Role Of Notch Signal Pathway In Breg Development And Allergic Responses

The balance of immune system depends on effector cells and regulatory cells.Regulatory T (Treg) cells, the counterpart of effector T cells, have been shown to playimportant roles in T cell-mediated immnue responses. B cells were thought to play centralroles in humoral immunity by producing antibodies and cytokines, and could also presentantigens and exert various functions essential for optimal immune responses. However,there is also a B cell subset that show a negative regulatory function in immune response.Recent studies revealed that a specific B cell subsets potently inhibit immuneresponses through the secretion of IL-10, which were termed B10cells. B10cells havebeen demonstrated to suppress immune responses and many immune-related diseases,such as experimental autoimmune encephalomyelitis(EAE), inflammatory bowel disease,lupus, contact hypersensitivity(CHS), inflammation and tumorigenesis. Recently studiesrevealed the phenptype of Bregs that producing IL-10. In2008, Tedder’s group showedthat in spleen B cells of mouse, the majority of IL-10producing B cells were found in theCD1dhiCD5+CD19+subset of WT mice. The IL-10producing B cells are only a smallportion of CD19+B cell in B cell subsets, but CD5+CD19+CD1dhighcell subsets are theprimary IL-10-producing B cells. The phenotype characterization of CD5+CD1dhighCD19+B10cells is an important advances in the field of Bregs research, which laid thefoundation for further investigation on the Bregs development and function. There are several signaling pathways participating in the development and functionof Bregs, including of TLR signaling, CD40signaling and BCR signaling. IL-21is also animportant regulatory molecular for Bregs. However, the precise mechanisms of Bregsdevelopment has not been elucidated so far.Notch signaling pathway has been shown to regulate cell subset development andbi-functional differentiation. Notch signaling not only regulates T/B cell differentiation,CD4/CD8T cell differentiation, but also regulate macrophages M1/M2polarization andDC immunization/tolerance. Thus it’s reasonable to speculate that Notch signalig mightalso be involved in Bregs development and in the control of positive/negative response ofB cells. Notch signaling pathway consists of ligands, receptors, downstream signaltransduction molecules and regulatory factors. RBP-J is a nuclear transcriptional factor,which is needed for the activation of all of the four Notch receptors. We have RBP-Jconditional knockout mice, in which the Notch signaling could be inactivated throughknocking out of RBP-J.Our previous study suggested that the number of marginal zone B cells (MZB) andT2-MZP cell decreased in gene-modified mice, in which the RBP-J gene wereknocked-out in B cells. Recent studies suggested that Bregs derived from T2-MZP cells.Thus we speculate that Notch signaling pathway plays a key role in Bregs developmentand function.Objectives: To exlpore the role of Notch signal pathway in Bregs development, and therole of Bregs in contact hypersensitivity and oral immune tolerance, by using thetransgenic mice in which the RBP-J gene were knocked-out specificially in B cells.Methods: We obtained the mice with the genotype as CD19-cre-RBP-Jflox/floxby crossingthe CD19-cre mice and RBP-J-flox mice. Flow cytometry was used to analyze thefrequencies of CD19+Cd5+CD1dhighBreg. Spleen cells from mice with different genotypeswere stimulated with different stimuli in vitro, and the frequencies of IL-10-producingB10and B10pro cells were analyzed by FCM. Then the model of contact hypersensitivityand oral tolerance were constructed to detect the role of Notch signaling in Breg function.Results: (1) Mice with RBP-J specificially knockout in B cells were obtained. Compared with theheterozygous mice, the frequencies of CD19+CD5+CD1dhighBregs were significantlyreduced in CD19-cre-RBP-Jf/fmice. After the cell surface and intra-cellular staining, FCMdata showed that the numbers of B10progenitor cells and B10cells were significantlylower than that of the control group.(2) In CHS model, the change of ears thickness of CD19-cre-RBP-Jf/fmice were moreobvious than that of heterozygous mice, and the HE staining displayed more pronouncedinflammation, indicating that B-cell specific RBP-J deficiency harboring more severeallergic reaction.(3) In the oral tolerance models, the serum IgE levels in CD19-Cre-RBP-Jf/fmice weresignificantly higher than the heterozygous mice, indicating that absence of Notch signalingattenuate oral tolerance.Conclusions: Notch signaling pathway regulates Breg development, and plays a pivotalrole in regulating contact hypersentivity and oral tolerance.