Protective Anti-RVFV NP Responses in Mice Figure 5. (a–f). Fold changes in expression of genes in the liver of experimental groups at 72 hours after RVFV infection. RecNP- immunized mice (n = 3) are indicated by solid black bars, adjuvant control mice (n = 3) by grey bars and PBS control mice (n = 3) by white bars. The horizontal dotted lines indicate the cut-off values for upregulation (+2) or downregulation (22). The asterisk (*) indicates where the P-value is smaller than or equal to 0.05 (statistically significant results). Standard deviation from the mean fold changes are indicated by the error bars. doi:10.1371/journal.pone.0025027.g005 survival, was downregulated in liver of non-immunized PBS control mice (Figure 5b) [78]. The expression of surfactant protein D (Sftpd), a member of the collectin family, important role player in innate immunity and inhibitor of T lymphocyte proliferation, was upregulated in liver of non-immunized control mice (Figure 5 d) [79]. The expression of the gene encoding the E3 Ubiquitin Ligase Cbl-b, capable of negatively regulating T-cell activation, was upregulated in liver of non-immunized control mice (Figure 5 d) [67,68,80]. The expression of the gene encoding interleukin-18, responsible for biasing immunity towards Th-1 cellular immunity and enhancing T-cell cytotoxicity, was downregulated in liver of non-immunized control mice (Figure 5 d) [81]. The expression of the suppressor of cytokine signaling 5 (Socs5), part of a family of proteins that negatively regulate cytokine signaling [82], was upregulated in liver of non-immunized control mice and the gene encoding the immunoglobulin binding protein 1 (Igbp1), a component of receptor cell signaling in B- and T-cells [45], was downregulated in liver of non-immunized control mice. Other genes (Cd81 and Pik3r1) involved in activation, signaling and differentiation of B- and T-cells were also downregulated in liver of non-immunized control mice (Table S1) [78,83]. An important role player in innate immunity, NK cells, was also activated in liver of non-immunized control mice (Figure 5 f) [64,84,85]. The gene encoding the histocompatibility 60 A protein, a ligand for an activating receptor on NK cells [85], was upregulated in non-immunized control mice. However, expression of two important genes in NK cell activation and maturation, IL-18 and the prolactin receptor (Prlr), were downregulated in liver of non-immunized control mice [81,84]. The expression of three genes encoding Toll-like receptors, a component of the innate immune system responsible for recognizing conserved structures of pathogens, were analyzed with only Tlr4 being upregulated in liver of non-immunized control mice (Table S1) [86]. In contrast to the liver, there was not such a clear bias towards apoptosis in the spleens of adjuvant and PBS control mice (Figure 4b, Figure 6a). Only two genes with pro-apoptotic effects were upregulated, and two genes with anti-apoptotic effects downregulated in spleen of control mice. Interestingly expression of Nos2 and Cd40, both with known pro-apoptotic effects and upregulated in liver of control mice, were downregulated in spleen of control mice. Similarly to liver, some genes with pro- inflammatory effects were upregulated in the spleens of adjuvant and PBS control mice (Figure 6b), except for the genes encoding Nos2, Cd40 and Cd40 ligand. The expression of various genes involved in the activation of B- and T-cell immunity were, PLoS ONE | www.plosone.org 6 September 2011 | Volume 6 | Issue 9 | e25027