Both Notch1 And Its Ligands In B Cells Promote Antibody Production

BackgroundAs a part of humoral immunity,B cells and antibodies play vital roles in infectious diseases,tumorigenesis,transplant rejection and autoimmune diseases.Antibodies are produced by plasma cells,which are differentiated by B cells.The development and activation of B cells are regulated by many signaling pathways,such as the phosphoinositide 3-kiase(PI3K)signaling pathway,the nuclear factor of kappa B(NF-?B)signaling pathway,the B-cell receptor(BCR)signaling pathway and the Notch signaling pathway.The function of Notch signaling pathway in B cells development and activation is one of the current research hotspots.In 1917,for the first time,geneticist Thomas Hunt Morgan and his colleagues described the Notch gene in fruit flies with defective wings.Notch is highly conserved transmembrane protein that is structurally divided into extracellular(NEC),transmembrane region(NTM)and intracellular domain(NICD).The mammalian Notch receptor family has four members,Notch 1-4,which can bind to five ligands(Delta-like 1,3,4 and Jagged 1,2).Activation of the Notch signaling pathway initiated by the ligand-receptors of adjacent cell surfaces bind to each other.The current study has confirmed that Notch 1,an important member of the Notch family,play a vital role in T cell development and differentiation,for example,in the mouse bone marrow,if Notch 1 gene is knocked-out,The thymus T cell development is blocked,accompanied by the production of a large number ectopic B cells.Notch signaling pathway can promote the development of marginal zone(MZ)and is very important for the advanced stage of B cell development and differentiation.In addition,ligands of Notch 1 also play an important role in the activation of B cells.These study show that Notch 1 may play crucial part in the proliferation of plasma cells and the antibodies production.Therefore,the main content of this study is the effect of Notchl and its ligands on the development of B cells and the production of antibodies.ObjectivesTo investigate whether Notch1 and its ligands have an effect on antibody production.Methods1)To investigate the expression of Notch 1 during the mouse B cell activation process,the splenocytes of mice were isolated and activated with LPS in vitro.The expression of Notch 1 during B-cell activation was detected by Fluorescence activated cell sorting(FACS);2)To Obtain B cell-specific knocked-out Notch 1 model mice.Mice expressing Cre recombinase under control of the CD19 promoter(CD19cre),and the Notchl flox/flox mice(Notch1f/f)were in which loxP sites flank exon 1 of the Notchl gene.Notchlf/f mice were crossed to CD19cre mice to produce Notch1f/f,CD19creNotch1f/+and CD19creNotch1f/f mice.3)The protein expression levels of Notchl receptor and its ligands Dkll and Jag1 in splenocytes of mice were detected by Western blotting.4)Enzyme-linked immune sorbent assay(ELISA)was used to examine the role of Notchl signaling pathway in antibody secretion in vitro B-cell activation system;5)Notch1f/f,CD19cre Notch1f/+and CD19cre Notch1f/f group mice received a s.c.injection of 125 mg myelin oligodendrocyte glycoprotein(MOG)35-55 peptide(Mimotopes)emulsified 1:1(v/v)in CFA containing 4 mg/ml Mycobacterium tuberculosis H37Ra,to the base of the tail and both flanks.Pertussis toxin(300 ng pertussis toxin in PBS)was injected i.p.at the time of induction,and a second dose was administered 3 days later.The same volume of CFA and pertussis toxin was injected into each mouse in the control group.ELISA was used to detect the level of antibody in serum.Results1)Notchl expression was up-regulated on the surface of LPS-activated B cells on the day 1,reached the peak on the day 2,visibly decreased on the day 3.These results suggest that Notchl expresses mainly during B cells are activated;2)The level of Notchl expression was lower in CD19hi B cells from CD19creNotch1f/f mice than that from Notch1f/f mice and CD19creNotch1f/+ mice.3)The splenic B cells of Notchlf/f CD19creNotchlf+and CD19creNotchlf/f mice were selected and stimulated with LPS in vitro.The results showed that the antibody levels of Notch1f/f and CD19creNotch1f+ mice were significantly increased,While there was almost no change in the level of CD19creNotch1f/f antibody.The ELISA results showed that B cell-specific depletion of Notchl decreased the secretion of antibodies induced by LPS in B cells;4)The splenocytes of Notchlf/f,CD19creNotchlf/+and CD19creNotchlf/f mice were stimulated by LPS in vitro.The FACS detection results showed that the antibody levels of the three genotypes were significantly increased.B cell-specific depletion of Notchl did not affect antibody production in LPS-stimulated splenocytes;5)EAE model was induced in Notchlf/f,CD19creNotch1f/+and CD19creNotch1f/f mice.ELISA assay showed that B cell-specific depletion of Notch1 did not reduce antibody production in EAE mice;6)The FACS detection and Western blotting assay showed that Notch1 was also expressed on other cells except B cells in spleen,Dll1 and Jagl were expressed on B cells,Notch1 in other cells may promote antibody production by binding its ligands such as Jagl in B cells.ConclusionsBoth Notchl and its ligands in B cells play an important role in antibody production.