Jak2 Modulates Innate Immunity Figure 6. Jak2/STAT5 signaling is indispensable for DC development and maturation, while the capacity of DC for secretion of proinflammatory cytokines is regulated by both Jak2/STAT5 and Jak2/STAT6 signaling. (A) Stat5-Tg mice show larger size for the spleen and higher number of splenocytes. (B) Transgenic Stat5 expression enhances BMDC yield and increases the percentage of splenic DCs in total splenocytes (C) DCs with transgenic Stat5 expression show higher potency in response to LPS stimulation. Significant higher percentage of DCs show matured phenotype as characterized by high levels of MHC II and costimulatory molecule expressions before/after LPS stimulation. (D) ELISA analysis of cytokines secreted by DCs with transgenic Stat5 expression. Stat5-Tg BMDCs showed similar capacity for secretion of TNFa, while the production of IL-12 was significantly higher than that of control BMDCs after LPS stimulation. (E) ELISA analysis of cytokine secretion by BMDCs generated from Stat42/2 and Stat62/2 mice. Stat62/2 BMDCs showed significant lower capacity for secretion of TNFa and IL-12 upon LPS stimulation. In contrast, Stat42/2 BMDCs showed similar capacity for secretion of above indicated cytokines as control BMDCs. (F) Down-regulation of Stat3 in DC2.4 cell by siRNA. DC2.4 cells were first transfected with a Stat3-specific siRNA or a scramble RNA followed by LPS stimulation for analysis of cytokine secretion. Left panel: Western blot results showing significant down-regulation of pSTAT3 in cells transfected with siRNA. Right panel: ELISA analysis of cytokine secretion. IL-12 was undetectable in both siRNA and scramble transfected DC2.4 cells, while siRNA transfected DC2.4 cells showed similar capacity for secretion of TNFa. Data shown in the figure are presented as mean 6 SE. *, p,0.05; **, p,0.01; ***, p,0.001. doi:10.1371/journal.pone.0009593.g006 ranging from 24 h to 7 days which implicates both altered innate immune response and maladaptive immune response. Since our focus is to determine Jak2 deficiency in DC-mediated innate immune response, it would be crucial to ultimately exclude the implication of adaptive immune response. As a result, we have chosen lethal dose of LPS-induced septic shock for the study. As expected, 78% of control mice died from septic shock within 36 h of lethal dose of LPS challenge, while 85% of Jak22/2 mice have survived from the same dose of LPS challenge (Figure 4A), indicating that Jak22/2 mice were protected from LPS-induced septic shock. Adoptive transfer studies were then applied to further address that this protective effect is DC-dependent. Since macrophages deficient for Jak2 only showed defective activation for STAT3 and 4 (Figure S7), we selected adoptive transfer of DCs for the study, in which 16107 normal DCs were transferred into irradiated Jak22/2 mice. As expected, Jak22/2 mice after PLoS ONE | www.plosone.org 8 March 2010 | Volume 5 | Issue 3 | e9593