adoptive transfer of normal DCs showed restored susceptibility to LPS-induced septic shock (Figure 4B). These results demonstrate that DCs with reduced capacity for initiation of innate immune response rendered Jak22/2 mice more resistant to LPS-induced septic shock. In consistent with this conclusion, ELISA analysis of serum cytokines revealed significant lower levels of proinflamma- tory cytokines such as TNFa and IL-12 in Jak22/2 mice after 12 h of nonlethal dose of LPS challenge (Figure 4C), while the production of IL-10 was similar between Jak22/2 and control mice (Figure S5). Of note, unlike TNFa and IL-12, we noticed much higher levels of serum IL-2 in Jak22/2 mice after LPS challenge as compared with control mice, although the absolute IL-2 level was relatively low in both strains of mice (Figure 4C). Recent studies demonstrated that microbial stimuli (not proinflammatory cyto- kines) are able to induce IL-2 secretion by DCs [42–44], while we failed to detect IL-2 secretion in DCs following LPS stimulation which could be due to the differences of our culture system or lower sensitivity of our detection system. Given the important regulatory role exerted by IL-2 in the immune system, it has been suggested to be tightly regulated by the STAT5-dependent negative feedback signal [45]. Therefore, the reduced Stat5 activity probably contributes to the observed higher levels of serum IL-2 in Jak22/2 mice. Studies have shown that DC-derived IL-2 plays a pivotal role in their capability to prime alloreactive T cells [42,46], and therefore, enhanced IL-2 production in Jak22/2 DCs probably contributes to their unchanged potency to stimulate allogenic or antigen specific T cell activation as well. Given the important role of Jak2 in cell signaling, we anticipate that Jak2 deficiency would affect the activation of multiple signaling pathways of DCs. Unexpectedly, we only detected altered activity for Stat3, 4, 5 and 6 (Figure 5). It seems that loss of Jak2 in DCs has negligible impact on Stat1 and NFkB signaling, which could be due to the compensated effect from other redundant Jak kinases. For example, other than Jak2, Stat1 can be activated by either Jak1 or Tyk2 [47]. Using Stat5-Tg mice, we have demonstrated evidence indicating that Jak2/STAT5 is indispensable for DC development and maturation in response to inflammatory stimulation. In opposite to Jak22/2 mice, Stat5- Tg mice displayed higher DC yield, larger spleen size and higher potency in response to LPS stimulation (Figure 6). However, Jak2/ STAT5 only partly regulates DCs secretion of IL-12, indicating additional signaling pathway(s) that are also implicated in the regulation of DCs secretion of proinflammatory cytokines. To examine the possible implication of additional signaling pathway(s), we performed studies in Stat62/2 mice and demon- strated a pivotal role for Jak2/STAT6 signaling in the regulation of cytokine secretion in DCs. We noticed a significant impaired production of TNFa and IL-12 in Stat62/2 DCs following LPS stimulation, although Stat62/2 DCs secreted relatively higher baseline of TNFa (Figure 6E). Based on the results for TNFa and IL-12 production, there is no doubt that Jak2/STAT6 signaling regulates the capacity of DCs secretion of proinflammatory cytokines. We further performed similar studies in Stat42/2 mice and DC2.4 cells transfected with a Stat3 siRNA. Surprisingly, we failed to detect any perceptible impact for STAT3 and STAT4 on the capacity of DCs for cytokine secretion (Figures 6E & F). Given the importance of STAT4 pathway in T cell polarization [48–51], Jak2/STAT4 signaling could be implicated in the regulation of DCs to mediate T cell polarization. On the other hand, STAT3 has been recently suggested to be important for DC development and differentiation [37–39], and therefore, Jak2/STAT3 could work together with Jak2/STAT5 signaling to implicate in DC development. Jak2 Modulates Innate Immunity Extensive studies have been conducted for decades to demonstrate the function of Jak2 in immune cells including DCs by using different Jak2 inhibitors. Results derived from these studies, however, remained controversial, because of non-specific- ity of each Jak2 inhibitor. To our knowledge, the present report is the first study carried out in adult mice deficient for Jak2. Due to the complexity of Jak2 in the regulation of immune responses, we have limited our current study to APCs, particularly, to DC- mediated innate immune response. The implication of Jak2 in the regulation of adaptive immunity is yet to be determined. We are also aware that Jak2 is essential for the development and functionality of other immune cells such as T and B cells. Future studies focused on demonstrating these questions would shed light to the development of novel therapeutic approaches to diseases such as cancer and autoimmune disorders [52]. In conclusion, we have demonstrated the function of Jak2 in the regulation of DC development, maturation and cytokine secretion. Our data strongly support that Jak2 only selectively regulates the capacity of DCs to initiate innate immune response, while their capacity to stimulate T cell activation is not impacted. As a result, mice deficient for Jak2 show a DC-dependent resistance to lethal dose of LPS-induced septic shock, a deadly disorder caused by the excessive innate immune response. Together, our data suggest that Jak2 could be a unique therapeutic target for the intervention and treatment of clinical septic shock. Materials and Methods Mice Stat42/2 (H-2d), Stat62/2 (H-2d) and BALB/cJ (H-2d) mice were purchased from the Jackson’s Laboratory. Cre-ERT2 transgenic mice under the control of human ubiquitin C promoter (H-2b), Jak2fl/fl mice (H-2b), C57/BL6 (H-2b) mice, Stat5b transgenic (Stat5-Tg) mice (H-2d) and OT-1 transgenic mice (H-2b) were bred and housed in the SPF facility of Medical College of Georgia (MCG). All studies were carried out in compliance with MCG and Tongji Medical College Animal Care and Use Committee guidelines. Induction of Jak2 Deficiency Cre+/+Jak2fl/fl mice were intraperitoneally (i.p.) injected with tamoxifen (25 mg/kg body weight) for 5 consecutive days. Tamoxifen was freshly dissolved in corn oil supplemented with 10% ethanol before injection. Cre+/+Jak2fl/fl littermates injected with equal volume of corn oil with 10% ethanol were served as controls. Generation of BMDCs Bone marrow cells were flushed from femurs and tibias, and 56105/ml cells were plated in 150-mm Petri dishes and cultured with RPMI-1640 supplemented with 10% FCS, GM-CSF (10 ng/ ml) and IL-4 (5 ng/ml) (PeproTech, Rocky Hill, NJ). Suspended cells were discarded at day-4 and the cultures were replaced with fresh media at day-7. BMDCs were then stimulated with LPS (0.5 mg/ml, Sigma, St Louis, MO) overnight and harvested on day-10 for experimental purpose [53]. Isolation of PEM PEM were collected by peritoneal lavage as reported with minor modifications [54]. Briefly, mice were i.p. injected with 5 ml sterilized cold RPMI 1640. PEM were harvested by washing peritoneal lavage twice with 5 ml cold RPMI 1640. After lysis of red blood cells, the cells were incubated for 3 h at 37uC in 35615 mm culture dishes. Non-adherent cells were removed by PLoS ONE | www.plosone.org 9 March 2010 | Volume 5 | Issue 3 | e9593