Study On The Treatment Of ITP Mice With DSP-IVIG And Its Regulation Of Macrophagic Function

Backgroud:Intravenous Immunoglobulin(IVIG),a first-line treatment for autoimmune diseases(AID),especially Immune Thrombocytopenia(ITP),is prepared from raw plasma.There are differences in protein types and expression levels between male and female plasma,and the prevalence of certain AIDs is disparate in different gender groups.At the same time,the curative effect of IVIG from diverse manufacturers and different batches varies.Are these differences related to sex differences in raw plasma?In other words,are there any differences between IVIG sourced from distinct sex-specific plasma(DSP-IVIG),including IVIG derived from female plasma(Female IVIG),IVIG derived from male plasma(Male IVIG),and IVIG derived from male and female plasma(Mix IVIG)?This study aims to figure out the differences of DSP-IVIG treating ITP and regulating macrophagic functions.There are no relevant research reports at home and abroad.Objective:Clarify the differences in immune regulatory effects of DSP-IVIG on ITP mice and its impact on macrophagic function.Methods:1.(1)Use HuProtTM 20K chips to detect the autoantibody spectrum of DSP-IVIG;(2)Perform bioinformatics analysis on differentially expressed autoantibody proteins.2.(1)Establish an ITP mouse model and evaluate platelets,spleen index,blood routine,pathological sections,etc.(2)DIA proteomics technology was used to analyze the discrepant improvements of DSP-IVIG on ITP mice.(3)Use flow cytometry and fluorescence slice staining to detect mouse immune cells including FcyRs,M1/M2 differentiation,Th,CTL,Thl,Th2,Th17,Treg,Tfh.etc.(4)Luminex was used to determine the cytokines of mouse spleen.3.(1)Establish an inflammatory model using monocytes/macrophages.(2)Establish a method for THP-1 or human peripheral blood monocytes to differentiate into M1/M2 macrophages(THP-1_M1/M2 and HU_Mono_M1/M2).(3)Use flow cytometry to detect the ability of Fc segment of DSP-IVIG binding to the FcγRs,the phagocytosis of monocytes/macrophages,the differentiation of M1/M2 macrophages,the apoptosis,the expression of FcyRs.(4)Luminex was used to detect cytokines.Results:1.There were differences in the autoantibody spectrum of DSP-IVIG.The differences mainly involved the immune regulatory network consisting of T cell line differentiation,T cell receptor binding,IL-17 signaling pathway,FcyRs mediated phagocytosis,cytokines,and chemokines.2.(1)After injecting antibodies(AB),the PLT of mice significantly decreased(P<0.0001)and the spleen index significantly increased(P<0.0001),which significantly improved after IVIG treatment(P<0.0001),and the spleen index of Mix IVIG group was higher than that of Male IVIG group(P<0.05).(2)Compared with PBS groups,the mice of AB groups showed increase in RDW-SD,MPV,PDW,and P-LCR(P<0.05),while decrease in RBC,HGB,HCT,and PCT(P<0.05).Meanwhile,IVIG can improve these changes(P<0.05).(3)The proteomics of ITP mice suggested that we should focus on FcyRs mediated phagocytosis,apoptosis and autophagy,platelet activation,fibrinogen complex,complement cascade reaction,immune response,antigen processing and presentation,immunoglobulin receptor binding,macrophage homeostasis,Th17 cell differentiation and Th17 signaling pathway,white blood cell transmembrane migration,regulation of red blood cell differentiation,NK cell mediated cytotoxicity,and neutrophil extracellular trap formation,chemokines including TGF-β,TNF、IFN-β、IFN-γ and IL13,signaling pathway of JAK-STAT,PI3K-Akt,NF-κB,Toll like receptor,B cell receptor,and T cell receptor.(4)Compared to the AB group,the mice of IVIG group showed weaker FcγRⅢ(CD16),less monocytes(P<0.05)and M1 macrophages(P<0.05),more M2 macrophages(P<0.05).All these changes and the expression of IL-6,IL-27,and IL-13 showed a consistent rule of the Male IVIG was stronger than Mix IVIG in improving ITP(P<0.05).(5)Compared to the PBS groups,the mice of AB groups showed more M1,CTL,Th1,Tfh,Th17,less M2,Th2,Treg.After administration of IVIG,these changes improved although these changes and improvements were not always statistically significant.3.(1)Male IVIG had weaker ability to activate complement and inhibit phagocytosis of monocytes and macrophages(P<0.05).(2)The ability of Fc segment of Male IVIG binding to the surface FcyRs was stronger(P<0.05).(3)Male IVIG enabled macrophages to have higher cell viability and less cell death(P<0.05).(4)Male IVIG can better downregulate CD36,CD68,and CD16(P<0.05),which showed the better inhibition to the pro-inflammatory effect of M1 macrophages.Conclusions:1.There were differences in the autoantibody spectrum of DSP-IVIG.2.DSP-IVIG can effectively improve ITP,but there were different advantages and disadvantages in different detection items.However,Male IVIG seemed to have advantages in improving spleen index and regulating the number and differentiation of Ml and M2 macrophages.3.DSP-IVIG can improve the inflammatory model,but the inhibitory effect of Male IVIG on inflammation was better than that of Female IVIG and Mix IVIG.In summary,this study indicated that there were differences in the immune regulation of DSP-IVIG on ITP mice and its impact on macrophage function was also discrepant.Male IVIG can better improve ITP mice and its inhibition effect on macrophagic inflammation was better.