The Modulatory Effects Of Ang ? Type 2 Receptors Signaling On Activation Of Synovium Macrophage With Rheumatoid Arthritis

Rheumatoid arthritis?RA?is an autoimmunity disease with an yet unknown autoimmune trigger which presents with a destructive inflammation of the joints and shows,in severe cases,an involvement of organs.In this pathology joints are exposed consistently to an inflammatory environment resulting in abnormal activation and display proliferation of synovial cells,irregular differentiation of osteoclasts and the release of a large number of inflammatory factors.One of these factors,angiotensin?Ang II?,is a catalytic product of the angiotensin-converting enzyme?ACE?and its main receptors are angiotensin II type 1 receptor?AT1R?and angiotensin II type 2receptor?AT2R?,which have similar affinity for Ang II.Ang II is involved in the contraction of blood vessels,the promotion of inflammation and fibrosis.Furthermore,it can trigger oxidative stress through AT1R.However,it can also produce opposite physiological effects through AT2R,such as a relaxation of blood vessels and anti-inflammatory,anti-fibrotic and anti-oxidative responses.Inflammatory immune cells including dendritic cells,macrophages,and lymphocytes have their own independent RAS.As a consequence,receptors of Ang II have a strong regulatory effect on cells of the immune system.Specifically,Ang II interacts via AT1R which is present on inflammatory immune cells,in an autocrine or paracrine fashion and modulates the inflammatory immune response by stimulating inflammatory immune cells.It has been reported that Ang II and AT1R play an important role in maintaining the autoimmune inflammatory immune response.Applying AT1R blockers in RA patients and the corresponding animal models not only attenuate synovitis but also improve cardiovascular abnormalities.After blocking of AT1R,the free Ang II in the body interacts predominantly with AT2R,thereby activating downstream signaling and exerting the protective function of this receptor.Thus,it is a vital pharmacological effect of ARBs in the treatment of RA.Synovium macrophages?SM?exhibit a range phenotypes and functions when encountering different factors,that generally are categorized as M1 and M2macrophages,two different polarization stages of macrophages.The quantity and proportion of M1/M2 macrophages vary in the different stages of RA disease development.In the active phase of RA,pro-inflammatory M1 macrophages are increased.In contrast,M2 macrophages which promote inflammation resolution and tissue repair are decreased.This imbalance of M1 and M2 exacerbates the inflammatory response.The question whether AT1R/AT2R imbalance promotes M1 macrophages polarization in RA synovitis,or whether activation of AT2R signaling inhibits an abnormal activation of SM has not been addressed yet.Previous studies have shown that intra-articular injection of the AT2R agonist CGP42112 in the adjuvant arthritis?AA?rat model can improve joint synovitis,inhibit synovial cell proliferation.This indicates that the inflammatory immune response can be relieved by activation of AT2R.This finding may provide a potential target for RA treatment.This project used collagen-induced arthritis?CIA?of rats and synovial tissue of RA patients,and applied immunofluorescence,immunohistochemistry,western blot,flow cytometry and AT2R pharmacological tools to investigate the role of SM and their response during RA.Furthermore,we explored the effects of AT2R signal transduction and MAPKs cross-talk in this disease.Our results provide an experimental basis for the regulation of angiotensin receptor signaling in abnormal activation of SM in RA patients,which may result in a new concept that uses AT2R as a therapeutic target for RA synovitis.Purpose:Our group aimed to explore the relationship between the imbalance of AT1R/AT2R signal transduction and the abnormal activation and function of SM.AT2R and its signal inhibited the abnormal activation of SM in RA,which may be associated with the inhibition of GRK2 transmembrane translocation and down-regulation of the MAPKs-NF-?B signal pathway.Method:1.The synovial tissues of the normal control and RA patients were collected,and the pathologicalfeaturesofRAsynoviumwerestudiedbyHEstaining.Immunohistochemistry was used to detect specifically the distribution of macrophages.Tissue immunofluorescence was used to observe the expression and co-localization of AT1R,AT2R,the M1 macrophage marker iNOS and the M2 macrophage marker TGM2and GRK2.Peripheral blood mononuclear cells?PBMCs?were collected from 15healthy volunteers and 32 RA patients.The levels of AT1R and AT2R in PBMC were detected by enzyme-linked immuno sorbent assay?ELISA?.Correlation analysis was used to analyze the correlation between AT1R and AT2R protein expression in human PBMC and RA index.2.The experimental rat CIA model was induced by using chicken type II collagen and Freund's incomplete adjuvants,followed by an intra-articular injection of AT2R agonist CGP42112?5?g/kg,10?g/kg,20?g/kg?.The volume of the secondary hind paw and the number of swollen hind paws were measured and calculated by using an animal hind paw instrument.HE staining was used to observe joint pathology.Luminex liquid chip analysis was applied to detect the levels of IL-1?,IL-6,IL-10,TNF-?,IFN-?and MIP-2in the rat serum.Flow cytometry was used to detect the expression of AT1R,AT2R and the macrophage markers CD86 and CD163 in a single cell suspension of rat synovial tissues.3.Bone marrow-derived macrophages?BMDM?were generated by culturing bone marrow cells with 10 ng/ml macrophage colony stimulating factor?M-CSF?for 4 days.To explore the optimal concentration of drugs a Griess reagent kit was used to detect NO release from bone marrow-derived macrophages under treatment conditions with different AT2R agonists or antagonists.The Luminex liquid chip was used to detect the levels of IL-1?,IL-6,IL-10,TNF-?and MIP-2 in BMDM supernatants treated with AT2R agonists at different time points.The phagocytic ability of BMDM was examined using FITC-Dextran.Western blot was used to detect the effect of AT2R agonist on the MAPK pathway.Flow cytometry and western blot were used to detect the expression of GRK2 in the cell membrane and cytoplasm,respectively.Co-expression of GRK2 and p-ERK was analysed by Co-IP.Nucleoproteins were extracted and analysed to detect the nuclear import of the nuclear transcription factors P65 and P50.Result:1.Compared to the healthy control group,the synovium of RA patients were significantly enlarged.The pathological examination showed pannus formation.Immunohistochemistry results showed the number of macrophages was significantly increased in the synovial lining of RA patients close to the joint cavity.Double fluorescent staining showed that the expression of AT1R and AT2R was significantly increased in macrophages,and macrophage polarization markers iNOS and Arg1 were both elevated.The ELISA results showed the levels of AT1R and AT2R of patients were significantly higher than those of healthy volunteers.Correlation analysis showed that the expression of AT1R protein in PBMCs was positively correlated with the polyarthritis index,which indicated the up-regulation of protein expression was associated with the increase of erythrocyte sedimentation rate?ESR?and C-Reactive Protein?CRP?.While,the expression of AT2R protein was negatively correlated with the ESR and CRP.2.Injection with CGP42112 into the joint cavity of rats relieved RA symptoms by activation of AT2R.Compared with the normal group,the volume of the secondary hind paw of the the model group was significantly increased as well as the number of swollen hind paws.However,the body weight was significantly lower than that of the normal group.Compared with the model group,the symptoms of secondary arthritis in CGP42112-treated and TNF antagonist-treated CIA rats were significantly reduced.Histopathological results showed that the synovitis indicators in the CGP42112-treated and TNF antagonist-treated CIA rats,including synovial cell hyperplasia,inflammatory cell infiltration,pannus formation,as well as cartilage and bone erosion were significantly reduced,compared to the model group.The proportion of M2 macrophages in the synovium of CGP42112-treated and TNF antagonist-treated CIA rats was higher than that of the model group.In addition,the expression of AT1R was found to be decreased,while the expression of AT2R was increased in SM.3.To investigate the underlying mechanisms how activation of AT2R alleviated the inflammatory immune response in rats,bone marrow derived macrophages were employed.Western blot analysis showed that CGP42112(10^-6M)treatment of macrophages in vitro significantly decreased the expression of AT1R increased the expression of AT2R and inhibited LPS-induced M1-type polarization.Administration of CGP4112 inhibited ERK phosphorylation but had no effect on P38 and JNK phosphorylation.Flow cytometry and western blot results showed CGP42112 can attenuate macrophage phagocytosis and inhibit the transmembrane translocation of GRK2 thereby promoting the binding of GRK2 to p-ERK in the cytoplasm.Conclusion:1.The expression of AT1R and AT2R were significantly increased in synovial tissues and PBMCs of RA patients.The imbalance of AT1R/AT2R may be involved in the polarization of M1 macrophages.2.Intra-articular injection of CGP42112 in a CIA rat model improved secondary synovitis,reduced serum cytokines and inhibited the M1-type polarization of macrophages in the synovia.This indicates that activation of AT2R may promote the polarization of macrophages to anti-inflammatory M2 macrophages by reversing the imbalance of AT1R/AT2R.3.AT2R and its signal inhibited the abnormal activation of macrophages in RA,which may be associated with the inhibition of GRK2 transmembrane translocation and down-regulation the p-ERK-NF-?B signal pathway.