DataSheet_1_Lipopolysaccharide-responsive beige-like anchor is involved in regulating NF-κB activation in B cells.pdf

IntroductionLipopolysaccharide-responsive and beige-like anchor (LRBA) is a scaffolding protein that interacts with proteins such as CTLA-4 and PKA, the importance of which has been determined in various cell types, including T regulatory cells, B cells, and renal cells. LRBA deficiency is associated with an inborn error in immunity characterized by immunodeficiency and autoimmunity. In addition to defects in T regulatory cells, patients with LRBA deficiency also exhibit B cell defects, such as reduced cell number, low memory B cells, hypogammaglobulinemia, impaired B cell proliferation, and increased autophagy. Although Lrba-/- mice do not exhibit the immunodeficiency observed in humans, responses to B cell receptors (BCR) in B cells have not been explored. Therefore, a murine model is for elucidating the mechanism of Lrba mechanism in B cells.
AimTo compare and evaluate spleen-derived B cell responses to BCR crosslinking in C57BL6 Lrba-/- and Lrba+/+ mice.
Materials and methodsSpleen-derived B cells were obtained from 8 to 12-week-old mice. Subpopulations were determined by immunostaining and flow cytometry. BCR crosslinking was assessed by the F(ab’)2 anti-μ chain. Activation, proliferation and viability assays were performed using flow cytometry and protein phosphorylation was evaluated by immunoblotting. The nuclear localization of p65 was determined using confocal microscopy. Nur77 expression was evaluated by Western blot.
ResultsLrba-/- B cells showed an activated phenotype and a decreased proportion of transitional 1 B cells, and both proliferation and survival were affected after BCR crosslinking in the Lrba-/- mice. The NF-κB pathway exhibited a basal activation status of several components, resulting in increased activation of p50, p65, and IκBα, basal p50 activation was reduced by the Plcγ2 inhibitor U73122. BCR crosslinking in Lrba-/- B cells resulted in poor p50 phosphorylation and p65 nuclear localization. Increased levels of Nur77 were detected.
DiscussionThese results indicate the importance of Lrba in controlling NF-κB activation driven by BCR. Basal activation of NF-κB could impact cellular processes, such as, activation, differentiation, proliferation, and maintenance of B cells after antigen encounter.

引言 脂多糖应答与米色样锚定蛋白（LRBA）是一种脚手架蛋白，可与细胞毒性T淋巴细胞相关抗原4（CTLA-4）、蛋白激酶A（PKA）等蛋白相互作用，其生物学重要性已在多种细胞类型中得到验证，包括调节性T细胞（T regulatory cells）、B细胞及肾细胞。LRBA缺陷与一类以免疫缺陷和自身免疫为特征的先天性免疫异常密切相关。除调节性T细胞功能缺陷外，LRBA缺陷患者还可出现多种B细胞异常，包括细胞数量减少、记忆B细胞比例降低、低丙种球蛋白血症、B细胞增殖受损及自噬水平升高。尽管Lrba基因敲除（Lrba-/-）小鼠未表现出人类患者中观察到的免疫缺陷表型，但目前尚未有研究探讨B细胞中B细胞受体（B cell receptors, BCR）的应答情况。因此，本研究构建小鼠模型，旨在阐明LRBA在B细胞中的作用机制。
研究目的 比较并评估C57BL/6背景下Lrba基因敲除（Lrba-/-）与野生型（Lrba+/+）小鼠脾脏来源B细胞经BCR交联后的应答差异。
材料与方法 从8至12周龄小鼠体内分离脾脏来源B细胞。通过免疫染色与流式细胞术鉴定细胞亚群。采用F(ab’)2抗μ链抗体诱导BCR交联。通过流式细胞术完成B细胞激活、增殖与活性检测，免疫印迹法（immunoblotting）评估蛋白磷酸化水平。使用共聚焦显微镜检测p65的核定位情况，蛋白质印迹法（Western blot）检测Nur77的表达水平。
结果 Lrba-/- B细胞呈现激活表型，且过渡型1 B细胞比例降低；在BCR交联刺激后，Lrba基因敲除小鼠的B细胞增殖与存活能力均受到影响。核因子κB（NF-κB）通路的多个组分呈现基础激活状态，导致p50、p65及IκBα的激活水平升高，磷脂酶Cγ2（Plcγ2）抑制剂U73122可抑制基础状态下的p50激活。Lrba-/- B细胞经BCR交联后，p50磷酸化水平及p65核定位能力均受损，同时检测到Nur77表达水平升高。
讨论 本研究结果证实了LRBA在调控BCR介导的NF-κB激活过程中的关键作用。NF-κB的基础激活可影响多种细胞生物学过程，包括抗原接触后B细胞的激活、分化、增殖及存活维持。