CN114286684B - Prevention and/or treatment of STAT 3-related diseases - Google Patents
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Abstract
本发明涉及通过调节14‑3‑3σ预防和/或治疗与STAT3相关的疾病。The present invention relates to the prevention and/or treatment of STAT3-related diseases by modulating 14-3-3σ.
Description
技术领域Technical field
本发明涉及通过调节14-3-3σ预防和/或治疗与STAT3相关的疾病。The present invention relates to the prevention and/or treatment of STAT3-related diseases by modulating 14-3-3σ.
背景技术Background technique
信号转导及转录激活蛋白3(STAT3)是STAT蛋白家族的成员,由多个转录因子组成,这些转录因子对参与许多细胞功能的基因进行调节。STAT蛋白由JAK激酶(JAK)激活。在细胞因子或生长因子的刺激后,STAT3被膜受体相关的JAK磷酸化,进而形成同源或异源二聚体,并转运到细胞核中进行转录。由于STAT3控制多种基因的表达,并在许多细胞过程(包括细胞生长和细胞凋亡1)中发挥关键作用,因此STAT3-KO在小鼠模型中具有胚胎致死性2。Signal transducer and activator of transcription 3 (STAT3) is a member of the STAT protein family and consists of multiple transcription factors that regulate genes involved in many cellular functions. STAT proteins are activated by JAK kinase (JAK). After stimulation by cytokines or growth factors, STAT3 is phosphorylated by membrane receptor-associated JAKs, forming homo- or heterodimers and transported to the nucleus for transcription. Because STAT3 controls the expression of multiple genes and plays a key role in many cellular processes, including cell growth and apoptosis , STAT3-KO is embryonic lethal in mouse models.
STAT3在免疫细胞中的功能已得到广泛研究。STAT3对于TH17细胞的分化是不可或缺的3-6。在病毒感染期间,T细胞中缺乏STAT3的小鼠完全丧失了产生滤泡辅助T细胞(Tfh)和抗体的能力7。人体内显著的STAT3突变是功能丧失性(LOF)突变,可导致高免疫球蛋白E综合征(HIES)。患有该疾病的患者会出现反复感染以及骨骼和牙齿发育异常8,9。另一方面,STAT3基因的功能获得性突变(GOF)导致自身免疫性疾病10。有报告表明,诸如Dock8、PGM3、SPINK5和TYK2等其他基因中的突变也可诱发HIES11-14。我们培育了DOCK8突变小鼠来模拟DOCK8患者体内的突变,并发现记忆B细胞的早期激活被破坏。此外,DOCK8缺乏导致CD19和WASP表达减少15。先前的研究表明,在TLR9介导的B细胞激活中,STAT3在DOCK8的下游,并且DOCK8通过激活STAT3来抑制IgE的产生16。然而,STAT3与DOCK8之间是否存在调节IgE产生的相关性尚不清楚。The function of STAT3 in immune cells has been extensively studied. STAT3 is indispensable for the differentiation of TH17 cells3-6. During viral infection, mice lacking STAT3 in their T cells completely lost the ability to produce follicular helper T cells (Tfh) and antibodies7 . Significant STAT3 mutations in humans are loss-of-function (LOF) mutations that lead to hyperimmunoglobulin E syndrome (HIES). Patients with this disease develop recurrent infections and abnormal bone and tooth development 8,9 . On the other hand, gain-of-function mutations (GOF) in the STAT3 gene cause autoimmune diseases 10 . There are reports that mutations in other genes such as Dock8, PGM3, SPINK5, and TYK2 can also induce HIES 11-14 . We generated DOCK8 mutant mice to mimic the mutations found in DOCK8 patients and found that early activation of memory B cells was disrupted. Furthermore, DOCK8 deficiency results in reduced expression of CD19 and WASP 15 . Previous studies have shown that STAT3 is downstream of DOCK8 in TLR9-mediated B cell activation, and DOCK8 inhibits IgE production by activating STAT316 . However, whether there is a correlation between STAT3 and DOCK8 in regulating IgE production is unclear.
近期发现微RNA(miRNA)与高IgE之间存在关联。使用miRNA146A转基因小鼠可以证实,miRNA146A通过上调14-3-3σ来促进IgE类别转换,14-3-3σ是B细胞中免疫球蛋白类别转换DNA重组(CSR)的关键因素17-19。Jurkat T细胞中miRNA146A的异位表达导致STAT3表达适度下调20。此外,当STAT3的激活在肝细胞癌(HCC)细胞中被阻断时,miRNA146A的表达降低21。另外,在丙型肝炎感染期间,miRNA146A控制单核细胞中SOCS1/STAT3和细胞因子的表达22。然而,尤其是在B细胞中,STAT3突变所致的HIES的潜在机制仍不清楚。Recently, a correlation between microRNA (miRNA) and high IgE has been discovered. Using miRNA146A transgenic mice, it was confirmed that miRNA146A promotes IgE class switching by upregulating 14-3-3σ, which is a key factor in immunoglobulin class switching DNA recombination (CSR) in B cells17-19 . Ectopic expression of miRNA146A in Jurkat T cells resulted in modest downregulation of STAT3 expression20. Furthermore, when activation of STAT3 is blocked in hepatocellular carcinoma (HCC) cells, the expression of miRNA146A is reduced 21 . Additionally, during hepatitis C infection, miRNA146A controls the expression of SOCS1/STAT3 and cytokines in monocytes 22 . However, the underlying mechanisms of HIES caused by STAT3 mutations remain unclear, especially in B cells.
B细胞受体(BCR)信号传导对于调节B细胞过程和功能至关重要。BCR信号传导决定B细胞的命运及其抗体应答的程度。当抗原与BCR结合时,可引发受体的构象变化并触发信号传导级联,如CD19、Lyn、Syk、Btk、PLCγ以及BCR信号传导中负向调节剂(包括SHIP和PTEN)的磷酸化23-25。BCR信号传导还可通过诸如WASP、abp1和N-WASP等若干肌动蛋白调节剂诱导肌动蛋白重组,而肌动蛋白重组通过调节BCR的移动为BCR信号传导提供反馈26-29。STAT3如何在STAT3 LOF和GOF患者B细胞中影响BCR信号传导尚不清楚。B cell receptor (BCR) signaling is critical for regulating B cell processes and function. BCR signaling determines the fate of B cells and the extent of their antibody response. When antigen binds to the BCR, it induces conformational changes in the receptor and triggers signaling cascades, such as phosphorylation of CD19, Lyn, Syk, Btk, PLCγ, and negative regulators of BCR signaling, including SHIP and PTEN23 -25 . BCR signaling can also induce actin reorganization through several actin regulators such as WASP, abp1 and N-WASP, and actin reorganization provides feedback to BCR signaling by regulating BCR movement26-29 . How STAT3 affects BCR signaling in STAT3 LOF and GOF patient B cells remains unclear.
在一项研究中,STAT3缺乏阻碍了骨髓B细胞的早期发育,但stat3的缺失是由并非B细胞特有的Mx1Cre驱动的30。相比之下,CD19Cre Stat3 fl/fl小鼠未观察到发育缺陷,因此stat3的缺失发生在B细胞发育后期,并且只有T细胞依赖性(TD)IgG应答严重受损,而TD-IgM、IgE、和IgA应答或非T细胞依赖性(TI)IgM和IgG3应答未受损30,31。STAT3信号传导对维持生发中心(GC)至关重要,并通过调节GC活性解释系统性红斑狼疮(SLE)的发病机制32。STAT3患者的记忆B细胞(包括转换和未转换的免疫球蛋白)减少33-35。为了使用小鼠模型精确研究STAT3突变仅在B细胞中引发HIES的潜在机制,我们培育了在B细胞发育早期就已缺失stat3的Mb1Cre stat3 flox/flox小鼠(STAT3KO)。In one study, STAT3 deficiency impeded early bone marrow B cell development, but loss of stat3 was driven by Mx1 Cre that was not specific to B cells 30 . In contrast, no developmental defects were observed in CD19 Cre Stat3 fl/fl mice, such that loss of stat3 occurs late in B cell development and only T cell-dependent (TD) IgG responses are severely impaired, whereas TD-IgM, IgE, and IgA responses or T cell-independent (TI) IgM and IgG3 responses were not impaired 30,31 . STAT3 signaling is critical for the maintenance of germinal centers (GCs) and explains the pathogenesis of systemic lupus erythematosus (SLE) by regulating GC activity32 . STAT3 patients have reduced memory B cells (including switched and unswitched immunoglobulins) 33-35 . To use a mouse model to precisely study the underlying mechanism by which STAT3 mutations trigger HIES only in B cells, we generated Mb1 Cre stat3 flox/flox mice (STAT3KO) in which stat3 is deleted early in B cell development.
发明内容Contents of the invention
在第一方面,本发明提供了一种预防和/或治疗需要这种预防和/或治疗的患者的与STAT3相关的疾病的方法,所述方法包括在所述患者体内调节14-3-3σ。In a first aspect, the present invention provides a method of preventing and/or treating a STAT3-related disease in a patient in need of such prevention and/or treatment, said method comprising modulating 14-3-3σ in said patient. .
在第一方面的一个实施方案中,所述STAT3是功能丧失性STAT3或功能获得性STAT3。在第一方面的另一个实施方案中,所述STAT3是功能丧失性STAT3。In one embodiment of the first aspect, the STAT3 is a loss-of-function STAT3 or a gain-of-function STAT3. In another embodiment of the first aspect, the STAT3 is a loss-of-function STAT3.
在第一方面的另一个实施方案中,所述与STAT3相关的疾病是高IgE综合征。In another embodiment of the first aspect, the STAT3-associated disease is hyperIgE syndrome.
在第一方面的另一个实施方案中,调节14-3-3σ是下调14-3-3σ或上调14-3-3σ。在第一方面的另一个实施方案中,调节是下调14-3-3σ。在第一方面的另一个实施方案中,下调14-3-3σ是用14-3-3σ抑制剂进行的。在第一方面的另一个实施方案中,所述14-3-3σ抑制剂是肽。在第一方面的另一个实施方案中,所述肽包含氨基酸序列NH2-YGRKKKRQRRRPHCVPRDLSWLDLEANMCLP-COOH。In another embodiment of the first aspect, modulating 14-3-3σ is down-regulating 14-3-3σ or up-regulating 14-3-3σ. In another embodiment of the first aspect, the modulating is down-regulating 14-3-3σ. In another embodiment of the first aspect, down-regulation of 14-3-3σ is performed using a 14-3-3σ inhibitor. In another embodiment of the first aspect, the 14-3-3σ inhibitor is a peptide. In another embodiment of the first aspect, the peptide comprises the amino acid sequence NH2 -YGRKKKRQRRRPHCVPRDLSWLDLEANMCLP-COOH.
在第二方面,本发明提供了一种14-3-3σ调节剂、一种包含所述14-3-3σ调节剂的药物组合物或者一种包含所述14-3-3σ调节剂的试剂盒,用于预防和/或治疗需要这种预防和/或治疗的患者的与STAT3相关的疾病。In a second aspect, the invention provides a 14-3-3σ modulator, a pharmaceutical composition comprising the 14-3-3σ modulator or an agent comprising the 14-3-3σ modulator. A cartridge for the prevention and/or treatment of a STAT3-related disease in a patient in need of such prevention and/or treatment.
在第二方面的一个实施方案中,所述14-3-3σ调节剂是14-3-3σ下调剂或14-3-3σ上调剂。在第二方面的另一个实施方案中,所述14-3-3σ调节剂是14-3-3σ下调剂。在第二方面的另一个实施方案中,所述14-3-3σ下调剂是14-3-3σ抑制剂。在第二方面的另一个实施方案中,所述14-3-3σ抑制剂是肽。在第二方面的另一个实施方案中,所述肽包含氨基酸序列NH2-YGRKKKRQRRRPHCVPRDLSWLDLEANMCLP-COOH。In one embodiment of the second aspect, the 14-3-3σ modulator is a 14-3-3σ down-modulator or a 14-3-3σ up-modulator. In another embodiment of the second aspect, the 14-3-3σ modulator is a 14-3-3σ down-modulator. In another embodiment of the second aspect, the 14-3-3σ down-modulator is a 14-3-3σ inhibitor. In another embodiment of the second aspect, the 14-3-3σ inhibitor is a peptide. In another embodiment of the second aspect, the peptide comprises the amino acid sequence NH2 -YGRKKKRQRRRPHCVPRDLSWLDLEANMCLP-COOH.
在第二方面的另一个实施方案中,所述STAT3是功能丧失性STAT3或功能获得性STAT3。在第二方面的另一个实施方案中,所述STAT3是功能丧失性STAT3。In another embodiment of the second aspect, the STAT3 is a loss-of-function STAT3 or a gain-of-function STAT3. In another embodiment of the second aspect, the STAT3 is a loss-of-function STAT3.
在第二方面的另一个实施方案中,所述与STAT3相关的疾病是高IgE综合征。In another embodiment of the second aspect, the STAT3-associated disease is hyperIgE syndrome.
在第二方面的另一个实施方案中,所述药物组合物还包含药学上可接受的赋形剂。In another embodiment of the second aspect, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.
在第二方面的另一个实施方案中,包含在试剂盒中的14-3-3σ调节剂处于药物组合物的形式。在第二方面的另一个实施方案中,所述试剂盒还包含应用所述14-3-3σ调节剂的说明书。In another embodiment of the second aspect, the 14-3-3σ modulator included in the kit is in the form of a pharmaceutical composition. In another embodiment of the second aspect, the kit further comprises instructions for using the 14-3-3σ modulator.
在第三方面,本发明提供了14-3-3σ调节剂在预防和/或治疗需要这种预防和/或治疗的患者的与STAT3相关的疾病中的用途。In a third aspect, the present invention provides the use of a 14-3-3σ modulator in the prevention and/or treatment of STAT3-related diseases in a patient in need of such prevention and/or treatment.
在第三方面的一个实施方案中,所述14-3-3σ调节剂是14-3-3σ下调剂或14-3-3σ上调剂。在第三方面的另一个实施方案中,所述14-3-3σ调节剂是14-3-3σ下调剂。在第三方面的另一个实施方案中,所述14-3-3σ下调剂是14-3-3σ抑制剂。在第三方面的另一个实施方案中,所述14-3-3σ抑制剂是肽。在第三方面的另一个实施方案中,所述肽包含氨基酸序列NH2-YGRKKKRQRRRPHCVPRDLSWLDLEANMCLP-COOH。In one embodiment of the third aspect, the 14-3-3σ modulator is a 14-3-3σ down-modulator or a 14-3-3σ up-modulator. In another embodiment of the third aspect, the 14-3-3σ modulator is a 14-3-3σ down-modulator. In another embodiment of the third aspect, the 14-3-3σ down-modulator is a 14-3-3σ inhibitor. In another embodiment of the third aspect, the 14-3-3σ inhibitor is a peptide. In another embodiment of the third aspect, the peptide comprises the amino acid sequence NH2 -YGRKKKRQRRRPHCVPRDLSWLDLEANMCLP-COOH.
在第三方面的另一个实施方案中,所述STAT3是功能丧失性STAT3或功能获得性STAT3。在第三方面的另一个实施方案中,所述STAT3是功能丧失性STAT3。In another embodiment of the third aspect, the STAT3 is a loss-of-function STAT3 or a gain-of-function STAT3. In another embodiment of the third aspect, the STAT3 is a loss-of-function STAT3.
在第三方面的另一个实施方案中,所述与STAT3相关的疾病是高IgE综合征。In another embodiment of the third aspect, the STAT3-associated disease is hyperIgE syndrome.
在第四方面,本发明提供了14-3-3σ调节剂在制造药物中的用途,所述药物用于预防和/或治疗需要这种预防和/或治疗的患者的与STAT3相关的疾病。In a fourth aspect, the present invention provides the use of a 14-3-3σ modulator in the manufacture of a medicament for the prevention and/or treatment of STAT3-related diseases in a patient in need of such prevention and/or treatment.
在第四方面的一个实施方案中,所述14-3-3σ调节剂是14-3-3σ下调剂或14-3-3σ上调剂。在第四方面的另一个实施方案中,所述14-3-3σ调节剂是14-3-3σ下调剂。在第四方面的另一个实施方案中,所述14-3-3σ下调剂是14-3-3σ抑制剂。在第四方面的另一个实施方案中,所述14-3-3σ抑制剂是肽。在第四方面的另一个实施方案中,所述肽包含氨基酸序列NH2-YGRKKKRQRRRPHCVPRDLSWLDLEANMCLP-COOH。In one embodiment of the fourth aspect, the 14-3-3σ modulator is a 14-3-3σ down-modulator or a 14-3-3σ up-modulator. In another embodiment of the fourth aspect, the 14-3-3σ modulator is a 14-3-3σ down-modulator. In another embodiment of the fourth aspect, the 14-3-3σ down-modulator is a 14-3-3σ inhibitor. In another embodiment of the fourth aspect, the 14-3-3σ inhibitor is a peptide. In another embodiment of the fourth aspect, the peptide comprises the amino acid sequence NH2 -YGRKKKRQRRRPHCVPRDLSWLDLEANMCLP-COOH.
在第四方面的另一个实施方案中,所述STAT3是功能丧失性STAT3或功能获得性STAT3。在第四方面的另一个实施方案中,所述STAT3是功能丧失性STAT3。In another embodiment of the fourth aspect, the STAT3 is a loss-of-function STAT3 or a gain-of-function STAT3. In another embodiment of the fourth aspect, the STAT3 is a loss-of-function STAT3.
在第四方面的另一个实施方案中,所述与STAT3相关的疾病是高IgE综合征。In another embodiment of the fourth aspect, the STAT3-associated disease is hyperIgE syndrome.
附图说明Description of drawings
图1:STAT3对维持外周血B细胞的稳态至关重要,但对骨髓B细胞则不然。Figure 1: STAT3 is critical for maintaining homeostasis of peripheral blood B cells, but not bone marrow B cells.
(A-C)来自WT和STAT3 KO小鼠(n=8)骨髓细胞的前祖B细胞(A)、祖B细胞(B)、早期前B细胞(C)、晚期前B细胞(D)、未成熟B细胞(E)和再循环B细胞(F)的流式细胞术数据以及这些细胞各自的百分比和绝对数量。(D)从A和B门控的不同群体中CD127的MFI。(E-H)来自WT和STAT3KO小鼠脾细胞(n=8)的T1、T2、FO细胞群体的流式细胞术数据以及这些细胞各自的百分比和绝对数量。(I和J)来自小鼠脾细胞(n=8)的MZ B细胞的流式细胞术数据以及这些细胞的百分比和绝对数量。(K和L)来自小鼠脾细胞(n=8)的GC细胞的流式细胞术数据以及这些细胞的百分比和绝对数量。(M-O)来自WT和STAT3 KO小鼠(n=6)的B1a和B1b细胞的流式细胞术分析以及这些细胞各自的百分比和绝对数量。(P-R)来自经过或未经5天抗CD40和IL-4刺激的WT和STAT3 KO小鼠(n=4)的IgE+脾脏B细胞的流式细胞术数据以及这些细胞的百分比和绝对数量。(S)通过ELISA得出的WT和STAT3 KO小鼠血清(n=6)IgE的滴度。所示为三个独立实验的代表性结果。*P<0.05并且**P<0.01。(A-C) Pre-progenitor B cells (A), progenitor B cells (B), early pre-B cells (C), late pre-B cells (D), and late pre-B cells from bone marrow cells of WT and STAT3 KO mice (n=8). Flow cytometry data of mature B cells (E) and recycling B cells (F) and the respective percentages and absolute numbers of these cells. (D) MFI of CD127 in different populations gated from A and B. (EH) Flow cytometry data of T1, T2, FO cell populations from WT and STAT3KO mouse splenocytes (n=8) and the respective percentages and absolute numbers of these cells. (I and J) Flow cytometry data of MZ B cells from mouse splenocytes (n=8) and the percentage and absolute number of these cells. (K and L) Flow cytometry data of GC cells from mouse splenocytes (n=8) and the percentage and absolute number of these cells. (MO) Flow cytometric analysis of B1a and B1b cells from WT and STAT3 KO mice (n=6) and the respective percentages and absolute numbers of these cells. (PR) Flow cytometry data of IgE + splenic B cells from WT and STAT3 KO mice (n=4) with or without 5 days of anti-CD40 and IL-4 stimulation and the percentage and absolute number of these cells. (S) IgE titers in WT and STAT3 KO mouse serum (n=6) by ELISA. Shown are representative results from three independent experiments. *P<0.05 and **P<0.01.
图2:STAT3正向调节近侧BCR信号传导。Figure 2: STAT3 positively regulates proximal BCR signaling.
(A和B)来自WT和STAT3 KO小鼠的脾脏B细胞经过标记,并使用AF546-单生物素化Fab'-抗IgG加链霉亲和素通过可溶性抗原(sAg)在37℃下经不同时间长度的刺激。在固定和透化后,将细胞针对pCD19进行染色,通过共聚焦显微术成像,并对BCR与pCD19之间的相关系数进行定量。(C)通过蛋白质印迹分析的经sAg刺激的WT和STAT3 KO小鼠脾脏B细胞中的pCD19/CD19水平。(D和E)经sAg刺激的小鼠脾脏B细胞中pY和pBtk的共聚焦显微图像,并对BCR与pY/pBtk之间的相关系数进行定量。(F)通过蛋白质印迹分析的经sAg刺激的小鼠脾脏B细胞中的pY和pBtk/Btk水平。(G和H)通过共聚焦显微术分析的经sAg刺激的小鼠脾脏B细胞中的pSHIP,并对BCR与pSHIP之间的相关系数进行测量。(I)经sAg刺激的小鼠脾脏B细胞中pSHIP/SHIP水平的蛋白质印迹。所示为三个独立实验的代表性结果。比例尺,2.5μm。使用NIS Elements AR 3.2软件对多于50个细胞的相关系数进行定量。曼-惠特尼U检验(图2B、图2E、图2H)。**P<0.05;***P<0.01。(A and B) Splenic B cells from WT and STAT3 KO mice were labeled and differentially expressed with soluble antigen (sAg) using AF546-monobiotinylated Fab'-anti-IgG plus streptavidin at 37°C. Duration of stimulation. After fixation and permeabilization, cells were stained for pCD19, imaged by confocal microscopy, and the correlation coefficient between BCR and pCD19 was quantified. (C) pCD19/CD19 levels in sAg-stimulated splenic B cells of WT and STAT3 KO mice analyzed by Western blot. (D and E) Confocal microscopy images of pY and pBtk in mouse splenic B cells stimulated with sAg, and the correlation coefficient between BCR and pY/pBtk was quantified. (F) pY and pBtk/Btk levels in sAg-stimulated mouse splenic B cells analyzed by Western blot. (G and H) pSHIP in sAg-stimulated mouse splenic B cells analyzed by confocal microscopy, and the correlation coefficient between BCR and pSHIP was measured. (I) Western blot of pSHIP/SHIP levels in sAg-stimulated mouse splenic B cells. Shown are representative results from three independent experiments. Scale bar, 2.5 μm. Correlation coefficients for more than 50 cells were quantified using NIS Elements AR 3.2 software. Mann-Whitney U test (Figure 2B, Figure 2E, Figure 2H). **P<0.05; ***P<0.01.
图3:STAT3的缺乏减少了由WASP和WIP介导的F-肌动蛋白的积累以及BCR簇集和正向信号复合体募集。Figure 3: Lack of STAT3 reduces F-actin accumulation as well as BCR clustering and forward signaling complex recruitment mediated by WASP and WIP.
(A)WT和STAT3 KO小鼠脾脏B细胞中的pWASP和肌动蛋白的共聚焦显微术分析。B细胞在37℃下经不同时间长度的sAg刺激,然后固定、透化并针对pWASP和肌动蛋白(使用鬼笔环肽)进行染色。(B)将来自WT和STAT3KO小鼠的脾脏B细胞用BV510-抗B220标记,在37℃下经不同时间长度的sAg刺激,然后如A所述进行固定、透化和染色。通过流式细胞术测量pWASP和肌动蛋白的MFI。(C)通过蛋白质印迹分析的经sAg刺激的WT和STAT3 KO小鼠脾脏B细胞中的pWASP/WASP水平。(D)来自WT和STAT3 KO小鼠(左小图)的脾脏B细胞中以及健康对照(HC)和功能丧失性(LOF)STAT3患者PBMC(右小图)中WIP和DOCK8的表达。将GAPDH用作上样对照。(E-J)将来自WT和STAT3 KO小鼠的脾脏B细胞使用AF546-单生物素化Fab'-抗IgG在膜拴系抗原(membrane-tethered antigen,mAg)上经不同时间长度的刺激,然后固定、透化并针对pWASP和肌动蛋白进行染色。使用TIRFm分析细胞,并测量BCR、pWASP和肌动蛋白的MFI,以及使用干涉反射显微术(IRM)测量B细胞接触面积。所示为代表性图像和平均值(±SD),其中使用NIS Elements AR 3.2软件分析了多于50个细胞。比例尺,2.5mm。*P<0.05;**P<0.01。曼-惠特尼U检验(图2G-图2J)。(A) Confocal microscopy analysis of pWASP and actin in splenic B cells of WT and STAT3 KO mice. B cells were stimulated with sAg for varying lengths of time at 37°C, then fixed, permeabilized, and stained for pWASP and actin (using phalloidin). (B) Splenic B cells from WT and STAT3KO mice were labeled with BV510-anti-B220, stimulated with sAg for varying lengths of time at 37°C, and then fixed, permeabilized, and stained as described in A. The MFI of pWASP and actin was measured by flow cytometry. (C) pWASP/WASP levels in sAg-stimulated splenic B cells of WT and STAT3 KO mice analyzed by Western blot. (D) Expression of WIP and DOCK8 in splenic B cells from WT and STAT3 KO mice (left panel) and in healthy control (HC) and loss-of-function (LOF) STAT3 patient PBMC (right panel). GAPDH was used as a loading control. (E-J) Splenic B cells from WT and STAT3 KO mice were stimulated with AF546-monobiotinylated Fab'-anti-IgG on membrane-tethered antigen (mAg) for varying lengths of time and then fixed. , permeabilized, and stained for pWASP and actin. Cells were analyzed using TIRFm and MFI of BCR, pWASP and actin were measured, as well as B cell contact area using interference reflection microscopy (IRM). Shown are representative images and means (±SD) where more than 50 cells were analyzed using NIS Elements AR 3.2 software. Scale bar, 2.5mm. *P<0.05; **P<0.01. Mann-Whitney U test (Figure 2G-Figure 2J).
图4:STAT3缺乏导致pCD19、pBtk和pY的募集减少,但pSHIP的募集增加。Figure 4: STAT3 deficiency results in reduced recruitment of pCD19, pBtk and pY, but increased recruitment of pSHIP.
(A和D)将来自WT和STAT3 KO小鼠的脾脏B细胞在37℃下经不同时间长度的mAg刺激,然后固定、透化并针对pCD19进行染色。使用TIRFm拍摄图像,并对B细胞接触区内pCD19的MFI进行分析。(B、E、F)使用TIRFm对经mAg刺激及针对pBtk和pY进行染色的小鼠脾脏B细胞进行成像,并对接触区内pBtk和pY的MFI进行测量。(C和G)使用TIRFm对经mAg刺激及针对pSHIP进行染色的小鼠脾脏B细胞进行成像,并对接触区内pSHIP的MFI进行测量。所示为代表性图像和平均值(±SD),其中从3个独立实验使用NIS Elements AR 3.2软件分析了多于50个细胞。比例尺,2.5mm。*P<0.05;**P<0.01。曼-惠特尼U检验(图2D、图2E、图2F、图2G)。(A and D) Splenic B cells from WT and STAT3 KO mice were stimulated with mAg for varying lengths of time at 37°C, then fixed, permeabilized, and stained for pCD19. Images were taken using TIRFm and the MFI of pCD19 within the B cell contact zone was analyzed. (B, E, F) TIRFm was used to image mouse spleen B cells stimulated with mAg and stained for pBtk and pY, and the MFI of pBtk and pY in the contact zone was measured. (C and G) TIRFm was used to image mouse splenic B cells stimulated with mAg and stained for pSHIP, and the MFI of pSHIP in the contact zone was measured. Shown are representative images and means (±SD) from 3 independent experiments where >50 cells were analyzed using NIS Elements AR 3.2 software. Scale bar, 2.5mm. *P<0.05; **P<0.01. Mann-Whitney U test (Figure 2D, Figure 2E, Figure 2F, Figure 2G).
图5:STAT3缺乏降低PI3K-Akt-mTORC1介导的代谢信号传导通路。Figure 5: STAT3 deficiency reduces the PI3K-Akt-mTORC1-mediated metabolic signaling pathway.
(A)将来自WT和STAT3 KO小鼠的脾脏B细胞经不同时间的sAg刺激,裂解,并使用蛋白质印迹探测pPI3k/PI3k、pAkt/Akt、pFoxo1/Foxo1、pMtor/Mtor和pS6/S6。(B)来自WT和STAT3 KO小鼠的脾脏B细胞响应于抑制剂寡霉素、FCCP(羰基氰对三氟甲氧基苯腙)和鱼藤酮治疗的基础耗氧率(OCR)。(C和D)将来自WT和STAT3 KO小鼠(n=4)的脾脏B细胞(1x106)用Cell Trace Violet标记,并用LPS和CpG刺激72小时。通过流式细胞术检测B220+细胞的增殖,并对增殖细胞的百分比进行定量。(E和F)用LPS和CpG刺激72小时后,小鼠脾脏B220+细胞中膜联蛋白V的表达。对膜联蛋白V+细胞的百分比(n=4)进行定量。所示为三个独立实验的代表性结果。**P<0.05;***P<0.01。(A) Splenic B cells from WT and STAT3 KO mice were stimulated with sAg for different times, lysed, and probed for pPI3k/PI3k, pAkt/Akt, pFoxo1/Foxo1, pMtor/Mtor, and pS6/S6 using Western blotting. (B) Basal oxygen consumption rate (OCR) of splenic B cells from WT and STAT3 KO mice in response to treatment with the inhibitors oligomycin, FCCP (carbonyl cyanide p-trifluoromethoxyphenylhydrazone), and rotenone. (C and D) Splenic B cells (1x10 6 ) from WT and STAT3 KO mice (n=4) were labeled with Cell Trace Violet and stimulated with LPS and CpG for 72 hours. The proliferation of B220 + cells was detected by flow cytometry, and the percentage of proliferating cells was quantified. (E and F) Annexin V expression in mouse spleen B220 + cells after 72 h of stimulation with LPS and CpG. The percentage of Annexin V + cells (n=4) was quantified. Shown are representative results from three independent experiments. **P<0.05;***P<0.01.
图6:来自STAT3 LOF患者的记忆B细胞破坏了早期BCR激活。Figure 6: Memory B cells from STAT3 LOF patients disrupt early BCR activation.
(A、D-F)将从HC和STAT3 LOF患者外周血(P1-P4)中分选的B细胞经不同时间长度的mAg刺激,固定、透化并针对CD27(以标记记忆B细胞)和肌动蛋白(使用鬼笔环肽)进行染色。使用TIRFm拍摄图像,使用IRM分析B细胞接触面积,并测量接触区内BCR和肌动蛋白的MFI。(B和G)使用TIRFm对经mAg刺激及针对CD27和pCD19进行染色的HC和STAT3 LOF患者B细胞进行成像,并对接触区内pCD19的MFI进行测量。(C和H)使用TIRFm对经mAg刺激及针对CD27和pY进行染色的HC和STAT3 LOF患者B细胞进行成像,并对接触区内pY的MFI进行测量。所示为代表性图像和平均值(±SD),其中从3个独立实验使用NIS Elements AR 3.2软件分析了多于50个细胞。比例尺,2.5mm。*P<0.05;**P<0.01。曼-惠特尼U检验(图2D-图2H)。(I和J)HC以及STAT3 LOF(P1-P6)和GOF(P7)患者PBMC(n=6)的B细胞亚型的流式细胞术数据,以及初始B细胞、记忆B细胞、过渡B细胞和浆母细胞的定量百分比。(K和L)将从HC、STAT3 LOF(P1-P4)和GOF(P7)患者PBMC中分选的B细胞经不同时间长度的sAg刺激,并使用流式细胞术对初始和记忆B细胞(n=4)的百分比进行分析。所示为三个独立实验的代表性结果。*P<0.05;**P<0.01。(A, D-F) B cells sorted from the peripheral blood of HC and STAT3 LOF patients (P1-P4) were stimulated with mAg for varying lengths of time, fixed, permeabilized, and targeted to CD27 (to label memory B cells) and actin Proteins (using phalloidin) were stained. Take images using TIRFm, analyze the B cell contact area using IRM, and measure the MFI of BCR and actin within the contact area. (B and G) TIRFm was used to image HC and STAT3 LOF patient B cells stimulated with mAg and stained for CD27 and pCD19, and the MFI of pCD19 in the contact zone was measured. (C and H) TIRFm was used to image HC and STAT3 LOF patient B cells stimulated with mAg and stained for CD27 and pY, and the MFI of pY within the contact zone was measured. Shown are representative images and means (±SD) from 3 independent experiments where >50 cells were analyzed using NIS Elements AR 3.2 software. Scale bar, 2.5mm. *P<0.05; **P<0.01. Mann-Whitney U test (Figure 2D-Figure 2H). (I and J) Flow cytometry data of B cell subtypes in HC and STAT3 LOF (P1-P6) and GOF (P7) patient PBMCs (n=6), as well as naive B cells, memory B cells, and transitional B cells and quantitative percentage of plasmablasts. (K and L) B cells sorted from HC, STAT3 LOF (P1-P4), and GOF (P7) patient PBMCs were stimulated with sAg for different lengths of time, and flow cytometry was used to analyze naïve and memory B cells ( The percentage of n=4) was analyzed. Shown are representative results from three independent experiments. *P<0.05; **P<0.01.
图7:STAT3缺乏通过增强miRNA146A-14-3-3σ轴导致高IgE。Figure 7: STAT3 deficiency leads to high IgE by enhancing the miRNA146A-14-3-3σ axis.
(A)对来自WT和STAT3-KO小鼠(n=6)的脾脏B细胞中miRNA146A和14-3-3σmRNA的分析。(B)对来自HC和LOF患者PBMC(n=6)(P1-P7)的B细胞中miRNA146A和14-3-3σmRNA的分析。(C)使用蛋白质印迹检测的来自WT和STAT3 KO小鼠脾脏(左小图)以及HC和LOF患者PBMC(右小图)(P4-P6)的B细胞中14-3-3σ的表达。将GAPDH用作上样对照。(D)对来自WT小鼠的B细胞中STAT3与14-3-3σ启动子结合的PCR分析。将IgG用作阴性对照。(E)将来自WT和STAT3KO小鼠的脾脏B细胞用或不用10μM R18处理1小时,用sAg刺激,然后裂解并使用蛋白质印迹探测pAkt和pFoxo1。将β-肌动蛋白用作上样对照。(F)对WT和STAT3 KO小鼠用或不用R18(0.2μg/kg体重)腹膜内连续注射14天,然后将脾脏B细胞进行分离并用sAg刺激,裂解,并使用蛋白质印迹探测pAkt和pFoxo1。将β-肌动蛋白用作上样对照。(G-J)对WT和STAT3 KO小鼠(n=3)用或不用R18(0.2μg/kg体重)腹膜内连续注射14天,并通过流式细胞术分析FO和GCB细胞的百分比。(K和L)通过流式细胞术对经过或未经R18处理且经过或未经刺激的WT和STAT3 KO小鼠测量IgE+B细胞和IgE转换B细胞的百分比。(M)通过ELISA对经过或未经R18处理的WT和STAT3 KO小鼠(n=5)血清中的IgE滴度进行测量。所示为三个独立实验的代表性结果。*P<0.05并且**P<0.01。(A) Analysis of miRNA146A and 14-3-3σ mRNA in splenic B cells from WT and STAT3-KO mice (n=6). (B) Analysis of miRNA146A and 14-3-3σ mRNA in B cells from HC and LOF patient PBMCs (n=6) (P1-P7). (C) Expression of 14-3-3σ in B cells from WT and STAT3 KO mouse spleens (left panels) and HC and LOF patient PBMCs (right panels) (P4-P6) detected using Western blot. GAPDH was used as a loading control. (D) PCR analysis of STAT3 binding to the 14-3-3σ promoter in B cells from WT mice. IgG was used as a negative control. (E) Splenic B cells from WT and STAT3KO mice were treated with or without 10 μM R18 for 1 h, stimulated with sAg, then lysed and probed for pAkt and pFoxo1 using Western blotting. β-Actin was used as a loading control. (F) WT and STAT3 KO mice were injected intraperitoneally with or without R18 (0.2 μg/kg body weight) for 14 days, and then splenic B cells were isolated and stimulated with sAg, lysed, and probed for pAkt and pFoxo1 using Western blotting. β-Actin was used as a loading control. (GJ) WT and STAT3 KO mice (n=3) were injected intraperitoneally with or without R18 (0.2 μg/kg body weight) for 14 consecutive days, and the percentages of FO and GCB cells were analyzed by flow cytometry. (K and L) Percentages of IgE + B cells and IgE-switched B cells were measured by flow cytometry in WT and STAT3 KO mice treated with or without R18 and stimulated with or without R18. (M) IgE titers were measured by ELISA in the serum of WT and STAT3 KO mice (n=5) with or without R18 treatment. Shown are representative results from three independent experiments. *P<0.05 and **P<0.01.
图8Figure 8
(A)对来自WT和STAT3-KO小鼠(n=4)的脾脏B细胞中STAT3 mRNA的分析。(B和C)来自WT和STAT3 KO小鼠的脾脏B220、MZ、FO、T1和T2细胞中膜联蛋白V表达的流式细胞术数据以及细胞群(n=8)的定量百分比。(D和E)来自WT和STAT3 KO小鼠的脾脏B220、MZ、FO、T1和T2细胞中Ki67表达的流式细胞术数据以及细胞群(n=8)的定量百分比。所示为三个独立实验的代表性结果。(F和G)对WT和STAT3 KO小鼠用或不用R18(0.2μg/kg体重)腹膜内连续注射14天。数据显示对MZ细胞的流式细胞术分析及其百分比(n=3)。所示为三个独立实验的代表性结果。*P<0.05并且**P<0.01。(A) Analysis of STAT3 mRNA in splenic B cells from WT and STAT3-KO mice (n=4). (B and C) Flow cytometry data of annexin V expression in splenic B220, MZ, FO, T1, and T2 cells from WT and STAT3 KO mice and quantitative percentage of cell populations (n=8). (D and E) Flow cytometry data of Ki67 expression in splenic B220, MZ, FO, T1, and T2 cells from WT and STAT3 KO mice and quantitative percentage of cell populations (n=8). Shown are representative results from three independent experiments. (F and G) WT and STAT3 KO mice were injected intraperitoneally with or without R18 (0.2 μg/kg body weight) for 14 consecutive days. Data show flow cytometric analysis of MZ cells and their percentages (n=3). Shown are representative results from three independent experiments. *P<0.05 and **P<0.01.
实施例Example
如下将详细解释本申请。This application will be explained in detail below.
本研究中,我们发现在STAT3 KO B细胞中,近侧正向BCR信号传导有所减少,但负向调节剂SHIP有所增强。在STAT3 KO B细胞中,WASP的激活和由此产生的肌动蛋白重组减少了,并且B细胞的早期激活(包括BCR簇集、B细胞铺展和信号复合体募集)也受损。通过使用来自STAT3 LOF患者的外周血单个核细胞(PBMC),我们发现记忆B细胞的早期激活和肌动蛋白的积累也有所减少。值得注意的是,我们发现记忆B细胞和浆母细胞减少了,但STAT3LOF和GOF细胞二者中的初始B细胞却增加了。另外,来自STAT3 LOF的初始B细胞易分化为记忆B细胞。在机制上,STAT3 KO B细胞具有更多且在体外易形成IgE+B细胞。最后,我们发现在STAT3 KO B细胞和STAT3 LOF患者中miRNA146A和14-3-3σ的表达均有增强。In this study, we found that proximal positive BCR signaling is reduced but the negative regulator SHIP is enhanced in STAT3 KO B cells. In STAT3 KO B cells, WASP activation and resulting actin reorganization are reduced, and early B cell activation, including BCR clustering, B cell spreading, and signaling complex recruitment, is also impaired. By using peripheral blood mononuclear cells (PBMC) from STAT3 LOF patients, we found that early activation of memory B cells and actin accumulation were also reduced. Notably, we found a decrease in memory B cells and plasmablasts, but an increase in naïve B cells in both STAT3LOF and GOF cells. In addition, naive B cells derived from STAT3 LOF readily differentiate into memory B cells. Mechanistically, STAT3 KO B cells have more and readily form IgE + B cells in vitro. Finally, we found that the expression of both miRNA146A and 14-3-3σ was enhanced in STAT3 KO B cells and STAT3 LOF patients.
转录因子在维持正常发挥功能的免疫系统中起着至关重要的作用;因此,转录因子突变可导致免疫失调。STAT3或Dock8功能缺失性突变导致免疫缺陷和IgE过度产生,称为高IgE综合征(HIES),这种疾病的潜在机制很多尚不清楚。在本研究中,我们使用来自STAT3功能丧失性(LOF)和功能获得性(GOF)患者的样品以及来自我们培养的STAT3 B细胞特异性缺失小鼠(STAT3 KO)的样品来研究HIES的机制。有趣的是,我们发现STAT3 KO小鼠的外周血B细胞稳态与STAT3 LOF患者的表型相似,其中的滤泡(FO)和生发中心(GC)B细胞均有减少,但边缘区(MZ)B细胞、静息B细胞和激活IgE+B细胞却有所增加。另外,在抗原刺激下STAT3 KO B细胞的B细胞受体(BCR)信号传导减少,这是因为BCR簇集以及WASP和F-肌动蛋白的积累均有减少。令人兴奋的是,一种中心枢纽蛋白14-3-3σ在STAT3 KO小鼠和STAT3LOF患者的B细胞中增多,14-3-3σ是增加IgE产生所必不可少的。14-3-3σ的增加与上游介导物miRNA146A的表达增加有关。在STAT3 KO小鼠中用R18肽抑制14-3-3σ可将BCR信号传导以及FO、GC和IgE+B细胞分化挽救到野生型(WT)小鼠的程度。总之,我们的研究建立了STAT3-miRNA146A-14-3-3σ的新型调节途径,以调节BCR信号传导、外周血B细胞分化和IgE产生,可为HIES治疗提供靶标。Transcription factors play a critical role in maintaining a properly functioning immune system; therefore, mutations in transcription factors can lead to immune dysregulation. Loss-of-function mutations in STAT3 or Dock8 lead to immunodeficiency and overproduction of IgE, called hyperIgE syndrome (HIES). Many of the underlying mechanisms of this disease remain unclear. In this study, we used samples from STAT3 loss-of-function (LOF) and gain-of-function (GOF) patients and samples from our cultured STAT3 B cell-specific deletion mice (STAT3 KO) to investigate the mechanisms of HIES. Interestingly, we found that peripheral blood B cell homeostasis in STAT3 KO mice was similar to the phenotype of STAT3 LOF patients, in which both follicular (FO) and germinal center (GC) B cells were reduced, but marginal zone (MZ) ) B cells, resting B cells and activated IgE+ B cells increased. Additionally, B cell receptor (BCR) signaling was reduced in STAT3 KO B cells upon antigen stimulation, due to reduced BCR clustering and accumulation of WASP and F-actin. Excitingly, a central hub protein, 14-3-3σ, is increased in B cells of STAT3 KO mice and STAT3LOF patients, and 14-3-3σ is essential for increased IgE production. The increase in 14-3-3σ is associated with the increased expression of the upstream mediator miRNA146A. Inhibition of 14-3-3σ with R18 peptide in STAT3 KO mice rescued BCR signaling and FO, GC and IgE+B cell differentiation to the extent of wild-type (WT) mice. In conclusion, our study established a novel regulatory pathway of STAT3-miRNA146A-14-3-3σ to regulate BCR signaling, peripheral blood B cell differentiation, and IgE production, which may provide targets for HIES treatment.
材料与方法Materials and Methods
小鼠mice
通过将Mb1Cre小鼠(Jackson Laboratory)与stat3 flox/flox小鼠36杂交获得具有C57/BL6背景的STAT3条件性敲除小鼠(在这里称为STAT3 KO小鼠),具有相同遗传背景的stat3 flox/flox小鼠用作野生型对照(WT)。根据重庆医科大学附属儿童医院动物中心的方案,所有小鼠均保存在单独的通风笼中。除非另有说明,否则在小鼠8-12周龄时对其进行分析。如先前所述对骨髓、脾脏单细胞悬浮液和B细胞进行分离26。对于体内用R18治疗,对WT和STAT3 KO小鼠用R18(0.2μg/kg体重)腹膜内(IP)连续注射14天。所有动物实验均按照重庆医科大学儿童医院机构动物护理和使用委员会批准的方案进行。STAT3 conditional knockout mice with a C57/BL6 background (referred to here as STAT3 KO mice) were obtained by crossing Mb1 Cre mice (Jackson Laboratory) with stat3 flox/flox mice36 , stat3 with the same genetic background flox/flox mice were used as wild-type controls (WT). All mice were kept in individual ventilated cages according to the protocol of the Animal Center of Children's Hospital of Chongqing Medical University. Unless otherwise stated, mice were analyzed when they were 8–12 weeks of age. Bone marrow, spleen single cell suspensions and B cells were isolated as previously described26. For in vivo treatment with R18, WT and STAT3 KO mice were injected intraperitoneally (IP) with R18 (0.2 μg/kg body weight) for 14 consecutive days. All animal experiments were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee of Chongqing Medical University Children's Hospital.
患者patient
从2017年到2018年,共有7名中国患者参与本研究,包括6名STAT3功能丧失性(LOF)患者和1名STAT3功能获得性(GOF)患者(表1)。From 2017 to 2018, a total of 7 Chinese patients participated in this study, including 6 STAT3 loss-of-function (LOF) patients and 1 STAT3 gain-of-function (GOF) patient (Table 1).
表1.患者信息,包括性别、年龄和STAT3突变类型。Table 1. Patient information, including gender, age, and STAT3 mutation type.
STAT3 LOF和GOF患者的诊断是以临床体征、症状和STAT3突变为依据做出。健康对照受试者由7名年龄相仿的受试者组成。收集人外周血单个核细胞(PBMC),并使用EasySepTM人B细胞分离试剂盒(17954;Stemcell)从人PBMC中分离B细胞。经重庆医科大学儿童医院伦理委员会批准,已获得所有儿童父母的签字同意。Diagnosis of patients with STAT3 LOF and GOF is based on clinical signs, symptoms, and STAT3 mutations. Healthy control subjects consisted of 7 subjects of similar age. Human peripheral blood mononuclear cells (PBMC) were collected, and B cells were isolated from human PBMC using EasySep ™ Human B Cell Isolation Kit (17954; Stemcell). It was approved by the Ethics Committee of Children's Hospital of Chongqing Medical University, and signed consent was obtained from the parents of all children.
R18肽合成R18 peptide synthesis
通过GL Biochem合成R18氨基酸序列NH2-YGRKKKRQRRRPHCVPRDLSWLDLEANMCLP-COOH。The R18 amino acid sequence NH2 -YGRKKKRQRRRPHCVPRDLSWLDLEANMCLP-COOH was synthesized by GL Biochem.
细胞培养cell culture
获得WT和STAT3 KO小鼠脾脏B细胞(1x105),并用在含有10%FCS(Hyclone)的5mlRPMI 1640中的10μg/ml抗小鼠CD40(BE0016-2,BioXcell)和8ng/ml IL-4(R&D Systems)刺激5天,然后通过流式细胞术分析细胞的IgE表达。为了检测B细胞增殖,将小鼠脾脏B细胞(1x105)分离并用Cell Trace Violet标记,然后在含有10%FCS(Hyclone)、10mmol/Lβ-巯基乙醇和1%链霉素-青霉素的RPMI 1640中与5mg/mL LPS(Sigma-Aldrich)或10μg/ml B类CpG寡核苷酸(ODN1826,Invivogen)以200μl/孔的体积在96孔圆底板中培养5天,然后通过细胞计量术进行分析。WT and STAT3 KO mouse splenic B cells (1x10 5 ) were obtained and treated with 10 μg/ml anti-mouse CD40 (BE0016-2, BioXcell) and 8 ng/ml IL-4 in 5 ml RPMI 1640 containing 10% FCS (Hyclone) (R&D Systems) were stimulated for 5 days, and then cells were analyzed for IgE expression by flow cytometry. To detect B cell proliferation, mouse splenic B cells (1x10 5 ) were isolated and labeled with Cell Trace Violet, then incubated in RPMI 1640 containing 10% FCS (Hyclone), 10 mmol/L β-mercaptoethanol, and 1% streptomycin-penicillin. Neutralized with 5 mg/mL LPS (Sigma-Aldrich) or 10 μg/ml class B CpG oligonucleotide (ODN1826, Invivogen) in a volume of 200 μl/well and cultured in 96-well round bottom plates for 5 days, then analyzed by cytometry. .
流式细胞术和抗体Flow Cytometry and Antibodies
为进行流式细胞术分析,从骨髓和脾脏分离单细胞悬浮液,并将所述单细胞悬浮液与Fcγ受体(FcγR)阻断性抗体(抗小鼠CD16/CD32;BD Bioscience)在冰上孵育,然后在具有2%FBS的PBS中用以下抗体(Ab)染色。对于表面标记物,所使用的抗小鼠抗体包括FITC-抗CD127(135008;BioLegend)、APC-抗CD43(143208)、PE-抗BP-1(108307;BioLegend)、亮紫510-抗B220(103247;BioLegend)、PE/Cy7-抗CD24(101822;BioLegend)和亮紫421-抗IgM(406518;BioLegend)、FITC-抗CD19(101506;BioLegend)、FITC-抗CD95(554257;BD Biosciences)、FITC-抗膜联蛋白V(640906;BioLegend)、PE-抗CD23(101608;BioLegend)、PerCP/Cy5.5-抗IgD(405710;BioLegend)、APC-抗CD21(123412;BioLegend)、Alexa Fluor 647-抗GL7(144606;BioLegend)、亮紫421-抗IgM(406518;BioLegend)、PE-抗CD5(100608;BioLegend)和APC-抗CD11b(101212;BioLegend)。为进行细胞内染色,根据制造商说明书,使用Foxp3染色缓冲液套件(eBiosciense)对细胞进行固定和透化,并用Ki67(25-56698-82,ebioscience)和PE-抗IgE(406907;BioLegend)对细胞染色。用于对人B细胞表面标记物染色的抗人抗体和试剂包括FITC-抗CD19(302206;BioLegend)、太平洋蓝-抗CD38(356628;BioLegend)、BV510-抗IgD(348220;BioLegend)、PE-抗CD24(311106;BioLegend)和AF647-抗CD27(302812;BioLegend)。For flow cytometry analysis, single cell suspensions were isolated from bone marrow and spleen and incubated on ice with an Fcγ receptor (FcγR) blocking antibody (anti-mouse CD16/CD32; BD Bioscience). Incubate on and then stain with the following antibodies (Ab) in PBS with 2% FBS. For surface markers, anti-mouse antibodies used included FITC-anti-CD127 (135008; BioLegend), APC-anti-CD43 (143208), PE-anti-BP-1 (108307; BioLegend), Brilliant Violet 510-anti-B220 ( 103247; BioLegend), PE/Cy7-anti-CD24 (101822; BioLegend) and Brilliant Violet 421-anti-IgM (406518; BioLegend), FITC-anti-CD19 (101506; BioLegend), FITC-anti-CD95 (554257; BD Biosciences), FITC-anti-Annexin V (640906; BioLegend), PE-anti-CD23 (101608; BioLegend), PerCP/Cy5.5-anti-IgD (405710; BioLegend), APC-anti-CD21 (123412; BioLegend), Alexa Fluor 647 - anti-GL7 (144606; BioLegend), Brilliant Violet 421-anti-IgM (406518; BioLegend), PE-anti-CD5 (100608; BioLegend) and APC-anti-CD11b (101212; BioLegend). For intracellular staining, cells were fixed and permeabilized using the Foxp3 Staining Buffer Kit (eBiosciense) according to the manufacturer's instructions and stained with Ki67 (25-56698-82, ebioscience) and PE-anti-IgE (406907; BioLegend). Cell staining. Anti-human antibodies and reagents for staining human B cell surface markers include FITC-anti-CD19 (302206; BioLegend), Pacific Blue-anti-CD38 (356628; BioLegend), BV510-anti-IgD (348220; BioLegend), PE- anti-CD24 (311106; BioLegend) and AF647-anti-CD27 (302812; BioLegend).
先前已经描述了磷酸化信号传导蛋白水平的检测37。简单来说,将脾脏B细胞分离并用亮紫510-抗B220(103247;BioLegend)染色,然后与生物素缀合F(ab)2抗小鼠IgG的可溶性抗原(sAg)孵育30分钟,再与链霉亲和素孵育10分钟,随后在37℃下进行不同时间长度的BCR信号传导诱导。在BCR激活后,立即用磷酸化流式细胞术(Phosflow)裂解/固定缓冲液(BD Biosciences)固定细胞,并用磷酸化流式细胞术透化缓冲液III(BD Biosciences)透化细胞。将细胞用针对pWASP(S483/S484)(A300-205A;Bethyl)和F-肌动蛋白(R37110;Thermo Fisher)的抗体染色,二抗使用Alexa Fluor 405-山羊抗兔IgG抗体(A-31556;Thermo Fisher)。在FACS Canto(BD Biosciences)上采集流式细胞术数据,并使用FlowJo软件进行分析。Detection of phosphorylated signaling protein levels has been described previously37. Briefly, splenic B cells were isolated and stained with Brilliant Violet 510-anti-B220 (103247; BioLegend), then incubated with biotin-conjugated F(ab) 2 anti-mouse IgG soluble antigen (sAg) for 30 min, and then with Streptavidin incubation was performed for 10 min, followed by induction of BCR signaling for varying lengths of time at 37°C. Immediately after BCR activation, cells were fixed with Phosflow lysis/fixation buffer (BD Biosciences) and permeabilized with Phosflow permeabilization buffer III (BD Biosciences). Cells were stained with antibodies against pWASP(S483/S484) (A300-205A; Bethyl) and F-actin (R37110; Thermo Fisher), and the secondary antibody was Alexa Fluor 405-goat anti-rabbit IgG antibody (A-31556; Thermo Fisher). Flow cytometry data were collected on a FACS Canto (BD Biosciences) and analyzed using FlowJo software.
全内反射荧光显微术和共聚焦显微术Total internal reflection fluorescence microscopy and confocal microscopy
如先前所述,获得全内反射荧光显微术(TIRFm)和共聚焦荧光显微术图像26。简单来说,将来自人PBMC和小鼠脾脏的B细胞在37℃下在膜拴系抗原(mAg)上进行不同时间点的孵育。在固定和透化后,将细胞针对pCD19(ab203615;Abcam)、pBtk(5082S;SignalingTechnology,丹佛市,马萨诸塞州)、pY(05-321;Merck-Millipore)、pSHIP(3941S;CellSignaling Technology,丹佛市,马萨诸塞州)和pWASP(A300-205A;Bethyl Laboratories)进行染色。使用NIS Elements AR 3.2软件进行TIRFm分析。使用干涉反射显微术(IRM)图像测定B细胞接触面积。为进行平均荧光强度(MFI)分析,减去背景荧光,并在B细胞接触区内测量MFI。针对每组数据,对来自2个或3个独立实验的>20个单独细胞进行分析。Total internal reflection fluorescence microscopy (TIRFm) and confocal fluorescence microscopy images were obtained as previously described26 . Briefly, B cells from human PBMC and mouse spleen were incubated on membrane-tethered antigen (mAg) at 37°C for different time points. After fixation and permeabilization, cells were raised against pCD19 (ab203615; Abcam), pBtk (5082S; Signaling Technology, Denver, MA), pY (05-321; Merck-Millipore), pSHIP (3941S; CellSignaling Technology, Denver, MA) , Massachusetts) and pWASP (A300-205A; Bethyl Laboratories) for staining. TIRFm analysis was performed using NIS Elements AR 3.2 software. B cell contact area was determined using interference reflection microscopy (IRM) images. For mean fluorescence intensity (MFI) analysis, background fluorescence was subtracted and MFI was measured within the B cell contact zone. For each set of data, >20 individual cells from 2 or 3 independent experiments were analyzed.
为进行共聚焦显微术分析,分离来自WT和STAT3 KO小鼠的脾脏B细胞,并通过将细胞与AF546-单生物素化Fab'-抗IgG及链霉亲和素在4℃下孵育对细胞进行sAg刺激,然后将细胞进行洗涤并加温至37℃保持不同的时间点。在固定和透化后,将细胞针对pCD19(ab203615;Abcam)、pBtk(5082S;Signaling Technology)、pY(05-321;Merck-Millipore)、pSHIP(3941S;Cell Signaling Technology,丹佛市,马萨诸塞州)和pWASP(A300-205A;Bethyl Laboratories)进行染色,然后通过共聚焦显微术成像,并使用NIS ElementsAR3.2软件进行分析。For confocal microscopy analysis, splenic B cells from WT and STAT3 KO mice were isolated and treated by incubating the cells with AF546-monobiotinylated Fab'-anti-IgG and streptavidin at 4°C. The cells were stimulated with sAg, then washed and warmed to 37°C for different time points. After fixation and permeabilization, cells were raised against pCD19 (ab203615; Abcam), pBtk (5082S; Signaling Technology), pY (05-321; Merck-Millipore), pSHIP (3941S; Cell Signaling Technology, Denver, MA) and pWASP (A300-205A; Bethyl Laboratories) were stained and then imaged by confocal microscopy and analyzed using NIS ElementsAR3.2 software.
免疫印迹Western blot
如先前所述进行免疫印迹分析37。为检测磷酸化信号传导蛋白,通过将来自WT和STAT3 KO小鼠的脾脏B细胞与生物素化抗小鼠IgG F(ab)2在冰上孵育30分钟,然后与链霉亲和素在冰上孵育10分钟来对所述细胞进行sAg刺激,然后将细胞进行洗涤并加温至37℃保持指定的时间,并进行细胞裂解。将细胞裂解物在SDS-PAGE凝胶上运行,并进行针对pAkt(Ser473;Cell Signaling Technology)、pBtk(5082S;Cell Signaling Technology)、pCD19(3571;Cell Signaling Technology)、pY(05321;Merck-Millipore)、pSHIP(3941S;Cell Signaling Technology)、pWASP(ab5278;abacm)、p-mTOR(5536S;Cell SignalingTechnology)、pS6(4856S;Cell Signaling Technology)、pFoxo1(9461S;Cell SignalingTechnology)和pPI3K(17366S;Cell Signaling Technology)的免疫印迹。为检测总蛋白表达,将人或小鼠B细胞进行裂解,并进行针对Btk(8547S;Cell Signaling Technology)、SHIP(2728S;Cell Signaling Technology)、WASP(sc-13139;Santa CruzBiotechnology)、Akt(9272S;Cell Signaling Technology)、S6(2217S;Cell SignalingTechnology)、Foxo1(2880S;Cell Signaling Technology)、mTOR(7C10;Cell SignalingTechnology)、PI3K(4292;Cell Signaling Technology)、CD19(3574;Cell SignalingTechnology)、Btk(8547;Cell Signaling Technology)、DOCK8(sc-292124;Santa CruzBiotechnology)、WIP(sc-271113;Santa Cruz Biotechnology)、人/小鼠/大鼠14-3-3σ抗体(AF4424;Bio-Techne)的免疫印迹,并将抗小鼠GAPDH和β-肌动蛋白用作上样对照。对于体外用R18处理,将来自WT和KO小鼠的脾脏B细胞在与sAg孵育之前与媒介物或10μM R18孵育1h。Immunoblot analysis was performed as previously described 37 . To detect phosphorylated signaling proteins, splenic B cells from WT and STAT3 KO mice were incubated with biotinylated anti-mouse IgG F(ab) 2 for 30 min on ice followed by streptavidin on ice. The cells were incubated for 10 minutes for sAg stimulation, and then the cells were washed, warmed to 37°C for the specified time, and lysed. Cell lysates were run on SDS-PAGE gels and analyzed for pAkt (Ser473; Cell Signaling Technology), pBtk (5082S; Cell Signaling Technology), pCD19 (3571; Cell Signaling Technology), pY (05321; Merck-Millipore ), pSHIP (3941S; Cell Signaling Technology), pWASP (ab5278; abacm), p-mTOR (5536S; Cell Signaling Technology), pS6 (4856S; Cell Signaling Technology), pFoxo1 (9461S; Cell Signaling Technology) and pPI3K (17366S; Cell Signaling Technology). To detect total protein expression, human or mouse B cells were lysed and assayed for Btk (8547S; Cell Signaling Technology), SHIP (2728S; Cell Signaling Technology), WASP (sc-13139; Santa Cruz Biotechnology), Akt (9272S ; Cell Signaling Technology), S6 (2217S; Cell Signaling Technology), Foxo1 (2880S; Cell Signaling Technology), mTOR (7C10; Cell Signaling Technology), PI3K (4292; Cell Signaling Technology), CD19 (3574; Cell Signaling Technology), Btk ( 8547; Cell Signaling Technology), DOCK8 (sc-292124; Santa Cruz Biotechnology), WIP (sc-271113; Santa Cruz Biotechnology), human/mouse/rat 14-3-3σ antibody (AF4424; Bio-Techne) Blot, and anti-mouse GAPDH and β-actin were used as loading controls. For in vitro treatment with R18, splenic B cells from WT and KO mice were incubated with vehicle or 10 μM R18 for 1 h before incubation with sAg.
Seahorse测定Seahorse assay
使用生物素化抗小鼠IgG F(ab)2在37℃下将从WT和STAT3 KO小鼠中分离的脾脏B细胞与sAg孵育1小时,然后在XF培养基(含有5mM葡萄糖、2mM L-谷氨酰胺和1mM丙酮酸钠的无缓冲剂DMEM)中孵育细胞。使用XF-24细胞外流量分析仪(Seahorse Bioscience)测量响应于1M寡霉素、2M氟羰基氰化苯腙(FCCP)和1M鱼藤酮的耗氧率(OCR)。Splenic B cells isolated from WT and STAT3 KO mice were incubated with sAg for 1 hour at 37°C using biotinylated anti-mouse IgG F(ab) 2 and then incubated in XF medium (containing 5mM glucose, 2mM L- Cells were incubated in unbuffered DMEM (glutamine and 1 mM sodium pyruvate). Oxygen consumption rate (OCR) in response to 1 M oligomycin, 2 M fluorocarbonyl cyanide phenylhydrazone (FCCP), and 1 M rotenone was measured using an XF-24 extracellular flux analyzer (Seahorse Bioscience).
ELISAELISA
从WT和STAT3 KO小鼠中收集血清,并根据制造商说明书使用ELISA试剂盒(eBioscience)测量IgE水平。Serum was collected from WT and STAT3 KO mice, and IgE levels were measured using an ELISA kit (eBioscience) according to the manufacturer's instructions.
定量RT-PCRQuantitative RT-PCR
为检测WT和STAT3 KO小鼠以及健康对照(HC)和LOF患者中STAT3、miRNA146A和14-3-3δmRNA表达的差异,从小鼠脾脏和患者PBMC中分离B细胞,然后通过RNAPURE试剂盒(RP1202;BioTeke)分离RNA,并用PrimeScript RT试剂盒(RR037A;Takara)进行逆转录。使用转录的cDNA以在CFX96 Touch Real-Time System(Bio-Rad)上用Advanced SYBR GreenSupermix(Bio-Rad)分析不同基因的表达。STAT3(小鼠)F:5′-TGTCAGATCACATGGGCTAAAT-3′;STAT3(小鼠)R:5′-GGTCGATGATATTGTCTAGCCA-3′;14-3-3σ(小鼠)σF:5′-AGAACCCAGCGTTACTCTCGA-3′;14-3-3σ(小鼠)σR:5′-CCA CCACGTTCTTGTAAGCT-3′;14-3-3σ(人)σF:5′-GAAGTTGCAGCTGATTAAGGAC-3′;14-3-3σ(人)σR:5′-TCTGGATTAGTTGCATTGGCTA-3′。根据制造商方案,使用Taqman微RNA转录和微RNA测定试剂盒分析miRNA146A的表达。To detect the differences in STAT3, miRNA146A and 14-3-3δ mRNA expression in WT and STAT3 KO mice as well as healthy controls (HC) and LOF patients, B cells were isolated from mouse spleens and patient PBMCs and then analyzed by RNAPURE kit (RP1202; RNA was isolated from BioTeke) and reverse transcribed using PrimeScript RT kit (RR037A; Takara). Transcribed cDNA was used to analyze the expression of different genes with Advanced SYBR GreenSupermix (Bio-Rad) on a CFX96 Touch Real-Time System (Bio-Rad). STAT3 (mouse) F: 5'-TGTCAGATCACATGGGCTAAAT-3'; STAT3 (mouse) R: 5'-GGTCGATGATATTGTCTAGCCA-3'; 14-3-3σ (mouse) σF: 5'-AGAACCCAGCGTTACTCTCGA-3'; 14 -3-3σ(mouse)σR:5′-CCA CCACGTTCTTGTAAGCT-3′; 14-3-3σ(human)σF:5′-GAAGTTGCAGCTGATTAAGGAC-3′; 14-3-3σ(human)σR:5′- TCTGGATTAGTTGCATTGGCTA-3′. The expression of miRNA146A was analyzed using the Taqman MicroRNA Transcription and MicroRNA Assay Kit according to the manufacturer's protocol.
染色质免疫沉淀(ChIP)测定Chromatin Immunoprecipitation (ChIP) Assay
根据制造商方案,使用酶解染色质IP试剂盒(琼脂糖珠)(9002S;Cell Signaling Technology)进行ChIP研究。在室温下用甲醛对来自WT C57BL/6小鼠的B细胞处理10分钟。细胞裂解物经过超声处理产生长度为100-1000碱基对的剪切交联染色质。将可溶性染色质用针对STAT3的抗体(9139S;Cell Signaling Technology)进行免疫沉淀,并在Bio-Rad PCR仪器上进行PCR检测。所使用的引物如下:小鼠14-3-3σ启动子正向5’-CACACCCACACTACCTCACA-3’和反向5’-GTGGTAGTGCTGTCCAGGTG-3。According to the manufacturer's protocol, use ChIP studies were performed using enzymatic chromatin IP kit (agarose beads) (9002S; Cell Signaling Technology). B cells from WT C57BL/6 mice were treated with formaldehyde for 10 min at room temperature. Cell lysates are sonicated to produce sheared, cross-linked chromatin 100-1000 base pairs in length. Soluble chromatin was immunoprecipitated with an antibody against STAT3 (9139S; Cell Signaling Technology) and detected by PCR on a Bio-Rad PCR instrument. The primers used were as follows: mouse 14-3-3σ promoter forward 5′-CACACCCACACTACCTCACA-3′ and reverse 5′-GTGGTAGTGCTGTCCAGGTG-3.
统计学分析Statistical analysis
除非另有说明,否则使用Prism 7软件通过双尾非配对学生t检验评估统计学显著性(*p<0.05;**p<0.01;***p<0.001)。数据表示为平均值+/-标准差(SD)。Unless otherwise stated, statistical significance was assessed by two-tailed unpaired Student's t test using Prism 7 software (*p<0.05; **p<0.01; ***p<0.001). Data are expressed as mean +/- standard deviation (SD).
结果result
STAT3对维持外周血B细胞的稳态至关重要,但对骨髓B细胞则不然。STAT3 is critical for maintaining homeostasis of peripheral blood B cells, but not bone marrow B cells.
为研究骨髓B细胞发育过程中是否需要STAT3,我们通过将Mb1Cre小鼠与stat3 flox /flox小鼠杂交产生STAT3 B细胞特异性敲除小鼠Mb1Cre stat3flox/flox(STAT3 KO)。stat3mRNA在STAT3 KO B细胞中的表达显著降低,表明有效缺失(图8A)。用BP-1和CD24抗体对STAT3 KO小鼠的骨髓B细胞亚类进行染色,以确定前祖B细胞、祖B细胞和早期前B细胞,同时使用B220 IgM抗体鉴定晚期前B细胞、未成熟B细胞和再循环B细胞。除再循环B细胞频率呈下降趋势外,我们未观察到所有受检亚群有任何异常(图1A、图1B、图1C)。IL-7信号传导对BM B细胞发育至关重要,然而,在WT与STAT3 KO小鼠之间IL-7受体表达(CD127)并无差异(图1D)。这表明STAT3对于骨髓B细胞发育是不可或缺的。通过使用IgM IgD抗体对T1、T2和滤泡(FO)B细胞染色,使用CD23和CD21抗体定义边缘区(MZ)B细胞,以及使用GL7和CD95抗体鉴定生发中心(GC)B细胞,我们进一步确定了STAT3如何影响外周血B细胞。在STAT3 KO小鼠中,T1 B细胞的百分比和数量正常,但T2 B细胞的百分比和数量有所减少(图1E、图1F、图1G)。值得注意的是,在STAT3 KO小鼠中,FO的百分比和数量显著减少(图1E、图1H),但MZ B细胞的百分比和数量却增加了(图1I、图1J)。另外,STAT3 KO小鼠的自发性GC B细胞的比率和数量也有所减少(图1K、图1L)。我们通过用Ki67和膜联蛋白V染色检查了静息态B细胞的增殖和凋亡,并发现在WT与KO小鼠之间,Ki67和膜联蛋白V在FO、MZ、GC、T1和T2细胞中的表达均无差异(图8B-图8E)。其次,我们发现B1a细胞的百分比和数量正常,但B1b细胞的比率显著降低(图1M、图1N、图1O)。最后,我们检查了用IL-4和抗CD40刺激5天的静息和激活B细胞的IgE染色。有趣的是,我们发现STAT3 KO B细胞在静息态和激活态下的IgE百分比均显著增加(图1P-图1R)。我们还检测了WT和STAT3 KO小鼠血清中的IgE水平,并发现STAT3 KO小鼠中的IgE滴度也有所增加(图1S)。这些结果表明,STAT3对维持外周血B细胞的稳态至关重要。To investigate whether STAT3 is required during bone marrow B cell development, we generated STAT3 B cell-specific knockout mice Mb1 Cre stat3 flox /flox (STAT3 KO) by crossing Mb1 Cre mice with stat3 flox/ flox mice. Expression of stat3 mRNA was significantly reduced in STAT3 KO B cells, indicating efficient deletion (Fig. 8A). Staining of bone marrow B cell subsets from STAT3 KO mice with BP-1 and CD24 antibodies to identify progenitor B cells, progenitor B cells, and early pre-B cells, while using the B220 IgM antibody to identify late pre-B cells, immature pre-B cells B cells and recycling B cells. Except for a downward trend in the frequency of recycling B cells, we did not observe any abnormalities in any subpopulation examined (Figure 1A, Figure 1B, Figure 1C). IL-7 signaling is critical for BM B cell development, however, there was no difference in IL-7 receptor expression (CD127) between WT and STAT3 KO mice (Fig. 1D). This suggests that STAT3 is indispensable for bone marrow B cell development. By staining T1, T2, and follicular (FO) B cells using IgM IgD antibodies, defining marginal zone (MZ) B cells using CD23 and CD21 antibodies, and identifying germinal center (GC) B cells using GL7 and CD95 antibodies, we further Determined how STAT3 affects peripheral blood B cells. In STAT3 KO mice, the percentage and number of T1 B cells were normal, but the percentage and number of T2 B cells were reduced (Figure 1E, Figure 1F, Figure 1G). Notably, in STAT3 KO mice, the percentage and number of FOs were significantly reduced (Figure 1E, Figure 1H), but the percentage and number of MZ B cells were increased (Figure 1I, Figure 1J). In addition, the ratio and number of spontaneous GC B cells in STAT3 KO mice were also reduced (Figure 1K, Figure 1L). We examined the proliferation and apoptosis of resting B cells by staining with Ki67 and annexin V, and found that Ki67 and annexin V increased in FO, MZ, GC, T1, and T2 cells between WT and KO mice. There was no difference in expression (Figure 8B-Figure 8E). Second, we found that the percentage and number of B1a cells were normal, but the ratio of B1b cells was significantly reduced (Figure 1M, Figure 1N, Figure 1O). Finally, we examined IgE staining of resting and activated B cells stimulated with IL-4 and anti-CD40 for 5 days. Interestingly, we found that the IgE percentage of STAT3 KO B cells was significantly increased in both resting and activated states (Figure 1P-Figure 1R). We also examined IgE levels in the serum of WT and STAT3 KO mice and found that IgE titers were also increased in STAT3 KO mice (Fig. 1S). These results indicate that STAT3 is critical for maintaining peripheral blood B cell homeostasis.
STAT3正向调节近侧BCR信号传导。STAT3 positively regulates proximal BCR signaling.
由于STAT3对于维持外周血B细胞的稳态至关重要,而外周血B细胞的稳态与抗原BCR信号传导高度相关38-40,因此我们对STAT3如何调节BCR信号传导展开研究。我们使用共聚焦显微术检查STAT3缺失对CD19时空组织的影响,CD19是一种参与放大上游BCR信号传导的共同受体。在sAg刺激后的5分钟和10分钟,STAT3 KO B细胞中pCD19与BCR之间的共定位显著减少(图2A、图2B)。此外,通过对在不同时间长度内用可溶性抗原(sAg)刺激的脾脏B细胞的裂解物进行免疫印迹,确定了STAT3 KO B细胞中pCD19水平降低(图2C)。总之,这意味着STAT3影响CD19的时空组织和激活。接下来,我们使用与上文类似的条件通过共聚焦显微术和免疫印迹检查酪氨酸磷酸化蛋白(pY)和pBtk(CD19的直接下游激酶)造成的总BCR信号传导水平。在STAT3 KO B细胞中,pY/pBtk与BCR之间的相关系数在10min时显著降低(图2D、图2E)。另外,STAT3 KO B细胞中的pY和pBtk水平显著降低(图2F)。最后,我们使用与上文相同的条件确定了STAT3缺乏对pSHIP与BCR之间的共定位以及SHIP(其为BCR信号传导的近侧负向调节剂)激活的影响。STAT3 KO B细胞中BCR与pSHIP之间的共定位在5min时显著降低(图2G、图2H),并且STAT3 KO B细胞中SHIP的磷酸化增强(图2I)。总之,这些结果表明STAT3缺乏破坏了正向与负向BCR信号传导的平衡。Since STAT3 is crucial for maintaining the homeostasis of peripheral blood B cells, and the homeostasis of peripheral blood B cells is highly related to antigen BCR signaling38-40 , we studied how STAT3 regulates BCR signaling. We used confocal microscopy to examine the impact of STAT3 deletion on the spatiotemporal organization of CD19, a coreceptor involved in amplifying upstream BCR signaling. Colocalization between pCD19 and BCR was significantly reduced in STAT3 KO B cells at 5 and 10 minutes after sAg stimulation (Fig. 2A, Fig. 2B). Furthermore, reduced pCD19 levels in STAT3 KO B cells were determined by immunoblotting lysates of splenic B cells stimulated with soluble antigen (sAg) for different lengths of time (Fig. 2C). Taken together, this means that STAT3 affects the spatiotemporal organization and activation of CD19. Next, we examined total BCR signaling levels by confocal microscopy and immunoblotting using conditions similar to those above, resulting from tyrosine-phosphorylated protein (pY) and pBtk, a direct downstream kinase of CD19. In STAT3 KO B cells, the correlation coefficient between pY/pBtk and BCR decreased significantly at 10 min (Figure 2D, Figure 2E). In addition, pY and pBtk levels were significantly reduced in STAT3 KO B cells (Fig. 2F). Finally, we determined the effect of STAT3 deficiency on colocalization between pSHIP and BCR and activation of SHIP, a proximal negative regulator of BCR signaling, using the same conditions as above. The colocalization between BCR and pSHIP in STAT3 KO B cells was significantly reduced at 5 min (Figure 2G, Figure 2H), and the phosphorylation of SHIP was enhanced in STAT3 KO B cells (Figure 2I). Taken together, these results indicate that STAT3 deficiency disrupts the balance of positive and negative BCR signaling.
STAT3缺乏减少了由WASP和WIP介导的F-肌动蛋白的积累以及BCR簇集和正向信号复合体募集。STAT3 deficiency reduces F-actin accumulation as well as BCR clustering and forward signaling complex recruitment mediated by WASP and WIP.
先前的报道证实了巨噬细胞系中STAT3与肌动蛋白之间的相关性41。因此,我们试图确定STAT3 KO B细胞中的异常BCR信号传导是否会导致肌动蛋白的破坏。为检查这一点,将经sAg刺激的B细胞进行固定、透化,并分别使用特异性抗体和鬼笔环肽针对肌动蛋白调节剂pWASP和F-肌动蛋白进行染色,然后通过共聚焦显微术、免疫印迹和磷酸化流式细胞术进行分析。在STAT3 KO B细胞的免疫印迹和磷酸化流式细胞术中,只有pWASP的表达降低,而总WASP的表达未降低(图3A-图3C),这与肌动蛋白积累减少相关。为进一步探讨STAT3如何促进pWASP表达的潜在机制,通过免疫印迹检查了WIP的表达,WIP可稳定WASP的磷酸化并防止WASP降解。不出所料,STAT3KO鼠B细胞和STAT3 LOF患者的PBMC中的WIP表达减少了(图3D)。这表明pWASP水平降低可能是由WIP的表达减少所致。DOCK8在WASP的上游15,并且有报道表明DOCK8缺乏患者也患有HIES42。因此,我们检查了DOCK8在STAT3 KO小鼠和LOF患者中的表达。有趣的是,STAT3 KO鼠B细胞和STAT3 LOF患者PBMC中的DOCK8表达显著减少(图3D)。我们先前的研究表明,质膜上存在缺陷的F-肌动蛋白重组会影响BCR的速度以及后续的BCR簇集和BCR信号传导43。为研究STAT3缺失对B细胞早期激活(包括BCR簇集和信号复合体积累)的影响,我们使用全内反射荧光显微术(TIRFm)观察经膜拴系抗原(mAg)刺激的B细胞。脾脏B细胞经不同时间长度的mAg激活后,用鬼笔环肽、pWASP、pCD19、pBtk、pY和pSHIP染色。在STAT3 KO B细胞中,通过接触区内的平均荧光强度(MFI)测量的B细胞铺展和BCR簇集显著减少(图3E、图3G、图3H)。在STAT3 KO B细胞的接触区内,F-肌动蛋白和pWASP的募集也显著减少(图3F、图3I、图3J)。此外,在B细胞接触区内,pCD19(图4A、图4D)、pBtk和pY(图4B、图4E、图4F)信号复合体的募集显著减少,但在STAT3 KO B细胞中,pSHIP的募集有所增强(图4C、图4G)。综上所述,这些结果表明STAT3缺乏减少了由WASP在质膜上介导的F-肌动蛋白的募集,从而导致BCR簇集和BCR信号传导的减少,而这可能是由于缺乏将BCR隔离成簇的驱动力。Previous reports confirmed the association between STAT3 and actin in macrophage cell lines41 . Therefore, we sought to determine whether aberrant BCR signaling in STAT3 KO B cells leads to actin disruption. To examine this, sAg-stimulated B cells were fixed, permeabilized, stained for the actin regulators pWASP and F-actin using specific antibodies and phalloidin, respectively, and then visualized by confocal Analyzes were performed by microsurgery, immunoblotting, and phosphorylation flow cytometry. In immunoblotting and phospho-flow cytometry of STAT3 KO B cells, only the expression of pWASP, but not total WASP, was reduced (Figure 3A-Figure 3C), which correlated with reduced actin accumulation. To further explore the potential mechanism of how STAT3 promotes pWASP expression, the expression of WIP, which stabilizes WASP phosphorylation and prevents WASP degradation, was examined by Western blotting. As expected, WIP expression was reduced in B cells from STAT3KO mice and in PBMCs from STAT3 LOF patients (Fig. 3D). This suggests that reduced pWASP levels may be caused by reduced expression of WIP. DOCK8 is upstream of WASP 15 , and it has been reported that patients with DOCK8 deficiency also suffer from HIES 42 . Therefore, we examined the expression of DOCK8 in STAT3 KO mice and LOF patients. Interestingly, DOCK8 expression was significantly reduced in B cells of STAT3 KO mice and PBMC of STAT3 LOF patients (Fig. 3D). Our previous studies showed that defective F-actin reorganization at the plasma membrane affects the speed of BCR and subsequent BCR clustering and BCR signaling43 . To study the impact of STAT3 loss on early B cell activation, including BCR clustering and signaling complex accumulation, we used total internal reflection fluorescence microscopy (TIRFm) to observe B cells stimulated by membrane-tethered antigen (mAg). Splenic B cells were stained with phalloidin, pWASP, pCD19, pBtk, pY and pSHIP after being activated by mAg for different lengths of time. B cell spreading and BCR clustering, measured by mean fluorescence intensity (MFI) within the contact zone, were significantly reduced in STAT3 KO B cells (Figure 3E, Figure 3G, Figure 3H). The recruitment of F-actin and pWASP was also significantly reduced within the contact zone of STAT3 KO B cells (Figure 3F, Figure 3I, Figure 3J). Furthermore, the recruitment of pCD19 (Figure 4A, Figure 4D), pBtk, and pY (Figure 4B, Figure 4E, Figure 4F) signaling complexes was significantly reduced within the B cell contact zone, but in STAT3 KO B cells, the recruitment of pSHIP Enhanced (Figure 4C, Figure 4G). Taken together, these results indicate that STAT3 deficiency reduces F-actin recruitment to the plasma membrane mediated by WASP, resulting in reduced BCR clustering and BCR signaling, which may be due to a lack of sequestration of the BCR Clustering driving force.
STAT3缺乏降低PI3K-Akt-mTORC1介导的代谢信号传导通路。STAT3 deficiency reduces the PI3K-Akt-mTORC1-mediated metabolic signaling pathway.
PI3K参与代谢信号传导通路,并且是Btk激活的直接下游效应子44。因此,我们检查了由PI3K介导的mTORC1和mTORC2信号传导。首先,将经sAg刺激的STAT3 KO B细胞进行裂解并用pPI3K特异性抗体探测,并发现pPI3K水平降低(图5A)。接下来,我们使用类似条件检查了mTORC2信号传导分子,包括pAkt和pFoxo1。经刺激后的STAT3 KO B细胞中的pAkt和pFoxo1水平也有所降低(图5A)。此外,经刺激后的STAT3 KO B细胞中mTORC1和mTORC2的mTOR共同亚基的磷酸化水平也有所降低(图5A)。最后,通过检查pS6水平确定了Akt-mTORC1的下游激酶的激活。我们发现pS6水平也降低了(图5A)。由于mTORC活性与细胞代谢高度相关,我们利用Seahorse XF技术对氧化磷酸化进行实时分析。耗氧率(OCR)用作氧化磷酸化的量度。首先用寡霉素处理细胞以阻断线粒体ATP的产生,然后用羰基氰4-(三氟甲氧基)苯腙(FCCP)处理细胞以诱导最大耗氧量,再用鱼藤酮加抗霉素处理细胞以抑制电子传递链。用sAg刺激细胞后,STAT3 KO B细胞的基础和最大呼吸速率均低于WT B细胞(图5B)。我们还使用膜联蛋白V和Cell Trace Violet染色通过流式细胞术检查受LPS或CpG脉冲的B细胞的增殖和凋亡,并发现STAT3KO B细胞的增殖有所增加(图5C、图5D),但膜联蛋白V的表达未见差异(图5E、图5F)。因此,STAT3 KO B细胞在受到刺激时不存在增殖缺陷,也不会衰弱和因凋亡而死亡。总之,这些结果意味着STAT3缺乏抑制了B细胞代谢来对BCR刺激作出响应。PI3K is involved in metabolic signaling pathways and is a direct downstream effector of Btk activation44. Therefore, we examined mTORC1 and mTORC2 signaling mediated by PI3K. First, sAg-stimulated STAT3 KO B cells were lysed and probed with pPI3K-specific antibodies, and pPI3K levels were found to be reduced (Fig. 5A). Next, we examined mTORC2 signaling molecules, including pAkt and pFoxo1, using similar conditions. The levels of pAkt and pFoxo1 were also reduced in stimulated STAT3 KO B cells (Fig. 5A). In addition, the phosphorylation levels of the mTOR common subunits of mTORC1 and mTORC2 were also reduced in stimulated STAT3 KO B cells (Fig. 5A). Finally, activation of downstream kinases of Akt-mTORC1 was determined by examining pS6 levels. We found that pS6 levels were also reduced (Fig. 5A). Since mTORC activity is highly correlated with cellular metabolism, we utilized Seahorse XF technology for real-time analysis of oxidative phosphorylation. Oxygen consumption rate (OCR) is used as a measure of oxidative phosphorylation. Cells were first treated with oligomycin to block mitochondrial ATP production, then treated with carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) to induce maximum oxygen consumption, and then treated with rotenone plus antimycin. cells to inhibit the electron transport chain. After stimulating cells with sAg, the basal and maximum respiratory rates of STAT3 KO B cells were lower than those of WT B cells (Fig. 5B). We also examined the proliferation and apoptosis of B cells pulsed with LPS or CpG by flow cytometry using Annexin V and Cell Trace Violet staining and found increased proliferation of STAT3KO B cells (Figure 5C, Figure 5D), However, no difference was found in the expression of annexin V (Figure 5E, Figure 5F). Therefore, STAT3 KO B cells do not have a proliferation defect when stimulated, nor do they weaken and die by apoptosis. Taken together, these results imply that STAT3 deficiency inhibits B cell metabolism in response to BCR stimulation.
来自STAT3 LOF患者的记忆B细胞破坏了早期激活。Memory B cells from STAT3 LOF patients have disrupted early activation.
CD19的激活对记忆B细胞的早期激活至关重要45,并且为确定STAT3缺乏对人记忆B细胞早期激活的影响,我们通过TIRFm检查了HC和STAT3 LOF患者的B细胞在经mAg刺激时的BCR簇集和B细胞铺展。与HC的记忆B细胞相比,STAT3 LOF患者的记忆B细胞(鉴定为CD27+)的接触面积显著减少(图6A、图6D)。与HC相比,STAT3 LOF患者的记忆B细胞接触区内的BCR簇集也显著减少(图6A、图6E)。接下来,我们通过鬼笔环肽染色来检查记忆B细胞中的肌动蛋白重组,并发现来自STAT3 LOF患者的记忆B细胞接触区内的肌动蛋白积累显著减少(图6A、图6F)。为进一步确定STAT3缺乏对记忆B细胞中信号复合体募集的影响,我们检查了接触区内的pY和pCD19水平。与HC相比,STAT3 LOF患者的记忆B细胞接触区内的pY和pCD19水平显著降低(图6B、图6C、图6G、图6H)。这些结果表明STAT3缺乏阻断了记忆B细胞的早期激活。我们通过不同的抗体染色组合进一步分析了STAT3 LOF和GOF患者的免疫表型。有趣的是,我们发现STAT3 LOF和GOF患者的记忆B细胞的百分比显著降低,而STAT3 LOF和GOF患者的初始B细胞的百分比却有所增加(图6I、图6J)。值得注意的是,STAT3 LOF患者的过渡B细胞的频率显著增加,而STAT3 GOF患者的过渡B细胞的频率显著降低。最后,STAT3LOF和GOF患者的浆母细胞的百分比均有所降低(图6I、图6J)。接下来,我们研究了在体外STAT3缺乏对初始B细胞向记忆B细胞过渡的影响。将来自STAT3 LOF患者的PBMC用sAg刺激5min,并针对IgD和CD27进行染色。我们发现在STAT3 LOF患者中,初始B细胞(IgD+CD27-)的频率显著降低,而记忆B细胞(IgD-CD27-)的频率显著升高(图6K、图6L)。这些结果表明,缺乏STAT3的初始B细胞易形成记忆B细胞,这可能符合STAT3 LOF患者的高IgE模型。Activation of CD19 is critical for early activation of memory B cells, 45 and to determine the impact of STAT3 deficiency on early activation of human memory B cells, we examined the BCR of B cells from HC and STAT3 LOF patients upon mAg stimulation by TIRFm Clustering and B cell spreading. Compared with the memory B cells of HC, the contact area of memory B cells (identified as CD27 + ) in STAT3 LOF patients was significantly reduced (Figure 6A, Figure 6D). BCR clustering within the memory B cell contact zone was also significantly reduced in STAT3 LOF patients compared with HC (Figure 6A, Figure 6E). Next, we examined actin reorganization in memory B cells by phalloidin staining and found that actin accumulation within the contact zone of memory B cells from STAT3 LOF patients was significantly reduced (Figure 6A, Figure 6F). To further determine the impact of STAT3 deficiency on signaling complex recruitment in memory B cells, we examined pY and pCD19 levels within the contact zone. The levels of pY and pCD19 in the memory B cell contact zone were significantly reduced in STAT3 LOF patients compared with HC (Figure 6B, Figure 6C, Figure 6G, Figure 6H). These results indicate that STAT3 deficiency blocks early activation of memory B cells. We further analyzed the immune phenotypes of STAT3 LOF and GOF patients through different antibody staining combinations. Interestingly, we found that the percentage of memory B cells was significantly reduced in patients with STAT3 LOF and GOF, while the percentage of naïve B cells was increased in patients with STAT3 LOF and GOF (Figure 6I, Figure 6J). Notably, patients with STAT3 LOF had a significantly increased frequency of transitional B cells, whereas patients with STAT3 GOF had a significantly decreased frequency of transitional B cells. Finally, the percentage of plasmablasts was reduced in both STAT3 LOF and GOF patients (Figure 6I, Figure 6J). Next, we studied the effect of STAT3 deficiency on the transition of naive B cells to memory B cells in vitro. PBMC from STAT3 LOF patients were stimulated with sAg for 5 min and stained for IgD and CD27. We found that the frequency of naïve B cells (IgD + CD27 − ) was significantly decreased, while the frequency of memory B cells (IgD − CD27 − ) was significantly increased in STAT3 LOF patients (Figure 6K, Figure 6L). These results indicate that naive B cells lacking STAT3 are prone to form memory B cells, which may be consistent with the high IgE model of STAT3 LOF patients.
STAT3缺乏通过增强miRNA146A-14-3-3σ轴导致高IgE。STAT3 deficiency leads to high IgE by enhancing the miRNA146A-14-3-3σ axis.
先前的研究表明,miRNA146A通过上调14-3-3σ表达促进IgE转换18。为确定STAT3是否调节miRNA146A-14-3-3σ轴,我们使用RT-PCR和免疫印迹检查了STAT3 KO小鼠或STAT3LOF患者的B细胞中miRNA146A和14-3-3σ的表达。有趣的是,STAT3 KO B细胞中miRNA146a和14-3-3σ的mRNA水平增强(图7A),并且STAT3 KO小鼠B细胞中14-3-3σ蛋白的表达也显著增加(图7C)。在STAT3 KO小鼠中发现的表型在STAT3 LOF患者中得到重现(图7B、图7C)。在机制上,我们使用ChIP发现STAT3通过结合14-3-3σ启动子来调节14-3-3σ表达(图7D)。为进一步证实STAT3通过中心枢纽14-3-3σ调节BCR信号传导、IgE产生和B细胞分化,将STAT3 KO B细胞或小鼠分别在体外或体内用14-3-3σ抑制剂肽R18处理。我们发现,在STAT3 KO B细胞中,诸如pAkt和pFoxo1等BCR信号传导分子的水平被挽救到WT B细胞的程度(图7E、图7F)。另外,我们分析了经14-3-3σ抑制剂处理的STAT3 KO小鼠外周血B细胞亚类、IgE+B细胞和IgE血清的百分比,并且发现除MZ B细胞(图8F、图8G)外,FO(图7G、图7H)、GC(图7I、图7J)和IgE+B细胞(图7K、图7L)的百分比在静息态和激活态下均被挽救至WT小鼠的程度。此外,注射了14-3-3σ抑制剂的STAT3 KO小鼠的血清IgE滴度(图7M)也被挽救。这些结果表明STAT3-miRNA146A-14-3-3σ轴对BCR信号传导、IgE产生以及FO和GC B细胞分化是重要的。Previous studies have shown that miRNA146A promotes IgE switching by upregulating 14-3-3σ expression 18 . To determine whether STAT3 regulates the miRNA146A-14-3-3σ axis, we examined the expression of miRNA146A and 14-3-3σ in B cells from STAT3 KO mice or STAT3LOF patients using RT-PCR and immunoblotting. Interestingly, the mRNA levels of miRNA146a and 14-3-3σ were enhanced in STAT3 KO B cells (Fig. 7A), and the expression of 14-3-3σ protein was also significantly increased in B cells of STAT3 KO mice (Fig. 7C). The phenotype found in STAT3 KO mice was recapitulated in STAT3 LOF patients (Fig. 7B, Fig. 7C). Mechanistically, we used ChIP to find that STAT3 regulates 14-3-3σ expression by binding to the 14-3-3σ promoter (Fig. 7D). To further confirm that STAT3 regulates BCR signaling, IgE production, and B cell differentiation through the central hub 14-3-3σ, STAT3 KO B cells or mice were treated with the 14-3-3σ inhibitor peptide R18 in vitro or in vivo, respectively. We found that in STAT3 KO B cells, the levels of BCR signaling molecules such as pAkt and pFoxo1 were rescued to the extent of WT B cells (Figure 7E, Figure 7F). In addition, we analyzed the percentages of peripheral blood B cell subsets, IgE + B cells, and IgE serum in STAT3 KO mice treated with 14-3-3σ inhibitors, and found that in addition to MZ B cells (Figure 8F, Figure 8G) , the percentages of FO (Fig. 7G, Fig. 7H), GC (Fig. 7I, Fig. 7J) and IgE + B cells (Fig. 7K, Fig. 7L) were rescued to the extent of WT mice in both resting and activated states. In addition, serum IgE titers in STAT3 KO mice injected with 14-3-3σ inhibitors (Fig. 7M) were also rescued. These results indicate that the STAT3-miRNA146A-14-3-3σ axis is important for BCR signaling, IgE production, and FO and GC B cell differentiation.
讨论discuss
STAT3缺乏导致HIES,但其潜在分子机制尚不清楚。通过使用STAT3缺失的小鼠模型和患者样品研究B细胞发育的早期阶段,我们发现FO、MZ和GC B细胞的分化被破坏。令人振奋的是,STAT3 KO小鼠中在体外的IgE+B细胞生成和IgE同种型转换均有增强,从而证明该模型是研究STAT3缺乏引起的高IgE的分子机制的最佳模型。另外,我们发现STAT3破坏了正向和负向BCR信号传导的平衡,并减少了BCR信号传导。在机制上,我们发现在初始B细胞中,BCR簇集、B细胞铺展和BCR信号复合体募集减少,这可能是由于缺乏来自肌动蛋白的驱动力。此外,在STAT3缺乏的细胞中,记忆B细胞的早期激活(包括B细胞铺展、BCR簇集和BCR信号复合体募集)也有所减少。在机制上,我们发现决定IgE同种型转换的关键因素是STAT3KO B细胞中14-3-3σ表达的增强,以及其上游介导物miRNA146A。有趣的是,我们发现体外或体内抑制14-3-3σ可以将STAT3 KO B细胞或STAT3 KO小鼠的BCR信号传导和IgE产生挽救到正常水平。这是首次报道STAT3 KO B细胞中14-3-3σ表达增强。STAT3 deficiency causes HIES, but the underlying molecular mechanisms are unknown. By studying the early stages of B cell development using STAT3-deficient mouse models and patient samples, we found that differentiation of FO, MZ, and GC B cells was disrupted. Encouragingly, both in vitro IgE + B cell production and IgE isotype switching were enhanced in STAT3 KO mice, demonstrating that this model is the best model to study the molecular mechanisms of high IgE caused by STAT3 deficiency. Additionally, we found that STAT3 disrupted the balance of positive and negative BCR signaling and reduced BCR signaling. Mechanistically, we found that BCR clustering, B cell spreading, and BCR signaling complex recruitment are reduced in naïve B cells, likely due to the lack of driving force from actin. Furthermore, early activation of memory B cells, including B cell spreading, BCR clustering, and BCR signaling complex recruitment, was also reduced in STAT3-deficient cells. Mechanistically, we found that the key factor determining IgE isotype switching is the enhanced expression of 14-3-3σ in STAT3KO B cells, as well as its upstream mediator, miRNA146A. Interestingly, we found that inhibition of 14-3-3σ in vitro or in vivo rescued BCR signaling and IgE production in STAT3 KO B cells or STAT3 KO mice to normal levels. This is the first report of enhanced 14-3-3σ expression in STAT3 KO B cells.
14-3-3σ在B细胞中的作用此前已被研究过。14-3-3σ缺乏小鼠具有减少的外周血B细胞(如FO、MZ和再循环B细胞)以及非T细胞依赖性抗原应答46。在STAT3 KO小鼠中,虽然14-3-3σ表达增强了,但MZ B细胞却减少了,这表明14-3-3σ的最佳表达对外周血B细胞的稳态至关重要。14-3-3σ缺乏小鼠经抗IgM刺激后,pAkt和pErk1/2信号传导减少了46。我们的STAT3 KO小鼠在抗原刺激下,近侧和远侧BCR信号传导也减少了。这也表明14-3-3σ的最佳表达对正常BCR信号传导是重要的。The role of 14-3-3σ in B cells has been studied previously. 14-3-3σ-deficient mice have reduced peripheral blood B cells (such as FO, MZ and recycling B cells) and T cell-independent antigen responses46 . In STAT3 KO mice, although 14-3-3σ expression was enhanced, MZ B cells were reduced, suggesting that optimal expression of 14-3-3σ is critical for peripheral blood B cell homeostasis. 14-3-3σ-deficient mice have reduced pAkt and pErk1/2 signaling after anti-IgM stimulation46. Our STAT3 KO mice also had reduced proximal and distal BCR signaling upon antigen stimulation. This also suggests that optimal expression of 14-3-3σ is important for normal BCR signaling.
BCR信号传导与IgE产生之间的关系仍有待阐明。DOCK8患者患有HIES,并且我们发现DOCK8缺乏的B细胞中BCR信号传导水平降低15。类似地,在STAT3患者中,我们还发现DOCK8表达减少且BCR信号传导水平降低。这些结果意味着,BCR信号传导的减少可导致IgE同种型转换。先前的研究表明,BCR交联通过添加具有IL-4的LPS来阻断IgG1和IgE的类别转换47。The relationship between BCR signaling and IgE production remains to be elucidated. DOCK8 patients suffer from HIES, and we found reduced levels of BCR signaling in DOCK8-deficient B cells15. Similarly, in STAT3 patients, we also found reduced DOCK8 expression and reduced BCR signaling levels. These results imply that reduced BCR signaling can lead to IgE isotype switching. Previous studies have shown that BCR cross-linking blocks IgG1 and IgE class switching by adding LPS with IL- 447 .
在我们的研究中,14-3-3σ抑制剂R18对STAT3 KO B细胞中BCR信号传导的抑制减少了IgE的产生。因此,我们确定低BCR信号传导有利于IgE同种型转换。我们还发现STAT3表达对记忆B细胞的早期激活至关重要。值得注意的是,STAT3 LOF和GOF患者的记忆B细胞均有减少而初始B细胞均有增加,这表明STAT3的最佳表达对于维持B细胞的记忆应答至关重要。In our study, inhibition of BCR signaling in STAT3 KO B cells by the 14-3-3σ inhibitor R18 reduced IgE production. Therefore, we determined that low BCR signaling favors IgE isotype switching. We also found that STAT3 expression is critical for early activation of memory B cells. Notably, both STAT3 LOF and GOF patients had a decrease in memory B cells and an increase in naïve B cells, suggesting that optimal expression of STAT3 is critical for maintaining B cell memory responses.
剩下的一个问题就是STAT3调节BCR信号传导的详细分子机制。STAT3KO B细胞中CD19、BTK和SHIP的转录水平完全没有改变,这表明STAT3不会调节近侧BCR信号传导基因的转录。关键在于,要使用ChIP测定和STAT3抗体作为诱饵来寻找目标基因的调节区。STAT3已被证明与诸如NF-kB、RelA、HIF1a、Jun、STAT1、mTOR和ICOS等若干蛋白质相互作用26、48-53。One remaining question is the detailed molecular mechanism by which STAT3 regulates BCR signaling. Transcript levels of CD19, BTK, and SHIP were completely unchanged in STAT3KO B cells, indicating that STAT3 does not regulate the transcription of proximal BCR signaling genes. The key is to use a ChIP assay and a STAT3 antibody as bait to find the regulatory region of the gene of interest. STAT3 has been shown to interact with several proteins such as NF-kB, RelA, HIF1a, Jun, STAT1, mTOR and ICOS26,48-53 .
一个矛盾是STAT3患者的记忆B细胞减少了,但STAT3 KO初始B细胞却毫无缘由地易形成记忆B细胞,这可能是由于一种补偿机制。先前的研究表明,记忆B细胞可能需要较少的STAT3活性来对细胞因子作出响应,从而进行浆母细胞分化35。我们的结果证明,STAT3-KO初始B细胞易于变成记忆B细胞和IgE转换型浆母细胞;这可能是由于miRNA146A和14-3-3σ蛋白具有较高表达。我们需要进一步确定STAT3 KO B细胞中激活诱导脱氨酶(AID)的表达。但STAT3患者的记忆B细胞为何减少仍不明确。最近的一项研究表明,mTORC1缺乏导致Ag特异性记忆B细胞以及浆细胞和GC B细胞减少54。我们的结果表明,STAT3 KO B细胞中mTORC1和mTORC2活性均降低,并且STAT3-mTORC轴调节记忆B细胞的形成。此外,STAT3 KO初始B细胞的代谢活性较低,这可能是初始B细胞向记忆B细胞转换的原因。One paradox is that STAT3 patients have reduced memory B cells, but STAT3 KO naïve B cells are prone to form memory B cells for no reason, possibly due to a compensatory mechanism. Previous studies have suggested that memory B cells may require less STAT3 activity to undergo plasmablast differentiation in response to cytokines 35 . Our results demonstrate that STAT3-KO naïve B cells readily become memory B cells and IgE-switched plasmablasts; this may be due to the higher expression of miRNA146A and 14-3-3σ protein. We need to further determine the expression of activation-induced deaminase (AID) in STAT3 KO B cells. But why patients with STAT3 have fewer memory B cells remains unclear. A recent study showed that mTORC1 deficiency leads to a decrease in Ag-specific memory B cells as well as plasma cells and GC B cells 54 . Our results indicate that both mTORC1 and mTORC2 activities are reduced in STAT3 KO B cells and that the STAT3-mTORC axis regulates the formation of memory B cells. In addition, STAT3 KO naïve B cells have lower metabolic activity, which may be the reason for the conversion of naïve B cells into memory B cells.
总体来说,我们的研究通过使用优化的小鼠模型和患者样品揭示了STAT3患者高IgE综合征的潜在机制。STAT3 KO小鼠中14-3-3σ表达的增强导致增加的IgE+B细胞生成和较低的BCR信号传导。Overall, our study reveals the underlying mechanism of hyperIgE syndrome in STAT3 patients by using optimized mouse models and patient samples. Enhanced 14-3-3σ expression in STAT3 KO mice leads to increased IgE + B cell production and lower BCR signaling.
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