Cytokine Property of Tongue Sole ISG15 Figure 7. Effect of rCsISG15 and rCsISG15M on the expression of immune genes in lymphocytes. Tongue sole head kidney lymphocytes were treated with or without (control) rCsISG15 or rCsISG15M, and the expression of immune relevant genes was determined by quantitative real time RT-PCR at various time points. The assay was performed three times, and the results are shown as means6SE. *P,0.05; **P,0.01. doi:10.1371/journal.pone.0044884.g007 (ii) Effect on lymphocyte immune response. To examine the potential effect of rCsISG15 on lymphocyte immune response, HK lymphocytes were treated with or without rCsISG15 or rCsISG15M and then subjected to qRT-PCR analysis of the expression of IL-1b, CsIL8 (a tongue sole homologue of IL-8), TLR9, MHC IIb, CsCCK1 (a CC chemokine), and CsCXCe1 (a CXC chemokine), which are the major immune relevant genes known in tongue sole. The results showed that treatment with rCsISG15 induced significant inductions of all the examined genes in a time-dependent manner, with drastic inductions (maximum levels of 21–73 fold) occurring in CsIL8, CsCCK1, and CsCXCe1 expression, and moderate inductions (maximum levels of 5–7 fold) occurring in IL-1b, TLR9, and MHC IIb expression (Fig. 7A). In cells treated with rCsISG15M, the induction patterns of CsIL8, CsCCK1, and CsCXCe1 were similar to those in rCsISG15- treated cells but the induction folds were in general significantly lower than those in rCsISG15-treated cells. No significant difference was observed between the inductions of TLR9 effected by rCsISG15M and rCsISG15. For MHC IIb and IL-1b, the induction patterns induced by rCsISG15M differed from those induced by rCsISG15. The peak induction of MHC IIb induced by rCsISG15M was ,2-fold lower than that induced by rCsISG15, while the peak induction of IL-1b induced by rCsISG15M was comparable to that induced by rCsISG15. (iii) Effect on lymphocyte resistance against viral infection. The above results led us to wonder whether the immunoregulatory effect observed with rCsISG15 on lympho- cytes would have any effect on cellular resistance against viral infection. To examine this question, HK lymphocytes were treated with or without rCsISG15, rCsISG15M, or E. coli preparation before being infected with megalocytivirus. Viral copy numbers in the infected cells were then determined at 2 h and 4 h post-infection. The results showed that at both time points, the viral copies in rCsISG15- and rCsISG15M -treated cells were significantly lower than those in the control cells (Fig. 8A). However, compared to rCsISG15-treated cells, PLOS ONE | www.plosone.org 5 September 2012 | Volume 7 | Issue 9 | e44884