Immunoregulatory Mechanisms Of Pim-1Kinase In Pathogenesis Of Mouse Model Of Ulcerative Colitis

BackgroundInflammatory bowel disease (IBD) is characterized by chronic relapsing inflammatory disorders of the gastrointestinal tract. Although the etiology of IBD remains unclear, accumulating evidence has indicated that dysfunction of the mucosal immune system plays an important role in the pathogenesis of IBD, Pim-1is a serine/threonine kinase that was first discovered as a preferential proviral insertion site in Moloney Murine Leukemia Virus (MoMuLV) induced T-cell lymphomas, Pim-1kinase is involved in the control of cell growth, differentiation and apoptosis. Accumulating data support that Pim-1are essential components of an endogenous pathway that regulates T cell growth and survival. The crucial role of Pim kinase in immune cells activation and proliferation makes it an attractive target for immunomodulatory therapy. Indeed, the pharmacological inhibition of Pim kinases exhibits immunomodulatory benefits, However, it remains unclear whether pim-1kinase is involved in the pathology of ulcerative colitis and whether pim-1kinase could modulate T help cell differentiation and macrophage activation during the development of ulcerative colitis.The aims of this study were to explore the role of Pim-1kinase in the pathology of ulcerative colitis and to assess whether Pim-1kinase may act to regulate innate and adaptive immune responses,inhibiting Pim-1kinase may be of therapeutic benefit as a treatment regimen for ulcerative colitis. In vitro effects of Pim-1on LPS-induced macrophage activation as well as in vivo effects of PIM-Inhibitor (PIM-Inh) in mice with DSS-induced colitis, were explored.Colon inflammation, production of proinflammatory cytokines and NF-κB activation in colon tissues,and The expression of T cell master transcription factors were were assayed.In summary, our data may provide evidence that Pim-1may act as a central regulator in the immune response by regulates macrophages activation and Th cells differentiation.These findings may offer a promising alternative to our current approaches of managing ulcerative colitis.The first chapter:Expression of Pim-1kinases in Mice Colitis Model that Acute Colitis Induced with DSSObjective:To explore the role of Pim-1in the initiation and development of ulcerative colitis Methods:We examined Pim-1mRNA and protein expression in colonic samples by real-time quantitative reverse transcription-PCR, Western blot and Immunohistochemical Staining Methods, at0,1,4and7days after mice were induced by giving5%DSS, The correlation between the two parameters was tested using the Pearson correlation test,P<0.05was considered to be statistically significant. Result:①The animals treated with5%DSS for7days developed symptoms of acute colitis at an incidence of100%, exhibited body weight loss and showed watery or bloody diarrhea beginning on day4of the DSS administration,②Increased amounts of Pim-1were observed as soon as1day after starting DSS,the difference was not, however, significant,The Pim-1protein expression were significantly higher on day7than on day4,or on day1, Consistent with the Western blot result,Relatively high concentrations of Pim-1mRNA occurred in colon tissue at4days of DSS treatment,reaching their highest point at day7,③Pearson correlation analysis revealed a strong positive correlation between DAI and expression of Pim-1(R=0.868,P<0.01), between histological disease scores and expression of Pim-1(R=0.851, P<0.01) Conclusion:We observed that the expression of pim-1correlated with the degree of mucosal inflammation in vivo,These data suggest that pim-1kinase may be involved in the development of ulcerative colitis.The second chapter:Research on mechanisms of Pim-1kinase in modulation of T-cell differentiation and Macrophage Activation in DSS-Induced Acute Colitis.Objective:The aim of this study was to understand more about the role of Pim-1kinase in contributing to the pathology of ulcerative colitis and to assess whether blocking Pim-1kinase may be of therapeutic benefit as a treatment regimen for ulcerative colitis. Methods:Mice with acute colitis induced by5%DSS were treated with or without PIM-Inh,Body weight and colon inflammation were evaluated,and production of lymphocytes cytokines (IFN-γ,IL-4, TGF-β,IL-17) in colon tissues was determined by ELSIA.The expression of T cell master transcription factors(T-bet, ROR-yt,GATA-3,Foxp3)were measured by Real-Time qRT-PCR and Western Blot. Nuclear factorκB (NF-κB) and inducible nitric oxide synthase(iNOS) activation in colon tissues was assayed. Result:①the PIM-Inh had protective and therapeutic potentials in acute DSS colitis in vivo②treatment with PIM-Inh inhibited the production of IFN-y and IL-17in a dose-dependent manner,The production of TGF-β was higher in PIM-Inh (10mg/kg/d)treated mice as compared to either DSS or PIM-Inh (5mg/kg/d) treated mice(P<0.05), Production of IL-4in PIM-Inh treated mice was intermediate between that of control group and DSS treated mice, the difference was not, however, significant③In this study,T-bet,ROR-yt was significantly up-regulated in acute ongoing DSS colitis,In contrast,The expression of Foxp3was inhibited in DSS group mice,Administration of PIM-Inh resulted in inhibition of T-bet,ROR-yt expression in a dose-dependent manner and the induction of FOXP3expression,whereas PIM-Inh did not cause any significant change in GATA-3expression compared with DSS colitis group,④in Acute Colitis mice, pNF-κB and iNOS expression in colon tissues are significantly increased. PIM-Inh treatment significantly inhibited NF-κB activation and thus down-regulated iNOS expression in colon tissues of mice in a dose-dependent manner,compared with mice without PIM-Inh treatment.Conclusion:we found that PIM-Inh reduced the proinflammatory immune response through the inhibition of the overactivation of macrophages and the down-regulation of excessive Thl-and Th17-type immune responses, Furthermore, PIM-Inh could skew T-cell differentiation towards a Treg phenotype.These results indicate that Pim-1kinase is involved in the pathophysiology of ulcerative colitis.The third chapter:Effects of Pim-1on TLR4/NF-kBsignaling in vitroObjective:To investigate pim-1activation induced by LPS in macrophages and to explore whether PIM-Inh inhibits LPS-induced macrophage activation in vitro.Methods:①we identified the expressions of Pim-1in RAW264.7cells incubated with different doses of LPS by Western blotting and real-time PCR, respectively at different time points (12,24h),②RAW264.7were pretreated with different doses of PIM-Inh2h before addition of LPS (1ug/mL). The expression of pNF-kB P65was detected by Western blot1.5h after LPS activation. The levels of TNFa in the supernatants, collected24h after LPS stimulation, were assayed by ELISA.Result:①treatment of RAW264.7with LPS increases expressions of mRNA Pim-1in a dose-dependent manner, expressions of Pim-1was significantly increased at12hours after incubation with LPS,which was slightly higher at12hours than that detected at24hours,p<0.05,Significantly different from normol group②When the macrophages were treated with PIM-Inh,the expression of phosphorylated-NF-kB P65and TNFa production were inhibited in a dose-dependent pattern, Treatment with100umol/L PIM-Inh achieves the highest inhibitory effect on TNFa production,p<0.05,Significantly different from the group with LPS treatment. Conclusion:data suggest that treatment with LPS increases Pim-1production in a dose-dependent manner,PIM-Inh administration inhibited NF-κB activation,which subsequently resulted in a down-regulation of iNOS expression in macrophages and an inhibition of TNF production.