The Effect Of Angiotensin Ⅱ Type 2 Receptor In The Rheumatoid Arthritis-Associated Interstitial Lung Injury

Rheumatoid arthritis（RA）is a systemic autoimmune disease that primarily accumulates in the joints.Due to long-term inflammatory environment stimulation,RA also affects multiple organs and exhibits extra-articular manifestations.Interstitial lung disease（ILD）is one of the most common manifestations.About 60%of RA patients showed interstitial lung injury,which usually occurred within 5 years after the diagnosis of RA,and was positively correlated with the severity of RA.There are two theories about the immune pathogenesis of ILD:One theory believes that ILD originates from synovial tissue injury and that the immune response to articular citrulline protein presents a similar antigenic cross-reaction in the lung.This hypothesis is based on the fact that articular symptoms occur before lung disease in RA patients.Another hypothesis suggests that the symptoms that precede RA joints may originate from one or more mucosal lesions,and then involve synovial joints,which could explain that about 20%of the lung symptoms of RA patients precede the occurrence of joint symptoms.The semi-permeable barrier formed by endothelial cells between the blood and the interstitial tissue helps to maintain homeostasis.The injury of endothelial cells leads to the destruction of the integrity of the barrier,which increases the permeability of blood vessels and promotes the infiltration of inflammatory cells.In turn,inflammatory cells will also secrete a variety of pro-inflammatory mediators,aggravating endothelial cell damage.AngiotensinⅡtype 2 receptor（Agtr2）binding with angiotensinⅡcan produce anti-inflammatory and other physiological functions that are opposite to those mediated by Agtr1.we previously found that Agtr2 plays an important role in autoimmune diseases such as RA.After the administration of Agtr2 agonist CGP42112,adjuvant induced synovitis in rats with rheumatoid arthritis was significantly relieved.In addition,the activation of Agtr2 can also promote the polarization of synovial M1-type macrophages to M2-type macrophages,thus alleviating the progression of arthritis.In recent years,more and more studies have shown that Agtr2 has a protective effect on lung injury.Agtr2 is expressed at low levels in normal lung tissue,with increased reactivity in inflammation and injury.Its elevation reduces inflammation in chronic obstructive pulmonary disease,inhibits the development of idiopathic pulmonary fibrosis,and significantly improves the symptoms of pulmonary hypertension symptoms.Therefore,the role of Agtr2 in RA-ILD was worth further study.Purpose:1.To investigate the potential pathological mechanism of RA-ILD.2.To determine whether Agtr2 activation could alleviate the prog ression of lung inflammation in collagen-induced arthritis（CIA）.3.To explore the mechanism of Agtr2 activation in relieving CIA-lung inflammation.Method:1.The CIA mouse model was established by injection of an emulsion of type Ⅱ collagen（1 mg/ml）in complete Freund’s adjuvant and incomplete Freund’s adjuvant on days 0 and 21,respectively.From day 28 after the first immunization,arthritis index scores were performed on the mice,and the mice were divided into two groups according to the severity of arthritis development:the peak period of arthritis（day 42）and remission period of arthritis（day 56）.The mice were sacrificed at different time points and lung tissues were collected for further investigation.hematoxylin-eosin（H&E）staining was used to observe lung pathology.q PCR was employed for rheumatoid factor（RF）,lung injury marker（KL-6 and SP-D）,inflammatory marker（IL-1β,TNF-α,Arg-1 and Ym-1）and endothelial cells（ECs）injury index（ICAM-1and VCAM-1）.The titers of anti-CCP antibody in lung tissue were detected by ELISA.The changes of ECs and monocytes in lung tissue were detected by immunofluorescence and flow cytometry.The expression of Ly6C in monocytes of lung tissue was detected by flow cytometry.Agtr2 protein levels in lung tissues were detected by Western blot.2.CX3CR1^lo(Ly6C^hi)and CX3CR1^hi(Ly6C^lo)monocytes were sorted by flow cytometer according to the fluorescence intensity of CX3CR1.Gene expression of inflammatory markers in Ly6C^hi and Ly6C^lo monocytes was detected by QPCR.The expressions of i NOS,CD206 and Agtr2 in Ly6C^hi and Ly6C^lo monocytes were detected by immunofluorescence.3.Mice endothelial C166 cells pretreated with LPS（100 ng/ml）were used to simulate the damaged ECs in vitro.The effects of C166 cells on the migration ability of monocytes were detected by Transwell assay.The co-localization of sorted cells and pre-stimulated C166 cells was detected by living cell workstation.CCK-8,angiogenesis assay and QPCR were used to detect the proliferation,tube formation and surface adhesion molecule expression of Ly6C^hi and Ly6C^lo monocytes to C166 cells,respectively.4.Agtr2 agonist C21(10^-6M)was administered to pre-stimulated C166 cells.Transwell assay was used to detect the effect of Agtr2 activation on monocyte migration.CCK-8,angiogenesis assay and q PCR were used to detect the proliferation,tube formation and surface adhesion molecule expression of C166 cells after Agtr2activation,respectively.Treated sorted Ly6C^hi monocytes with C21(10^-6M),and the transition of Ly6C^hi into Ly6C^lo monocytes was detected by flow cytometry.5.CIA mice were given C21（0.3 mg/kg）by intraperitoneal injection every 2-3 days from day 28 until day 42.On day 42,the mice were sacrificed and lung tissue was taken.H&E staining was used to observe pulmonary pathology.The levels of markers related to rheumatoid factor（RF）,lung injury marker（KL-6 and SP-D）,inflammatory marker（IL-1β,TNF-α,Arg-1 and Ym-1）and ECs injury index（ICAM-1 and VCAM-1）were determined by q PCR.The titers of anti-CCP antibody in lung tissue were detected by Elisa.The number of ECs and the expression of Ly6C on monocytes were detected by flow cytometry.Results:1.With the progression of the arthritis,CIA mice developed lung damage,characterized by interstitial thickening,infiltration of inflammatory cells,and,more seriously,lymphocyte follicular formation.Especially on day 42（the peak of arthritis）,the number of ECs decreased,the expression of CD31 in the vascular lumen was significantly reduced,and endothelial integrity was impaired.At the same time,mononuclear cells increased,mainly Ly6C^hi mononuclear cells with a pro-inflammatory phenotype.On day 56,the number of ECs gradually increased and CD31expression recovered.Notably,when infiltrating monocytes decreased,the proportion of Ly6C^lo monocytes with an anti-inflammatory phenotype increased.2.Compared with Ly6C^hi monocytes,Ly6C^lo monocytes expressed higher CD206,Ym-1,Arg-1 and Agtr2.Compared with Ly6C^lo monocytes,Ly6C^hi monocytes expressed higher levels of i NOS,IL-1βand TNF-α.3.C166 cells have a strong ability to recruit monocytes after damage.Compared with Ly6C^himonocytes,sorted Ly6C^lo monocytes are more likely to locate around damaged ECs,promote ECs proliferation,vascular tube formation,and reduce the expression of adhesion molecules VCAM-1 and ICAM-1.4.After C21 treatment,the proliferative and tube forming capacity of the damaged C166 cells was restored,the expression of adhesion molecules VCAM-1 and ICAM-1was decreased,and monocyte migration mediated by it was inhibited.C21 promotes the transition of Ly6C^hi monocytes into Ly6C^lo monocytes.5.After administration of C21,the lung inflammation in mice was relieved,the number of ECs increased,and the ratio of Ly6C^hi to Ly6C^lo monocytes decreased.Conclusion:1.Lung injury in CIA mice is mainly related to ECs injury and monocyte infiltration.2.ECs injury may be an initial event of CIA-ILD and a potential pathogenesis of RA-ILD.3.The activation of Agtr2 alleviates lung inflammation in CIA mice by alleviating ECs damage and promoting the transition of Ly6C^hi into Ly6C^lo monocytes.4.Agtr2 may be a new target for drug development and treatment of RA-ILD.