Transcriptome Analysis of Human Peripheral Blood Mononuclear Cells Exposed to Lassa Virus and to the Attenuated Mopeia/Lassa Reassortant 29 (ML29), a Vaccine Candidate

<div><p>Lassa virus (LASV) is the causative agent of Lassa Fever and is responsible for several hundred thousand infections and thousands of deaths annually in West Africa. LASV and the non-pathogenic Mopeia virus (MOPV) are both rodent-borne African arenaviruses. A live attenuated reassortant of MOPV and LASV, designated ML29, protects rodents and primates from LASV challenge and appears to be more attenuated than MOPV. To gain better insight into LASV-induced pathology and mechanism of attenuation we performed gene expression profiling in human peripheral blood mononuclear cells (PBMC) exposed to LASV and the vaccine candidate ML29. PBMC from healthy human subjects were exposed to either LASV or ML29. Although most PBMC are non-permissive for virus replication, they remain susceptible to signal transduction by virus particles. Total RNA was extracted and global gene expression was evaluated during the first 24 hours using high-density microarrays. <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002406#s3" target="_blank">Results</a> were validated using RT-PCR, flow cytometry and ELISA. LASV and ML29 elicited differential expression of interferon-stimulated genes (ISG), as well as genes involved in apoptosis, NF-kB signaling and the coagulation pathways. These genes could eventually serve as biomarkers to predict disease outcomes. The remarkable differential expression of thrombomodulin, a key regulator of inflammation and coagulation, suggests its involvement with vascular abnormalities and mortality in Lassa fever disease.</p></div

Thrombomodulin (THBD) in LASV infection.

<p>LASV infects endothelial cells, which are the main THBD producers. Increased THBD expression will capture thrombin through its 4, 5 and 6 domains, activating protein C (aPC), inhibiting the coagulation pathway and activating PAR1 membrane protein. PAR-1 has cytoprotective and anti-inflammatory affects, also inhibiting platelet activation.</p

Genes most differentially-expressed between LASV and ML29.

<p>Genes most differentially-expressed between LASV and ML29.</p

Gene Ontology (GO) analysis of coagulation-related genes.

<p>Gray figures represent the coagulation related genes [THBD, HBEGF, PLAU, ITGAM, ITGB3, PD-ECGF (TYMP)], affected by LASV exposure and the nodes with arrows represent key genes from the immune-response (Akt, ERK, NFkB, p38MAPK and IL1β).</p

Heat map of the genes affected by ML29 and Lassa exposure in all three sets of human donor PBMC with respect to gene expression in non-exposed PBMC.

<p>RNA was extracted for analysis at 4, 8 and 24 hours after exposure. For a complete listing of all 122 genes, see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002406#pntd.0002406.s002" target="_blank">Table S1</a> in the supplemental material. Green color represents down-regulated genes (two-fold or more when compared with non-exposed control). Red color represents up-regulated genes (two-fold or more when compared with non-exposed control).</p

Coagulation pathway genes affected by Lassa virus.

<p>Coagulation pathway genes affected by Lassa virus.</p

Validation of expression patterns of selected genes by quantitative RT-PCR.

<p>There was 76% correlation between microarray and qRT-PCR.</p>a<p>Two different readings for a particular gene in the same chip.</p

Membrane and soluble thrombomodulin (THBD) measurement.

<p><b>A</b>). Cell surface THBD was detected by flow cytometry on gated CD14+ cells. This experiment was repeated with PBMC from 4 different healthy donors with similar results. After exposure to either LPS or ML29, cell-surface THBD was down-regulated by approximately 30%, at both 8 and 24 hours post exposure (hpe). According to a paired Student's t test, this down-regulation was significant (p<0.05) at both time-points. <b>B</b>). Soluble THBD measured by ELISA in dendritic cell supernatants exposed to LPS inhibitor, ML29, LASV and LASV plus LPS 24 hpe. Standard deviation of THBD concentrations are shown from three different experiments. THMD from ML29 cells is significantly depressed with respect to THBD from non-exposed control cells. Lassa-exposed cells express significantly more THBD than ML29-exposed cells.</p