Influenza H5N1 virus infection of polarized human alveolar epithelial cells and lung microvascular endothelial cells

Background

Highly pathogenic avian influenza (HPAI) H5N1 virus is entrenched in poultry in Asia and Africa and continues to infect humans zoonotically causing acute respiratory disease syndrome and death. There is evidence that the virus may sometimes spread beyond respiratory tract to cause disseminated infection. The primary target cell for HPAI H5N1 virus in human lung is the alveolar epithelial cell. Alveolar epithelium and its adjacent lung microvascular endothelium form host barriers to the initiation of infection and dissemination of influenza H5N1 infection in humans. These are polarized cells and the polarity of influenza virus entry and egress as well as the secretion of cytokines and chemokines from the virus infected cells are likely to be central to the pathogenesis of human H5N1 disease.

Aim

To study influenza A (H5N1) virus replication and host innate immune responses in polarized primary human alveolar epithelial cells and lung microvascular endothelial cells and its relevance to the pathogenesis of human H5N1 disease.

Methods

We use an in vitro model of polarized primary human alveolar epithelial cells and lung microvascular endothelial cells grown in transwell culture inserts to compare infection with influenza A subtype H1N1 and H5N1 viruses via the apical or basolateral surfaces.

Results

We demonstrate that both influenza H1N1 and H5N1 viruses efficiently infect alveolar epithelial cells from both apical and basolateral surface of the epithelium but release of newly formed virus is mainly from the apical side of the epithelium. In contrast, influenza H5N1 virus, but not H1N1 virus, efficiently infected polarized microvascular endothelial cells from both apical and basolateral aspects. This provides a mechanistic explanation for how H5N1 virus may infect the lung from systemic circulation. Epidemiological evidence has implicated ingestion of virus-contaminated foods as the source of infection in some instances and our data suggests that viremia, secondary to, for example, gastro-intestinal infection, can potentially lead to infection of the lung. HPAI H5N1 virus was a more potent inducer of cytokines (e.g. IP-10, RANTES, IL-6) in comparison to H1N1 virus in alveolar epithelial cells, and these virus-induced chemokines were secreted onto both the apical and basolateral aspects of the polarized alveolar epithelium.

Conclusion

The predilection of viruses for different routes of entry and egress from the infected cell is important in understanding the pathogenesis of influenza H5N1 infection and may help unravel the pathogenesis of human H5N1 disease.     

Long-term water table manipulations alter peatland gaseous carbon fluxes in Northern Michigan

Northern peatland water table position is tightly coupled to carbon (C) cycling dynamics and is predicted to change from shifts in temperature and precipitation patterns associated with global climate change. However, it is uncertain how long-term water table alterations will alter C dynamics in northern peatlands because most studies have focused on short-term water table manipulations. The goal of our study was to quantify the effect of long-term water table changes (~80 years) on gaseous C fluxes in a peatland in the Upper Peninsula of Michigan. Chamber methods were utilized to measure ecosystem respiration (ER), gross primary production (GPP), net ecosystem exchange (NEE), and methane (CH₄) fluxes in a peatland experiencing levee induced long-term water table drawdown and impoundment in relation to an unaltered site. Inundation raised water table levels by approximately ~10 cm and resulted in a decrease in ER and GPP, but an increase of CH₄ emissions. Conversely, the drained sites, with water table levels ~15 cm lower, resulted in a significant increase in ER and GPP, but a decrease in CH₄ emissions. However, NEE was not significantly different between the water table treatments. In summary, our data indicates that long-term water table drawdown and inundation was still altering peatland gaseous C fluxes, even after 80 years. In addition, many of the patterns we found were of similar magnitude to those measured in short-term studies, which indicates that short-term studies might be useful for predicting the direction and magnitude of future C changes in peatlands.     

Upregulation of intracellular antioxidant enzymes in brain and heart during estivation in the African lungfish Protopterus dolloi

The African slender lungfish, Protopterus dolloi, is highly adapted to withstand periods of drought by secreting a mucous cocoon and estivating for periods of months to years. Estivation is similar to the diapause and hibernation of other animal species in that it is characterized by negligible activity and a profoundly depressed metabolic rate. As is typically observed in quiescent states, estivating P. dolloi are resistant to environmental stresses. We tested the hypothesis that P. dolloi enhances stress resistance during estivation by upregulating intracellular antioxidant defences in brain and heart tissues. We found that most of the major intracellular antioxidant enzymes, including the mitochondrial superoxide dismutase, cytosolic superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase, were upregulated in brain tissue of lungfish that had estivated for 60 days. Several of these enzymes were also elevated in heart tissue of estivators. These changes were not due to food deprivation, as they did not occur in a group of fish that were deprived of food but maintained in water for the same period of time. We found little evidence of tissue oxidative damage in estivators. Products of lipid peroxidation (4-hydroxynonenal adducts) and oxidative protein damage (carbonylation) were similar in estivating and control lungfish. However, protein nitrotyrosine levels were elevated in brain tissue of estivators. Taken together, these data indicate that estivating P. dolloi have enhanced oxidative stress resistance in brain and heart due to a significant upregulation of intracellular antioxidant capacity.     

Transbilayer movement of Glc-P-dolichol and its function as a glucosyl donor: protein-mediated transport of a water-soluble analog into sealed ER vesicles from pig brain

The results described in the accompanying article support the model in which glucosylphosphoryldolichol (Glc-P-Dol) is synthesized on the cytoplasmic face of the ER, and functions as a glucosyl donor for three Glc-P-Dol:Glc0-2Man9-GlcNAc2-P-P-Dol glucosyltransferases (GlcTases) in the lumenal compartment. In this study, the enzymatic synthesis and structural characterization by NMR and electrospray-ionization tandem mass spectrometry of a series of water-soluble beta-Glc-P-Dol analogs containing 2-4 isoprene units with either the cis - or trans - stereoconfiguration in the beta-position are described. The water- soluble analogs were (1) used to examine the stereospecificity of the Glc-P-Dol:Glc0-2Man9GlcNAc2-P-P-Dol glucosyltransferases (GlcTases) and (2) tested as potential substrates for a membrane protein(s) mediating the transbilayer movement of Glc-P-Dol in sealed ER vesicles from rat liver and pig brain. The Glc-P-Dol-mediated GlcTases in pig brain microsomes utilized [3H]Glc-labeled Glc-P-Dol10, Glc-P-(omega, c )Dol15, Glc-P(omega, t,t )Dol20, and Glc-P-(omega, t,c )Dol20as glucosyl donors with [3H]Glc3Man9GlcNAc2-P-P-Dol the major product labeled in vitro. A preference was exhibited for C15-20 substrates containing an internal cis -isoprene unit in the beta-position. In addition, the water-soluble analog, Glc-P-Dol10, was shown to enter the lumenal compartment of sealed microsomal vesicles from rat liver and pig brain via a protein-mediated transport system enriched in the ER. The properties of the ER transport system have been characterized. Glc- P-Dol10was not transported into or adsorbed by synthetic PC-liposomes or bovine erythrocytes. The results of these studies indicate that (1) the internal cis -isoprene units are important for the utilization of Glc-P-Dol as a glucosyl donor and (2) the transport of the water- soluble analog may provide an experimental approach to assay the hypothetical "flippase" proposed to mediate the transbilayer movement of Glc-P-Dol from the cytoplasmic face of the ER to the lumenal monolayer.