The Regulatory Mechanism Of MFG-E8 On The Immune Response Of Systemic Lupus Erythematosus-derived Neutrophils And Associated Tissue Damage

Objective:Systemic erythematosus lupus(SLE)is a chronic autoimmune disease,which is characterized by the overproduction of autoantibodies in sera with multisystem and multiple organs of the patients are involved,and the disease has servere impact on the body and psyche.Abnonnal features of the SLE-derived neutrophils,had pay attention by more and more reserchers,which promoted aberrant immune response,such as increased infiltration,increased apoptosis,and released extracellular traps,eventually leading to autoantibody production and immune complex deposition in the tissues and organs.Milk fat globule-EGF factor 8(MFG-E8)is a lipophilic glycoprotein,ubiquitously expressed various organs and cells in a variety of species,which was originally discovered as a bridge molecule that interacted with phagocytes to stimulate the uptake of the apoptotic cells.MFG-E8-deficient mice cause impaired clearance of apoptotic cells,and then these apoptotic cells undergo secondary necrosis and release proinflammatory or immunogenic self-antigens such as high mobility group box-1 protein(HMGB-1)and dsDNA,ultimately leading to autoimmune disease similar to SLE.However,whether MFG-E8 can alleviate tissue damage by improving the aberrant immune reaponse of neutrophils in SLE is still not clear.Therefore,the aim of this study was to explore the mechanism of MFG-E8 regulating the abnormal immune response of neutrophils in innate immunity in SLE.Methods:(1)Determine the levels of MFG-E8 in patients with SLE and Pristane-induced mice:sera from 51 active treated or non-treated young female SLE patients and 35 healthy individuals(20-45 years old),who were admitted to Wuhan No.1 Hospital were used to detect the MFG-E8 levels by ELISA.The level of MFG-E8 in sera from mice treated with Pristane(0.5 mL)for 24 weeks were detected with ELISA.Meanwhile,in the early inflammation phase(Pristane-treatment for 2 weeks),the levels of MFG-E8 in bronchoalveolar lavage fluid(BALF)and peritoneal lavage fluid were determined with ELISA,and the expression of MFG-E8 in lung tissue was tested by western blot.(2)The effect of MFG-E8 on the early lung and peritoneal inflammation in response to Pristane:For lupus induction,female constructed MFG-E8 knockout(Mfge8-/-)mice and age-matched wild-type(WT)littermates received a single 0.5 mL i.p.injection of Pristane.The total BAL leukocyte counts and peritoneal leukocyte counts including CD11b+ cells,neutrophils(polymorphonuclear neutrophils,PMNs)and macrophages at 16 h,5 days,10 days and 14 days after the Pristane injection were detected by flow cytometry.Meanwhile,the levels of cytokines IL-6,IL-12/IL-23p40,TNFa,IL-1?,IL-10,TGF-?1,and chemokine MIP-2 in BALF and peritoneal lavage fluid from mice with Pristane treatment were examined with ELISA.Moreover,the peritoneal monocyte counts in Mfge8-/-and WT mice treated with Pristane for 2 weeks were tested by flow cytometry,and the peritoneal levels of IFN-a were detected with ELISA.As the i.p.Pristane injection fiequently resulted in diffuse pulmonary hemorrhage(DPH)within a few weeks in C57BL/6 mice,the DPH ratio,DPH score and neutrophil infiltration were analyzed by H&E straining,in addition,total protein in BALF was determined with ELISA.Meanwhile,histopathological analysis and inflammatory cell infiltration were evaluated in spleen,liver,and kidney tissues with H&E or PAS staining.(3)Explore the regulatory mechanism of MFG-E8 on neutrophil recruitment in early inflammation of Pristane-induced mice:In the early lung and peritoneal inflammation,neutrophil recruitment was increased in MFG-E8-deficient mice of Pristane treatment.To further inveatgate the effect of MFG-E8 on neutrophil migration,in this study,the chemotaxis efficiency of Mfge8-/-and WT neutrophils were admitted in vivo and in vitro.Bone marrow-derived neutrophils(BMDNs)isolated from the WT and Mfge8-/-mice with Percoll gradients were labeled with fluorescent PKH26 and PKH67 respectively,and the labeled WT and Mfge8-/-BMDNs were i.v.injected together in equal numbers(4×106)into WT mice 1 h after Pristane treatment,and 16 h later,the migration of the transfused BMDNs was observed in the peritoneal cavity and lung by flow cytometry.Meanwhile,the migration of WT and Mfge8-/-BMDNs was conducted in Transwell polycarbonate inserts in vitro.Pretreatment with i.p.injection of recombinant murine MFG-E8(rmMFG-E8,20 ?g/kg),the peritoneal and BAL neutrophil counts from Pristane-induced Mfge8-/-or WT mice were tested by flow cytometry after 16 h.Moreover,with rmMFG-E8(500 ng/ml)pretreatment,the effect of MFG-E8 on neutrophil migration were detected in vitro.The levels of chemokine MIP-2 in BALF and peritoneal lavage fluid from Pristane-treated Mfge8-/-and WT mice were determined with ELISA,and the expression of chemokine receptor CXCR2 on WT and Mfge8-/-BMDNs were determined by flow cytometry.Furthermore,neutrophil counts and the expression of CXCR2 on neutrophil in peripheral blood cells(PBCs)from 52 active treated or non-treated young female SLE patients and 36 healthy individuals.Neutrophils in PBCs from 12 SLE patients and healthy individuals respectively were isolated and cultured in vitro,and pretreated with 500 ng/mL recombinant human(rhMFG-E8),then the expression of CXCR2 on neutrophils were determined with flow cytometry.In Pristane-induced lupus mice,the expression of CXCR2 on BMDNs from WT mice,mice with Pristane treatment for 16 h,mice pretreated with rmMFG-E8 which were induced by Pristane for 16 h,were examined by flow cytometry.(4)The effect of MFG-E8 on apoptosis in Pristane-induced mice:After staining with Annexin V and PI,BAL and peritoneal apoptotic cells from WT and Mfge8-/-mice treated with Pristane for 5 d were detected by flow cytometry.Moreover,the dead cells in lung tissues from WT and Mfge8-/-mice treated with Pristane for 14 days were determined with terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling method(TUNEL),and the expression of apoptosis-related protein,such as Bcl-2,Bcl-xL,and Bax in lung tissues were examined with western blot.Apoptotic neutrophils and phosphatidylserine(PS)-coated carboxylated beads were utilized to determine phagocytosis efficiency of WT and Mfge8-/-activated peritoneal macrophages by immunofluorescence.(5)The effect of MFG-E8 on neutrophil extracellular traps(NETs)release in Pristane-induced mice:NETosis acts as a special form of death for neutrophils,releasing a large number of autoantigens associated with autoantibodies in SLE.The spontaneous NET formation or stimulation with PMA,LPS,and MIP-2 from WT and Mfge8-/-BMDNs were determined with immunofluorescence.The sera from WT and Mfge8-/-mice with Pristane treatment for 24 weeks were collected and utilized to stimulate NET release from WT neutrophils.We further detected the factors which directly stimulated NETs formation,such as anti-NET antibody,circulating immune caomplexes(CICs),TNFa,and MIP-2.The NETs lodged into lung and kidney tissues were examined with immunofluorescence.(6)Detect autoantibody production and immune complex deposit:After Pristane exposure for 24 weeks,the spleen size of WT and Mfge8-/-mice were tested with anatomical analysis,and the pathological analysis of spleen were examined with H&E staining.Moreover,the pathological change of liver,lung,and kidney tissues were observed by H&E or PAS staining.Autoantibody titers(ANA,anti-dsDNA antibody,and ANCA)in sera from WT and Mfge8-/-mice were estimated with immunofluorescence.For pulmonary and glomerular immune complex deposition,IgG and C3 were quantified by direct immunofluorescence.Moreover,urine albumin and Kim-1 were determined with ELISA and urine creatinine were detected by sarcosine oxidase enzymatic(SOE)assay.Results:(1)MFG-E8 is essential for SLE patients and mice with Pristane-induced lupus:The levels of MFG-E8 in sera from patients with SLE and WT mice treated with Pristane for 24 weeks were significantly higher than that in normal control.In the early inflammatory phase(14 days later),the levels of MFG-E8 in BALF and peritoneal lavage fluid of Pristane-induced mice were markedly elevated,and the expression of MFG-E8 in lung tissues from WT mice with Pristane treatment were obviously upregulated compared with WT mice without treatment.These results showed that MFG-E8 played an important role in the pahogenesis of SLE patients ans SLE model mice.(2)MFG-E8 deficiency enhanced early pulmonary and peritoneal inflammation in response to Pristane:The levels of MFG-E8 were increased in patients with SLE and mice with Pristane exposure.However,the early inflammation response was aggravated in Pristane-induced MFG-E8-deficient mice.For example,the numbers of BAL leucocytes,PMNs and macrophages were significantly increased at 16 h,5 days,10 days,and 14 days in the lungs of the Mfge8-/? mice of Pristane treatment than that in WT mice;Meanwhile,the numbers of total cells,CD11b + cells,PMNs were markedly increased in the peritoneal cavity of the Mfge8-/-mice with Pristane treatment,while macrophages were decreased.Moreover,along with a large number of inflammatory cell infiltration in lung and peritoneal cavity,the BAL and peritoneal pro-inflammatory cytokines such as IL-6?IL-12/IL-23p40?TNFa were significantly elevated in Pristane-induced Mfge8-/-mice than that in WT mice,and peritoneal anti-inflammatory cytokines IL-10 and TGF-?1 were obviously downregulated in Mfge8-/-mice of Pristane treatment.After 14 days of injection of Pristane,the number of monocytes in peritoneal cavity of Mfge8-/--mice was significantly increased than that in WT micen,and the secretion of IFN-? was markedly upregulated.As the i.p.Pristane injection for 2 weeks frequently resulted in diffuse pulmonary hemorrhage(DPH)in C57BL/6 mice,in this study,the Mfge8-/-mice developed more complete DPH than WT mice after a 2-week Pristane treatment,displaying a higher DPH prevalence and DPH score,accompanied by neutrophil infiltration was significantly increased and BAL protein concentration sharply elevated.In addition,varying degrees of pathological changes were observed in the liver,kidney,and spleen tissues in Mfge8-/-and WT mice.(2)MFG-E8 modulates neutrophil recruitment by regulating CXCR2 expression on neurophils in Pristane-induced mice:The results of this study showed that the migrated numbers of i.v.injected Mfge8-/-BMDNs into lung tissues and peritoneal cavity were significantly raise compared with WT BMDNs.And the neutrophil chemotaxis was sharply increased enhanced in Mfge8-/-BMDNs than that in WT mice in vitro.After pretreatment with rmMFG-E8,the neutrophil recruitment were significantly reduced in the peritoneal cavities and lungs from WT or Mfge8-/-mice of Pristane exposure compared with the mice that were not treated with rmMFG-E8.Moreover,the chemotaxis efficiency of Mfge8-/-and WT BMDNssin vitiro were markedly decreased after the rmMFG-E8 treatment compared with the BMDNs without rmMFG-E8 treatment when exogenous MIP-2 was added as a chemotactic stimulus.The further explore and the results showed that the levels of MIP-2 were not significantly increased in BALF and peritoneal cavity of Pristane-induced Mfge8-/-and WT mice,however,as the receptor for MIP-2-dependent chemotaxis,its expression CXCR2 on BMDNs from the Mfge8-/-mice was higher than WT BMDNs.In addition,the expression of CXCR2 on neutrophils in PBCs of SLE patients was detected,and and found that an increased in the neutrophil ratio and CXCR2 expression were apparent in the PBCs of SLE patients compared with the healthy controls.Furthermore,the rhMFG-E8 treatment induced a significant downregulation of CXCR2 expression on the neutrophils from SLE patients in vitro.Moreover,the expression of CXCR2 on BMDNs from WT mice of Pristane treatment was higher than the WT without treatment,and pretreated with rmMFG-E8,its expression was decreased.(4)MFG-E8 deficiency enhances accumulation of apoptotic cells in Pristane-induced mice:The proportions of early apoptotic and late apoptotic/necrotic cells were significantly increased in the BALF and peritoneum of Mfge8-/-mice compared with WT mice after Pristane injection for 5 d.After Pristane treatment for 2 weeks,a marked increase in the number of dead cells trapped in lung tissue of Mfge8-/-mice was observed with H&E staining and TUNEL detection compared with WT mice.Moreover,the expression of Bcl-2 and Bcl-xL were markedly decreased while Bax was increased,and Bcl-2/Bax ratio was significantly decreased in the Mfge8-/-lung tissues compared with the WT tissues.Moreover,this study confirmed that the activated peritoneal fge8-/-macrophages showed impairments in phagocytosing the apoptotic neutrophils and phosphatidylserine(PS)-coated carboxylated beads.Therefore,these results indicated that the aggravated accumulation of dead cells in lung and peritoneum was partially caused by the increased neutrophil infiltration and abnormal apoptosis in the Mfge8-/? mice and partially was caused by the inefficient phagocytosis of apoptotic cells by the Mfge8-/-macrophages.(5)MFG-E8-deficient neutrophils are primed to release NETs:Neutrophils isolated fr-om bone marrow of Mfge8-/? mice showed significantly enhanced spontaneous NET formation compared with the WT mice,and released higher NETs stimulated with LPS and MIP-2.In this study,compared with the serum from WT mice of 24-week Pristane treatment,the serum from Pristane-treated Mfge8-/-mice induced increased NET formation.Further detection,and founded that the increase in the CICs,TNF-a levels,and a trend toward increased MIP-2 levels in sera of Mfge8-/-mice than that in WT mice,and the sera levels of anti-NET antibodies in the Mfge8-/--mice were elevated than that in WT mice,which combined with NETs to avoid being degraded.Furthermore,immunofluorescence results showed that after Pristane exposure for 2 weeks,the NETs began to produce NETs in lung tissues of Mfge8-/-mice,and after 24 weeks,a large number of NETs were lodged in the interstitial alveoli of Mfge8-/-mice.Furthermore,after 24 weeks of Pristane stimulation,the NETs were deposited in the kidney tissues around the glomeruli of the Mfge8-/-mice.(6)The lack of MFG-E8 exacerbates the induction of autoantibody production and immune complex deposition following Pristane exposure:After 24 weeks,splenomegaly was observed in the Mfge8-/? mice treated with or without Pristane;in parallel,obvious pathological features were visualized,including enlarged B cell follicles(white pulps),the infiltration of oil droplets and increased numbers of megakaryocytes.These abnormalities were also aggravated in the lung and liver of the Pristane-treated Mfge8-/-mice.H&E staining revealed that the bronchial wall was markedly thickened,ectopic lymphoid cells were abnormally accumulated around the bronchus,and haemosiderin-laden macrophages were deposited within the alveoli;similarly,there was an increased in the number of neutrophils,oil droplets,ectopic lymphoid cells or lipogranulomas in the liver.Mfge8-/-mice showed higher serum anti-nuclear antibody(ANA),anti-dsDNA antibody,and ANCA levels than the WT mice at 24 weeks.There was also an effect on the autoantibody specificity:Pristane-injected WT mice frequently generated an antibody that commonly presented as a c-ANCA antibody(proteinase 3-ANCA,PR3-ANCA):in contrast.most of the Pristane-injected Mfge8-/-mice predominantly exhibited p-ANCA antibodies(myeloperoxidase-ANCA,MPO-ANCA).MFG-E8-deficient mice also showed an increase of immune complex deposit in lung and kidney tissues.In addition,the lack of MFG-E8 resulted in the deposition immune complexes within the interstitial alveoli,but the immune complexes deposited within blood vessels of WT lung tissue.Moreover,the Pristane-treated Mfge8-/-mice showed a marked enhancement in the albuminuria/creatinine ratio and a trend toward increased Kim-1 levels.Conclusion:MFG-E8 attenuates aberrant immune response of SLE-derived neutrophils through regulating the expression of CXCR2,which decreases neutrophil recruitment,promoted the phagocytosis of apoptotic neutrophils,and suppresses NETs release,and ultimately results in a significant downregulation of the type-I IFN response,autoantibody(ANA,anti-dsDNA antibody,and ANCA)synthesis and immune complex deposit,protecting lupus-related tissue damage.Therefore,MFG-E8 may represent a therapeutic target to control early inflammation to efficiently treat environmental exposure lupus.