Roles And Mechanisms Of MTOR Signaling Pathway In The Maturation And Antibody Production Of B Lymphocytes In Mice

Background and aimsThe mechanistic target of rapamycin (mTOR) is a highly conserved serine/threonine protein kinase. There are two different kinds of complexes in the body, including mTORC1 (mTOR complex 1) and mTORC2 (mTOR complex 2). mTORC1 has five components:mTOR,the catalytic subunit of the complex; regulatory-associated protein of mTOR (Raptor); mammalian lethal with Sec 13 protein 8 (mLST8); pralinerich AKT substrate 40kDa(PRAS40); and DEP-domain-containing mTOR-interacting protein(Deptor). As a central regulatory molecule, it could integrate signals in and out of cells, thereby regulating cell metabolism, growth, proliferation and survival. Bacterial derivatives of rapamycin can inhibit mTORCl, and can be used as a potential immunosuppressant. Tuberous sclerosis complex (TSC) is the most signoficant upstream regulator of mTORC1.It could activate GTP enzyme (GTP activating protein, GAP),enabling GTP decomposing into GDP in the small GDP enzyme Rheb, thereby in the inactiviated state. As immediate upstream of the mTORCl, the activation of Rheb could activate mTORC1, resulting in activation of its downstream target of phosphorylation of ribosomal S6 kinase (S6K) and the transcription initiation factor 4E binding protein 1 (4E-BP1), thus promoting cell ribosomal protein translation and the occurrence.mTORC2 includes mTOR, mLST8/GβL, Rictor (rapamycin-insensitive companion of mTOR), mSinl (SAPK interacting protein 1) and PRR5/Protor. mSinl, Rictor and PRR5/Protor are specific subunits in mTORC2. The main difference between them is that mTORC2 doesn’t contain raptor but contains the newly discovered protein Rictor (rapamycin-insensitive companion of mTOR).In mammalian cells, deletion of Rictor will lead to the damage and loss of function of mTORC2 complexes. The function of mTORC2 still needs to be clarified. In yeast and mammalian cells, it has been demonstrated that mTORC2 plays key roles in various biological processes, including cell survival, metabolism, proliferation and cytoskeleton organization.mTORC2 was able to phosphorylate hydrophobic motif (HM) of AKT (Ser473 site), so that making it in fully activation. AKT is one of key kinases regulating cell survival.More and more evidences indicated that mTOR plays an important role in the regulation of immune response. mTOR could identificate and integrate signals inside and outside the cell,thus regulate the immune microenvironment.In addition, it plays a key role in regulating neutrophils, mast cells, dendritic cells, T cells and B cells.B lymphocytes are mainly found in the blood,lymph node, spleen, tonsils and other mucosal tissue. B cells produce antibodies (immunoglobulins) and present antigen,becoming the main participants in non-specific and specific humoral immune response. The function of B cell is closely related to its development,which could be divided into two phases:the antigen-dependent phase and antigen-independent phase. The phase of differentiation from the early hematopoietic progenitors to mature B lymphocytes is carried out in the bone marrow,which is independent of the stimulation of antigen, so that it is called the antigen independent phase. We can identify five different stages in this phase,which are early progenitors B cell (Pro-B), late Pro-B cell, Precursor B cell(Pre-B),immature B cell (Immature B) and mature B cell. The antigen-dependent phase appeared accompanied the B cells response to antigens,which generally occurs in the peripheral immune organs. After encountering antigen, B cells become activated and differentiate into memory B cells or plasma cells. When B cells leave the bone marrow, its function was not yet mature and it located in the transitional stage 1 (T1). When flowing into the spleen accompanied blood, B cell gradually developed into the transition stage 2 (T2), mature (Mature B) and marginal zone B cells (marginal zone B cell, MZB). T2 b cells differentiate to mature follicular B cells in the follicular of spleen and lymphatic node, while differentiate in to marginal zone B cells in the spleen.B cell differentiation failure and proliferation disorder, or plasma cell dysfunction will result in deficiency of antibody synthesis or secretion, which are clinically known as the primary humoral immunodeficiency syndrome. For different antibody deficiency, abnormal B cells maturation or response to antigen stimulation are different. Typical clinical features of this kind of disease are suppurative bacterial infection repeatedly,and if not accept active treatment, Patients will die before reaching adulthood. There are plenty of reports indicated that B cells are closely related to various autoimmune diseases,such as rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), idiopathic thrombocytopenic purpura (ITP), Hashimoto’s thyroiditis,Graves’ disease,ankylosing spondylitis,myasthenia gravis,insulin-dependent diabetes mellitus (DM) and RF negative adolescent arthritis,etc.As an important immunoregulatory molecule,mTOR plays a key role in humoral immunity and formation of germinal center. It is reported that mTOR participates in humoral immune response, formation of germinal center in the spleen, maturation of antibody affinity, somatic hypermutation (SHM) and class switch recombination (CSR) and many other aspects.It is indicated that pten,a tumor-inhibitory gene in the upstream of mTORC1, was involved in differentiation of B1 and marginal zone B cell population, as well as the proliferation and survival of B cell in vivo. Similarly, another study found that tscl, an upstream inhibitor of mTORCl,was involved in the maturation of B cells and formation of marginal zone B cells (MZB)’s.Concerning mTORC2, the researchers found that, deletion of sinl result in the reduces of differentiation from progenitor B cells into immature IgM+ B cells, the increases in levels of il-7r and rag recombinase (ragl and rag2), thus the elevated rearranging activity of progenitor B cell V (D) J.Since mTORC2 regulates the phosphorylation of AKT,we infer that AKT is the main point in the process of the RAG expression regulated by mTORC2. deletion of Akt2 and sinl in progenitor B cells appear similar increases in Rag. Meanwhile konckout AKT in B cells confirmed its role in formation of MZB and B1 and survival of FOB. Thus, mT0RC2 may regulate the survival and proliferation of mature B cell, as well as the development of marginal zone B cells and B1 cell.However, with respect to Raptor and Rictor, which were the respective core protein of mTORC1 and mTORC2, there is no literature reported regarding whether they participate in the development of B lymphocytes. Thus,this study aims to create mice with B lymphocyte-specific deletion of raptor and rictor using the cre-loxp system to carry out related researches.Methods1.Using Cre-loxp system, we have successfully created mice with B lymphocyte-specific deletion of raptor and rictor respectively. The effect of deletion was identified by PCR and agarose gel electrophoresis for mice genotypes, while western blot for protein expression in B cells.2. The impact of rictor deletion on mouse B lymphocyte development was analysed by flow cytometry.3. ELISA assay to determine the effect of raptor and rictor activation on the antibody production of B lymphocytes in mice respectively.4.Analysis on the effect of rictor deletion on mice B lymphocytes apoptosis by flow cytometry. 5.qPCR technology to examine the effects of rictor knockout on the expression of bcl-2, rag2 and il-7r gene in B lymphocytes.Results1.The mice model of B lymphocyte specificity knockout raptor was constructed successfully.We generated mice (B cells-raptor KO) with a conditionally ablated raptor gene in B lymphocytes using a Cre expression cassette under the control of the CD 19 promoter. Western blotting showed that the expression of Raptor was decreased in B cells-raptor KO mice, but not in control littermates. mTORC1 activity was monitored by assaying the level of phospho-S6(s235/236). The specific Reduction of phospho-S6(s235/236) in B lymphocytes was observed in B cells-raptor KO mice. In summary, the mice model of B lymphocyte specificity knockout raptor was constructed successfully.2. Specific deletion of raptor in B lymphocyte affects the humoral immune function of B cells.Then we investigate how the knockout of raptor influence the humoral immune function of B cells. And we found that lackinng raptor in B cells can weaken the immune response to OVA, thymus dependent antigen, suggesting that the generation of OVA-specific antibody was reduced.3.The mice model with B lymphocyte-specific deletion of rictor was constructed successfully.We generated mice (B cells-rictor KO) with a conditionally ablated rictor gene in B lymphocytes using a Cre expression cassette under the control of the CD 19 promoter. Western blotting showed that the expression of Rictor was decreased in B cells-rictor KO mice, but not in control littermates. mTORC2 activity was monitored by assaying the level of phospho-AKT(s473). The specific reduction of phospho-AKT(s473) in B lymphocytes was observed in B cells-rictor KO mice. In summary, the mice model of B lymphocyte specificity knockout rictor was constructed successfully.4. B lymphocyte-specific deletion of rictor affects the maturation of B cells and the increase of rag2 and il-7r.There is no significant difference for the number of B cells in the bone marrow between of B cells-rictor KO mice and control mice..However further analysis of the various stages of B lymphocytes in bone marrow showed that Pre B cells increased significantly, while Immature B cells decreased obviously. The results suggested that B lymphocytes may be partially blocked in Pre B cells stage and cannot provide a stage of further differentiation and maturation.Then we found that rag2 and il-7r were up-regulated in the knockout mice. B cells of KO mice in spleen and peritoneal cavity have significantly reduced compared to control mice. Further analysis of all B cell subsets in the spleen showed that there were no significant differences of Transitional B, Follicular B cells or Marginal zone B cells between the knockout mice and the control. While B1 cells decrease in the peritoneal cavity.5. The deletion of rictor in B cells resulted in increased apoptosis of B cells and decreased expression of bcl-2.We used the AnnexinV staining, and detected the percentage of Annexin V-FITC positive cells by flow cytometry, results showed that spleen B cells in knockout mice had an increased cell apoptosis compared with control. We investigated the molecular mechanism of how rictor deletion effects apoptosis. We used qPCR to analyze the gene expression level of bcl-2, then we found that bcl-2 expression was reduced.6. B lymphocyte-specific deletion of rictor affects the humoral immune function in vivo.Then we investigate how the deletion of rictor influence the humoral immune function of B cells. And we found that lacking Rictor in B cells can weaken the immune response of OVA, thymus dependent antigen, which represented the generation of OVA specific antibody reduced.Conclusion1.We have successfully constructed two kinds of mice models for raptor and rictor specific knockout in B cells respectively and using PCR and western blot to verified the knockout effect.2.mT0R singaling pathway effects the maturation of B cells. In B cell-rictor KO mice, the Pre B cells in bone marrow increased, accompanied by increases in the mRNA level of rag2 and il-7r. B1 cells reduced,and peripheral mature B cells reduced, but the distribution of spleen B cell subsets was not changed.3. The knockout of rictor resulted in increases in apoptosis of spleen B lymphocytes, accompanied by decreases in the mRNA level of bcl-2.4.mTOR singaling pathway effects the humoral immune function of B cells.We found that the levels of specific antibody immuned to OVA (OVA-IgG,OVA-IgM) were decreased in raptor knockout mice, and they were the same as the levels of OVA-IgM and OVA-IgG in rictor knockout mice.