Hilchey Shannon P, Palshikar Mukta G, Mendelson Eric S, Shen Shichen, Rasam Sailee, Emo Jason A, Qu Jun, Thakar Juilee, Zand Martin S
Department of Medicine, Division of Nephrology, University of Rochester Medical Center, Rochester, NY 14642, USA.
Biophysics, Structural, and Computational Biology Program, University of Rochester Medical Center, Rochester, NY 14642, USA.
Life (Basel). 2022 Aug 22;12(8):1284. doi: 10.3390/life12081284.
Coordinated migration of B cells within and between secondary lymphoid tissues is required for robust antibody responses to infection or vaccination. Secondary lymphoid tissues normally expose B cells to a low O2 (hypoxic) environment. Recently, we have shown that human B cell migration is modulated by an O2-dependent molecular switch, centrally controlled by the hypoxia-induced (transcription) factor-1α (HIF1A), which can be disrupted by the immunosuppressive calcineurin inhibitor, cyclosporine A (CyA). However, the mechanisms by which low O2 environments attenuate B cell migration remain poorly defined. Proteomics analysis has linked CXCR4 chemokine receptor signaling to cytoskeletal rearrangement. We now hypothesize that the pathways linking the O2 sensing molecular switch to chemokine receptor signaling and cytoskeletal rearrangement would likely contain phosphorylation events, which are typically missed in traditional transcriptomic and/or proteomic analyses. Hence, we have performed a comprehensive phosphoproteomics analysis of human B cells treated with CyA after engagement of the chemokine receptor CXCR4 with CXCL12. Statistical analysis of the separate and synergistic effects of CyA and CXCL12 revealed 116 proteins whose abundance is driven by a synergistic interaction between CyA and CXCL12. Further, we used our previously described algorithm BONITA to reveal a critical role for Lymphocyte Specific Protein 1 (LSP1) in cytoskeletal rearrangement. LSP1 is known to modulate neutrophil migration. Validating these modeling results, we show experimentally that LSP1 levels in B cells increase with low O2 exposure, and CyA treatment results in decreased LSP1 protein levels. This correlates with the increased chemotactic activity observed after CyA treatment. Lastly, we directly link LSP1 levels to chemotactic capacity, as shRNA knock-down of LSP1 results in significantly increased B cell chemotaxis at low O2 levels. These results directly link CyA to LSP1-dependent cytoskeletal regulation, demonstrating a previously unrecognized mechanism by which CyA modulates human B cell migration. Data are available via ProteomeXchange with identifier PXD036167.
为了对感染或疫苗接种产生强大的抗体反应，B细胞在二级淋巴组织内以及在不同二级淋巴组织之间的协调迁移是必需的。二级淋巴组织通常使B细胞暴露于低氧（缺氧）环境中。最近，我们已经表明，人类B细胞迁移受一种氧依赖性分子开关调节，该开关由缺氧诱导（转录）因子-1α（HIF1A）集中控制，而免疫抑制性钙调神经磷酸酶抑制剂环孢素A（CyA）可破坏这种调节。然而，低氧环境减弱B细胞迁移的机制仍不清楚。蛋白质组学分析已将CXCR4趋化因子受体信号传导与细胞骨架重排联系起来。我们现在假设，将氧感应分子开关与趋化因子受体信号传导和细胞骨架重排联系起来的途径可能包含磷酸化事件，而这些事件在传统的转录组学和/或蛋白质组学分析中通常会被遗漏。因此，在用趋化因子CXCL12激活趋化因子受体CXCR4后，我们对用CyA处理的人类B细胞进行了全面的磷酸化蛋白质组学分析。对CyA和CXCL12的单独及协同作用的统计分析揭示了116种蛋白质，其丰度受CyA和CXCL12之间的协同相互作用驱动。此外，我们使用我们之前描述的算法BONITA揭示了淋巴细胞特异性蛋白1（LSP1）在细胞骨架重排中的关键作用。已知LSP1可调节中性粒细胞迁移。为了验证这些建模结果，我们通过实验表明，B细胞中的LSP1水平随着低氧暴露而增加，而CyA处理导致LSP1蛋白水平降低。这与CyA处理后观察到的趋化活性增加相关。最后，我们直接将LSP1水平与趋化能力联系起来，因为LSP1的短发夹RNA敲低导致低氧水平下B细胞趋化性显著增加。这些结果直接将CyA与LSP1依赖性细胞骨架调节联系起来，证明了一种以前未被认识的CyA调节人类B细胞迁移的机制。数据可通过ProteomeXchange获得，标识符为PXD036167。
