IgD Enhances The Release Of Neutrophil Extracellular Traps（NETs） Via FcδR In Rheumatoid Arthritis Patients

Rheumatoid arthritis（RA）is a common autoimmune,high morbidity,and disability disease with numerous inflammatory cells infiltrating the interstitial of articular cartilages and bones.Neutrophils play an important role in regulating RA which participates in the pathogenesis of RA.Neutrophil extracellular traps（NETs）are released by activated neutrophils and can generation of modified autoantigens to induce autoimmunity reaction,which causes inflammation of the synovium.There are a large number of immunoglobulins and immune complexes in the peripheral blood and synovial fluid of RA patients,which can promote the recruitment of neutrophils and the formation of NETs.But the role of immunoglobulins that induce the NET formation in the peripheral blood and synovial fluid of RA is largely unknown.Our group previously found the correlation between high levels of IgD expression and clinical diagnostic indicators of RA.Abnormally high levels of IgD participate in the development of RA by activating T cells.IgD can bind to Fc receptors（FcδR or IgDR）that were originally discovered on T cells in human peripheral blood.Our group further found that the expression of FcδR was also higher in RA patients than in healthy controls.At the onset of the RA inflammatory response,eighty to 90%of the leukocytes infiltrating the synovial fluid are neutrophils.Whether FcδR is expressed on the neutrophils,and whether high levels of IgD in RA patients are associated with the formation of NETs and exacerbate synovial inflammation in RA?This study intends to further explore the correlation between the formation of NETs and the levels of IgD,anti-CCP,and FcδR in RA patients;Can IgD specifically promote the formation of NETs through FcδR on neutrophils?Our group synthesized the IgD-Fc-Ig fusion protein that can target IgD-FcδR with the potential to treat RA（patent license numbers:ZL201510600762X,ZL201810692531X）.Our group’s previous results showed that the IgD-Fc-Ig has a therapeutic effect on arthritis in animal experiments.Can IgD-Fc-Ig play a role in improving synovial inflammation in RA by blocking IgD binding to FcδR on neutrophils,reducing the formation of NETs?So,blood samples were collected from RA patients and healthy controls,to clarify the correlation between Cit H3 and IgD,anti-CCP and FcδR in RA patients;to explore the effect of IgD on the formation of NETs;confocal microscopy,scanning electron microscopy and q PCR were used to detect the effects of IgD-Fc-Ig fusion protein on the formation of NETs induced by IgD.The model of AA rat was established,IgD-Fc-Ig was administered subcutaneously to observe the function of neutrophils,and changes in the function of fibroblast-like synoviocytes（FLS）to confirm that IgD-Fc-Ig can improve synovial inflammation in AA rats.This study intends to clarify the role of IgD-FcδR in promoting the release of NETs in RA and to provide the experimental basis for the development of IgD-Fc-Ig as an innovative drug for the highly selective treatment of high IgD RA.ObjectiveTo clarify the correlation between Cit H3 and IgD,anti-CCP and FcδR in RA patients;to clarify the effect of IgD promotes the release of NETs from neutrophils by binding to FcδR,and NETs stimulated with IgD can promote the abnormal proliferation of FLS;to study the relevant mechanisms to reveal the therapeutic effect of IgD-Fc-Ig on improving RA synovial inflammation and reducing the formation of NETs by blocking IgD-FcδR.MethodsBlood samples were collected from RA patients and healthy controls,and plasma was collected by centrifugation.Isolating neutrophils and detecting the expression of FcδR on neutrophils by flow cytometry;ELISA was used to detect the levels of Cit H3,IgD,and anti-CCP in the plasma of RA patients;Flow cytometry was used to detect the expression of FcδR（CD16/CD15/FcδR）and NETs（MPO/Cit H3）induced by IgD in healthy controls;Western blot was used to detect the expressions of MPO,NE and Cit H3proteins induced by IgD in healthy controls;Imaging flow cytometry to detect the effect of IgD-Fc-Ig on NETs（MPO/Cit H3）generation induced by IgD in healthy people;q PCR was used to detect the m RNA expression of MPO,NE and PAD4 in neutrophils induced by IgD;CCK-8 detects the proliferation of FLS after co-culture of NETs with RA FLS;the expression of MHCⅡon RA FLS detected by flow cytometry.The model of AA rats was established,to study the effect of IgD-Fc-Ig on the overall indexes and pathological changes of spleen and ankle joints in AA rats;ELISA detection the levels of IgD,anti-CCP2,and Cit H3 in the plasma of AA rats;to explore the effect of IgD-Fc-Ig on neutrophil function and the mechanism of symptom relief in AA rats;the effect of IgD-Fc-Ig on the expression of ROS and apoptosis of neutrophils were detected by flow cytometry;the effect of IgD-Fc-Ig on the infiltration of neutrophils in ankle joints of AA rats was detected by immunofluorescence;q PCR detection the m RNA expression of MPO,NE,and PAD4 in rat spleen;CCK-8 was used to detect the proliferation of FLS after co-culture of AA FLS and neutrophils from different groups.Results1.Clinical indicators and data of RA patients44 RA and physical examination were collected,the ratio of male to female was12:32,clinical indicators including age（age,years）,rheumatoid factor（RF,IU/ml）,C-reactive protein（CRP,mg/L）,Erythrocyte sedimentation rate（ESR,mm/h）and（anti-CCP,RU/ml）.The mean±standard error were 57.45±1.62,68.85±10.18,21.22±3.36,34.44±4.83,858.33±101.99 respectively.2.Correlation between IgD,Cit H3,anti-CCP,and FcδR on neutrophils and in plasma of RA patients and healthy controlsCompared with healthy controls IgD（43.860±7.708μg/m L）and Cit H3（2.268±0.211ng/m L）,the plasma levels of IgD（87.475±34.385μg/m L）and Cit H3（5.8289±1.692ng/m L）were significantly higher in RA patients;compared with healthy c-ontrols,the percentage of FcδR on neutrophils in RA patients was significantly increased.The data showed that s IgD and Cit H3 demonstrated a significant corre lative relationship（r=0.4889,P=0.0008）,the association between FcδR and Cit H3（r=0.5546,P=0.0007）,Cit H3 and anti-CCP（r=0.7505,P<0.0001）were significantl y correlated as well.3.The effect of IgD on neutrophils in healthy controlsThe expression of FcδR（CD16⁺CD15⁺FcδR⁺）and NETs（MPO⁺Cit H3⁺）were detected by flow cytometry.IgD stimulated neutrophils in healthy controls,flow cytometry results showed that IgD（3,10μg/m L）could significantly increase the percentage of FcδR（CD15⁺CD16⁺FcδR⁺）and NETs（MPO⁺Cit H3⁺）in healthy controls.4.The effect of IgD on the protein expression of MPO,NE,and Cit H3 in healthy controlsCompared with the control group,MPO,NE,and Cit H3 protein expressions were increased in IgD（3μg/m L）group.5.The effect of IgD-Fc-Ig on neutrophils in healthy controls induced by IgDThe neutrophils isolated from the peripheral blood of healthy controls were divided into IgD（3μg/m L）,IgD（3μg/m L）+IgD-Fc-Ig（1,3,10μg/m L）groups.The flow cytometry and imaging flow cytometry results showed that IgD（3μg/m L）group significantly promoted the expression of FcδR（CD15⁺CD16⁺FcδR⁺）and NETs（MPO⁺Cit H3⁺）in healthy controls,and IgD-Fg-Ig（10μg/m L）group could down-regulate the expression of FcδR（CD15⁺CD16⁺FcδR⁺）and NETs（MPO⁺Cit H3⁺）in healthy controls.6.The effects of IgD-Fc-Ig on MPO,NE,PAD4 m RNA expression of neutrophils induced by IgD in healthy controlsNeutrophils isolated from the peripheral blood of healthy controls were treated with IgD（3μg/m L）and IgD（3μg/m L）+IgD-Fc-Ig（1,3,10μg/m L）,and the RNA of neutrophils were extracted.Compared with the control group,IgD-Fc-Ig（3,10μg/m L）down-regulated NE m RNA induced by IgD,IgD-Fc-Ig（10μg/m L）down-regulated the expression of PAD4 m RNA induced by IgD.7.Effects of IgD-Fc-Ig on the expression of MPO,NE,Cit H3 fluorescence and the morphology of neutrophils in healthy control neutrophils induced by IgDCompared with the control group,confocal microscopy and scanning electron microscopy showed the formation of NETs in the IgD（3μg/m L）group.And IgD-Fc-Ig（10μg/m L）can significantly reduce NETs generation.8.The effect of NETs from IgD induced neutrophils on FLS function and the role of IgD-Fc-Ig8.1 Effects of NETs on FLS proliferationCompared with the control group,NETs stimulated with IgD（3μg/m L）and PMA（20nmo L）groups could induce FLS proliferation,and NETs stimulated with IgD（3μg/m L）with IgD-Fc-Ig（3,10μg/m L）group could down-regulate proliferation of FLS.8.2 The effect of NETs on the expression of MHC II on FLSCompared with the control group,the NETs from IgD（3μg/m L）and PMA（20nmo L）groups could significantly increase the expression of MHCII（HLA-DR+）on FLS.NETs from IgD（3μg/m L）with IgD-Fc-Ig（10μg/m L）group down-regulated the expression of MHCⅡ.9.Effects of IgD-Fc-Ig on inflammatory index assessment of ratCompared with the AA group,the IgD-Fc-Ig group had significantly increased weight,improved global scoring,decreased paw volume,arthritis index,and swollen joint count.10.The effect of IgD-Fc-Ig on the destruction of the rat ankle boneCompared with the normal group,the AA rats showed tissue swelling and bone erosion.After treatment with IgD-Fc-Ig（9mg/kg）,the X-ray scores of the rats were decreased,and the tissue swelling and bone erosion were improved in the rats.11.The effect of IgD-Fc-Ig on the changes of serum IgD,anti-CCP2,and Cit H3 levels in AA ratsCompared with the normal group,the serum levels of IgD,Cit H3,and anti-CCP2were significantly increased in the AA group;With the administration of IgD-Fc-Ig（9mg/kg）and etanercept（3mg/kg）,s IgD,Cit H3,and anti-CCP2 levels were significantly reduced in AA rats.12.Effects of IgD-Fc-Ig on the spleen and joint pathology in AA ratsThe histopathology of the spleen,which was characterized by the density of periarterial lymphatic sheath,the emergence of germinal centers,marginal zone hyperplasia,and red pulp proliferation was significantly alleviated by IgD-Fc-Ig（9mg/kg）treatment.Etanercept treatment exhibited similar effects to that observed with IgD-Fc-Ig treatment.Results from the H＆E staining and Safranin O-fast green cartilage staining of the ankle joint sections showed synoviocyte proliferation and inflammatory infiltration,pannus formation,as well as cartilage erosion,were significantly attenuated following administration of IgD-Fc-Ig（3,9mg/kg）.The effects of IgD-Fc-Ig（3,9mg/kg）on AA rats were similar to the effects of the positive control drug etanercept.13.Effects of IgD-Fc-Ig on ROS production and apoptosis in neutrophils of AA ratsIn comparison with the normal group,the model group showed a significant increase in ROS levels from neutrophils.Administration of IgD-Fc-Ig（3,9mg/k g）significantly reduced the neutrophil ROS production.The levels of apoptosis n eutrophils in AA rats were significantly decreased,IgD-Fc-Ig（9mg/kg）administra tion could promote neutrophil apoptosis.Etanercept（3mg/kg）could downregulate the expression of ROS and increase the apoptosis rate of neutrophils in AA rats.14.The effect of IgD-Fc-Ig on the expression of MPO,NE,and PAD4 m RNA in the spleen of AA ratsThe spleen RNA was extracted from different groups.Compared with the normal group,IgD-Fc-Ig（9mg/kg）could significantly down-regulate the expressions of MPO,NE,and PAD4 m RNA in AA rats.Etanercept（3mg/kg）exhibited similar effects to that observed in IgD-Fc-Ig treatment groups.15.The effect of neutrophils on the proliferation of FLS in AA ratsCCK8 results showed that compared with the control group,the proliferation of AA FLS increased significantly after coculturing with neutrophils from the AA model rats group,and neutrophils from IgD-Fc-Ig（9mg/kg）and etanercept groups could significantly decrease the abnormal proliferation of FLS.Conclusions1.The result showed that Cit H3 and IgD,anti-CCP demonstrated a significant correlative relationship in the plasma of RA patients,the association between Cit H3 and FcδR was significant as well,indicating IgD-FcδR may be involved in neutrophil activation and NETs formation.2.IgD can induce the activation of neutrophils,increase the expression of FcδR,and the overproduction of NETs.IgD-Fc-Ig can neutralize abnormally expressed IgD-FcδR and decreased the activation of neutrophils and the formation of NETs induced by IgD.3.IgD-Fc-Ig has a therapeutic effect on AA rats and reduces the plasma levels of IgD,Cit H3,and anti-CCP2 in AA rats.IgD-Fc-Ig can improve the overall indexes of AA rats,reduced ROS production of neutrophils,promote the apoptosis of neutrophils,and down-regulated the expression of PAD4 m RNA.4.There is a correlation between IgD and the formation of NETs in RA patients.IgD-Fc-Ig has the potential to be a new biological agent that can alleviate the symptoms of RA by blocking IgD-FcδR and reducing the formation of NETs with high IgD levels.