Paeoniflorin-6’-O-benzene Sulfonate Restrains Th17 Cell Differentiation Through Inhibiting STAT3 Phosphorylation In Mouse With Collagen-induced Arthritis

Rheumatoid arthritis（RA）is one of the common systemic autoimmune diseases,the main clinical manifestation is limb joints inflammatory,however its exact pathogenesis is still unknown.Studies have shown that T cells dysfunction,especially abnormal activation of CD4 positive helper T（Th）cells play a crucial role in the development of RA.Th0 cells activate specific transcription factors under different stimuli,and then differentiate into different Th subsets:Th1,Th2,Th17,and regulatory T（Treg）cells.Th1/Th2 and Th17/Treg restrict each other and maintain the balance of immune system,if the balance is destruct,immune deviation will be induced,leading to occurrence and development of RA.Th17 cells have become an important therapeutic target for inflammatory immune diseases due to its strong inflammatory effect through interleukine-17（IL-17）.The level of IL-17 in serum and synovial fluid of RA patients is significantly increased,and the level of IL-17 in serum of collagen induced arthritis（CIA）and adjuvant arthritis（AA）rat models is also abnormally increased.IL-17 activates dendritic cells,macrophages and other immune cells in synovial tissue of joints,and further promotes the production of more pro-inflammatory cytokines such as IL-6,activates fibroblast-like synoviocyte（FLS）and osteoclasts,thus destroys joints and cartilage.The differentiation of Th17 cells is regulated by Janus protein tyrosine kinase（JAK）-signal transducers and activators of transcription（STAT）signaling pathway.Under the stimulation of IL-6,IL-23,transforming growth factor-b（TGF-b）and other cytokines,JAK is activated by IL-6receptor（IL-6R）and subsequently phoaphorylate downstream STAT3,which promotes the transcription of retinoid-related orphan receptor gammat（RORgt）,and finally drives Th0 cells differentiate into Th17 cells.Therefore,inhibition of Th17 cell differentiation will help to improve the joints inflammation of RA.Molecules that negatively regulate Th17 cell differentiation are mainly including suppressor of cytokine signaling（SOCS）protein,protein inhibitors of activated STATs（PIAS）and protein tyrosine phosatases（PTPs）,among which tyrosine-protein phosphatase non-receptor type 6（PTPN6）,also known as SHP1 of PTP family plays a particularly prominent role in dephosphorylation of STAT3,resulting in preventing Th17 cell differentiation through inhibiting JAK-STAT signaling pathway.Researches have also indicated that the function of SHP1 requires the scaffold proteinb-arrestin2（arrb2）.In natural killer（NK）cells,arrb2 binds to SHP1/2 to inhibit the cytotoxic effect of NK cells.The classic function of arrb2 is to mediate the desensitization and endocytosis of G protein coupled receptors（GPCRs）.When GPCRs are activated by corresponding ligands,GPCRs kinase 2（GRK2）phosphorylates the activated receptor and then recruits arrb2 to desensitise and internalize the receptor,thereby terminating downstream signals.It is well known that T cells express a variety of GPCRs,which play important roles in regulating the activation and differentiation of T cells.Especially,physiological concentration endogenous A₃ adenosine receptor（A₃AR）activation by adenosine prevents Th17 cell production.The proportion of Th17 cells were significantly increased in the spleen and mesenteric lymph node of arrb2knockout mice with colitis.However,the role and mechanism of the cross-regulation of GPCRs and its regulatory molecule arrb2 with JAK-STAT pathway in the differentiation of Th17 cells of RA still remains unclear.Paeoniflorin-6’-O-benzene sulfonate（CP-25）developed by our research group significantly improves joint inflammation of CIA rats and reduces the percentage of Th17 cells in spleen and peripheral blood.We have reported that CP-25 is able to regulate the desensitization and endocytosis of the prostaglandin receptor EP4,restore the signaling of EP4 in FLS,and inhibit the abnormal proliferation of FLS.Although it is demonstrated that the regulation of GPCRs by CP-25 is due to the inhibition of GRK2 activity,whether CP-25 affects arrb2 function through its upstream molecule GRK2,leading to the negative modulation of JAK-STAT signal pathway and consequently prevent Th17 cell differentiation need to be illuminated.On the basis of previous studies,by establishing an arthritis model using arrb2knockout mice,and treating CIA mice with CP-25,we explored the role of arrb2 in the differentiation of Th17 cells,as well as the mechanism of CP-25 in reducing Th17differentiation.This work will shed light on the the pathogenesis of RA and pharmacological effect of CP-25.OBJECTIVETo investigate the regulatory role of arrb2 in Th17 cell differentiation;To illuminate the mechanism of CP-25 in reducing the formation of Th17 cells in CIA mice.METHODSCIA model was established with DBA/1 mice,and CP-25（50mg/kg/d）,GRK2inhibitor paroxetine（PAR,15mg/kg/d）or methotrexate（MTX,2 mg/kg,every three days）was gavaged for 2 weeks continuously.The overall indicators and joint pathology were observed.The proportion of Th17 cells in peripheral blood and spleen was detected by flow cytometry.The level of phosphorylated STAT3 was determined by immunofluorescence.The colocalization of arrb2 with SHP1,SHP1 with STAT3,and arrb2 with A₃AR in CD4⁺T cells was detected by COIP or immunofluorescence.Splenic lymphocytes from C57BL/6J background of wild type（WT）mice and arrb2knockout(arrb2^-/-)mice were induced into Th17 cells in vitro with the stimulation of IL-6,IL-23 and TGF-b,with or without SHP-1 inhibitor,A₃AR agonist,CP-25(1×10^-5mol/L)or PAR(1×10^-5mol/L),the percentage of Th17 cells and the phosphorylation of STAT3 in CD4⁺T cells was determined.The collagen antibody induced arthritis（CAIA）model was established with arrb2^-/-and WT mice,the overall clinical manifestation and joint pathology were observed,and the frenquency of Th17cells in peripheral blood and spleen was also detected.RESULTS1.CP-25 significantly improved the overall indicators of CIA miceCIA mice showed a significant joint inflammation,joint swelling,which was effectively restored by CP-25 or PAR treatment.2.CP-25 effectively reduced the proportion of Th17 cells in spleen and peripheral blood of CIA miceTh17 cells in the spleen and peripheral blood of CIA mice were significantly increased,and CP-25 or PAR significantly decreased the proportion of splenic and circulating Th17 cells.3.CP-25 significantly inhibited the phosphorylation of STAT3 in splenic CD4⁺T cells of CIA miceImmunofluorescence data showed that CP-25 administration significantly reduced p STAT3 level in CD4⁺T cells in the spleen of CIA mice.4.Inhibition of SHP1 further promoted the differentiation of Th17 cells in vitroIL-6,IL-23 and TGF-bwere used to induce splenic cell from WT mice to differentiate into Th17 cells in vitro,with or without SHP1 inhibitor.The result showed that SHP1 inhibitor further increase the proportion of Th17 cells induced by combined cytokines.5.Activation offurther increased the phosphorylation of STAT3 in CD4⁺T lymphocyteIL-6,IL-23 and TGF-bwere used to induce splenic cell from WT mice to differentiate into Th17 cells in vitro,with or without high concentration of A₃AR receptor agonist.Immunofluorescence results showed that STAT3 phosphorylation was significantly increased in induced CD4⁺T cells,but is further elevated with the treatment of A₃AR agonist.6.Activation ofdecreased the colocalization of SHP1 with STAT3 and arrb2 with SHP1,and increased the combination of arrb2 andIL-6,IL-23 and TGF-bwere used to induce splenic cell from WT mice to differentiate into Th17 cells in vitro,the colocalization of SHP1 with STAT3 and arrb2 with SHP1 in CD4⁺T cells was reduced,and the colocalization of arrb2 with A₃AR was not significantly affected.With the stimulation of A₃AR agonist,the interactions between SHP1 and STAT3,arrb2 and SHP1 were further reduced,but the combination of arrb2 and A₃AR was markedly improved.7.Depletion of arrb2 significantly promoted in vitro Th17 cell differentiationIL-6,IL-23 and TGF-bwere used to induce splenic cell from WT or arrb2^-/-mice to differentiate into Th17 cells in vitro.The result showed that the proportion of Th17cells induced with splenic cell from arrb2^-/-mice was significantly higher than that from WT mice.8.Depletion of arrb2 further increased the phosphorylation of STAT3 in CD4⁺T cell during Th17 cell differentiationIL-6,IL-23 and TGF-bwere used to induce splenic cell from WT or arrb2^-/-mice to differentiate into Th17 cells in vitro.The ratio of p STAT3/STAT3 was significantly increased in CD4⁺T cells from WT or arrb2^-/-mice under induction,deficiency of arrb2 further increased the phosphorylation rate of STAT3 comparing with that of the WT group.9.The CAIA model of arrb2^-/-mice had significantly more severe joint inflammation than the WT-CAIA miceBoth arrb2^-/-and WT mice reached peak arthritis around day 10 after antibodies injection,with significant joint swelling,increased arthritis index and weight loss,but the overall manifestation of arthritis in arrb2^-/--CAIA mice was significantly worse than that in WT-CAIA mice.10.Depletion of arrb2 did not significantly affect spleen and thymus index in CAIA miceCAIA mice of different genotypes had significantly higher spleen indexes and slightly lower thymus indexes which were not significant.The thymus and spleen indexes of arrb2^-/--CAIA mice were not significantly different from WT-CAIA mice11.Arrb2^-/--CAIA mice had more severe joint pathological performance than that of WT-CAIA miceComparing with normal mice,the H&E staining of the joints from CAIA mice showed definite hyperplasia of FLS,lymphocytes infiltration in synovial tissue,pannus formation and cartilage distruction.The pathological performance of joints from arrb2^-/--CAIA was more severe than that of the WT-CAIA mice.12.Deficiency of arrb2 further increased the proportion of Th17 cells in peripheral blood and spleen of CAIA miceFlow cytometry data indicated that the proportion of Th17 cells in the spleen and peripheral blood of CAIA mice was obviously upregulated in relative to the normal mice with same genotype.However,Th17 cell percentage was significantly higher in arrb2^-/--CAIA mice than that in WT-CAIA mice.13.CP-25 treatment significantly increased the colocalization of arrb2 with SHP1 in CD4⁺splenic T cells of CIA miceThe expression of arrb2 was increased significantly in CD4⁺T cells from CIA mice,but CP-25 did not obviously change it.The colocalization of arrb2 with SHP1was decreased in CD4⁺T cells from CIA mice,and was rescued by the administration of CP-25 or PAR,the result was confirmed by COIP.14.CP-25 restored the colocalization of SHP1 with STAT3 in CD4⁺T cells treated by Th17 induction combo in vitroIL-6,IL-23 and TGF-bwere used to induce WT splenic cells to differentiate into Th17 cells in vitro,with or without A₃AR agonist,CP-25 or PAR for 72 h.As detected,the colocalization of SHP1 with STAT3 in CD4⁺T cells was significantly reduced,but was recovered by CP-25 or PAR treatment.15.CP-25 decreased the colocalization of arrb2 within CD4⁺T cells treated by Th17 induction combo in vitroIL-6,IL-23 and TGF-bwere used to induce WT splenic cells to differentiate into Th17 cells in vitro,with or without A₃AR agonist,CP-25 or PAR for 72 h.The results showed that a large amount of arrb2 binded to A₃AR in differentiating splenic CD4⁺T cells,and CP-25 or PAR in vitro treatment significantly reduced the colocalization of arrb2 with A₃AR.CONCLUSIONS1.Arrb2 prevents Th17 cell differentiation through facilitating the interaction of SHP1 with STAT3 thus inhibiting the phosphorylation of STAT3.2.CP-25 inhibits IL-6R triggered JAK-STAT3 signaling by reducing the combination of arrb2 with A₃AR and rescuing the interaction of arrb2 with SHP1, thus prevents the differentiation of Th17 and finally alleviates the inflammation of joints.