| Objectives:Rheumatoid arthritis(RA)is a systemic autoimmune disease characterized by chronic joint inflammation.Although its pathogenesis is related to many factors such as genetics,environment etc.,recent studies have shown that the imbalance of immune cell function especially the imbalance of Th17 and Treg has become one of the causes of RA,that manifested by an increase in the number of Th17 cells,an increase of proinflammatory cytokines and a decrease in the function of Treg cells.The SNS is activated in ADs and acts on humoral and cellular immunity primarily through β2-AR.The adrenergic p2 receptor(β2-AR)is one of the typical G-protein coupled receptors(GPCRs).Upon initial stimulation,β2AR activates the Gs protein to increase adenylyl cyclasesignaling and enhance PKA activity to propagate the cellular response.Mechanistically,β2-AR signaling was identified to occur vectorially through heterotrimeric G proteins that are negatively regulated by GRK and arrestin effectors.In addition,β-arrestin2 also can activate other signaling molecules such as ERK etc.to amplify receptor response.Previous studies in our laboratory have shown norepinephrine inhibits Th17 cells via β2-AR signaling in a mouse model of rheumatoid arthritis.However,it is unclear how the β2-AR receptor signal regulates the function of Treg cells of CIA.To this end,we first observed the effect ofβ2-AR activation on the function of Treg cells in CIA mice,and explored its mechanism by investigating its signaling pathway.Then Treg cells with enhanced function were inputted to CIA mice and the effects of joint inflammation,bone destruction,and Th17/Treg imbalance in CIA mice were observed.It is hoped that through our research,we can better understand the regulation of P2-AR on immune cells under CIA conditions,and then further open up new ideas for the development of targeted drugs for RA.Methods:1.Preparation of CIA mouse model:DBA/1 mice(Male,6-8 weeks)were immunized by chicken type Ⅱ collagen.Mice were observed for clinical score of limbs.On day 41 after immunization,mice were sacrificed and the thickness of hind paws and ankle joints were measured,the change of histopathological was observed by HE stain and the quantity of anti-CII IgG antibody in serum was detected by ELISA.2.Signal molecules in Treg cells are detected by Western-blot or ELISA:2.1 On the 41st day after the initial immunization,Treg cells were isolated from the mouse spleen.There are 6 groups in this experiment:1.Intact Treg cells;2.Intact Treg cells+Terb(10-5M);3.Intact Treg cells.+Terb(10-4M);4.CIA Treg cells;5.CIA Treg cells+Terb(10-5M);6.CIA Treg cells+Terb(10-4M).The expressions of β2-AR,PKA,β-arrestin2 and pERK1/2 were detected by Western-blot,and the cAMP content and PKA activity were detected by ELISA.2.2 In order to observe the crosstalk between β2-AR-cAMP-PKA signaling pathway and β2-AR-β-arrestin2-ERK1/2 signaling pathway in Treg cells of CIA mice:On the 41st day after the initial immunization,Treg cells were isolated from the spleen of mice.The experimental groups were:1.Intact Treg cells;2.CIA Treg cells;3.CIA Treg cells+Terb(10-4M)4,CIA Treg cells+H-89(10-5M)+Terb(10-4M);5,CIA Treg cells+β-arrestin2-shRNA+Terb(10-4M)6,CIA Treg cells+U0126(1μM)+Terb(10-4M).The cAMP content and PKA activity of Treg cells were detected by ELISA.Western-blot assay was used to detect the expression levels of P-arrestin2,pERK1/2 and ERK1/2 in Treg cells.3.The production of IL-10 and TGF-β of Treg cells were detected by Real-time PCR and ELISA.The proliferation of Teff cells was evaluated by CFSE assay.3.1 In order to observe the effect of β2-AR-cAMP-PKA signaling pathway on Treg cell function in CIA mice:On the 41st day after the initial immunization,Treg cells of intact mice and CIA mice were isolated and divided into 6 groups:1.Intact Treg cells;2.CIA Treg cells;3.CIA Treg cells+Terb(10-5M)4,CIA Treg cells+Terb(10-4M);5.CIA Treg cells+PKA inhibitor H-89(10-6M)+Terb(10-4M);6.CIA Treg cells+PKA inhibitor H-89(10-5M)+Terb(10-4M).mRNA levels of IL-10 and TGF-β were detected by real-time PCR and the concentration of IL-10 and TGF-β in the cell culture supernatant was detected by ELISA.In addition,in order to detect the change of the ability of Treg cells inhibiting Teff cells,the Treg cells and Teff cells were also isolated from spleen of mice,and The Teff cells were stimulated with CD3/CD28 and stained with CFSE.Treg cells were mixed with Teff cells at a ratio of 1:3 after 48 hours of incubation alone.The proliferation of Teff cells was detected by flow cytometry.The experimental groups were:1,Teff cells;2.Intact Treg cells+Teff cells;3.Intact Treg cells+Terb(10-4M)+Teff cells;4.CIA Treg cells+Teff cells;5.CIA Treg cells+Terb(10-4M)+Teff cells;6.CIA Treg cells+H-89(10-5 M)+Terb(10-4 M).3.2 In order to observe the effect of β-AR-β-arrestin2-ERK1/2 signaling pathway on Treg cell function in CIA mice:Firstly,the Treg cells isolated from the spleen of intact mice were transfected with three pairs of shRNAs and one pair of negative shRNA targeting β-arrestin2 gene of mice.A pair of ShRANs with the highest efficiency of silencing β-arrestin2 gene were screened.On the 41st day after the initial immunization,Treg cells of intact mice and CIA mice were isolated from the spleen of mice and divided into 6 groups:1.Intact Treg cells;2.CIA Treg cells;3.CIA Treg cells+Terb(10-4M);4.CIA Treg cells+P-arrestin2-shRNA-mock+Terb(10-4M);5.CIA Treg cells+β-arrestin2-shRNA+Terb(10-4M)6.CIA Treg cells+ERK inhibitor U0126(1μM)+Terb(10-4M).mRNA expressions of IL-10 and TGF-β were detected by real-time PCR,and the secretion of IL-10 and TGF-β in the cell culture supernatant were determined by ELISA.In addition,in order to detect the change of the ability of Treg cells inhibiting Teff cells,the Treg cells and Teff cells were also isolated from spleen of mice,and The Teff cells were stimulated with CD3/CD28 and stained with CFSE.Treg cells were mixed with Teff cells at a ratio of 1:3 after After 48 hours of incubation alone.The proliferation of Teff cells was observed by flow cytometry.The experimental groups were:1,Teff cells;2.Intact Treg cells+Teff cells;3.CIATreg cells+Teff cells;4.CIA Treg cells+β-arrestin2-shRNA-mock+Terb(10-4M).5.CIA Treg cells+β-arrestin2-shRNA+Terb(10-4M)6.CIA Treg cells+U0126(1μM)+Terb(10-4 M).3.3 In order to know the effect of β-arrestin2 gene overexpression on the function of CIA Treg cells:On the 41st day after the initial immunization,Treg cells were isolated from the spleen of mice and infected with Lentiviral of overexpression of P-arrestin2 gene.The experimental groups were:1.Intact Treg cells;2,CIA Treg cells;3,CIA Treg cells+Terb(10-4M);4,CIA Treg cells+β-arrestin2-negative lentiviral+Terb(10-4M)5,CIA Treg cells+β-arrestin2-lentiviral+Terb(10-4M).Real-time PCR was used to detect the expression of IL-10 and TGF-β mRNA.The secretion of IL-10 and TGF-β in cell culture supernatant was detected by ELISA.Secondly,on the 41st day of the primary immunization,the ability of Treg cells to inhibit the proliferation of Teff cells was detected.Treg cells and Teff cells were isolated and cultured as above,and the proliferation of Teff cells was detected by flow cytometry.The experimental groups were:1,Teff cells;2.Intact Treg cells+Teff cells;3.CIA Treg cells+Teff cells;4.CIA Treg cells+Terb(10-4M)+Teff cells;5.CIA Treg cells+β-arrestin2 negative lentiviral+Terb(10-4M)+Teff cells;6,CIA Treg cells+β-arrestin2 lentiviral+Terb(10-4M)+Teff cells.4.After inputting of β-arrestin2 overexpressing Treg cells to CIA mice,multiple indicators were used to detect changes in Th17/Treg imbalance and disease severity:Treg cells were isolated from the spleen of intact mice,then the cells were divided into two groups:1.Treg cells of p-arrestin 2 overexpressing;2.The same number of untreated Treg cells.On the 40th day after the initial immunization,two groups of Treg cells were adoptively transferred to CIA mice via the tail vein respectively.The experimental groups were:1.Intact mice;2.CIA mice;3,CIA mice+untreated Treg cells;4,CIA mice+β-arrestin2 gene overexpressed Treg cells.The clinical scores were observed until the 55th day after the initial immunization;the width of the ankle joint and the thickness of the rear paws of mice were measured with micrometer.The histopathological changes of the ankle joint were observed by HE staining.Anti-CII-IgG antibody level in the serum was detected by ELISA.mRNAs of IL-10、TGF-β、IL-17、IL-22、Trap、Ctsk、Ctr、MMP-9、ALP、Osteocalcin、Colla1、Colla2 in ankle joint were detected by realtime-PCR.The concentration of IL-10、TGF-β、IL-17 and IL-22 in the supemant of joint and in serum of mice were determined by ELISA.The number of Th17 and Treg cells in spleen CD4+T cells were detected by flow cytometry.Results:1.Successful preparation of CIA mouse model:Compared with intact mice,the clinical score of mice that had received CII injection began to rise on day 31 after the first immunization and reached the peak at day 35,and remained high until the last day observed in this study.The thickness of both ankle joints and rear paws were augmented and anti-CII IgG antibody level was notably elevated in CIA mice.Histopathologic observation of ankle joints of CIA mice displayed an evident inflammatory change on day 41.2.CIA enhances P2-AR-cAMP-PKA signaling pathway in Treg cells,and β2-AR agonist further promotes activation of this signaling pathway:β2-AR were expressed by Treg cells and GIA upregulates β2-AR expression.Terb further enhances β2-AR expression by Treg cells either in intact or CIA condition,while the effect of Terb is weakened under CIA conditions.The cAMP-PKA pathway is activated in Treg cells of CIA and Terb further activates this pathway.In addition,we also compared the proportion of changes of intact Treg cells and CIA Treg cells and we found the activation ofβ-AR-cAMP-PKA signaling weakened in CIA.3.P2-AR agonist enhances Treg cell function in CIA,which is partially blocked by PKA inhibitors:the production of IL-10 and TGF-β were increased in CIA Treg cells.Terb further up-regulated the production of IL-10 and TGF-β in CIA Treg cells.PKA inhibitor H-89 partially blocked the effect of Terb.In addition,the ability of Treg cells inhibiting the proliferation of Teff cells diminished in CIA;Terb enhanced the function of CIA Treg cells to inhibit the proliferation of Teff cells,and this effect of Terb was partially antagonized by H-89.4.CIA enhances the β-arrestin2-ERK1/2 signaling pathway in Treg cells,and the P2-AR agonist further promotes activation of this signaling pathway:the expression ofβ-arrestin2 and the level of pERK1/2 were increased in CIA Treg cells.Terb can further up-regulate the expression of β-arrestin2 and the level of pERK1/2 in intact and CIA Treg cells.In addition,we also compared the proportion of changes of intact Treg cells and CIA Treg cells and we found the activation of β2-AR-β-arrestin2-ERK1 signaling weakened in CIA.5.β2-AR agonist enhances Treg cell function,which is partially blocked byβ-arrestin2 gene silencing or ERK1/2 inhibitor:the productionof TGF-β and IL-10 were elevated in Treg cells of CIA.The productionof TGF-β and IL-10 of Treg cells were further elevated after β2-AR had actived by Terb β-arrestin2 gene knockdown and ERK inhibition in Treg cells significantly down-regulated the productionlevels of TGF-β and IL-10.In addition,the ability of Treg cells inhibiting the proliferation of Teff cells diminished in CIA;Terb enhanced the function of CIA Treg cells to inhibit the proliferation of Teff cells,and this effect of Terb was partially antagonized by β-arrestin2 gene knockdown or inhibition of ERK1/2.6.β2-AR agonist promotes the activation of cAMP-PKA and β-arrestin2-ERK1/2 in Treg cells of CIA,but there is an interaction between the two signaling pathways:further increased cAMP content and PKA activity in CIA Treg cells were increaseded afterβ-arrestin2 gene knockdown in CIA Treg cells,and the expressions of β-arrestin2 and pERK1/2 of Treg cells were upregulated after PKA was inhibited by H-89 in CIA Treg cells.7.Overexpression of β-arrestin2 gene in Treg cells of CIA promotes the enhancement of Treg cell function induced by β2-AR agonists:β-arrestin2 gene overexpression further up-regulate the productionof IL-10 and TGF-β in CIA Treg cells.In the other hand,β-arrestin2 gene overexpression significantly enhances the inhibition ability of CIA Treg cells to Teff cells.8.Treg cell input reduces joint inflammation,joint bone destruction,elevated serum antibody levels,and Th17/Treg imbalance in CIA mice.Transfusion of Treg cells overexpressing the β-arrestin2 gene into CIA mice further improves the above phenomenon:After inputtting of Treg cells to CIA mice,joint redness and swelling were significantly reduced,clinical score decreased,and articular inflammatory cell infiltration decreased.Moreover,after inputtting Treg cells of β-arrestin gene overexpressing to CIA mice,joint redness and swelling were further alleviated,and clinical score was further reduced.After inputtting of Treg cells to CIA mice,the expression of osteoclast-specific genes in the joints downregulated,the expression of osteoblast-specific genes were upregulated,the productionof IL-10 and TGF-β in the joint and joint tissue supernatants were upregulated,IL-17 concentration of supernanant in joint were reduced.Moreover,after inputtting of Treg cells of overexpressing the β-arrestin gene to CIA mice,the expression of osteoclast-specific genes were further downregulated,and the expression of osteoblast-specific genes were further upregulated;the productionof IL-10 and TGF-βfurther upregulated.After inputtting of Treg cells to CIA mice,the levels of anti-CII-type collagen antibodies in serum decreased,the concentrations of TGF-β and IL-10 in serum increased,the concentration of IL-17 in serum decreased,and after inputtting of Treg cells of overexpressing β-arrestin gene to CIA mice,the concentration of IL-17 in serum further decreased.After inputtting of Treg cells to CIA mice,the number of Treg(CD25+FOXP3+)in CD4+T cells of spleens increased,after inputtting of Treg cells of P-arrestin gene overexpressing,the number of Tregs(CD25+FOXP3+)further increased.Conclution:1.32-AR-cAMP-PKA signaling pathway of the ankle joint attenuate in the RA mouse model.2.The β2-AR-cAMP-PKA signaling pathway and the β-AR-β-arrestin2-ERKl/2 signaling pathway are activated in Treg cells of RA model,and the β2-AR agonist further activate these two pathways.3.After β2-AR activation,the function of Treg cells enhances in RA model mice which is not only mediated by β2-AR-cAMP-PKA signaling pathway,but also mediated byβ2-AR-β-arrestin2-ERK1/2 signaling pathway.There is a reciprocal interaction between the two pathways.4.Overexpression of β-arrestin2 gene of Treg cells enhances Treg cell function in RA model mice.Inputtting of β-arrestin2 gene overexpressing Treg cells to CIA mice corrects Thl7/Treg imbalance,reduces joint inflammation and bone damage in CIA. |
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