Abstract Immune responses during infection with pandemic H1N1 2009 influenza A virus (2009-H1N1) are still poorly understood. Using an experimental infection model in ferrets, we examined the ...pathological features and characterized the host immune responses by using microarray analysis, during infection with 2009-H1N1 A/California/07/2009 and seasonal A/Brisbane/59/2007. Chemokines CCL2, CCL8, CXCL7 and CXCL10 along with the majority of interferon-stimulated genes were expressed early, correlated to lung pathology, and abruptly decreased expression on day 7 following infection of A/California/07/2009. Interestingly, the drop in innate immune gene expression was replaced by a significant increase of the adaptive immune genes for granzymes and immunoglobulins. Serum anti-influenza antibodies were first observed on day 7, commensurate with the viral clearance. We propose that lung pathology in humans occurs during the innate phase of host immunity and a delay or failure to switch to the adaptive phase may contribute to morbidity and mortality during severe 2009-H1N1 infections.
Abstract Type I interferons (IFNs) are essential to the clearance of viral diseases, however, a clear distinction between genes upregulated by direct virus–cell interactions and genes upregulated by ...secondary IFN production has not been made. Here, we investigated differential gene regulation in ferrets upon subcutaneous administration of IFN-α2b and during SARS-CoV infection. In vivo experiments revealed that IFN-α2b causes STAT1 phosphorylation and upregulation of abundant IFN response genes (IRGs), chemokine receptors, and other genes that participate in phagocytosis and leukocyte transendothelial migration. During infection with SARS-CoV not only a variety of IRGs were upregulated, but also a significantly broader range of genes involved in cell migration and inflammation. This work allowed dissection of several molecular signatures present during SARS-CoV which are part of a robust IFN antiviral response. These signatures can be useful markers to evaluate the status of IFN responses during a viral infection and specific features of different viruses.
In the present study, we explored the molecular mechanisms by which bacterial endotoxin (LPS) mediates the down‐regulation of CCR2 receptors on human monocytes. We found that LPS induced a marked ...reduction in CCR2 cell surface protein levels which was blocked by pretreatment with the tyrosine kinase inhibitors genistein and herbimycin A. The effector mechanism underlying LPS‐induced CCR2 down‐modulation appears to involve the enzymatic activity of proteinases since Western blot analysis of LPS‐stimulated monocytes revealed the degradation of a 38‐kDa species corresponding to the CCR2B monomer. In RBL cells expressing the CCR2B‐green fluorescent protein (GFP) fusion chemokine receptor, LPS stimulated the internalization and degradation of CCR2. The serine proteinase inhibitor N‐α‐p‐tosyl‐L‐lysine chloromethyl ketone blocked LPS‐induced down‐modulation of CCR2 in monocytes and CCR2B‐GFP in RBL cells. This work describes a previously uncharacterized mechanism for CC chemokine receptor down‐modulation that is dependent upon tyrosine kinase activation and serine proteinase‐mediated receptor degradation and may provide further insight into the mechanisms of leukocyte regulation during immunological and inflammatory responses.
Abstract Pandemic H1N1 influenza A (H1N1pdm) elicits stronger pulmonary inflammation than previously circulating seasonal H1N1 influenza A (sH1N1), yet mechanisms of inflammatory activation in ...respiratory epithelial cells during H1N1pdm infection are unclear. We investigated host responses to H1N1pdm/sH1N1 infection and virus entry mechanisms in primary human bronchial epithelial cells in vitro . H1N1pdm infection rapidly initiated a robust inflammatory gene signature (3 h post-infection) not elicited by sH1N1 infection. Protein secretion inhibition had no effect on gene induction. Infection with membrane fusion deficient H1N1pdm failed to induce robust inflammatory gene expression which was rescued with restoration of fusion ability, suggesting H1N1pdm directly triggered the inflammatory signature downstream of membrane fusion. Investigation of intra-virion components revealed H1N1pdm viral RNA (vRNA) triggered a stronger inflammatory phenotype than sH1N1 vRNA. Thus, our study is first to report H1N1pdm induces greater inflammatory gene expression than sH1N1 in vitro due to direct virus–epithelial cell interaction.
Background: CD4 super(+) T cells are critically important in HIV infection, being both the primary cells infected by HIV and likely playing a direct or indirect role in helping control virus ...replication. Key areas of interest in HIV vaccine research are mechanisms of viral escape from the immune response. Interestingly, in HIV infection it has been shown that peptide sequence variation can reduce CD4 super(+) T cell responses to the virus, and small changes to peptide sequences can transform agonist peptides into antagonist peptides. Results: We describe, at a molecular level, the consequences of antagonism of HIV p24-specific CD4 super(+) T cells. Antagonist peptide exposure in the presence of agonist peptide caused a global suppression of agonist-induced gene expression and signaling molecule phosphorylation. In addition to down-regulation of factors associated with T cell activation, a smaller subset of genes associated with negative regulation of cell activation was up-regulated, including KFL-2, SOCS-1, and SPDEY9P. Finally, antagonist peptide in the absence of agonist peptide also delivered a negative signal to T cells. Conclusions: Small changes in p24-specific peptides can result in T cell antagonism and reductions of both T cell receptor signaling and activation. These changes are at least in part mediated by a dominant negative signal delivered by antagonist peptide, as evidenced by up-regulation of negative regulatory genes in the presence of agonist plus antagonist stimulation. Antagonism can have dramatic effects on CD4 super(+) T cell function and presents a potential obstacle to HIV vaccine development.
HIV infection is characterized by a host response composed of adaptive and innate immunity that partially limits viral replication; however, it ultimately fails in eradicating the virus. To model ...host gene expression during acute HIV infection, we infected cynomolgus macaques with the SIV/HIV-1 chimeric virus, SHIV89.6P, and profiled gene expression in peripheral blood over a 5-wk period using a high density cDNA microarray. We demonstrate that viral challenge induced a widespread suppression of genes regulating innate immunity, including the LPS receptors, CD14 and TLR4. An overexpression of 16 IFN-stimulated genes was also observed in response to infection; however, it did not correlate with control over viral titers. A statistical analysis of the dataset identified 10 genes regulating apoptosis with differential expression during the first 2 wk of infection (p < 0.004). Quantitative real-time PCR verified transcriptional increases in IFN-alpha-inducible genes and decreases in genes regulating innate immunity. Therefore, the persistence of high viral loads despite an extensive IFN response suggests that HIV can resist in vivo IFN treatment despite published reports of in vitro efficacy. The transcriptional suppression of genes regulating innate immunity may allow HIV to evade acute host responses and establish a chronic infection and may reduce innate host defense against opportunistic infections.
CD4+ T cells are critically important in HIV infection, being both the primary cells infected by HIV and likely playing a direct or indirect role in helping control virus replication. Key areas of ...interest in HIV vaccine research are mechanisms of viral escape from the immune response. Interestingly, in HIV infection it has been shown that peptide sequence variation can reduce CD4+ T cell responses to the virus, and small changes to peptide sequences can transform agonist peptides into antagonist peptides.
We describe, at a molecular level, the consequences of antagonism of HIV p24-specific CD4+ T cells. Antagonist peptide exposure in the presence of agonist peptide caused a global suppression of agonist-induced gene expression and signaling molecule phosphorylation. In addition to down-regulation of factors associated with T cell activation, a smaller subset of genes associated with negative regulation of cell activation was up-regulated, including KFL-2, SOCS-1, and SPDEY9P. Finally, antagonist peptide in the absence of agonist peptide also delivered a negative signal to T cells.
Small changes in p24-specific peptides can result in T cell antagonism and reductions of both T cell receptor signaling and activation. These changes are at least in part mediated by a dominant negative signal delivered by antagonist peptide, as evidenced by up-regulation of negative regulatory genes in the presence of agonist plus antagonist stimulation. Antagonism can have dramatic effects on CD4+ T cell function and presents a potential obstacle to HIV vaccine development.
Ferrets (
Mustela putorius furo) develop symptoms upon influenza infection that resemble those of humans, including sneezing, body temperature variation and weight loss. Highly pathogenic strains of ...influenza A, such as H5N1, have the capacity to cause severe illness or death in ferrets. The use of ferrets as a model of influenza infection is currently limited by a lack of species-specific immunological reagents. Interferon gamma (IFN-
γ) plays a key role in the development of innate and adaptive immunity and the regulation of Th1-type immune responses. Here we describe the cloning of the full-length cDNA for ferret IFN-
γ. Multiple sequence alignment of the predicted amino acid sequence with those of other species indicates that the predicted ferret protein shares the highest identity with Eurasian badger IFN-
γ. We raised two hybridoma clones expressing monoclonal antibodies against recombinant ferret IFN-
γ capable of detecting IFN-
γ protein derived from mitogen-stimulated ferret PBMCs by immunoblotting, ELISA and ELISPOT assay. Finally, an ELISA utilizing the ferret-specific antibodies detected elevated levels of IFN-
γ in serum samples from H3N2 influenza A-infected ferrets.
Chemokines and their receptors function in the recruitment and activation of cells of the immune system to sites of inflammation. As such, chemokines play an important role in mediating ...pathophysiological events during microbial infection. In particular, CXCL9, CXCL10 and CXCL11 and their cognate receptor CXCR3 have been associated with the clinical course of several infectious diseases, including severe acute respiratory syndrome (SARS) and influenza. While CXCL9, CXCL10 and CXCL11 share the same receptor and have overlapping functions, each can also have unique activity in host defense. The lack of a preferred characterized animal model for SARS has brought our attention to ferrets, which have been used for years in influenza studies. The lack of immunological reagents for ferrets prompted us to clone CXCL9, CXCL10, CXCL11 and CXCR3 and, in the case of CXCL10, to express the gene as a recombinant protein. In this study we demonstrate that endogenous ferret CXCL10 exhibits similar mRNA expression patterns in the lungs of deceased SARS patients and ferrets experimentally infected with SARS coronavirus. This study therefore represents an important step towards development of the ferret as a model for the role of CXCL9, CXCL10 and CXCL11:CXCR3 axis in severe viral infections.
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