Jak2 Modulates Innate Immunity Figure 5. Western blot analysis of the activation states of Jak2 downstream molecules. 26106/ml Jak22/2 and control BMDCs were harvested after 30 min of LPS stimulation (1 mg LPS/ml) and cell lysates were prepared for Western blot analysis of the phosphorylated form of signaling molecules. (A) pIkBa; (B) phosphorylated STAT3 (pSTAT3); (C) pSTAT4; (D) pSTAT5; (E) pSTAT6; and (F) pSTAT1. Four independent experiments were performed and consistent results were obtained. doi:10.1371/journal.pone.0009593.g005 presentation [35], it is possible that Jak2/STAT4 signaling implicates in DC-mediated T cell polarization. Consistent with this assumption, we failed to detect DCs originated from both Jak22/2 and control mice secretion of IFNc after LPS stimulation despite lower percentage of DCs carrying high levels of MHC-II and costimulatory molecules in Jak22/2 mice (data not shown). As mice deficient for Stat3 lead to embryonic lethality [36], we used DC2.4 cells, a B6-derived DC line, for the study. DC2.4 cells were transfected with a Stat3 siRNA or a scramble RNA as described. Western blot analysis confirmed a significant reduction for the active form of STAT3 in response to LPS stimulation in cells transfected with siRNA (Figure 6F, left panel). Culture supernatants were then collected and subjected to analysis of proinflammatory cytokine secretion. To our surprise, we failed to detect IL-12 production in all cultures either before or after LPS stimulation. However, DC2.4 cells secreted high levels of TNFa, but no significant difference was noted between cells transfected with siRNA and scramble RNA (Figure 6F, right panel), indicating that Jak2/STAT3 signaling may not affect the capacity of DCs secretion of proinflammatory cytokines. Several recent studies suggested a role for Stat3 in DC development and differentiation [37–39], and therefore, Jak2/STAT3 is possibly to work together with Jak2/STAT5 signaling involved in DC development. Discussion Given the importance of Jak2 in both physiological and pathological conditions, a critical role for Jak2 in immune cells especially DCs has long been proposed. However, despite past extensive studies, its exact function in DCs remains controversial [26,27,35,40]. The main reason for the discrepant results is that most studies were conducted using Jak2 inhibitors as loss of Jak2 is embryonic lethal, while those inhibitors could non-specifically target other molecules. In this study, we have generated an inducible Jak2 deficient model by crossing Jak2fl/fl mice with Cre- ERT2 transgenic mice. We first performed studies to examine the implication of Jak2 in DC development. We have demonstrated that loss of Jak2 impairs DC development as manifested by the reduced BMDC yield, smaller spleen size, and reduced percentage of DCs in total splenocytes (Figures 1B–D). Other than this recognized role, we further noticed a pivotal role for Jak2 in the regulation of the capacity for DCs to mediate innate immune response. Jak22/2 DCs were found to be less potent in response to inflammatory stimuli (Figures 1E & 1F), and furthermore, they showed significant reduced capacity to secrete proinflammatory cytokines such as TNFa and IL-12 upon LPS stimulation (Figure 2A). As a result, Jak22/2 mice were defective for the early clearance of Listeria after infection as compared with that of control mice (Figure 2C). However, their potency to mediate adaptive immune response such as the capacity to activate allogenic or antigen specific T cells was not affected (Figures 3A– C). Very interestingly, it appears that Jak2 regulation of innate immune response is DC-dependent as macrophages deficient for Jak2 showed similar capacity secretion of proinflammatory cytokines such as TNFa and IL-12 (Figure 2B). This discrepancy could be caused by the difference for the STAT activation profile between DCs and macrophages. For example, Jak2 deficiency affected the activation of STAT3, 4, 5 and 6 in DCs (Figure 5), while defective activation was only noted for STAT3 and 4 in Jak22/2 macrophages (Figure S7). Together, our data suggest that Jak2 selectively modulates innate immune response which appears to be DC-dependent. To further dissect the role of Jak2 in DC-mediated innate immune response, we used septic shock, a typical disorder characterized by the uncontrolled innate immune response, as a model for the study. Septic shock is a severe sepsis due to excessive release of proinflammatory cytokines such as TNFa and IL-12, which then lead to vasodilation, increased vascular permeability, hypotension, multiple organ failure and ultimately shock and death. Unlike septic death resulted from late phase of sepsis, septic shock is manifested by excessive innate immune response such as copious amount of proinflammatory cytokine release. Therefore, Jak2 signaling might be a potential therapeutic target for controlling excessive innate immune response during septic shock. Cecal ligation and puncture (CLP) is a widely used experimental model for sepsis [41]. However, animals die after CLP usually PLoS ONE | www.plosone.org 7 March 2010 | Volume 5 | Issue 3 | e9593