have not yet been identified.     

During viral infection of the respiratory tract, CD27, 4-1BB, and OX40 collectively determine formation of CD8+ memory T cells and their capacity for secondary expansion

Independent studies have shown that CD27, 4-1BB, and OX40 can all promote survival of activated CD8+ T cells. We have therefore compared their impact on CD8+ memory T cell formation and responsiveness within one, physiologically relevant model system. Recombinant mice, selectively lacking input of one or two receptors, were challenged intranasally with influenza virus, and the immunodominant virus-specific CD8+ T cell response was quantified at priming and effector sites. Upon primary infection, CD27 and (to a lesser extent) 4-1BB made nonredundant contributions to accumulation of CD8+ virus-specific T cells in draining lymph nodes and lung, while OX40 had no effect. Interestingly though, in the memory response, accumulation of virus-specific CD8+ T cells in spleen and lung critically depended on all three receptor systems. This was explained by two observations: 1) CD27, 4-1BB, and OX40 were collectively responsible for generation of the same memory CD8+ T cell pool; 2) CD27, 4-1BB, and OX40 collectively determined the extent of secondary expansion, as shown by adoptive transfers with standardized numbers of memory cells. Surprisingly, wild-type CD8+ memory T cells expanded normally in primed OX40 ligand- or 4-1BB ligand-deficient mice. However, when wild-type memory cells were generated in OX40 ligand- or 4-1BB ligand-deficient mice, their secondary expansion was impaired. This provides the novel concept that stimulation of CD8+ T cells by OX40 and 4-1BB ligand during priming imprints into them the capacity for secondary expansion. Our data argue that ligand on dendritic cells and/or B cells may be critical for this.