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

Zapata, Juan Carlos; Carrion, Ricardo; Patterson, Jean L.; Crasta, Oswald; Zhang, Yan; Mani, Sachin; Jett, Marti; Poonia, Bhawna; Djavani, Mahmoud; White, David M.; Lukashevich, Igor S.; Salvato, Maria S.

Due to the nature of LF disease, six LASV-upregulated genes related to the coagulation pathway were analyzed (Table 3) . Thrombomodulin (THBD), heparin-binding growth factor (HBGF), and integrin alpha M (ITGAM), were up-regulated after LASV exposure at all time-points in contrast to ML29 that downregulated them. Plasminogen activator urokinase (PLAU) and integrin beta3 (ITGB3) were mostly up-regulated with LASV exposure and remained unchanged or down-regulated with ML29. PD-ECGF (TYMP) was slightly up-regulated after exposure to both viruses. The analysis of those coagulation-related genes using the Gene Ontology (GO) software showed interaction between them and genes related to immune response ( Figure 3 ). In order to validate cDNA array results, THBD expression was measured on the surface of ML29 exposed-monocytes by flow cytometry, or quantified by ELISA in supernatants of DC exposed to LASV or ML29. There were no significant changes in thrombomodulin expression in CD14+ cells from PBMC treated with LPS or ML29 when compared with non-treated cells at 4 hpe (data not shown). However, as shown in figure 4A , there was a significant reduction in THBD expression on the surface of CD14+ cells after LPS (from ,74% control to 52% at 8 hpe and 47% at 24 hpe) or ML29 stimulus (from ,74% control to 57% at 8 hpe and 51% at 24 hpe), all in agreement with microarray results showing lower THBD RNA in ML29-exposed PBMC ( Figure 1 and Table S1 ). DC cultures exposed to LASV secreted significantly higher levels of THBD than DC exposed to ML29 (Figure 4B ), in agreement with transcriptome data. Control DC secreted 0.502 pg/ml THBD, whereas Lassa-exposed DC expressed 0.724 pg/ml THBD, significantly more than ML29-exposed DC (0.161 pg/ml). Treatment with LASV and LPS together diminished the THBD expression from 0.724 pg/ml after LASV exposure to 0.481 pg/ml. The other coagulation-related genes could also contribute to LF disease: Plasminogen activator urokinase (PLAU) expression could be contributing to the anti-clotting effect mediated by LASV exposure. Heparin-binding epidermal growth factor-like (HB-EGF) expression is involved in several blood vessel physiologies [58] , in kidney pathology [59] , [60] , inhibition of NF-kappa B activation [61] , in cardiac hypertrophy, and together with PD-ECGF promotes vascular maturation [62] , [63] . The ITGB3 (CD61, platelet marker) was 12-fold up-regulated in cells exposed to LASV at 24 h. Integrins are integral cell-surface proteins composed of a and b chains. A given chain may combine with multiple partners resulting in different integrins. The ITGB3 is found along with the aIIb chain in platelets working as a receptor for fibrinogen, von Willebrand factor, plasminogen, prothrombin, thrombospondin, fibronetin, osteospontin (SPP1), and vitronectin [64] , [65] , [66] . Integrins are known to participate in cell adhesion as well as cell-surface mediated signaling. High affinity LASV binding to cellular alpha-dystroglycan (a-DG) receptor perturbs the signaling cross- 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, cellsurface 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). Soluble THBD measured by ELISA in dendr