The Expression Of Interleukin-22,25 In Periodontitis And The Construction Of Integrin α5β1

Periodontitis is a disease characterized by a chronic bacterial infection of the periodontal supporting tissues.The interaction between microorganisms and the host defense is involved in the pathological process of periodontitis.This topic started from the distribution and temporal expression of a newly discovered cytokine,interleukin-22(IL-22),and its receptors in the rat periodontitis model.Then,we confirmed that another newly discovered interleukin,interleukin-2(IL-25)can induce matrix metalloproteinase 2(metalloproteinase-2,MMP-2)and matrix metalloproteinase 9(metalloproteinase-9,MMP-9)expression,increase cell migration and expression of fibros actin(F-actin)in human periodontal ligament cells(PDLCs).By using associated inhibitor,the effect of IL-25 was found to be involved in extracellular-signal-regulated kinase(ERK)and p38 fissure activated protein kinase(p38MAPK)signal pathways.Finally,in order to interpret the relationship between periodontitis and integrin α5β1 at a more microscopic level,we constructed and screened a functional integrin β1 biosensor.Hopefully,we would explore the relationship between α5β1 activation and periodontitis from sub-protein levels in subsequent experiments.In summary,we selected three distinct proteins as interests,tried to explore the mechanisms of host factors behind periodontitis.Part I Temporal expression of interleukin-22,interleukin-22 receptor 1 and interleukin-22-binding protein during experimental periodontitis in ratsObjectives: The purpose of this study was to investigate histological changes and the levels of interleukin-22(IL-22),interleukin-22 receptor1(IL-22R1)as well as interleukin-22 binding protein(IL-22BP)in experimental periodontitis.Material and Methods: Sixty male 8-week-old Sprague-Dawley rats were randomly allocated to 6 groups.Experimental periodontitis was established and the rats were sacrificed on day 0(baseline),3,5,7,11,and 15.Histopathologic changes were detected by hematoxylin and eosin(H&E)staining while alveolar bone loss was determined by micro-computed tomography radiographic(micro-CT)examinations.Tartrate-resistant acid phosphatase(TRAP)was used to investigate osteoclast activity.Real-time quantitative polymerase chain reaction(q PCR)and Immunohistochemistry were used to investigate the expression and location of IL-22,IL-22R1,and IL-22 BP in gingival tissues.Results: 1)H&E staining showed that there was an increasingly severe destruction of the epithelial layer occurred between days 3 and 7,and the hyperplasia of pocket epithelium and the formation of periodontal pockets could be detected from day 11 to 15.2)Micro-CT examinations indicated there was an exponential increase in alveolar bone loss from day 3 to day 11(p<0.01).TRAP staining showed the number of osteoclasts peaked at day 3.3)IL-22 BP expressed strongly under steady-states in epithelial cells.IL-22 positive cells could be clearly observed both in the epithelial layer and around the lamina propria while the IL-22R1 was mainly localized in the epithelium layer of the damage period.4)q PCR revealed up-regulation of IL-22 and IL-22R1 as well as down-regulation of IL-22 BP in the destructive gingival tissues.Conclusions: This study shows the expression and localization of IL-22,IL-22R1,and IL-22 BP during the development of periodontitis.The results suggest that the upregulation of IL-22,IL-22R1 and down-regulation of IL-22 BP might play a key role in the hyperplasia of pocket epithelium and the formation of periodontal pockets.Part II Interleukin-25 regulates matrix metalloproteinase-2 and-9 expression in human periodontal ligament cells through extracellular-signal-regulated kinase(ERK)and p38 mitogen-activated protein kinase(p38MAPK)pathwaysObjective: Interleukin-25(IL-25)has been implicated in various immune disease and inflammatory pathology.We aimed to investigate the effects of IL-25 on the expression of metalloproteinase-2(MMP-2),-8,-9 in periodontal ligament cells(PDLCs),cell migration,fibros actin(F-actin),and to explore the effects of extracellular-signalregulated kinase(ERK),c-Jun N-terminal kinase(JNK)and p38 mitogen-activated protein kinase(p38MAPK)signaling pathways during periodontitis.Methods: To evaluate the expression of MMP-2,-8,-9 and F-actin,PDLCs were treated by various doses of IL-25(0,20,50,100,and 500 ng/ml).Protein expression of extracellular metalloproteinase inducer(EMMPRIN)was also evaluated by western blot.Cell scratches experiment was preformed to test the cell migration ability.ERK,JNK,p38 MAPK pathways and related expression of P-ERK and P-p38 MAPK were examined after treating with different doses of IL-25 and after treating with inhibitors of ERK and p38 MAPK respectively.Immunofluorescence(IF)of MMP-2,-9,and Factin were also evaluated after inhibitor treatment.Results: IL-25 increased the protein expression of MMP-2 and MMP-9.MMP-8 and EMMPRIN expressions were not regulated by IL-25 in PDLCs.F-actin was strongly expressed in IL-25-treated group,and the location extended from the central part to the whole cell,compared with control group.p38 MAPK and ERK pathways were involved in IL-25 mediated MMP-2,-9,F-actin expressions and cell migration.SB203580 and U0126 blocked the effects of IL-25 through the inhibition of ERK,p38 MAPK,P-ERK,P-p38 MAPK.Conclusion: The data indicated that IL-25 could regulate cell migration,MMP-2 and-9 expression,but not MMP-8 and EMMPRIN expression in PDLCs.Moreover,ERK and p38 MAPK pathways might be involved in the regulatory effect of IL-25 on PDLCs.Part III Construction,selection and functional analysis of fluorescence resonance energy transfer-based integrin β1 tension sensorsObjectives: The aim of this program is to create and find functional integrin β1 tension sensors.In the future,we would like to apply the functional tension sensor to research such as the pathogenesis of periodontitis.Material and Methods: After analyzing the sequence of integrin β1 cytoplasmic tail,10 positions outside functional domain were chosen for sensor insertion,each construct was named based on its amino acid sequence.β1-798 was used as internal control.He La cells were transfected with all integrin constructs using Lipofectamine 3000 for preliminary selection.Construct expression and morphological changes in focal adhesions(FAs)were investigated.Based on the results of this experiment,β1-767 and β1-765 were chosen for further analysis.Fluorescence resonance energy transfer(FRET)imaging and overlap percentage analysis in β1-null GD25 cells.An activation-specific anti-β1 antibody 9EG7 was utilized to identify the state of recombinant integrin β1 in GD25 cells.Results: 1)Based on the results of first round selection,β1-767 and β1-765 were selected because they had high expression and did not exert an obvious effect on focal adhesion properties,comparing to β1-798.2)FRET ratio was quantified in each group,only β1-767 displayed a significant decrease in FRET efficiency(p <0.0001,compared with β1-798).3)The percentage overlap between paxillin and monomeric teal fluorescent protein(m Teal FP)was compared.β1-767,β1-765 as well as β1-798 had colocalization with FAs,no difference was detected when comparing to β1-798 in overlap area measurement.4)The state of integrin β1 was tested using β1-activating antibody.GD25 cell line did not express endogenous integrin β1,but after electroporation with β1-767,β1-765 and β1-798,GD25 cell line recovered its functional,active β1 expression.Conclusion: we successfully constructed integrin β1 containing tension sensors,screened out a functional integrin β1-767 which acts well as both tension sensor and β1 integrin and does not affect the normal biological activities of cells.In future experiments,we will use β1-767 as a tool to explore the role of intracellular tensionregulated integrin β1 activation in the development of periodontitis.