Jak2 Modulates Innate Immunity Figure 3. DCs deficient for Jak2 show similar potency to stimulate T cell activation. (A) Allogenic MLR results. (B) Antigen-specific MLR results. (C) ELISA analysis of cytokines produced by the activated T cells. MLR were carried out using either BALB/c T cells (allogenic) or OT-1 T cells (antigen-specific) as responders in the condition of DC/T ratio 1:10. T cell proliferation was estimated by 3H thymidine incorporation. Data shown in the figure are mean 6 SE of three independent experiments performed. doi:10.1371/journal.pone.0009593.g003 showed similar survival rate after lethal dose of LPS challenge (Figure 4B), demonstrating that DCs with reduced capacity to initiate innate immune response protected Jak22/2 mice from septic shock. To further address above question, we examined serum TNFa and IL-12 production in mice challenged with nonlethal dose of LPS (150 mg/mouse). Serum samples were collected from both Jak22/2 and control mice after 12 h of LPS challenge. Sera pooled from 3 mice of each study group were subjected to ELISA analysis of cytokine production. Consistent with above observa- tions, Jak22/2 mice produced significant lower levels of serum TNFa and IL-12 as compared with that of control mice (Figure 4C). Interestingly, we observed much higher levels of serum IL-2 in Jak22/2 mice although IL-2 levels for both Jak22/2 and control mice were relatively low (Figure 4C). Similar as in vitro studies, we failed to detect significant differences for serum IL-10 between Jak22/2 and control mice after LPS challenge (Figure S5B). As DC apoptosis is a potential causative factor for post-septic death, we examined DC apoptosis by flow cytometry. BMDCs were treated with 10 mg/ml LPS for 96 h and then stained with Annexin-V and PI for flow cytometry analysis. We failed to detect a significant difference of apoptosis between Jak22/2 and control BMDCs (Figure S6A). We further analyzed splenic DC apoptosis in Jak22/2 mice after 20 h of LPS challenge (150 mg/ mouse), and similar results were obtained (Figure S6B). Loss of Jak2 Impairs the Activation of STAT3, 4, 5, and 6 To investigate the underlying mechanisms by which Jak2 regulates DC-mediated innate immune response, 26106/ml Jak22/2 or control BMDCs were stimulated with 1 mg/ml LPS for 30 min and then harvested for Western blot analysis of target molecules. Given the importance of NFkB signaling in LPS-induced immune response, we first examined phosphorylated IkBa (pIkBa) in BMDC lysates. To our surprise, we failed to detect a significant difference for pIkBa between Jak22/2 and control BMDCs (Figure 5A). Therefore, Jak2 regulation of DCs for secretion of proinflammatory cytokines seems to be independent of NFkB signaling. We next examined the activation states of downstream signaling molecules. As STAT signaling has been demonstrated to be crucial for cytokine receptors to transduce intracellular signals, we therefore checked the phosphorylation states of transcription factors STAT1, STAT3, STAT4, STAT5 and STAT6. It was found that loss of Jak2 significantly attenuated STAT3, 4, 5 and 6 activation in BMDCs as manifested by the significant decreased levels for phosphorylated STAT3 (pSTAT3, Figure 5B), pSTAT4 (Figure 5C), pSTAT5 (Figure 5D) and pSTAT6 (Figure 5E). In contrast, Jak2 did not show a perceptible effect on STAT1 activation (Figure 5F). To demonstrate why macrophages deficient for Jak2 showed similar capacity secretion of proinflammatory cytokines, we further examined the activation states of all STAT factors in Jak22/2 macrophages as above. Unlike DCs, loss of Jak2 in macrophages only affected the activation of STAT3 and 4 (Figure S7). Jak2/STAT5 Signaling Is Indispensable for DC Development and Maturation We next sought to examine the functional relevance of Jak2/ STAT5 signaling in DCs. Stat5-Tg mice and their control PLoS ONE | www.plosone.org 5 March 2010 | Volume 5 | Issue 3 | e9593