An ADAM-10 dependent EPCR shedding links meningococcal interaction with endothelial cells to purpura fulminans
Fig 5
ADAM17 is not responsible for the meningococcus-induced EPCR shedding.
(A) ADAM17 membranous expression of HDMEC cells transfected with a siRNA against ADAM17 (blue) or control (green). Grey: isotype control. For the quantification of ADAM17 expression see supplemental data S3 Fig. (B) EPCR expression in HDMEC cells transfected with either a control siRNA (left) or ADAM17 siRNA (right) and infected with a WT N. meningitidis strain for 4 hours (red) or left non-infected (green). Grey: isotype control. For the quantification of EPCR expression see supplemental data S3 Fig. A Crispr/Cas9-mediated knockdown of ADAM17 expression was performed as described in Material and Methods in the Human Cerebral Microvessels Endothelial Cells line hCMEC/D3. (C) ADAM17 expression in the resulting cell line (blue) compared to that of the parental cell line (green). (D) EPCR expression in parental hCMEC/D3 cell line (left) or ADAM17-negative-hCMEC/D3 cell line (right) infected with a WT N. meningitidis strain for 4 hours (red) or left non-infected (green). The adhesion of N. meningitidis on hCMEC/D3 is reduced compared to the adhesion on HDMEC explaining the slighty reduced shedding phenotype observed with hCMEC/D3. Data are representative of 3 independent experiments. (grey: isotype control). For the quantification of EPCR expression see supplemental data S5 Fig. (E) Soluble EPCR in the supernatant of ADAM17-negative-hCMEC/D3 cells after infection with a WT strain or non-infected (control) cells. Data are mean (+/- SEM) of 3 independent experiments. *:p = 0.007 (t-test).