The Regulatory Role Of ?-arrestin2 In Plasma Cell Differentiation In Murine Rheumatoid Arthritis Model And The Effect Of CP-25

IntroductionRheumatoid arthritis?RA?is an autoimmune disease characterized by joint involvement.Its main pathological feature is abnormal activation of immune cells,a variety of inflammatory immune cells infiltrate into synovium,causing synovium cells to proliferate violently like tumor,forming pannus,eroding cartilage and bone tissue,eventually leading to joint deformity and loss of function.In addition to the main complaints and physical signs,the early diagnosis of RA mainly depends on ESR,rheumatoid factor?RF?and anti-cyclic citrullinated peptide?CCP?antibodies and other laboratory test indicators,in which both RF and anti-CCP antibodies are autoantibodies,suggesting that B cell activation,excessive plasma cell cloning,and a large number of autoantibodies production are one of the important pathological mechanisms of RA.Under the condition of RA disease,the B lymphocyte stimulated by antigen can develop and differentiate into memory B cell and plasma cell secreting high affinity antibody.Blimp-1,as the"primary regulator of ultimate differentiation of B lymphocytes",is an important transcription factor promoting phenotypic differentiation and antibody secretion in plasma cells.Autoantibodies produced by autoreactive B lymphocytes attack their tissues and cause an antibody-mediated immune response that involves multiple organs,damages the body and mediates the development of RA.Therefore,excessive plasma cells produced by excessive activation and differentiation of B lymphocytes play an important pathological role in the development of RA and are an important target for anti-RA therapy.Studies have found that toll-like receptor4?TLR4?-mediated signaling plays an important role in the differentiation of B lymphocytes into plasma cells.TLR4 is an important member of the toll like receptor?TLR?family to recognize pathogenic microorganisms.It is the most important receptor to mediate the response of lipopolysaccharide?LPS?,the main component of Gram-negative bacteria cell wall,which is expressed in almost all cells and distributed on the cell surface.When TLR4 receptor receives the stimulation of its ligand,TNFR-associated factor?TRAF6?and interleukin-1 receptor associated kinase?IRAK1/4?are collected together to myeloiddifferentiationfactor88?My D88?to form a complex,so that TRAF6 will undergo ubiquitination oligomerization,activate TAK1,and then TAK1will activate inhibitor of nuclear factor-?B kinases?IKKS?,the activated IKKS will phosphorylate and degrade inhibitor of nuclear factor-?B?I?B?,and nuclear factor-?B?NF-?B?will transfer into the nucleus to activate Blimp-1 transcription.Since TLR4 on the cell membrane is the promoter of signal transduction in plasma cells,during the course of RA,it continuously receives ligand stimulation to promote the excessive differentiation of B lymphocytes.the classical animal model of RA is established by injecting LPS or BCG-activated TLR4,so the regulation of TLR4 function is important in maintaining immune balance.In addition to the role of TLR4,the regulation of immune cell function by G-protein-coupled receptors?GPCRs?as the largest receptor superfamily has been paid more attention in recent years.GPCRs determines the activation and function of immune cells by regulating intracellular cyclic adenosine monophosphate?c AMP?levels.Ligands including prostaglandins,adenosine,chemokines and other levels of various GPCR in RA patients were significantly increased and receptors were activated.GPCRs is mainly negatively regulated by G protein coupled receptor kinase?GRK?and?-arrestin2??-arr2?.When the receptor is phosphorylated by GRK,?-arr2 mediates the vast majority of GPCRs endocytosis,desensitization,etc.It has been found that?-arr2 not only mediates the endocytosis of GPCRs,but also mediates the endocytosis of some non-GPCRs,such as growth factor receptor,so as to negatively regulate its signal pathway.A number of studies have shown that?-arr2 has anti-inflammatory effect,and the joint inflammation of?-arr2^-/-mice is more serious than that of wild type?WT?mice,but the specific mechanism is not clear.The results showed that the percentage of plasma cells of collagen antibody induced arthritis?CAIA?in?-arr2^-/-mice was significantly higher than that in WT-CAIA mice,suggesting that?-arr2 participated in the differentiation of plasma cells,but the molecular mechanism needs to be clarified.At the same time,we found that the new active monomer CP-25,which was synthesized by the esterification modification of Pae,could effectively reduce the percentage of activated B cell subpopulation and the secretion of Ig G1and Ig G2A in CIA mice,suggesting that CP-25 has a clear inhibitory effect on the production of plasmacytes.But the specific mechanism of action is unclear.whether CP-25 regulates plasma cell formation by affecting?-arr2 function remains to be elucidated.In order to elucidate the regulatory effect of?-arr2 on the formation of plasma cells and the effect of CP-25 in arthritis mice,we established?-arr2^-/-and WT mice CAIA models,and observed the effect of?-arr2^-/-and WT mice CAIA models,TNP-LPS induced?-arr2^-/-and WT mice immune response in vivo and in vitro the spleen B cells were stimulated by LPS to study the regulation of?-arr2 on TLR4 signal of B lymphocytes and its mechanism in plasma cell differentiation.At the same time,CP-25 was given to treat CIA mice to clarify the effect of CP-25 on plasma cell differentiation of arthritis mice and the regulation of?-arr2.ObjectiveTo observe the effect of?-arr2^-/-on the progress of arthritis and the production of plasma cells,to reveal the regulatory effect of?-arr2 on TLR4 mediated plasma cell differentiation signal,and to clarify the pharmacological mechanism of CP-25 indirectly regulating?-arr2 through GRK2 to inhibit plasma cell differentiation.MethodsIn order to observe the role of?-arr2 in the course of arthritis and the production of plasma cells,a CAIA model of?-arr2^-/-and WT mice was established.The overall indexes of mice?body weight,thymus index,spleen index,joint swelling number and arthritis index?were observed.The pathology of spleen and ankle joint was examined by H&E staining.The swelling degree and blood flow signal of knee joint were detected by ultrasonic imaging Flow cytometry was used to detect the percentage of plasma cells?CD19^-CD138⁺?and memory B cells?CD19⁺CD27⁺?in the spleen of CAIA mice;ELISA assay for the detection of Ig G1 and Ig G2A levels in serum of CAIA mice.To further reveal the regulatory effect of?-arr2 on TLR4 mediated differentiation signal of plasma cells,immunofluorescence assay was performed to detect TLR4 binding to?-arr2 and the translocation of NF-?B in the spleen of?-arr2^-/-and WT-CAIA mice.Flow cytometry was used to detect the cytomembrane expression of TLR4 on B cells;Western blot method was used to detect the expression of TLR4 in cytoplasm of B cells;TNP-LPS was injected intraperitoneally to activate TLR4 receptor of B cells of?-arr2^-/-and WT mice,and the proportion of spleen plasma cells was detected by flow cytometry.?-arr2^-/-and WT mouse spleen B cells were isolated in vitro and stimulated by LPS respectively.CCK-8 method was used to detect the effect of LPS on B cell viability in vitro.Detection of the percentage of B cells differentiated into plasma cells?CD19^-CD138⁺?and memory B cells?CD19⁺CD27⁺?by flow cytometry,the expression level of the transcription factor Blimp-1 m RNA by q PCR,Using LPS+interleukin-4?IL-4?to induce Ig G1 in B lymphocytes or Ig G2A induced by LPS+interferon-??IFN-??in B lymphocytes.And the production level of Ig G1 and Ig G2A antibodies in the supernatant of B cell culture by ELISA.In order to clarify the pharmacological mechanism of CP-25 reduces plasma cell formation,the CIA model of DBA/1 mice was established.The effects of CP-25 on the overall indexes?joint swelling number,arthritis index?and pathological changes?H&E staining?of ankle joint in CIA mice were observed.The effect of CP-25 on the percentage of spleen plasma cells and TLR4 membrane expression on B cells in CIA mice by flow cytometry,The co-localization of?-arr2 and TLR4,?-arr2 and GRK2 in B cells of CIA mice was detected by immunofluorescence,and western blot assay to detect the effect of CP-25 on the cytoplasmic TLR4 protein distribution in CIA mouse B cells.Results1. Depletion of?-arr2 can aggravate the overall index of CAIA mice.To establish the CAIA model of?-arr2^-/-and WT mice,compared with WT-CAIA mice,the weight loss of?-arr2^-/--CAIA mice was more obvious from the beginning of model d 10;while in the course of d 9-10,the joint swelling number and arthritis index of?-arr2^-/--CAIA mice increased more significantly;ultrasonic imaging detection found that the knee joint swelling of?-arr2^-/--CAIA mice was more obvious;compared with WT-CAIA mice,the knee joint swelling of?-arr2^-/--CAIA mice was more obvious In the joints of?-arr2^-/--CAIA mice,there were more obvious inflammatory cell infiltration,pannus appearance,bone erosion and synovium hyperplasia,more serious pathological changes of spleen,higher spleen index and lower thymus index.The results suggest that?-arr2 knockout will lead to more serious arthritis,so?-arr2 plays an important role in the development of experimental arthritis.2. Depletion of?-arr2 increases plasma cell formation and produces more antibodies in CAIA mice.Compared with WT-CAIA mice,the percentage of spleen plasma cells?CD19^-CD138⁺?and memory B cells?CD19⁺CD27⁺?in?-arr2^-/--CAIA mice were significantly higher,and the levels of serum Ig G1 and Ig G2A were higher,suggesting that?-arr2 knockout can cause the increase of plasma cell formation and antibody production in arthritis mice,that is to say,?-arr2 can inhibit the differentiation of plasma cells in arthritis mice.3. Depletion of?-arr2 promotes TLR4 cell membrane distribution and and signal transduction in B lymphocytes of CAIA mice.Compared with normal WT mice,the expression of TLR4 in the B lymphocyte membrane of WT-CAIA mice was significantly increased,the co-localization of TLR4with?-arr2 was significantly increased,and the translocation of NF-?B nucleus was increased.Compared with WT-CAIA mice,?-arr2^-/--CAIA mice showed more expression of TLR4 in the cell membrane of B lymphocytes of spleen,but less cytoplasmic distribution.The above results suggest that TLR4 signaling in B lymphocytes is activated and?-arr2 is linked to TLR4 under inflammatory conditions combined increase,knockdown of?-arr2 makes the TLR4 cell membrane more distributed,and the downstream transcription factor NF-?B activation is more obvious.These results suggest that?-arr2 may reduce plasma cell production by binding to TLR4 by reducing the TLR4distribution on the B lymphocyte membrane and inhibiting its downstream NF-?B activation.4. Knockout of?-arr2 further increases the formation of plasma cells in TNP-LPS intraperitoneally injected mice.We used TNP-LPS intraperitoneal injection of?-arr2^-/-and WT mice to activate TLR4 signaling and induce in vivo B lymphocyte activation and differentiation.Detection after injection d14 found that compared with normal WT mice,the proportion of WT-TNP-LPS mouse plasma cells was significantly increased,and the levels of producing Ig G1 and Ig G2A were also significantly increased.Compared with WT-TNP-LPS mice,?-arr2^-/--TNP-LPS mice had more splenic plasma cells and higher levels of serum Ig G1 and Ig G2A,suggesting that knockdown of?-arr2 could promote LPS-induced plasma cell differentiation.this result further indicated that?-arr2 could inhibit plasma cell differentiation by regulating TLR4 signal.These results suggest that TLR4 signal-mediated plasma cell differentiation is significantly negatively regulated by?-arr2.5. Deletion of?-arr2 causes increased TLR4 cell membrane distribution and increased LPS in vitro stimulation of B lymphocyte differentiation into plasma cells.In vitro,LPS or LPS+IL-4/IFN-?were used to stimulate the isolated B lymphocytes of?-arr2^-/-and WT mice spleen,The results showed that compared with the non stimulated cells,the stimulation in vitro significantly increased the total TLR4 expression,cytoplasmic and membrane distribution,increased the nuclear translocation of NF-?B,promoted the co-localization of?-arr2 and TLR4,enhanced the activity of B cells,increased the percentage of plasma cells,and produced more Ig G1 and Ig G2A.Compared with the WT-LPS group,the TLR4 expression of B-lymphocytes stimulated by LPS or LPS+IL-4/IFN-?did not change significantly,but the cytoplasmic expression decreased and the membrane expression increased,the NF-?B nuclear translocation increased,the B cell activity increased,the percentage of plasma cells and the production of Ig G1 and Ig G2A increased.The above results showed that deletion of?-arr2 caused more TLR4distribution of B lymphocytes on the cell membrane under LPS and other stimulation.The downstream signal was highly activated and the differentiation of plasma cells increased significantly.suggesting that?-arr2 inhibits its downstream NF-?B activation and prevents LPS and other induced plasma cell differentiation by reducing TLR4distribution on the B lymphocyte membrane.6. CP-25 significantly inhibits joint inflammation and overall indicators in CIA mice.Compared with normal mice,CIA mice had more joint swelling,and the number of joint swelling and arthritis index were higher.CIA mouse joint H&E staining showed obvious pathological changes.CP-25 gavage administration could significantly inhibit joint inflammation and pathological changes,and improve the overall index mentioned above,suggesting that CP-25 obvious inhibitory effect on joint inflammation.7. CP-25 restores the regulation of TLR4 Signal by?-arr2 and reduces the production of plasma cells in CIA mice.Compared with normal mice,the proportion of spleen plasma cells in CIA mice was significantly increased,the expression of TLR4 in B lymphocyte cell membrane and cytoplasm was significantly increased,and?-arr2 was more co-localized with TLR4 and GRK2.CP-25 significantly reduced the plasma cell proportion to near normal level;immunofluorescence detection found that CP-25 reduced the co-localization of GRK2with?-arr2,promoted the colocalization of?-arr2 with TLR4,reduced the distribution of TLR4 cell membrane and made more TLR4 distribution in the cytoplasm.These results suggest that CP-25 may play an anti-inflammatory role by inhibiting GRK2 activity,reducing GRK2's recruitment of?-arr2 in B lymphocytes,and restoring?-arr2's negative modulation of TLR4 Signal.Conclusion:1.?-arr2 prevents plasma cell differentiation and overproduction of antibodies in inflammatory conditions and plays an improved role in the development of rheumatoid arthritis.2.?-arr2 prevents B lymphocytes from differentiating into plasma cells and improves joint inflammation by binding to TLR4 on B lymphocytes,reducing TLR4 cell membrane distribution and inhibiting its downstream signal transduction.3.CP-25 may reduce plasma cell production and antibody production and play an anti-inflammatory role by inhibiting GRK2 activity,reducing the recruitment of?-arr2 in B lymphocytes and restoring the negative regulation of?-arr2 on TLR4 signaling.