Immunomodulatory Effects Of IgD-Fc-Ig Fusion Protein On Lupus Nephritis Of MRL/lpr Mice

Systemic lupus erythematosus?SLE?is a kind of life-long systemic autoimmune disease,which seriously impair liver,kidney,lung,heart and other organs.Its pathological feature is abnormal activation of T,B cells and overproduction of autoantibodies.Although the pathological mechanism of SLE is still unclear,many studies have clues that the pathogenesis of SLE may be related to genetic factors,environmental factors and hormones.On the one hand,these factors induce inherent immune abnormalities such as apoptosis defects,on the other hand,the immune cells?such as T cells,B cells,dendritic cells and macrophages?and their produced aberrant cellular active substances,which also aggravate the disorder of adaptive immunity,and eventually lead to a mass of autoantibodies,complement activation and tissue damage.Due to the complexity of its pathogenesis,there is no specific drug in the clinical treatment of SLE,mainly to alleviate clinical symptoms and immunosuppression.Commonly used drugs are hormones,immunosuppressive agents and some biological drugs.Long-term application of these drugs will produce many adverse reactions.Therefore,it is of great clinical significance to understand the pathogenesis of the disease,explore more concrete therapeutic targets and innovative drugs for SLE.Immunoglobulin D?Ig D?is one of many immunoglobulins found in human body fluids in 1965,including secretory Ig D?s Ig D?and membrane-bound Ig D?m Ig D?.Most of the secretory Ig D are distributed in human peripheral blood and lacrimal gland.Some studies have revealed that s Ig D combines with membrane receptor,Ig D receptor?Ig DR?,which can modulate the tolerance and activation of B cells to enhance the immune surveillance,augment the innate immune response of basophils,promote the transformation and generation of mature B cells,and play an irreplaceable role in immunoregulation.Clinical studies have found that the levels of serum s Ig D of SLE patients,rheumatoid arthritis?RA?patients,and primary Sjogren's syndrome?p SS?patients are uplifted and the levels of s Ig D in chronic inflammation and B-lymphoma are also increased,suggesting that the excessive levels of s Ig D may contribute to the pathogenesis of these diseases.In addition to the expression of Ig DR on T cells and basophils,Ig DR was also detected on the synoviocytes?FLS?of RA patients.Moreover,Ig D can arouse the increase of Ig DR on CD4+T cells,and Ig D-Ig DR reaction can boost the interaction of homologous T and B cells and the production of antibodies.Previous in vitro experiments showed that Ig D could induce proliferation of peripheral blood mononuclear cells in RA patients and healthy controls and secretion of TNF-?and IL-6.The expression of CD69,a surface marker of early activation of T cells,CD154?CD40L?as a costimulatory signal molecule also upregulated after stimulation by s Ig D.Besides,s Ig D increased the percentage of CD19^-CD138⁺cell in peripheral blood.All of these data convince that Ig D play a pivotal role in the activation of T and B cells.Ig D-Fc-Ig?DG?was successfully fused with human Ig D-Fc and Ig G-Fc by using Ig DR as the target in the laboratory?national patent has been obtained?,aiming to specifically block the Ig D-Ig DR pathway.The paper has published consolidate that the DG has immunomodulatory and anti-inflammatory effects.Besides,DG can inhibit the proliferation and activation of T cells induced by s Ig D,reduce the level of inflammatory cytokines.In order to investigate the therapeutic effect of DG on SLE,we selected the spontaneous lupus model MRL/lpr mice to observe the therapeutic effect of DG on MRL/lpr mice.MRL/lpr mice have asystematic lymphocyte proliferation,emerging obvious lymphadenopathy with joint inflammation,skin damage and other signs.The level of autoantibody is significantly increased,and the kidney has lupus nephritis like lesions,which is in accord with the characteristics of human SLE disease.In this study,the therapeutic effect of DG on lupus nephritis in MRL/lpr mice and its immunoregulatory effect on lymphocyte function were studied,and the mechanism of Ig D involved in SLE was further clarified,so as to provide experimental basis for the development of DG as an innovative drug for the treatment of SLE.OBJECTIVETo explore the pathological mechanism of regulating immune function of Ig D in SLE,and to evaluate the therapeutic effect of DG on lupus erythematosus like MRL/lpr mice.METHODMRL/lpr mice were randomly divided into the following groups:model group,DG?2,4,8mg/kg?group,Prednisone?Pre,5mg/kg?group,and BALB/c mice as the normal control group,9 mice in each group.Pre was administrated by gavage.According to the body weight?0.2ml/kg?of mice in DG group,the drug was injected subcutaneously twice a week;In Pre group,the drug was given by gavage?0.2ml/kg?every day.The duration of administration was 6 weeks.Normal and model group were given saline by subcutaneous injection for parallel control.Every week,observed and recorded the abnormal changes of behavior activity,hair state,excreta and other physical signs of animals before and after administration;The changes of urine protein was detected with visual protein test paper;Kidney and spleen pathology were observed with HE staining;Expression of complement 3?C3?of mouse kidney immune complex was detected by immunohistochemistry;The index of thymus and spleen were calculated;The urine protein was quantitative determined by BCA method;CCK-8 method was used to detect the proliferation of B and T lymphocytes induced by LPS and Con A;The serum levels of immunoglobulin D?Ig D?,anti-ds DNA,tumor necrosis factor-??TNF-??,interleukin-6?IL-6?and interferon-??IFN-??in mice were detected by ELISA;The ratio of T lymphocyte subsets,B cells and plasma cells in spleen lymphocyte suspension were analyzed with flow cytometry:T cells?CD3⁺?,activated T cells?CD4⁺CD69⁺?,naive T cells?CD4⁺CD62L⁺?,TFH?CD4⁺CXCR5⁺PD-1⁺?,Th17?CD4⁺IL-17A⁺?,Treg?CD19^-CD138⁺?,T cells expressing costimulatory molecule ligand?CD4⁺CD154⁺?,marginal B cells?CD19⁺CD21⁺CD23^-?,follicular B cells?CD19⁺CD21^-CD23⁺?,mature B cells?CD19⁺Ig D⁺Ig M^-?,immature B cells?CD19⁺Ig D⁺Ig M^-?;TFH cell specific transcription factor Bcl6 and cytokine IL-21m RNA in mouse spleen were detected by q-PCR;Western blot for the expression of JAK2,p-JAK2,STAT3 and p-STAT3.RESULTS1. DG could reduce the proteinuria level of MRL/lpr miceSemi quantitative method was used to measure the weekly proteinuria level of mice after administration,and the hair state and behavior activity of mice were observed and recorded.The results indicated that at the 4th week of administration,the impact of DG on the proteinuria,hair state and behavior activity of MRL/lpr mice was significantly different from that of the control group,and BCA method was used to quantify the collected urine.The results showed that DG?2,4,8mg/kg?could significantly decrease the level of proteinuria.2. DG could relieve the pathology of lupus nephritis and reduce the kidney deposition of immune complex C3 in MRL/lpr miceHE staining was used to observe the renal pathology of mice.Compared with the normal group,model group exhibited the glomerular hyaline degeneration and sclerosis,mesangial cell proliferation,a large number of inflammatory cells infiltration around the capillaries and glomerulus,interstitial fiber proliferation,fibrous crescent formation;DG?2,4,8mg/kg?group can significantly lessen the infiltration of glomerular inflammatory cells and decrease the mesangial cell proliferation,remit inflammatory situation,ease glomerular fibrosis;Moreover,The results of C3 deposition in mouse kidney manifest that DG?2,4,8mg/kg?group also reduce C3 deposition of renal immune complex while C3 deposition expression diffused in the model group;3. DG significantly reduced spleen index and thymus index of MRL/lpr mice,and alleviated spleen pathologyCompared with the normal group,the spleen and thymus volume of MRL/lpr mice were enlarged.The spleen index and thymus index of the model group were increased dramatically while the spleen index and thymus index of the DG?2,4,8mg/kg?group were notably lower than those of the model group.HE staining of spleen displayed the proliferation of follicles,diffuse hyperplasia of white pulp and the expansion of germinal centers in the model group.DG?2,4,8mg/kg?administration group decreased the volume of spleen and improved the pathological condition compared with the model group.4. DG could inhibit the proliferation of thymus T cells and spleen B cells in MRL/lpr miceCCK-8 method was used to detect the proliferation of T and B lymphocytes in mice.The results revealed that the proliferation of T and B lymphocytes in the model mice was significantly enhanced.Administration of DG?4,8mg/kg?could significantly inhibit T cells induced by Con A and DG?2,4,8mg/kg?could significantly inhibit B cell proliferation induced by LPS.5. DG could regulate T and B cell subsets of MRL/lpr miceFlow cytometry was used to analyze T,B cell subsets.The results showed that the proportion of T cells(CD3⁺),activated T cells?CD4⁺CD69⁺?,Th17 cells?CD4⁺IL-17A⁺?,TFH cells?CD4⁺CXCR5⁺PD-1⁺?and plasma cells?CD19^-CD138⁺?in spleen of MRL/lpr mice were increased remarkably in model group.DG?2,4,8mg/kg?group can modulate aberrant CD3⁺T cells,CD4⁺CD69⁺T cells,Th17 cell,TFH cell and plasma cell subsets.Compared with normal group,the proportion of naive cells?CD4⁺CD62L⁺?and total B cells?CD19⁺?decreased in model group.DG?2,4,8mg/kg?increased naive cells?CD4⁺CD62L⁺?slightly,but the difference was not statistically significant.DG?2,4,8mg/kg?significantly recover the proportion of CD19⁺B cells in spleen of MRL/lpr mice.While DG?2,4,8mg/kg?had no conspicuous effect on Th cell expressing?CD19⁺Ig D^-Ig M⁺?subsets.6. DG could decrease serum secretory Ig D?s Ig D?and anti-ds DNA antibody levels in MRL/lpr miceThe serum s Ig D and anti-ds DNA levels of MRL/lpr mice were detected by ELISA.The results turn out that the serum s Ig D and anti-ds DNA levels of MRL/lpr mice were significantly higher than those of normal mice,and the serum s Ig D level of DG?2,4,8mg/kg?group was distinctly lower than that of model group.The level of anti-ds DNA antibody in the serum of MRL/lpr mice was decreased remarkably in the DG?2,4,8mg/kg?group.7. DG could decrease the level of TNF-?,IL-6 and IFN-?in serum of MRL/lpr miceThe results manifested that the levels of TNF-?,IL-6 and IFN-?in the serum of MRL/lpr mice increased dramatically,DG?2,4,8mg/kg?reduced the level of TNF-?in the serum of MRL/lpr mice,and the level of IFN-?in the serum of MRL/lpr mice was downregulated significantly by DG?2,4,8mg/kg?.IL-6 also decreased remarkably in DG?2,4,8mg/kg?group,the difference has significance.8. DG could reduce the m RNA expression of transcription factor Bcl6 and cytokine IL-21 in spleen of MRL/lpr miceq-PCR was used to detect the m RNA expression of Bcl6 and IL-21.The results showed that the m RNA levels of Bcl6 and IL-21m RNA in spleen of MRL/lpr mice were up-regulated,while the administration of DG?4,8mg/kg?could inhibit these processes.9. DG could down-regulate the levels of p-JAK2/JAK2 and p-STAT3/STAT3 in the kidney of MRL/lpr miceKidney tissue of MRL/lpr mice was used for Western blot.The results of Western blot indicated that the expression of p-JAK2 and p-STAT3 was significantly decreased in the DG?2,4,8mg/kg?group compared with the model group.DG?2,4,8mg/kg?group have no significant impact on expression of JAK2 and STAT3.CONCLUSIONS1.Subcutaneous administration of DG can decrease the increased s Ig D level in serum of MRL/lpr mice,relieve the pathology of spleen and kidney,reduce the deposition of renal immune complex,lessen the level of anti-ds DNA and exert therapeutic effect on lupus nephritis of MRL/lpr mice.2.DG can adjust the immune disorder of T and B lymphocytes in spleen of MRL/lpr mice,and reduce the levels of IL-21,IL-6,TNF-?and IFN-?in serum or tissues of mice,which may correlate with the inhibition of JAK2 and STAT3 phosphorylation.