Investigating the role of the actin cytoskeleton in regulating TLR-mediated B cell responses during inflammatory challenges

B细胞是维持免疫稳态的主要调节因子。正确的B细胞激活是通过B细胞受体（BCR）对抗原的特异性识别以及激活的T细胞提供的共同刺激来保证的。细胞因子信号传导或toll样受体（TLR）的参与也影响B细胞的功能。异常的B细胞活化可能导致自身免疫性疾病的发展，如Wiskott-Aldrich综合征（WAS）。WAS是一种免疫缺陷，与高水平的IgA和IgE抗体相关，对感染的易感性增加，免疫介导性疾病（包括结肠炎）的风险增加。疾病的原因是基因突变导致WAS蛋白（WASP）或WASP相互作用蛋白（WIP）的表达缺陷，这两种蛋白都是肌动蛋白细胞骨架的调节因子。正如我们和其他人最近所证明的那样，缺乏WASP或WIP的B细胞对BCR和TLR信号都具有超增殖性。通过这里描述的项目，我们希望了解并进一步阐明在炎症背景下，这些肌动蛋白调节因子在TLR信号传导中的作用。本研究的初步结果表明，在体外对TLR4刺激的反应中，WIP缺陷的B细胞（WIP KO）分泌大量的促炎细胞因子IL-6和TNF-a。同时，WIP KO B细胞显示浆细胞特异性转录因子（TF） IRF4和TF T-bet的表达减少，这可能影响抗体和细胞因子的产生。在脓毒症的急性环境或对共生微生物群的反应中，促炎细胞因子可以成为自我毁灭的。我们的初步结果确实表明，未免疫WIP的KO小鼠肠道中B细胞活化和IgA产生增加，并且在只有B细胞缺乏WIP的嵌合体中早期发生结肠炎。因此，向浆细胞分化的缺陷以及B细胞产生IL-6和TNF-a的增加可能最终增强了WAS的自身免疫表型。因此，我们假设在B细胞中，WIP参与TLR4参与后调节TF表达、抗体产生和细胞因子分泌的信号通路，从而影响肠道内的免疫稳态。为了验证这个假设,我们计划(1)分析肌动蛋白监管者的角色在制品和黄蜂TLR4信号感应,内吞作用,细胞因子和抗体生产的B细胞在体外和体内,(2)分析潜在的分子机制确定TF表达和B细胞命运决定,(3)分析体内不平衡细胞因子的影响生产、制品的浆细胞分化和IgA抗体分泌KO B细胞在脓毒症和结肠炎的发病。通过研究这些目标，我们将进一步了解像WAS这样的自身免疫性疾病，这可能有助于开发新的治疗方案，不仅适用于WAS，也适用于其他免疫相关疾病，如炎症性肠病。

B cells are a major regulator in maintaining immune homeostasis. Correct B cell activation is ensured by specific recognition of antigen by the B cell receptor (BCR) together with co-stimulation provided by activated T cells. Cytokine signalling or the engagement of Toll-like receptors (TLR) also influence B cell function. Aberrant B cell activation might lead to the development of autoimmune diseases, as seen in Wiskott-Aldrich Syndrome (WAS). WAS is an immunodeficiency associated with high levels of IgA and IgE antibodies, increased susceptibility to infections and heightened risk of immune-mediated disorders, including colitis. Cause of disease are genetic mutations leading to defective expression of the WAS protein (WASP) or the WASP interacting protein (WIP), both regulators of the actin cytoskeleton. B cells deficient for WASP or WIP are hyper-proliferative to both BCR and TLR signalling, as we and others recently demonstrated. With the here described project, we wish to understand and further elucidate the role of those actin regulators in TLR signalling in the context of inflammation. Preliminary findings for this grant application show that in response to TLR4 stimulation in vitro, B cells deficient in WIP (WIP KO) secrete high amounts of the pro-inflammatory cytokines IL-6 and TNF-a. Concomitantly, WIP KO B cells show a diminished expression of the plasma cell specific transcription factor (TF) IRF4 and also of the TF T-bet, which might influence antibody and cytokine production. Pro-inflammatory cytokines can become self-destructive in the acute setting of sepsis or in response to the commensal microbiota. Our preliminary results indeed show an increased B cell activation and IgA production in the gut of non-immunised WIP KO mice, and an early onset of colitis in chimeras in which only B cells lack WIP. Thus, a defective differentiation into plasma cells together with an increased IL-6 and TNF-a production by B cells might ultimately enhance the autoimmune phenotype in WAS. We therefore hypothesize that in B cells WIP is involved in the modulation of signalling pathways regulating TF expression, antibody production and cytokine secretion after TLR4 engagement thereby influencing immune homeostasis in the gut. To test this hypothesis, we plan to (1) analyse the role of the actin regulators WIP and WASP in TLR4 signal induction, endocytosis, cytokine and antibody production of B cells in vitro and in vivo, (2) analyse the underlying molecular mechanism determining TF expression and B cell fate decision and (3) analyse the in vivo impact of the imbalanced cytokine production, plasma cell differentiation and IgA antibody secretion of WIP KO B cells during sepsis and the onset of colitis. By investigating these aims, we will gain further understanding of autoimmune diseases like WAS which may be useful for the development of novel treatment options, not only in WAS but also other immune-related disorders, like inflammatory bowel disease.