Microvesicles from malaria-infected red blood cells activate natural killer cells via MDA5 pathway
Fig 5
Microvesicles derived from iRBCs prime NK cells.
(A-B) iMVs (1 μg/ml) were either labeled or not labeled with PKH26 and then cultured with R-NK cells for 24 h. Shown are representative PKH26 vs CD56 staining profiles. Numbers indicate the percentage of positive cells in the gated region (A). Comparison of percentages of PKH26-positive NK cells in cultures with unlabeled or labeled iMV (B). (C) Confocal microscopy of PKH67-labelled R-NK cells after 24 h incubation with PKH26-labeled iMVs. Shows are representative images of an NK cell with an internalized iMV. Z-stack of 1 μm slices were captured and 3D volume rendering was performed to demonstrate the location of the internalized iMV (white arrow). (D) Gel electrophoresis of cDNA derived from capped and uncapped RNAs of MVs and iMVs. Size markers are labeled on the left. No RNA: no RNA was added during the reverse transcription. (E-F) Representative CD69 histograms of R-NK cells cultured with RBC (NK+RBC), iRBCs (NK+iRBCs), MVs at 10 μg/ml (NK+MV10), iMVs at 1 (NK+iMV1) and 10 μg/ml (NK+iMV10) (E) and comparison of CD69 MFI on R-NK cells from different donors in the indicated culture conditions (F). (G) Comparison of CD69 MFI on R-NK cells cultured with iMVs (at 10 μg/ml) in the presence of DMSO (control) or Bx795 (10 nM). (H) IFN-γ levels in the culture supernatants of R-NK cells cultured with RBCs, iRBCs, and iMVs at 100 μg/ml for 48 h. (I-J) R-NK cells were cultured in the presence of MVs or iMVs at 10 μg/ml for 96 h, and LFA-1 activation was assessed using m24 antibody. Shown are representative LFA-1 (m24) vs CD56 staining profiles of NK cells (I), and comparison of percentages of LFA-1 (m24)-positive NK cells (J) in the indicated conditions. (K) Comparison of parasitemia reduction when NR-NK cells were co-cultured with iRBCs without iMVs, with 10 μg/ml iMVs (iMV10) or 100 μg/ml iMVs (iMV100) for 96 h. Each symbol represents a different individual. Error bars represent mean ± SD. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns: not significant.