Rossella Sgarbanti

Infectious agents and neurodegeneration.

A growing body of epidemiologic and experimental data point to chronic bacterial and viral infections as possible risk factors for neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s disease and amyotrophic lateral sclerosis. Infections of the central nervous system, especially those characterized by a chronic progressive course, may produce multiple damage in infected and neighbouring cells. The activation of inflammatory processes and host immune responses cause chronic damage resulting in alterations of neuronal function and viability, but different pathogens can also directly trigger neurotoxic pathways. Indeed, viral and microbial agents have been reported to produce molecular hallmarks of neurodegeneration, such as the production and deposit of misfolded protein aggregates, oxidative stress, deficient autophagic processes, synaptopathies and neuronal death. These effects may act in synergy with other recognized risk factors, such as aging, concomitant metabolic diseases and the host’s specific genetic signature. This review will focus on the contribution given to neurodegeneration by herpes simplex type-1, human immunodeficiency and influenza viruses, and by Chlamydia pneumoniae.

Bcl-2 Expression and p38MAPK Activity in Cells Infected with Influenza A Virus IMPACT ON VIRALLY INDUCED APOPTOSIS AND VIRAL REPLICATION

Previous reports have shown that various steps in the influenza A virus life cycle are impaired in cells expressing the antiapoptotic protein Bcl-2 (Bcl-2+ cells). We demonstrated a direct link between Bcl-2 and the reduced nuclear export of viral ribonucleoprotein (vRNP) complexes in these cells. However, despite its negative impact on viral replication, Bcl-2 did not prevent host cells from undergoing virally triggered apoptosis. The proteins reduced antiapoptotic capacity was related to phosphorylation of its threonine 56 and serine 87 residues by virally activated p38MAPK. In infected Bcl-2+ cells, activated p38MAPK was found predominantly in the cytoplasm, colocalized with Bcl-2, and both Bcl-2 phosphorylation and virally induced apoptosis were diminished by specific inhibition of p38MAPK activity. In contrast, in Bcl-2-negative (Bcl-2−) cells, which are fully permissive to viral infection, p38MAPK activity was predominantly nuclear, and its inhibition decreased vRNP traffic, phosphorylation of viral nucleoprotein, and virus titers in cell supernatants, suggesting that this kinase also contributes to the regulation of vRNP export and viral replication. This could explain why in Bcl-2+ cells, where p38MAPK is active in the cytoplasm, phosphorylating Bcl-2, influenza viral replication is substantially reduced, whereas apoptosis proceeds at rates similar to those observed in Bcl-2− cells. Our findings suggest that the impact of p38MAPK on the influenza virus life cycle and the apoptotic response of host cells to infection depends on whether or not the cells express Bcl-2, highlighting the possibility that the pathological effects of the virus are partly determined by the cell type it targets.

Influenza virus replication in lung epithelial cells depends on redox-sensitive pathways activated by NOX4-derived ROS.

An overproduction of reactive oxygen species (ROS) mediated by NADPH oxidase 2 (NOX2) has been related to airway inflammation typical of influenza infection. Virus‐induced oxidative stress may also control viral replication, but the mechanisms underlying ROS production, as well as their role in activating intracellular pathways and specific steps of viral life cycle under redox control have to be fully elucidated. In this study, we demonstrate that influenza A virus infection of lung epithelial cells causes a significant ROS increase that depends mainly on NOX4, which is upregulated at both mRNA and protein levels, while the expression of NOX2, the primary source of ROS in inflammatory cells, is downregulated. Inhibition of NOX4 activity through chemical inhibitors or RNA silencing blocks the ROS increase, prevents MAPK phosphorylation, and inhibits viral ribonucleoprotein (vRNP) nuclear export and viral release. Overall these data, obtained in cell lines and primary culture, describe a so far unrecognized role for NOX4‐derived ROS in activating redox‐regulated intracellular pathways during influenza virus infection and highlight their relevance in controlling specific steps of viral replication in epithelial cells. Pharmacological modulation of NOX4‐mediated ROS production may open the way for new therapeutic approaches to fighting influenza by targeting cell and not the virus.

Current Advances in Anti-Influenza Therapy

Every year, influenza epidemics cause numerous deaths and millions of hospitalizations, but the most frightening effects are seen when new strains of the virus emerge from different species (e.g. the swine-origin influenza A/H1N1 virus), causing world-wide outbreaks of infection. Several antiviral compounds have been developed against influenza virus to interfere with specific events in the replication cycle. Among them, the inhibitors of viral uncoating (amantadine), nucleoside inhibitors (ribavirin), viral transcription and neuraminidase inhibitors (zanamivir and oseltamivir) are reported as examples of traditional virus-based antiviral strategies. However, for most of them the efficacy is often limited by toxicity and the almost inevitable selection of drug-resistant viral mutants. Thus, the discovery of novel anti-influenza drugs that target general cell signaling pathways essential for viral replication, irrespective to the specific origin of the virus, would decrease the emergence of drug resistance and increase the effectiveness towards different strains of influenza virus. In this context, virus-activated intracellular cascades, finely regulated by small changes in the intracellular redox state, can contribute to inhibit influenza virus replication and pathogenesis of virus-induced disease. This novel therapeutic approach involves advanced cell-based antiviral strategies. In this review current advances in the anti-influenza therapy for both traditional virus-based antiviral strategies as well as for alternative cell-based antiviral strategies are described focusing on the last 10 years. Anti-influenza compounds are classified on the basis of their chemical structure with a special attention to describe their synthetic pathways and the corresponding structure activity relationships.

Pepstatin A alters host cell autophagic machinery and leads to a decrease in influenza A virus production.

Autophagy is a survival mechanism that can take place in cells under metabolic stress and through which cells can recycle waste material. Disturbances in autophagic processes appear to be associated with a number of human pathologies, including viral infections. It has been hypothesized that viruses can subvert autophagy in order to penetrate the host cell and replicate. Because it has been suggested that autophagy is involved in influenza A virus replication, we analyzed the effects of two inhibitors of lysosomal proteases on the cellular control of influenza A virus replication. In particular, we used biochemical and morphological analyses to evaluate the modulation of influenza A/Puerto Rico/8/34 H1N1 virus production in the presence of CA074 and Pepstatin A, inhibitors of cathepsin proteases B and D, respectively. We found that Pepstatin A, but not CA074, significantly hindered influenza virus replication, probably by modulating host cell autophagic/apoptotic responses. These results are of potential interest to provide useful insights into the molecular pathways exploited by the influenza in order to replicate and to identify further cellular factors as targets for the development of innovative antiviral strategies. J. Cell. Physiol. 226: 3368–3377, 2011.

Redox proteomics of the inflammatory secretome identifies a common set of redoxins and other glutathionylated proteins released in inflammation, influenza virus infection and oxidative stress

Protein cysteines can form transient disulfides with glutathione (GSH), resulting in the production of glutathionylated proteins, and this process is regarded as a mechanism by which the redox state of the cell can regulate protein function. Most studies on redox regulation of immunity have focused on intracellular proteins. In this study we have used redox proteomics to identify those proteins released in glutathionylated form by macrophages stimulated with lipopolysaccharide (LPS) after pre-loading the cells with biotinylated GSH. Of the several proteins identified in the redox secretome, we have selected a number for validation. Proteomic analysis indicated that LPS stimulated the release of peroxiredoxin (PRDX) 1, PRDX2, vimentin (VIM), profilin1 (PFN1) and thioredoxin 1 (TXN1). For PRDX1 and TXN1, we were able to confirm that the released protein is glutathionylated. PRDX1, PRDX2 and TXN1 were also released by the human pulmonary epithelial cell line, A549, infected with influenza virus. The release of the proteins identified was inhibited by the anti-inflammatory glucocorticoid, dexamethasone (DEX), which also inhibited tumor necrosis factor (TNF)-α release, and by thiol antioxidants (N-butanoyl GSH derivative, GSH-C4, and N-acetylcysteine (NAC), which did not affect TNF-α production. The proteins identified could be useful as biomarkers of oxidative stress associated with inflammation, and further studies will be required to investigate if the extracellular forms of these proteins has immunoregulatory functions.

The Environmental Pollutant Cadmium Promotes Influenza Virus Replication in MDCK Cells by Altering Their Redox State

Cadmium (Cd) is a toxic heavy metal that is considered an environmental contaminant. Several sources of human exposure to Cd, including employment in primary metal industries, production of certain batteries, foods, soil and cigarette smoke, are known. Its inhalation has been related to different respiratory diseases and toxic effects, among which alterations of the physiological redox state in individuals exposed to the metal have been described. Host-cell redox changes characteristic of oxidative stress facilitate the progression of viral infection through different mechanisms. In this paper, we have demonstrated that pre-treatment with CdCl2 of MDCK cells increased influenza virus replication in a dose-dependent manner. This phenomenon was related to increased viral protein expression (about 40% compared with untreated cells). The concentration of CdCl2, able to raise the virus titer, also induced oxidative stress. The addition of two antioxidants, a glutathione (GSH) derivative or the GSH precursor, N-acetyl-l-cysteine, to Cd pre-treated and infected cells restored the intracellular redox state and significantly inhibited viral replication. In conclusion, our data demonstrate that Cd-induced oxidative stress directly increases the ability of influenza virus to replicate in the host-cell, thus suggesting that exposure to heavy metals, such as this, could be a risk factor for individuals exposed to a greater extent to the contaminant, resulting in increased severity of virus-induced respiratory diseases.

Modulation of Th1/Th2 immune responses to HIV-1 Tat by new pro-GSH molecules

We have previously demonstrated that in Ova-immunized mice the increase in intra-macrophage thiol pool induced by pro-GSH molecules modulates the Th1/Th2 balance in favour of a Th1-type immune response. We show now that the same molecules can support a Th1-type over Th2-type immunity against Tat, which is an early HIV-1 regulatory protein and a Th1 polarizing immunomodulator that is increasingly considered in new anti-HIV vaccination strategies. Our results indicate that Tat-immunized mice pre-treated with the C4 (n-butanoyl) derivative of reduced glutathione (GSH-C4) or a pro-drug of N-acetylcysteine (NAC) and beta-mercaptoethylamine (MEA) (I-152), have decreased levels of anti-Tat IgG1 as well as increased levels of anti-Tat IgG2a and IgG2b isotypes suggesting a Th1-type response. Moreover, Th1-(IFN-γ and IL-2) Ag-specific cellular responses were detected by ELISPOT assay in splenocytes of the same animals as well as an increase of IL-12 levels in the plasma. These findings suggest that the Th1 immune response to HIV-1 Tat could be further polarized by these molecules. These results together with those previously reported suggest that pro-GSH molecules could be used to modulate the immune response towards different antigens and may be further exploited for inducing specific Th1 immune responses against other HIV antigens as well as other intracellular pathogens in new Tat-based vaccination protocols.

Therapeutic Activity of an Anti-Idiotypic Antibody-Derived Killer Peptide against Influenza A Virus Experimental Infection

ABSTRACT The in vitro and in vivo activities of a killer decapeptide (KP) against influenza A virus is described, and the mechanisms of action are suggested. KP represents the functional internal image of a yeast killer toxin that proved to exert antimicrobial and anti-human immunodeficiency virus type 1 (HIV-1) activities. Treatment with KP demonstrated a significant inhibitory activity on the replication of two strains of influenza A virus in different cell lines, as evaluated by hemagglutination, hemadsorption, and plaque assays. The complete inhibition of virus particle production and a marked reduction of the synthesis of viral proteins (membrane protein and hemagglutinin, in particular) were observed at a KP concentration of 4 μg/ml. Moreover, KP administered intraperitoneally at a dose of 100 μg/mice once a day for 10 days to influenza A/NWS/33 (H1N1) virus-infected mice improved the survival of the animals by 40% and significantly decreased the viral titers in their lungs. Overall, KP appears to be the first anti-idiotypic antibody-derived peptide that displays inhibitory activity and that has a potential therapeutic effect against pathogenic microorganisms, HIV-1, and influenza A virus by different mechanisms of action.

Viral hemagglutinin is involved in promoting the internalisation of Staphylococcus aureus into human pneumocytes during influenza A H1N1 virus infection

Secondary pneumonia caused by Staphylococcus aureus is reemerging as a primary cause of excess mortality associated with infection by the influenza A virus. We have investigated in vitro the cellular and molecular mechanisms underlying this synergism. Experimental data show a significant increase in the efficiency of internalisation of S. aureus into cultured pneumocytes during the early phases of viral infection, while a relevant increase in the efficiency of adhesion is evident only later during viral infection, suggesting that the 2 effects are based on different molecular mechanisms. Data reported in this paper show that S. aureus cells can bind the viral hemagglutinin (HA) and that this binding promotes enhanced bacterial internalisation by 2 mechanisms: binding to HA exposed at the surface of infected cells and binding to free extracellular virions, enabling internalisation at high efficiency also in cells that are not infected by the virus. The affinity of binding that involves S. aureus and HA was shown to be enhanced by the reducing extracellular environment that the virus can generate.