Department of Endocrinology, Peking University First Hospital, Beijing, China.
Key Laboratory of Interdisciplinary Research, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China.
Front Immunol. 2022 Jan 24;12:724379. doi: 10.3389/fimmu.2021.724379. eCollection 2021.
Changes in IgG glycosylation, as a novel pathological feature, are observed in various autoimmune diseases (AIDs). The glycosylation patterns of IgG play a critical role in regulating the biological function and stability of IgG involved in the pathophysiology of many AIDs. However, the intracellular regulatory mechanisms underlying the effects of disturbances in various cytokines on IgG glycosylation are poorly understood. Thus, we investigated the regulatory effects of elevated cytokines in AIDs on intracellular IgG glycosylation within B cells.
First, we established a controlled primary culture system to differentiate human CD19 B cells into antibody-secreting cells (ASCs). Then, the IgG concentrations in the supernatants were measured by enzyme-linked immunoassay (ELISA) under IFN-γ, TNF-α, IL-21, IL-17A, BAFF, or APRIL stimulation. Next, the glycosylation levels of IgG under different stimuli were compared a lectin microarray. The fine carbohydrate structures of IgG were confirmed by matrix-assisted laser desorption/ionization-quadrupole ion trap-time of flight-mass spectrometry (MALDI-TOF-MS). Finally, the expression of glycosyltransferases and glycosidases in B cells under stimulation with several cytokines was detected by real-time PCR and western blotting.
We found that cytokines significantly promoted IgG production and led to considerably different IgG glycan patterns. Specifically, the results of lectin microarray showed the galactose level of IgG was increased by IFN-γ stimulation (<0.05), and the sialylation of IgG was increased by IL-21 and IL-17A (<0.05). The MALDI-TOF-MS data showed that the frequency of agalactosylation was decreased by IFN-γ with the increased frequency of mono-galactosylation and decreased frequency of digalactosylation, accompanied by upregulation of β-1,4-galactosyltransferase 1. Both frequencies of mono-sialylated and disialylated N-glycans were increased by IL-21 and IL-17A with decreased frequency of asialylation, and the expression of β-galactoside α-2,6-sialyltransferase 1 was upregulated by IL-21 and IL-17A.
Abnormally elevated cytokines in the microenvironment regulates IgG glycan patterns by regulating intracellular glycosyltransferases in human B cells.
IgG 糖基化的改变，作为一种新的病理特征，在各种自身免疫性疾病（AIDs）中都有观察到。IgG 的糖基化模式在调节参与许多 AIDs 病理生理学的 IgG 的生物学功能和稳定性方面起着关键作用。然而，各种细胞因子干扰对 IgG 糖基化影响的细胞内调节机制还知之甚少。因此，我们研究了 AIDs 中升高的细胞因子对 B 细胞内 IgG 糖基化的调节作用。
首先，我们建立了一个受控的原代培养系统，使人类 CD19 B 细胞分化为分泌抗体的细胞（ASCs）。然后，通过酶联免疫吸附测定（ELISA）在 IFN-γ、TNF-α、IL-21、IL-17A、BAFF 或 APRIL 刺激下测量上清液中的 IgG 浓度。接下来，通过凝集素微阵列比较不同刺激下 IgG 的糖基化水平。通过基质辅助激光解吸/电离-四极离子阱-飞行时间质谱（MALDI-TOF-MS）确认 IgG 的精细碳水化合物结构。最后，通过实时 PCR 和 Western blot 检测几种细胞因子刺激下 B 细胞中转糖基酶和糖苷酶的表达。
我们发现细胞因子显著促进了 IgG 的产生，并导致了明显不同的 IgG 聚糖模式。具体来说，凝集素微阵列的结果表明 IFN-γ 刺激增加了 IgG 的半乳糖水平（<0.05），而 IL-21 和 IL-17A 增加了 IgG 的唾液酸化（<0.05）。MALDI-TOF-MS 数据显示，IFN-γ 下调了agalactosylation 的频率，同时增加了单半乳糖基化和降低了双半乳糖基化的频率，伴随着 β-1,4-半乳糖基转移酶 1 的上调。IL-21 和 IL-17A 增加了单唾液酸化和双唾液酸化 N-糖基化的频率，同时降低了无唾液酸化的频率，并且 IL-21 和 IL-17A 上调了β-半乳糖苷 α-2,6-唾液酸转移酶 1 的表达。
微环境中异常升高的细胞因子通过调节人 B 细胞内的细胞内糖基转移酶来调节 IgG 糖型模式。
