Disturbed B cell homeostasis of B cells in SLE: Delineation of intrinsic signaling abnormalities

在系统性红斑狼疮(SLE)中发现了许多B细胞稳态的紊乱(浆母细胞增多和异常记忆B细胞)，并初步归因于B细胞过度活动。与BCR信号相关的SLE风险基因(LYN、BLK、BANK1、PTPN22等)的Gwas数据提供了进一步的支持。与之形成鲜明对比的是，我们的研究小组发现了异常的BCR信号，特别是在最近被其他人证实的SLE患者的记忆B细胞中。这里发现蛋白酪氨酸激酶(PTK)的磷酸化程度降低，即脾酪氨酸激酶Syk与蛋白丝氨酸激酶Akt的磷酸化程度降低，参与了B细胞的生存。这种失衡与蛋白酪氨酸磷酸酶(PTP)活性增强有关，但尚不清楚这一发现是SLE B细胞特有的，还是反映了受调控的激活后或静止的记忆B细胞的特征。我们的工作假设是，SLE B细胞存在固有的BCR信号异常，包括PTK和PTP(即SHP-1和PTEN)活性异常，这些异常与Akt依赖的通路作为维持自身反应性记忆B细胞的机制而受损的生存调节相互关联。或者，细胞因子(I型干扰素)等外在因素是导致这些功能变化的原因。该项目将解决SLE患者的B细胞耐受性是否受到基于遗传预定义风险分子的BCR信号通路的内在异常的干扰，或者SLE B细胞的BCR活性降低代表激活后状态。在这方面，该项目将描述控制SLE和其他炎症性条件(感染、炎症性风湿病)的记忆B细胞激活和稳定状态的机制。这项研究将利用对照和SLE B细胞对BCR相关的PTKs(例如BTK、PI3K、PLC-G2、BLNK)、细胞内钙释放、PTPs(例如SHP-1、PTEN)以及BCR辅助受体(例如CD22、CD19、CD45)进行全面的磷酸化分析。功能性bcr激活分析将与所研究的SLE患者的遗传bcr风险基因进行直接比较。重要的是，除BCR外，B细胞激活途径(CD40或TLR)及其组合将解决SLE B细胞激活减弱是否限于BCR或更一般的特征。此外，还将研究促炎症细胞因子(IL-10、IL-35)和促炎性细胞因子(IL-10、IL-35)在SLE和对照B细胞中的能力及其对BCR信号和Akt依赖生存的影响。在生存研究方面，该项目将分析上述条件下的p-Akt以及转录因子(即Bim、Bcl2、FOXO-1)的下游失衡。该项目将描述参与系统性红斑狼疮B细胞激活和存活的内在因素的作用，这些因素可能是自身免疫B细胞的分子机制，并将为新的治疗方法提供信息。

A number of disturbances in B cell homeostasis (increased plasmablasts and abnormal memory B cells) have been identified in systemic lupus erythematosus (SLE) and initially ascribed to B cell hyperactivity. Further support has been provided by GWAS data with SLE risk genes linked to BCR signaling (Lyn, BLK, BANK1, PTPN22 etc.). In striking contrast, our group found abnormal BCR signaling in particular in memory B cells from SLE patients confirmed recently by others. Here a diminished phosphorylation of protein tyrosine kinases (PTK), i.e. spleen tyrosine kinase Syk versus protein serine kinase Akt involved in survival of B cells was found. This dysbalance was related to enhanced protein tyrosine phosphatase (PTP) activity while it remains unknown if this finding is specific of SLE B cells or reflect a characteristic of regulated post-activation or quiescent memory B cells. Our working hypothesis is that SLE B cells have intrinsic abnormalities of BCR signaling including abnormal PTK and PTP (i.e. SHP-1 and PTEN) activity that are interconnected with impaired survival regulation by Akt dependent pathways as mechanisms to maintain autoreactive memory B cells. Alternatively, extrinsic factors such as cytokines (type I interferons) are causing these functional changes.The project will address whether B cell tolerance in SLE patients is disturbed by instrinsic abnormalities of BCR signaling pathways based on genetically predefined risk molecules OR diminished BCR activation of SLE B cells represent a post-activation status. In this regard, the project will delineate the mechanisms to control the activatory and steady state of memory B cells in SLE and other inflammatory conditions (infections, inflammatory rheumatic diseases) as controls. The study will undertake comprehensive phosphorylation analyses of BCR associated PTKs (e.g. Btk, PI3K, PLC-g2, BLNK), intracellular Ca release, PTPs (e.g. SHP-1, PTEN), as well as BCR co-receptors (e.g. CD22, CD19, CD45) using control and SLE B cells. Functional BCR activation analyses will be combined with direct comparison with genetic BCR risk genes of the SLE patients studied. Importantly, other than BCR B cell activation pathways (CD40 or TLRs) and combinations thereof will address if the diminished SLE B cell activation is restricted to BCR or a more general characteristic. Further the capacity of pro- (type I interferons, TNF, IL-1) and antiinflamatory cytokines (IL-10, IL-35) and their impact on BCR signaling and Akt dependent survival in SLE and control B cells will be studied. With regard to survival studies, the project will analyze p-Akt under the conditions mentioned above and the downstream dysbalances of transcription factors (i.e. Bim, Bcl-2, Foxo-1). The project will delineate the role of intrinsic factors involved in B cell activation and survival in SLE which candidate as molecular mechanisms of autoimmune B cells and will inform about new therapeutic approaches.