Cyclosporine A Modulates LSP1 Protein Levels in Human B Cells to Attenuate B Cell Migration at Low O2 Levels

Abstract

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.