| Systemic lupus erythematosus(SLE)is an autoimmune,inflammatory and connective tissue disease which can involve multiple organs in the body,and is more common in women.The disease is highly heterogeneous,with complex and varied clinical manifestations,slow onset and strong concealment.In the SLE population,the incidence of childhood disease accounts for about 10%-20%,and has a rising trend year by year.Because of its rapid development,easy to lead to important organ involvement,clinical symptoms are often more dangerous than adults,prognosis is worse and mortality is higher,so the childhood onset of SLE has attracted more attention.Dysfunction of T and B lymphocytes in patients with SLE leads to dysfunction of cellular immunity,humoral immunity and complement system,abnormal secretion of cytokines in peripheral blood,production of a variety of autoantibodies and deposition of immune complexes in tissues and organs,resulting in the involvement of multiple systems throughout the body,including skin and mucous membrane,serosa,kidney,joint and nervous system.At present,the main treatment is corticosteroids and/or non-specific immunosuppressants,but for SLE patients,in addition to the physical damage caused by the side effects of these conventional treatments,the continuous activity of the disease,the adverse effects of repeated treatment and irreversible end-stage organ damage also bring serious mental stress to the patients.It has become a very thorny social problem.Due to a series of toxic and side effects caused by conventional treatment of SLE,in order to improve the therapeutic effect and patient compliance,biological targeting agents developed for the pathogenesis of SLE are emerging continuously.Most of the studies are targeted at B lymphocyte stimulator,surface molecules and immune tolerance,which have been applied in the clinical treatment of SLE,such as Bailimu monoclonal antibody,Rituximab and so on.It is well known that inducible costimulatory molecule(ICOS)is an important member of the CD28 costimulatory molecule family.It is usually expressed on activated T cells and NK cells,which mainly plays an important role in humoral immunity and is closely related to autoimmune diseases.ICOS can specifically bind with its inducible costimulatory molecule ligand(ICOSL)to mediate the interaction between T and B cells,promote T cell proliferation,secrete pro-inflammatory factors and participate in inflammatory diseases.At the same time,ICOS can promote T cells to differentiate into follicular helper T cell(Tfh),which in turn promotes the proliferation,survival and antibody secretion of B cells in germinal center(GC).It have confirmed that T cells of SLE patients express higher levels of ICOS than healthy people,suggesting that ICOS/ICOSL play a certain role in the pathogenesis of SLE.In conclusion,we believe that blocking antibodies targeting the ICOS/ICOSL signal pathway may inhibit the pathogenesis of SLE.In view of the important role of ICOS/ICOSL signaling pathway in inflammatory diseases,especially autoimmune diseases,this study detected the expression of ICOSL in childhood onset of SLE and discussed its clinical significance combined with laboratory detection indexes.Furthermore,using MRL/lpr lupus mice as an experimental animal model to simulate the pathogenesis of SLE.To investigate the effect of ICOS monoclonal antibody on ICOS/ICOSL signal pathway and its possible protective effect on SLE disease,and to explore the possible protective mechanism of ICOS monoclonal antibody on renal ferroptosis in mice with lupus nephritis(LN),so as to find a new target for immune intervention of SLE pathological damage and provide scientific basis for clinical treatment of SLE.Part Ⅰ Expression and clinical significance of ICOSL in peripheral blood of children with SLEObjective:To detect the expression of ICOSL in peripheral blood of children with SLE and healthy children so as to explore the clinical significance of ICOSL expression in peripheral blood of children with SLE.Methods:The peripheral blood samples of 48 children with SLE(cSLE group)and 42 healthy children(NC group)were collected from September 2018 to December 2020 in our hospital.The cSLE group was further divided into inactive group(Inactive cSLE)and active group(Active cSLE)according to SLEDAI-2000 score.The expression of ICOSL in peripheral blood of children with SLE and healthy children was detected by ELISA method,and the levels of C3,C4 and IgG in peripheral blood of children with SLE were detected by automatic biochemical detector.Results:1.The expression of ICOSL in peripheral blood was detected by ELISA method.There was no statistically significant difference in the expression of ICOSL between NC group and cSLE group(including Active cSLE and Inactive cSLE)(P>0.05).Further analysis showed that there was no statistical difference between the Inactive cSLE group and the NC group,while there was a statistical difference between the Active cSLE group and the NC group(P>0.05,P<0.05,respectively).In cSLE,there was a significant statistical difference in the level of ICOSL between the Active cSLE group and the Inactive cSLE group(P<0.05),and the concentration of ICOSL in peripheral blood of the Active cSLE group was higher than that of the Inactive cSLE group.The ICOSL values measured were(112.64±27.34)ng/L in the Active cSLE group and(95.37±25.36)ng/L in the Inactive cSLE group.2.Detection and analysis of C3,C4 and IgG expression in blood showed that there was a positive correlation between ICOSL and IgG in the Active cSLE group(P<0.05),but there was no correlation between ICOSL and IgG in the Inactive cSLE group(P>0.05).Simultaneously,there were no correlation between ICOSL and C3 or C4 in the Active cSLE group and Inactive cSLE group(P>0.05).Conclusion:The expression of ICOSL exists in the peripheral blood of both children with SLE and healthy children.The expression of ICOSL is significantly high in active SLE children and is related to the expression of IgG in peripheral blood,suggesting that ICOS/ICOSL signal pathway is involved in the pathogenesis of children with SLE and is related to disease activity.Part Ⅱ Study on the protective effect of ICOS monoclonal antibody on SLEObjective:To explore the intervention effect and possible mechanism of ICOS monoclonal antibody in MRL/lpr lupus erythematosus mouse model,and compare the intervention effect with ICOS monoclonal antibody homotype control antibody,so as to provide new ideas and theoretical basis for biological targeted therapy of SLE.Methods:1.Female C57BL/6 mice were selected as control group(Control group),female MRL/lpr mice were selected as disease model group(MRL/lpr group)and intervention group(Anti-ICOS group).Anti-ICOS group was treated with ICOS monoclonal antibody,Control group and MRL/lpr group were treated with ICOS monoclonal antibody homotype control IgG2b antibody.Each group of mice were treated by intraperitoneal injection of antibodies.2.The levels of serum creatinine,blood urea nitrogen,urinary microalbumin and urinary creatinine were detected by automatic biochemical detector to evaluate the changes of renal function in mice.3.The pathological changes of kidney,lung and joint were detected by HE staining,and the appearance changes of spleen and lymph nodes were observed.4.Immunofluorescence was used to detect the deposition of IgG immune complex in the kidney of mice in each group.5.The level of anti-dsDNA antibody in peripheral blood of mice in each group was detected by ELISA double antibody sandwich method.6.Multifactor detection kit and flow cytometry were used to detect T cytokines in the peripheral blood of mice in each group.7.The proportion of Tfh cells in spleen of each group was detected by flow cytometry,and the expression level of transcription factor mRNA of Tfh cells in spleen of each group was detected by RT-qPCR.8.Western Blot was used to detect the expression of cytokine-related inflammatory signal pathway proteins in each group.Results:1.General situation:within 30 days after the intervention of each group,the mice in the Control group were intervened with the same type control antibody of ICOS monoclonal antibody,and the survival rate was 100%.The survival rate of the mice in the ICOS control group was only 50%,while that in the ICOS group was 75%.Compared with MRL/lpr group,depilation,ulcer lesions and lymph node enlargement in Anti-ICOS group were inhibited to a certain extent.2.Changes of renal function:Urinary protein:urinary microalbuminuria/creatinine(albumin creatinine ratio,ACR)in MRL/lpr group was significantly higher than that in Control group(P<0.001),and gradually aggravated with time.There was no significant difference in ACR between AntiICOS group and MRL/lpr group within 1 week after antibody intervention,but ACR in MRL/lpr group increased progressively with time from the 2nd week,but there were no significant increase in ACR between Anti-ICOS group and MRL/lpr group(P<0.01,P<0.001,P<0.001,respectively).Creatinine and urea nitrogen:the levels of serum creatinine and blood urea nitrogen in MRL/lpr group were significantly higher than those in Control group(P<0.001),and the average levels of serum creatinine and urea nitrogen in Anti-ICOS group were significantly lower than those in MRL/lpr group(P<0.05,P<0.01).3.Histopathological changes:Kidney:the basic structure of kidney in Control group was normal.In MRL/lpr group,the increase of glomerular matrix,the formation of crescents,the infiltration of a large number of monocytes around renal interstitium and small vessels,and the formation of protein tubules in renal tubules were observed.The pathological changes of renal parenchyma and interstitium in Anti-ICOS group were significantly less than those in MRL/lpr group.Lung:the alveolar and interstitial structure of mice in Control group were normal;in MRL/lpr group,pulmonary interstitial congestion and edema,a large number of inflammatory cell infiltration,alveolar septum thickening and alveolar compression occlusion were observed,and a large number of inflammatory cell infiltration around bronchioles were seen;in Anti-ICOS group,pulmonary interstitium was slightly congested,alveolar structure was still intact,alveolar septum thickened slightly,alveoli were not obviously compressed and occluded,and there were a small amount of inflammatory cell infiltration around bronchioles.Joint:the articular surface of Control group was smooth and the structure of synovium was normal;synovium of MRL/lpr group was significantly thickened with proliferation of small blood vessels,with a large number of synovial cell proliferation and inflammatory cell infiltration,and hyperplastic synovial attachment was also seen on the diseased joint surface;smooth joint surface of Anti-ICOS group showed mild synovial thickening with a small amount of synovial cell proliferation,and no obvious vascular proliferation was found under the synovium.Changes of spleen and lymph nodes:the spleen and lymph nodes of Control group were smaller than those of MRL/lpr group,and the spleen and lymph nodes of Anti-ICOS group were smaller than those of MRL/lpr group.The spleen visceral body ratio of MRL/lpr group was significantly higher than that of Control group(P<0.001),and the spleen viscera body ratio of Anti-ICOS group was significantly lower than that of MRL/lpr group(P<0.05).4.IgG deposition in kidney:there was no obvious IgG deposition in Control group,the red fluorescence intensity in kidney tissue of MRL/lpr group was higher,and IgG deposition was more serious,the red fluorescence intensity of kidney tissue of Anti-ICOS group was weaker than that of MRL/lpr group,and the deposition of IgG was less than that of MRL/lpr group.5.The change of anti-dsDNA antibody:the titer of anti-dsDNA antibody in MRL/lpr group was significantly higher than that in Control group(P<0.001),while that in AntiICOS group was significantly lower than that in MRL/lpr group(P<0.05).6.Expression of cytokines:serum cytokines IL-2,IL-4,IL-5,IL-6,IL-9,IL-10,IL-17A,IL-21,IL-22,TNF-α and IFN-γ in mice of each group were detected.The results showed that IL-6,IL-21,IL-10,IFN-γ and TNF-α in MRL/lpr group were significantly higher than those in Control group,and the difference were statistically significant(P<0.001,P<0.01,P<0.05,P<0.01 and P<0.001 respectively).Compared with MRL/lpr group,IL-6 and IL21 of Anti-ICOS group decreased significantly,while IL-10 tended to increase,and the difference were statistically significant(P<0.05,P<0.05 and P<0.01,respectively).There were no significant difference in IL-2,IL-4,IL-5,IL-9,IL-17A and IL-22 among the three groups.7.The proportion of Tfh cells and the expression of transcription factors in spleen:The proportion of spleen Tfh cells in MRL/lpr group was significantly higher than that in Control group(P<0.001),while that in Anti-ICOS group was lower than that in MRL/lpr group(P<0.001).Expression of Tfh-related transcription factors:compared with Control group,the mRNA expression of Tfh-related transcription factors Bcl6,c-Maf,TCF-1,LEF-1 and NFATc1 in spleen of MRL/lpr group were significantly higher(P<0.001,P<0.001,P<0.001 and P<0.01,respectively),and that of Anti-ICOS group were significantly higher than that of MRL/lpr group(P<0.01 and P<0.01,respectively),and the expression of Tfhrelated transcription factors in spleen of Tfh group were significantly higher than that of MRL/lpr group(P<0.001,P<0.01 and P<0.01,respectively).Conclusion:ICOS monoclonal antibody can improve and inhibit lupus-like symptoms,histopathology and related immunological indexes in MRL/lpr lupus mice,suggesting that ICOS/ICOSL signal,Tfh cells and their related factors play an important role in the pathogenesis of SLE,and the regulation of ICOS/ICOSL signal pathway can ameliorates lupus symptoms in SLE.Part Ⅲ Study on the mechanism of ICOS monoclonal antibody on LNObjective:To explore the possible mechanism of ferroptosis in the pathogenesis of LN in MRL/lpr mice and the protective effect of ICOS monoclonal antibody on LN in MRL/lpr mice,so as to provide theoretical basis for clinical comprehensive understanding and further study of LN.Methods:1.Kidney immunohistochemistry:the expression of GPX4,the key molecule of ferroptosis in the kidney of mice in each group was detected.2.Detection of malondialdehyde(MDA),a metabolite related to renal ferroptosis:the kidneys of mice in each group were cracked and ground,and the content of MDA in kidneys was determined by colorimetry.3.Detection of ferroptosis related molecules in kidney:Western Blot and RT-qPCR techniques were used to detect the protein and mRNA expression of ferroptosis-related molecules in the kidney of mice in each group.Results:1.Immunohistochemical changes of kidney:the expression area of GPX4 in MRL/lpr group was significantly lower than that in Control group(P<0.001),while the expression area of GPX4 in Anti-ICOS group was significantly larger than that in MRL/lpr group(P<0.001).2.The content of MDA in kidney:the content of MDA in MRL/lpr group was significantly higher than that in Control group(P<0.001),and the content of MDA in AntiICOS group was lower than that in MRL/lpr group(P<0.001).3.Expression of ferroptosis related molecules in kidney:The expression of ferroptosis related protein:compared with Control group,the expression of GPX4,SLC7A11 and TFR in MRL/lpr group were down-regulated(P<0.001),while the expression of ACSL4,PTGS2 and FTH1 were up-regulated(P<0.001).Compared with MRL/lpr group,the expression of SLC7A11,GPX4 and TFR in Anti-ICOS group were up-regulated(P<0.01),while the expression of ACSL4,PTGS2 and FTH1 were downregulated(P<0.001,P<0.01 and P<0.001,respectively).The expression of ferroptosis related protein at molecular level:compared with Control group,the expression of GPX4,SLC7A11 and TFR mRNA in MRL/lpr group decreased significantly(P<0.001),while the expression of ACSL4,PTGS2 and FTH1 increased,and there were statistical difference(P<0.01,P<0.001 and P<0.001,respectively).Compared with MRL/lpr group,the expression of mRNA of GPX4,SLC7A11 and TFR in Anti-ICOS group increased significantly(P<0.01,P<0.01 and P<0.001,respectively),while the expression of mRNA of AC SL4,PTGS2 and FTH1 decreased significantly(P<0.01).The expression of mRNA were consistent with the expression trend of the corresponding proteins mentioned above.Conclusion:ICOS monoclonal antibody has a certain protective effect on the pathogenesis of LN in MRL/lpr mice,which may be achieved by regulating the ferroptosis in the kidney of mice. |
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