An epigenetic GPI anchor defect impairs TLR4 signaling in the B cell transdifferentiation model for primary human monocytes BLaER1

Julia Wegner, Thomas Zillinger, Thais Schlee-Guimaraes, Eva Bartok, Martin Schlee

    Research output: Contribution to journalA1: Web of Science-articlepeer-review

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    Abstract

    Antigen-presenting myeloid cells like monocytes detect invading pathogens via pattern recognition receptors (PRRs) and initiate adaptive and innate immune responses. As analysis of PRR signaling in primary human monocytes is hampered by their restricted expandability, human monocyte models like THP-1 cells are commonly used for loss-of-function studies, such as with CRISPR-Cas9 editing. A recently developed transdifferentiation cell culture system, BLaER1, enables lineage conversion from malignant B cells to monocytes and was found superior to THP-1 in mimicking PRR signaling, thus being the first model allowing TLR4 and inflammasome pathway analysis. Here, we identified an important caveat when investigating TLR4-driven signaling in BLaER1 cells. We show that this model contains glycosylphosphatidylinositol (GPI) anchor-deficient cells, which lack CD14 surface expression when differentiated to monocytes, resulting in diminished LPS/TLR4 but not TLR7/TLR8 responsiveness. This GPI anchor defect is caused by epigenetic silencing of PIGH, leading to a random distribution of intact and PIGH-deficient clones after single-cell cloning. Overexpressing PIGH restored GPI-anchored protein (including CD14) expression and LPS responsiveness. When studying CD14- or other GPI-anchored protein-dependent pathways, researchers should consider this anomaly and ensure equal GPI-anchored protein expression when comparing cells that have undergone single-cell cloning, e. g. after CRISPR-Cas9 editing.

    Original languageEnglish
    Article number14983
    JournalScientific Reports
    Volume11
    Issue number1
    Pages (from-to)14983
    Number of pages15
    ISSN2045-2322
    DOIs
    Publication statusPublished - 2021

    Keywords

    • DOUBLE-STRANDED-RNA
    • BIOSYNTHESIS
    • RECOGNITION
    • CD14
    • LIPOPOLYSACCHARIDE
    • EXPRESSION

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