Mechanism Of Pro-inflammatory Effect Of ACPA In Rheumatoid Arthritis

Rheumatoid arthritis(RA)is a common autoimmune disease with a high incidence.RA’s incidence rate in adults is about 0.5%-1%,and the genetic possibility is 40-65%.The number of new cases each year is 5-50 in 100,000 people.The clinical manifestations of RA are mainly multiple symmetric arthralgia,swollen joints,and dysfunction of the small joints.Besides,RA patients also have systemic complication.In the late 1990 s,researchers identified a novel type of RA-specific autoantibodies which demonstrated excellent diagnostic value via analyzing serum samples of patients with RA.Such autoantibodies are known as anti-citrullinated protein antibodies(ACPA).ACPA antibodies target at a cluster of proteins or polypeptides modified by peptide arginine deiminase(PAD)after translation.Studies of the pathogenic effects of ACPA have initially discovered that ACPA can exacerbate chronic inflammation in RA.Besides,ACPA enhances the activity of macrophages by stimulating the production of pro-inflammatory cytokines such as TNF-α,collaborating with IgA or IgM rheumatoid factor(RF).IL-1β is a key inflammatory mediator of the innate immune response and plays a vital role in RA’s immunopathological development leading to chronic inflammation.Elevation of IL-1β could be observed at various stages of RA and located mainly in the joint synovium.The production of IL-1β requires the activation of inflammasomes.Inflammasomes are a cluster of multimeric protein complexes composed of inflammasome sensor molecules(eg.NLRP3,NLRC4,etc.),adaptor proteins(apoptosis-associated speck-like protein containing CARD,ASC),and Caspase-1.Among different inflammasomes,the NLRP3 inflammasome is the most commonly studied in RA.Previous studies have shown that NLRP3 inflammasome-associated genes(ASC,MEFV,NLRP3-FL,NLRP3-SL,and CASP1)are up-regulated in PBMCs from RA patients.NLRP3-mediated secretion of IL-1β could be regulated by PTPN22,an important risk factor for the occurrence and development of RA.However,whether ACPA can promote IL-1β production in macrophages of RA patients remains to be elucidated.Our study is mainly divided into three partsPart I: ACPA induces the activation of NLRP3 in macrophage which promotes the production of IL-1β.We determined IL-1β levels by immunohistochemical staining(IHC staining)in the synovial tissue of ACPA positive RA patients,ACPA negative RA patients,and osteoarthritis(OA)patients.The results showed that ACPA positive RA patients have higher levels of IL-1β.To further investigate this phenomenon,we used purified ACPA antibody to directly stimulate PBMC-derived macrophages.Detection of IL-1β in the culture medium by ELISA showed that macrophages produced IL-1β in a time-and dose-dependent manner after ACPA stimulation.Finally,after interfering the NLRP3 molecule,macrophages were not able to produce IL-1β after ACPA treatment.This demonstrated that ACPA antibodies promote the production of IL-1β by activating NLRP3 inflammasome.After analyzing the expression levels of CD147 and NLRP3 in the synovial tissue of RA patients,we found that both the expression of NLRP3 and CD147 in ACPA postive RA patients increased and their expression level were highly correlated.Then,we use lentivirus to interfere the CD147 expression.After interfering CD147 molecule,IL-1β induced by ACPA is significantly reduced.Meanwhile,compared with the control cells,Akt and NF-κB signaling pathways were de-activated,while JNK signaling pathway remained unchanged.Part II: ACPA enhances the interaction of CD147 and integrin β1 to activate Akt/NF-κB signaling pathway.Immunofluorescence staining demonstrated that CD147 and ACPA antibodies co-localized on the cell surface.Co-IP and SPR experiments demonstrated an interaction of CD147 and ACPA.Subsequently,Co-IP experiments demonstrated that ACPA can lead to enhanced interactions between CD147 and integrin β1.The interaction between CD147 and integrin β1 could be blocked by GRGDS,causing a decrease in Akt signaling pathway and IL-1β production.By inhibiting the Akt signaling pathway with LY294002,NLRP3 inflammasome activation and IL-1β production were also reduced.These results indicated that ACPA promotes the interaction of CD147 with integrin β1 to activate the Akt signaling pathway in mediating NLRP3 inflamasome.The immunofluorescence experiments and Western blot experiments,further demonstrated that activation of the Akt signaling pathway by CD147 and integrin β1 could activate the downstream NF-κB signaling pathway which resulted in the nuclear translocation of transcription factor p65,promoting the transcription of NLRP3 molecule and of pro-IL-1β,and accomplishing the priming stage.Part III: ACPA induces ATP release to activate NLRP3 inflammasome in macrophage.After measuring the ATP levels in the cell culture fluid by Luciferase,we found that the extracellular ATP levels increase after the ACPA treatment which could be inhibited by CD147 or integrin β1 interference molecule or blocking the interaction between the two molecules.The subsequent studies demonstrated that ACPA could activate Pannexin channels by enhancing the interaction of CD147 with integrin β1,inducing the release of ATP and increasing extracellular ATP level.The extracellular ATP could further interact with P2X7 receptors on the cell surface,causing the activation of NLRP3.In conclusion,for the first time,we discovered that ACPA promotes IL-1β in synovial tissue by promoting the expression and activation of NLRP3 inflammasome.ACPA treatment results in enhanced interaction between CD147 and integrin β1 in macrophages,which in turn activates the downstream Akt/NF-κB signaling pathway leading to increased expression of NLRP3 and pro-IL-1β.Meanwhile,ACPA activates Pannexin channels,causing ATP secretion.The extracellular ATP binds to P2X7 receptors which subsequently activate NLRP3 inflammasome.The experimental results verify a new mechanism of IL-1β production in RA,exploring a new role of ACPA in RA pathogenesis,and provide a new target for RA treatment.