Loss Of Jak2 Selectively Suppresses DC-mediated Innate Immunity And Protects LPS-induced Septic Shock

1.IntroductionAntigen presenting cells(APCs) initiate adaptive immunity by uptaking antigens anddegrading them into peptides suitable for presentation by the major histocompatibilitycomplex(MHC).They provide both first and second activation signals to T and B cells byexpressing peptide-MHC complex and costimulatory molecules on the surface.They alsoproduce cytokines to help lymphocyte activation.Other than their recognized role inmediating adaptive immunity,APCs also play a vital role in innate immunity.Dendritic cells(DCs) are the most potent APCs known today.They serve as a key component of innateimmunity and bridge innate and adaptive immune responses to bacteria and other pathogens.For example,the pathogen associated molecular patterns(PAMPs) and damage associatedmolecular patterns(DAMPs) can be sensed by pathogen recognition receptors(PRRs)expressed on their surface,and thereby,initiating a serial prompt responses includingendocytosis and cytokine secretion.Immune disorders are considered to be a result ofimbalanced immune defense as manifested by either excessive or defective response.Forexample,appropriate innate immune response would protect host from infections,whileexcessive response would result in inflammatory disorders such as sepsis.Septic shock is severe sepsis with organ hypoperfusion and hypotension that are poorlyresponsive to initial fluid resuscitation.The mortality rate in patients with septic shock rangesfrom 20 to 80%,and in the USA alone it is estimated that more than 100,000 deaths occureach year.Therefore,septic shock has been accounted for the most common cause of death in the intensive care unit.Appropriate innate response followed by normal adaptive defensewould be pivotal for the clearance of microbes invaded into the hosts.There is compellingevidence that deaths resulted from septic shock is due to inappropriate increase in innateimmune response to bacterial infection,while maladaptive immunity is responsible for thepost-septic deaths.Given the importance of DCs in the vanguard of innate immune response.research into the development of new septic therapeutics has focused more and more on theircrucial role in orchestrating the initial host response to infection,but the advancement hasbeen painfully slow and fraught with difficulties.The ideal therapeutic target for septic shockwould be directed to selectively modulate innate immune response without affecting adaptivedefense.Nevertheless,it would be a formidable challenge to characterize such a target amongall immune regulatory molecules within the genome.Jak2 is one of the four janus kinase members identified in mammals.It acts as a crucialcomponent of signal pathways that are involved in cellular survival,proliferation,differentiation and apoptosis.Particularly,it has been suggested to play a pivotal role in theregulation of DC development and functionality.However,most of those studies wereconducted by using Jak2 inhibitors such as AG490 as mice deficient for Jak2 were embryoniclethal.In the present study,we generated an inducible Jak2 knockout model by crossingJak2^{fl fl} mice with tamoxifen-inducible Cre transgenic mice.It is worth noting that DCsdeficient for Jak2 show reduced MHC-â…¡and costimulatory molecule expressions along withsuppressed pro-inflammatory cytokine secretion upon LPS stimulation.However.theircapacity to initiate adaptive immune response is not affected.As a result,Jak2 mice areremarkably resistant to LPS-induced septic shock.Together,our results suggest that Jak2could be a unique therapeutic target for clinical septic shock.2.Loss of Jak2 function impairs DC developmentWe first crossed tamoxifen-inducible Cre transgenic mice with Jak2^{fl fl} mice,and theresulting F1 Cre Jak2^fl mice were then used to generate B6.Cre Jak2^{fl fl} mice.Forinduction of Jak2 deficiency.8wk-old male B6.Cre Jak2^{fl fl} mice were i.p.injected withtamoxifen(1.0 mg/40g body weight) for five consecutive days,while male littermatesadministered with carrier solution(10% ethanol in corn oil) were used as controls.To confirm Jak2 deficiency,the mice were sacrificed after 2wk of last injection.BMDCs and splenocyteswere prepared and subjected to Western blot analysis of Jak2 expression.As a result,BMDCsoriginated from control mice showed high levels of Jak2 expression,while Jak2 wasundetectable in BMDCs of tamoxifen induced mice.Similar results were also observed insplenocytes.Collectively,these results indicate that B6.Cre^+/+Jak2^fl/fl mice lost Jak2expression after tamoxifen induction.Since previous studies suggested a critical role for Jak2 in DC differentiation,we firstsought to address the impact of Jak2 deficiency on DC development.To this end,10 x 10⁶bone marrow cells originated from Jak2^-/- and control littermates were induced with GM-CSFand IL-4 to generate BMDCs,respectively.Jak2 deficiency significantly reduced DCproduction,a 1.3-fold decrease for BMDC yield was observed in Jak2^-/- mice as compared tothat of control mice.In an effort to further check the differences of splenic DCs,we noticedthat the size for spleens of Jak2^-/- mice was significantly smaller than that of controllittermates(52±8mg vs.157±10mg,P＜0.001).Consistent with this observation,totalsplenocytes in Jak2^-/- mice were significantly less than that of control littermates.Next,weexamined the differences of splenic DCs between Jak2^-/- and control mice.To our surprise,inaddition to reduced number of total splenic DCs,the percentage of DCs in total spenocyteshas also significantly decreased.In contrast,we failed to detect a significant alteration for thepercentage of CD4 and CD8 T cells in total spenocytes.Together,these data suggest that lossof Jak2 function significantly impaired DC development.3.Jak2 deficiency inhibits DC maturationand cytokine secretionNext,we examined DC phenotypic differences between Jak2^-/- and control mice.Similarly,BMDCs were generated from bone marrow cells with GM-CSF and IL-4 as above,and day-9 of DC cultures were stimulated with LPS(500 ng/ml,Sigma,St Louis,MO)overnight and harvested on day 10 for flow cytometric analysis of surface marker expressions.It was found that in our culture condition both Jak2^-/- and control bone marrow cells produced＞85% purity of DCs.CD11c⁺ cells were then gated for the analysis of surface MHC-â…¡,CD80,CD86,and CD54 expressions.One noteworthy and striking observation is that loss of Jak2function rendered DCs significant less potent in response to maturation stimulation. Significant lower expressions for MHC-â…¡and co-stimulatory molecules such as CD80.86and 54 were noticed in Jak2^-/- BMDCs in unstimulated condition as compared to that ofcontrol cells.Upon LPS stimulation,majority of control BMDCs became matured ascharacterized by high levels of MHC-â…¡,CD80,86 and 54 expressions.In sharp contrast,onlya small proportion of Jak2^- BMDCs became matured.To further confirm this observation,weanalyzed splenic DCs.Single splenic cells were pooled from 3 mice and then co-stained forCD11c and MHC-â…¡or one of the above indicated co-stimulatory molecules.Similar asBMDCs,Jak2^- splenic DCs also displayed significant lower expression of MHC-â…¡andco-stimulatory molecules.Finally,we examined the phenotypic differences of macrophages,another important type of professional APCs.Peritoneal exudate macrophages(PEM) werecollected by peritoneal lavage as described.After removing the contaminated non-adherentcells,the adherent macrophages were subjected to flow cytometric analysis as above.Similarly.Jak2^- macrophages showed a significant less matured phenotype characterized bymuch lower expressions for MHC-â…¡and co-stimulatory molecules,as compared to that ofmacrophage originated from control littermates.To further address the phenotypic differences between Jak2^{- -} and control DCs,weanalyzed the profile of cytokine secretion.The above BMDC culture supernatants werecollected and subjected to ELISA analysis of TNFa.IL-2,IL-6,IL-10.and IL-12 secretion.Before stimulation.IL-2.IL-6,IL-10,and IL-12 were undetectable in the supernatantsoriginated from both Jak2^{- -} and control BMDCs cultures,while low levels of TNFa werepresent in both cultures,but the amount of TNFa resulted from Jak2^-/- cultures was 5-foldlower than that of control cultures.Nevertheless,upon LPS stimulation BMDCs secretedcopious amounts of proinflammatory cytokines such as TNFa,IL-6,and IL-12.However,itwas noticed that Jak2^{- -} BMDCs secreted significant less amount of TNFa,IL-6 and IL-12 ascompared to that of control BMDCs.In contrast,LPS failed to stimulate both Jak2^{- -} andcontrol BMDCs secretion of IL-2 and IL-10.All of these data demonstrate that loss of Jak2impairs the capacity of DCs to initiate innate immune response as manifested by distortedmaturation and proinflammatory cytokine secretion upon stimulation. 4.Jak2 deficiency does not affect the capacity of DCs to mediate adaptiveimmunityThe above results prompted us to check whether Jak2 deficiency would affect thecapability of DCs to mediate adaptive immune response.We first performed allogenic MLRto examine the capacity of DCs to stimulate T cell proliferation.To this end.irradiated Jak2^{- -}and control BMDCs were cocultured with T cells originated from BALB/c mice,and T cellproliferation was determined by H thymidine incorporation.Unexpectedly,Jak2^- BMDCswere also as potent as control BMDCs to stimulate alloreactive BALB/c T cell proliferation.To confirm this observation,we performed similar studies using splenic DCs.Splenocytespooled from 5 Jak2^{- -} or control mice were used to enrich splenic DCs using a mouse DCnegative selection enrichment kit as described.Irradiated splenic DCs(2000 rad) were thencocultured with BALB/c T cells as above,respectively.Consistently,splenic DCs were potentto stimulate alloreactive T cell activation.We next examined the capacity of Jak2^{- -} DCs toinitiate antigen-specific T cell activation.For this purpose,irradiated Jakl^{- -} BMDCs orsplenic DCs were first pulsed with 1μM OVA peptide,and then cocultured with OT-1 T cellsin the presence of ³H thymidine.Similar as above,both Jak2^- BMDCs and splenic DCs werepotent to stimulate antigen-specific T cell activation,and we failed to detect a significantdifference for T cell proliferation between Jak2^{- -} and control DCs.Next,we analyzed cytokine profiles of each T cell subset after Jak2^{- -} DC stimulation.Weselectively analyzed the production of IFN-γ(Th1),IL-10(Th2) and IL-17(Th17) by ELISAanalysis of above collected culture supernatants.Once again,Jak2^{- -} DCs were potent tostimulate T cells secretion of IFN-γ,IL-10.and IL-17.More importantly,we failed to detect asignificant difference between Jak2^{- -} and control DC stimulated T cells for secretion of IFN-γ.IL-10 and IL-17.Taken all of these results together,Jak2 deficiency only selectively inhibitsthe potency of DC initiated innate immune response,while the capacity of DCs to mediateadaptive immune response is not affected.5.Mice deficient for Jak2 are protected from lethal septic shockSepsis is a classic animal model of inflammation disease.Innate immunity plays themajor role in initiation and progression phase of sepsis.Under some circumstance,many components of innate immune response can result in cell and tissue damage and thus causesepsis.To confirm the role of Jak2 in innate immunity,sepsis model was selected for the invivo study.Briefly,50 mg/kg body weight LPS was i.p.injected into Jak2-/- and Jak2+/+mice 4 weeks after tamoxifen injection.The wild type mice are much weaker compared toJak-/- mice after LPS injection.The septic shock survival rate is 27.27%(Jak2+/+ mice) and76.92%(Jak2-/- mice) respectively(P＜0.05).Only 3 out of 13 Jak2-/- mice died of sepsis,while 8 out of 11 Jak2+/+ mice died.Pro-inflammatory cytokines(especially TNFα) have been illustrated to be critical for themortality of septic shock.We employed an unlethal sepsis model to reveal the effect of Jak2defection on serum pro-inflammatory cytokines level elevation.Jak2-/- mice and wild typelittermates were i.p.injected with unlethal dose of LPS.Serum was then collected and pooledfrom 3 mice for serum pro-inflammatory cytokines detection after 12 hours of induction.AsTNFαand IL-6 were the most frequently detected circulating cytokines in clinic,severalserum cytokines level including TNFαand IL-6 were detected by ELISA assay.As a result,the serum TNFα.IL-6 and IL-12 level of Jak2-/- mice is much lower than that of wild typemice.Serum IL-2 level was elevated in Jak2 mice although the IL-2 levels of both Jak2-/- andwild type mice were not very high.DC lost is the main cause for the immune suppression post-sepsis.To illustrate the effectof Jak2 defection on DC death,Jak2-/- and +/+ BMDCs were treated with 10μg/ml LPS andstained with Annexin-V and PI after 96 hours.Interestingly,there's no significant differencebetween knockout and wild type group.To further confirm this result.Jak2-/- and +/+ micewere i.p.injected with 150μg LPS/mouse and sacrificed 20 hours after injection,spleen cellswere prepared and subjected to apoptosis assay.Similar result was obtained.6.Jak2 defection leads to suppressed STAT4,5,and 6 activationTo investigate the underlying mechanism of the phenotype and cytokine profile change inJak2 deficient DC,we checked some possible downstream molecules of Jak2.Several STATmolecules can be activated by Jak2 and thus initiate different downstream effect.Todetermine which downstream molecules were activated by Jak2 during LPS stimulation.2×10⁶/ml Jak2-/- or +/+ BMDCs were stimulated with 1μg/ml LPS for 30 min and then lysed in 100ul RIPA lysis buffer completed with 1 x protease inhibitor cocktail and 1×phosphataseinhibitor cocktail.Cell lysates were then subjected to western blot detection.As a resultphosphorylated form of STAT4,STAT5,and STAT6 decreased in Jak2-/- BMDCs lysate.There is no significant effect on STAT1 activation and no detectable activated STAT3.SinceNFkB signal pathway is the major pathway in LPS signaling,we checked phosphorylatedphosphorylated IkBαand total NFkB level.No significant difference of phosphorylated lkBαand total NFkB level was detected between Jak2-/- and +/+ group.7.Jak2-STAT5 signaling promotes DC development and maturationTo investigate role of STAT5 signaling in the impact of innate immune by Jak2 defection,we used a STAT5 over-expression mouse model for the study.STAT5 transgenic mice andcontrol mice were sacrificed for spleen cells isolation and BMDCs induction.Spleen size ofSTAT5 transgenic positive mouse is obviously larger than that of transgenic negativelittermate and total spleen cell number isolated from transgenic mice is also larger than that ofwild type mice.Then the single spleen cells were subjected to FACS for DC percentageanalysis.DC population in spleen was raised by STAT5 over-expression.DCs were inducedfrom bone marrow cells of STAT5 transgenic mice and wild type mice with GM-CSF andIL-4 and harvested at day-9.STATS over-expression increased BMDC production as expected.Half of the harvested cells were cultured for 24 hours without treatment.Another half wassubjected to LPS stimulation for 24 hours.Culture supernatant was collected forpro-inflammatory cytokines(TNFα.IL-2.IL-6 and IL-12) ELISA assay and cells weresubjected to FACS analysis.As shown in fig.6F & G MHC-II+ DC population.CD80+ DCpopulation.CD86+ DC population and CD54+ population is higher in STAT5 over-expressedBMDCs than in wild type BMDCs under both the circumstances of untreated and LPS-treated.TNFαand IL-6 secretion was not changed upon STAT5 over-expression under bothun-stimulated and LPS-stimulated condition.Yet IL-12 secretion was elevated by STAT5over-expression after LPS stimulation.So Jak2 deficiency suppresses DC development andmaturation via inhibition of Jak2-STAT5 signaling. 8.Jak2-STAT6 signaling disruption suppresses proinflammatory cytokinessecretion stimulated by LPSSince STAT5 signaling only impact IL-12 secretion upon LPS stimulation,there must beanother signal pathway implicated in pro-inflammatory cytokines secretion after LPSstimulation.So we next examined which downstream signaling molecule is involved in theinfluenced pro-inflammatory cytokines secretion by Jak2 deficiency after LPS stimulation.2.5 X 10⁶/ml BMDCs derived from STAT4-/-,STAT6-/-,and wild type mice were stimulatedwith 0.5μg/ml LPS for 24 hours at day-9 and culture supernatant was collected forpro-inflammatory cytokines ELISA assay.Surprisingly,STAT6 deficiency elevatespro-inflammatory cytokines secretion under un-stimulated condition.Both TNFαand IL-6secretion by STAT6-/- BMDCs were significantly higher than wild type control underun-treated condition.1L-12 was undetectable without treatment in all groups.As expected,STAT6 deficiency suppresses responsiveness of BMDCs to LPS.After 24 hours LPSstimulation,both TNFαand IL-12 secretion by STAT6-/- BMDCs were significantly lowerthan wild type control.IL-6 secretion by STAT6-/- BMDCs was a little bit lower but withoutsignificance(p=0.07).Yet concerning the baseline level before treatment,the elevation of allpro-inflammatory cytokines was much lower in STAT6-/- group than the other groups.AfterLPS stimulation,TNFαand IL-6 elevated 40 and 244 fold respectively in supernatant of wildtype BMDCs.There is only 6.5 and 12.9 fold elevation in STAT6-/- supernatant respectively.STAT4 deficiency did not affect pro-inflammatory cytokines secretion significantly althoughTNFa elevation is lower than wild type control due to the higher TNFa secretion baseline ofSTAT4-/- BMDCs.All 3 pro-inflammatory cytokines secretion by STAT4-/- BMDCs is at thesame level as wild type control after LPS stimulation.The above results indicate that Jak2defection inhibit pro-inflammatory cytokines secretion via Jak2-STAT6 pathway.9.Conclusion1.We demonstrated strong evidence for Jak2 in the DC-mediated innate immunity.2.Jak2 signaling defect prevents DC development.3.Loss of Jak2 attenuates DC maturation and cytokine secretion.4.Initiation of adaptive immune response is not affected by Jak2 defect in DC. 5.Jak2 knockout mice are resistant to lethal dose of LPS-induced septic shock.6.The protective effect of Jak2 deficiency is associated with reduced proinflammatorycytokine secretion by DC.7.Jak2 defect impairs the activation of STAT4,5,6.8.Jak2/STAT5 signaling is indispensable for DC development and maturation9.Jak2/STAT6 signaling regulates the capacity of DCs for cytokine secretion.In summary,Jak2 regulates DC development and maturation via STAT5 pathway andmodulates proinflammatory cytokines secretion through STAT6 signaling.Jak2 deficiency inDC suppresses innate immunity without impact on adaptive immunity initiation and canserves as a wonderful therapeutic approach for septic shock.