Jak2 Modulates Innate Immunity Figure 4. Jak22/2 mice show DC-dependent resistance to lethal dose of LPS-induced septic shock. (A) The survival rates of Jak22/2 and control mice were monitored for 4 days after lethal dose of LPS challenge. It was noticed that mice would completely recover from septic shock if they did not die within 36 h of lethal dose of LPS challenge. 85% of Jak22/2 mice (17 out of 20) have completely recovered from septic shock, while only 22% of control mice (4 out of 18) have survived (p,0.0001). (B) Adoptive transfer of normal DCs into irradiated Jak22/2 mice restored their susceptibility to LPS-induced septic shock. Irradiated Jak22/2 or control mice were first adoptively transferred with 16107 normal BMDCs followed by lethal dose of LPS challenge. Jak22/2 mice after adoptive transfer of normal DCs showed similar survival rate as that of control mice, indicating that the resistant phenotype for septic shock seen in Jak22/2 mice is DC-dependent. (C) ELISA analysis of serum cytokines of nonlethal dose of LPS challenged mice. Both Jak22/2 and control mice were first challenged with nonlethal dose of LPS (150 mg/mouse) and 12 h later the mice were sacrificed, respectively. Sera were pooled from three mice and then subjected to ELISA analysis of cytokine production. Three pools were analyzed for each study group. Jak22/2 mice produced significant lower levels of proinflammatory cytokines as compared with that of control mice. The data are present as mean 6 SE of three replicates. *, p,0.05; **, p,0.01; ***, p,0.001. doi:10.1371/journal.pone.0009593.g004 littermates were used for the study. In contrast to Jak22/2 mice, the weight of spleens and number of splenocytes were significantly higher in Stat5-Tg mice as compared with their control littermates (Figure 6A). Consistent with this observation, the yield of BMDCs from Stat5-Tg mice was 1.6-fold higher than that of their control littermates (Figure 6B, left panel). Similarly, the percentage of splenic DCs in total splenocytes was significantly higher than their control littermates (Figure 6B, right panel). We further examined the impact of Jak2/STAT5 signaling on DC maturation. Similar as above, DCs were generated from bone marrow cells originated from Stat5-Tg mice and control littermates, respectively, and then stimulated with LPS overnight. As shown in Figure 6C, significant higher percentage of BMDCs derived from Stat5-Tg mice expressing high levels of MHC-II and costimulatory molecules such as CD80, CD86 and CD54 before and after LPS stimulation, as compared with their control littermates. We next examined cytokine secretion using the above culture supernatants. Interest- ingly, BMDCs with transgenic Stat5 expression showed similar capacity to secret TNFa, however, much higher levels of IL-12 was noted (Figure 6D). Together, our results suggest that Jak2/ STAT5 signaling plays an indispensable role in DC development, maturation and IL-12 secretion. Jak2/STAT6 Is Essential for DCs Secretion of Proinflammatory Cytokines To delineate whether Jak2/STAT4 and Jak2/STAT6 signaling implicate in DC development and DC-mediated innate immune response, Stat42/2 and Stat62/2 mice were used for the study. It is noteworthy that we failed to detect significant alterations for DC development and maturation in both Stat42/2 and Stat62/2 mice (data not shown). As a result, our next focus is to examine their implications in the regulation of cytokine secretion. Before stimulation, BMDCs only secreted low levels of TNFa, while IL- 12 was undetectable in the cultures of all BMDCs (Figure 6E). However, high levels of TNFa and IL-12 were detected after LPS stimulation and Stat62/2 BMDCs showed significant lower capacity to secret TNFa and IL-12, although we observed relatively higher levels of TNFa in Stat62/2 BMDCs in the unstimulated condition (Figure 6E). All of these results demon- strate that Jak2/STAT6 signaling regulates the capacity of DCs to secrete cytokines implicated in innate immune response. To our surprise, we failed to detect a significant impact for Jak2/STAT4 signaling on either DC development or cytokine secretion. Previous studies demonstrated that STAT4 levels directly correlate with IL-12-dependent IFNc production by DCs during antigen PLoS ONE | www.plosone.org 6 March 2010 | Volume 5 | Issue 3 | e9593