An epigenetic GPI\xa0anchor defect impairs TLR4 signaling in the B cell transdifferentiation model for primary human monocytes BLaER1

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

doi:10.1038/s41598-021-94386-z

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.

Highlights

Monocytes and monocyte-derived cells are an essential part of the human innate immune system
The isolation and expansion of single cells results in monoclonal BLaER1 lines, with some of them demonstrating attenuated LPS responsiveness. We show that this phenotype originates from epigenetic silencing of phosphatidylinositol N-acetylglucosaminyltransferase subunit H (PIGH), a protein involved in GPI anchor biosynthesis, which is necessary for CD14 surface expression on transdifferentiated BLaER1 cells
The comparison of the LPS/TLR4 response of primary human peripheral blood mononuclear cells (PBMCs) with THP-1 and transdifferentiated BLaER1 cells revealed that BLaER1 monocytes but not THP-1 cells can elicit a proper interleukin 6 (IL-6) response to LPS (Fig. 1)

Summary

Introduction

Monocytes and monocyte-derived cells are an essential part of the human innate immune system. CRISPR-Cas9-mediated gene editing via non-homologous end joining induces spontaneous insertions or deletions (indels) in the targeted gene This approach generally results in a polyclonal cell population in which every individual cell (and allele) can harbor different indels. The isolation and expansion of single cells results in monoclonal BLaER1 lines, with some of them demonstrating attenuated LPS responsiveness We show that this phenotype originates from epigenetic silencing of PIGH, a protein involved in GPI anchor biosynthesis, which is necessary for CD14 surface expression on transdifferentiated BLaER1 cells. While the BLaER1 population contains a mixture of PIGH-expressing and -deficient cells, limiting-dilution seeding can result in monoclonal cell lines with dramatically different TLR4 signaling phenotypes. Our study provides an important caveat for the use of BLaER1 cells in investigating TLR4-driven pathways

Methods

Results

Conclusion