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细胞稳态紊乱（浆母细胞增加和B细胞记忆异常），最初归因于B细胞过度活跃。GWAS数据进一步支持了与BCR信号相关的SLE风险基因（Lyn、BLK、BANK1、PTPN22等）。与此形成鲜明对比的是，我们的研究小组发现异常的BCR信号，特别是最近被其他人证实的SLE患者的记忆B细胞。这里发现蛋白酪氨酸激酶（PTK）的磷酸化减少，即脾脏酪氨酸激酶Syk与蛋白丝氨酸激酶Akt参与B细胞的存活。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细胞特异性的，还是反映了活化后或静止记忆B细胞的调节特征。我们的工作假设是SLE B细胞具有内在的BCR信号异常，包括异常的PTK和PTP（即SHP-1和PTEN）活性，这些异常与Akt依赖通路的生存调节受损相关，这是维持自身反应性记忆B细胞的机制。或者，外部因素如细胞因子（I型干扰素）引起这些功能变化。该项目将研究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BCR激活的减少代表激活后状态。在这方面，该项目将描述控制记忆B细胞激活和稳定状态的机制在SLE和其他炎症条件（感染，炎症性风湿病）作为对照。该研究将在对照和SLE B细胞中对BCR相关的PTKs（如Btk、PI3K、PLC-g2、BLNK）、细胞内Ca释放、PTPs（如SHP-1、PTEN）以及BCR共受体（如CD22、CD19、CD45）进行全面的磷酸化分析。功能性BCR激活分析将结合与所研究的SLE患者的遗传BCR风险基因的直接比较。重要的是，除了BCR之外，B细胞激活途径（CD40或TLRs）及其组合将解决SLE B细胞激活减弱是否仅限于BCR或更普遍的特征。进一步研究前I型干扰素、TNF、IL-1和抗炎细胞因子（IL-10、IL-35）的能力及其对SLE和对照B细胞中BCR信号传导和Akt依赖生存的影响。在生存研究方面，本项目将分析上述条件下的p-Akt和下游转录因子（即Bim、Bcl-2、Foxo-1）的失衡。该项目将描述SLE中涉及B细胞活化和生存的内在因素的作用，这些因素可能是自身免疫性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.