Innate Immune Response of Human Alveolar Macrophages during Influenza A Infection

Alveolar macrophages (AM) are one of the key cell types for initiating inflammatory and immune responses to influenza virus in the lung. However, the genome-wide changes in response to influenza infection in AM have not been defined. We performed gene profiling of human AM in response to H1N1 influenza A virus PR/8 using Affymetrix HG-U133 Plus 2.0 chips and verified the changes at both mRNA and protein levels by real-time RT-PCR and ELISA. We confirmed the response with a contemporary H3N2 influenza virus A/New York/238/2005 (NY/238). To understand the local cellular response, we also evaluated the impact of paracrine factors on virus-induced chemokine and cytokine secretion. In addition, we investigated the changes in the expression of macrophage receptors and uptake of pathogens after PR/8 infection. Although macrophages fail to release a large amount of infectious virus, we observed a robust induction of type I and type III interferons and several cytokines and chemokines following influenza infection. CXCL9, 10, and 11 were the most highly induced chemokines by influenza infection. UV-inactivation abolished virus-induced cytokine and chemokine response, with the exception of CXCL10. The contemporary influenza virus NY/238 infection of AM induced a similar response as PR/8. Inhibition of TNF and/or IL-1β activity significantly decreased the secretion of the proinflammatory chemokines CCL5 and CXCL8 by over 50%. PR/8 infection also significantly decreased mRNA levels of macrophage receptors including C-type lectin domain family 7 member A (CLEC7A), macrophage scavenger receptor 1 (MSR1), and CD36, and reduced uptake of zymosan. In conclusion, influenza infection induced an extensive proinflammatory response in human AM. Targeting local components of innate immune response might provide a strategy for controlling influenza A infection-induced proinflammatory response in vivo

Effects of the simian virus 40 origin of replication on transcription from the human immunodeficiency virus type 1 promoter.

Positive and negative effects of DNA replication on gene transcription have been documented in a variety of systems. We examined the effects of the simian virus 40 (SV40) origin of replication on transcription from the human immunodeficiency virus type 1 (HIV-1) promoter, using a transient expression assay in COS-1 cells. The basal activity and Tat transactivation of the HIV promoter were greatly stimulated by the SV40 origin of replication independent of its position relative to the long terminal repeat. These effects were abolished by mutational inactivation of the SV40 origin and were reduced by a DNA replication inhibitor. The magnitude of promoter activation exceeded the increment expected from the increase in template number resulting from DNA replication. The SV40 T-antigen-induced DNA replication augmented the generation of both processive and nonprocessive HIV long terminal repeat-directed transcripts, and Tat primarily enhanced the initiation of those transcripts that were destined to be efficiently elongated. Our data suggest that the HIV promoter displays greater transcriptional activity on replicative DNA templates. This property may influence the activity of integrated HIV provirus and its transition from latency to productive infection

Transduction of the human immunodeficiency virus type 1 promoter into human chromosomal DNA by adeno-associated virus: effects on promoter activity

Transcription of the human immunodeficiency virus type 1 (HIV-1) genome takes place after integration of the provirus into human chromosomal DNA. HIV transcription is known to be modulated by viral and cellular factors but the influence of flanking chromosomal sequences on proviral gene expression has not been well defined. To investigate the activity of the integrated HIV promoter, we exploited the ability of recombinant adeno-associated virus (AAV-2) to transfer and stably integrate genes into the human genome at random or site-specifically. Chimeric AAV vectors were constructed containing an HIV-CAT reporter cassette; some vectors also contained the neomycin resistance gene to facilitate the isolation of positive clones. HeLa cells were infected with recombinant AAV, in some instances together with wild-type virus as a source of AAV rep function. We isolated 25 clones of G418-resistant cells which carried the integrated HIV-CAT cassette, generally occupying unique sites that did not correspond to the AAV-specific region of chromosome 19. The HIV promoter was transcriptionally active in most of the clones. Basal promoter activity varied substantially among the clones, and its responsivity to the HIV transactivator Tat was also variable. The integrated HIV promoter was transactivated to comparable degrees by the one-exon form and two-exon form of Tat. These findings provide evidence that the transcriptional activity of the HIV promoter can be greatly influenced by the site of proviral insertion

The top 25 genes up-regulated or down-regulated by PR/8 infection in human AM at 24 hpi.

<p>Human AM from 3 non-smoking donors were isolated, cultured, and infected by PR/8 virus at a MOI of 0.5. The gene profiling of infected and non-infected cells at 24 hpi from each donor was examined by microarray experiments using Affymetrix HG-U133 Plus 2.0 chips (Affymetrix, Santa Clara, CA). The filtered gene list was generated as described in the Section of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029879#s4" target="_blank">Methods</a>. The data show the top 25 genes up-regulated or down-regulated altered by viral infection.</p><p>*indicates similar results from multiple probes.</p

Innate immune response of both live and UV-inactivated contemporary H3N2 influenza viruses-infected AM.

<p>Human AM isolated from donor lungs were cultured and infected by live NY/238 virus at a MOI of 0.1 or the equal amount of UV-inactivated NY/238. Cells were harvested at designated times for evaluation of their innate immune response. Panel A. Alterations in mRNAs of innate immune response-related genes at 3 and 24 hpi by realtime RT-PCR. The data represent mean+SE of the relative expression levels of each gene in infected cultures compared to that of non-infected controls after normalization to the level of the constitutive probe cyclophilin B, N = 4. * indicates P<0.05 and ** indicates P<0.01 between live and UV-inactivated cells. Panel B. Kinetics of cytokine and chemokine response by ELISA. The data show representative release of TNF-α, IFN-α, CXCL10, CXCL8, and CCL5 from both live and UV-inactivated NY/238 virus-infected AM from one of 6 donors that all showed similar response.</p