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

B细胞是维持免疫动态平衡的主要调节者。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确保的。细胞因子信号或Toll样受体(TLR)的参与也会影响B细胞的功能。异常的B细胞激活可能导致自身免疫性疾病的发展，如Wiskott-Aldrich综合征(Wiskott-Aldrich综合征)。是一种免疫缺陷，与高水平的IgA和IgE抗体有关，增加了对感染的易感性，并增加了包括结肠炎在内的免疫介导性疾病的风险。疾病的原因是基因突变导致肌动蛋白细胞骨架的两种调节蛋白--was蛋白(Wasp)或wasp相互作用蛋白(Wip)的表达缺陷。缺乏WASP或WIP的B细胞对BCR和TLR信号通路都是过度增殖的，正如我们和其他人最近所证明的那样。通过这里描述的项目，我们希望了解并进一步阐明这些肌动蛋白调节因子在炎症背景下TLR信号传递中的作用。这项赠款申请的初步研究结果表明，在体外对TLR4刺激的反应中，WIP(WIP KO)缺陷的B细胞分泌大量的促炎细胞因子IL-6和TNF-a。同时，WIP KO B细胞浆细胞特异性转录因子(TF)IRF4和TF T-bet的表达减弱，这可能影响抗体和细胞因子的产生。促炎细胞因子在脓毒症的急性期或对共生微生物区系的反应中会变得自毁。我们的初步结果确实表明，未免疫的WIP KO小鼠肠道中B细胞激活和IgA产生增加，并且只有B细胞缺乏WIP的嵌合体中结肠炎发病较早。因此，B细胞向浆细胞分化的缺陷，以及IL-6和TNF-a的产生增加，可能最终会增强AS的自身免疫表型。因此，我们假设在B细胞中，WIP参与了调节TLR4结合后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平衡。为了验证这一假说，我们计划(1)分析肌动蛋白调节因子WIP和WASP在体外和体内B细胞TLR4信号诱导、吞噬、细胞因子和抗体产生中的作用；(2)分析决定TF表达和B细胞命运的潜在分子机制；(3)分析在体内WIP KO B细胞在脓毒症和结肠炎发病过程中细胞因子产生失衡、浆细胞分化和IgA抗体分泌的影响。通过研究这些目的，我们将进一步了解自身免疫性疾病，如WASE，这可能有助于开发新的治疗方案，不仅是WAAS，还包括其他与免疫相关的疾病，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.