impact on their capacity to mediate adaptive immune response. As a result, Jak22/2 mice are remarkably resistant to lethal dose of LPS-induced septic shock. Our results suggest that Jak2 could be a unique therapeutic target for the intervention and treatment of clinical septic shock. Results Jak2 Is Essential for DC Development and Maturation Cre+/+Jak2fl/fl mice were generated by crossing Cre-ERT2 transgenic mice with Jak2fl/fl mice as described. For induction of Jak2 deficiency, 8 wk-old male Cre+/+Jak2fl/fl mice were i.p. injected with tamoxifen (25 mg/kg body weight) for five consecutive days. Male littermates administered with carrier solution (10% ethanol in corn oil) were used as controls. To confirm Jak2 deficiency, the mice were sacrificed after 2 wk of last injection. Bone marrow derived dendritic cells (BMDCs) and splenocytes were prepared and subjected to Western blot analysis of Jak2 expression. As shown in Figure 1A, BMDCs originated from control mice showed high levels of Jak2 expression, while Jak2 was undetectable in BMDCs of tamoxifen induced mice. Similar results were also observed in splenocytes (data not shown). Collectively, these results indicate that tamoxifen efficiently induced Jak2 deficiency in Cre+/+Jak2fl/fl mice. We first sought to address the impact of Jak2 deficiency on DC development. To this end, 16107 bone marrow cells originated from Jak22/2 and control littermates were induced with GM-CSF and IL-4 to generate BMDCs, respectively. Jak2 deficiency significantly reduced DC production, a 1.3-fold decrease for BMDC yield was observed in Jak22/2 mice as compared with that of control mice (Figure 1B). We also noticed a significant smaller size for spleens in Jak22/2 mice than that of control mice (5268 mg vs. 157610 mg, p,0.001; Figure 1C). In line with this observation, total splenocytes in Jak22/2 mice were significantly less than that of controls (Figure 1D, left panel). Next, we examined splenic DCs. To our surprise, in addition to the reduced number of total splenocytes, the percentage of DCs in spenocytes has also significantly decreased (Figure 1D, right panel). In contrast, we failed to detect a significant alteration for the percentage of CD4 and CD8 T cells in total spenocytes (data not shown). Together, these data suggest that loss of Jak2 significantly impaired DC development. We next examined DC phenotypic differences between Jak22/2 and control mice. For this purpose, day-9 BMDC cultures were stimulated with 0.5 mg/ml LPS overnight and harvested on day-10 for flow cytometry analysis of surface marker expressions. Both Jak22/2 and control bone marrow cells produced .85% purity of DCs, which were then gated for the analysis of surface MHC-II, CD80, CD86, and CD54 expressions. In contrast to previous published data [26,27], loss of Jak2 rendered DCs significant less potent in response to maturation stimulation. We first noticed that much lower percentage of Jak22/2 BMDCs expressing MHC-II and co-stimulatory molecules such as CD80, CD86 and CD54 before stimulation (Figure 1E). Upon LPS stimulation, majority of control BMDCs became matured as characterized by high levels of MHC-II, CD80, CD86 and CD54 expressions. In sharp contrast, only a small proportion of Jak22/2 BMDCs became matured (Figure 1F). To further confirm this observation, we analyzed splenic DCs. Single splenic cells were prepared and then co-stained for CD11c and MHC-II or one of the above indicated costimulatory molecules. Similar as BMDCs, significant lower percentage of Jak22/2 splenic DCs expressed high levels of MHC- II and costimulatory molecules (Figure S1). To further confirm these observations, we treated BMDCs with AG490, a broadly Jak2 Modulates Innate Immunity used Jak2 inhibitor. In contrast to previously published data [27], but consistent with our current data observed on Jak22/2 mice, AG490 significantly suppressed DC maturation (Figure S2). We also tested the impact of tamoxifen on DC maturation and failed to detect any perceptible effect (Figure S3). We next examined the impact of Jak2 deficiency on macro- phages, another important type of professional APCs. Peritoneal exudate macrophages (PEM) were collected by peritoneal lavage as described. Western blot analysis confirmed the absence of Jak2 in PEM derived from Jak22/2 mice (data not shown). The cells were then subjected to flow cytometry analysis as above. To our surprise, we failed to observe a significant difference for the number of macrophages between Jak22/2 and control mice, and Western analysis also confirmed Jak2 deficiency in PEM derived from Jak22/2 mice (data not shown). However, similar as DCs, Jak22/2 macrophages showed a less matured phenotype charac- terized by the lower percentage of cells expressing MHC-II and costimulatory molecules as compared with that of control mice (Figure S4). Jak2 Deficiency Attenuates the Capacity of DCs to Initiate Innate Immune Response Next, we examined the capacity of Jak22/2 DCs secretion of proinflammatory cytokines such as TNFa and IL-12 using the above culture supernatants. Before stimulation IL-12 was undetectable in both wild-type and Jak22/2 cultures, while low levels of TNFa were detected, but the amount in Jak22/2 cultures was 12-fold lower than that of control cultures (Figure 2A). Upon LPS stimulation BMDCs secreted copious amounts of TNFa and IL-12, and consistently, Jak22/2 BMDCs secreted much lower levels of TNFa and IL-12 as compared with that of control BMDCs (Figure 2A). However, both wild-type and Jak22/2 BMDCs showed similar capacity for secretion of IL-10 (Figure S5A).Similarly, we analyzed cytokine secretion from Jak22/2 macrophages. Supernatants harvested from above PEM cultures were examined for TNFa and IL-12 production by ELISA assay. Very surprisingly, unlike Jak22/2 DCs, macrophages deficient for Jak2 showed similar capacity to secrete TNFa and IL-12 as that of wild-type cells (Figure 2B). All together, these data suggest that loss of Jak2 only selectively impairs the capacity of DCs to initiate innate immune response. To further confirm above observations, we checked Listeria clearance in Jak22/2 mice as innate immunity especially the production of proinflammatory cytokines plays a pivotal role in the early clearance of Listeria after infection [28–31]. For this purpose, Mice were systemically infected by i.v. injection with 16106 CFU (,1 LD50) of Listeria, and organs from Jak22/2 and control mice were harvested after 2-day of infection. Dilutions of spleen and liver homogenates were plated on TSB-agar and colonies were counted after 2-day of culture, respectively. In line with above results, Jak22/2 mice showed significant lower capacity for early clearance of Listeria as manifested by 50–100 times more colonies observed in Jak22/2 mice as compared with that of control mice (Figure 2C). Jak22/2 DCs Possess Similar Capacity to Mediate Adaptive Immune Response The above results prompted us to check whether Jak2 deficiency would affect the capability of DCs to mediate adaptive immune response. We first performed allogenic MLR to examine the capacity of DCs to stimulate T cell proliferation. To this end, irradiated Jak22/2 and control BMDCs were cocultured with T cells originated from BALB/c mice, and T cell proliferation was determined by 3H thymidine incorporation. Unexpectedly, PLoS ONE | www.plosone.org 2 March 2010 | Volume 5 | Issue 3 | e9593