Skip to main content
. 2019 Dec 17;10:2913. doi: 10.3389/fimmu.2019.02913

Figure 4.

Figure 4

PGL-I targeting of CR3 triggers nuclear translocation of NFATc2 downstream of Syk that controls a specific mediator signature. (A,B) IL-2 produced in DCs supernatants, IL-10 in PMNs and IL-1β in MPs were measured by ELISA and (C) PGE2 by competition ELISA after overnight incubation the three recombinant BCG strains at MOI of 5. Data are presented as mean ± SEM. **P < 0.01; ***P < 0.001. (A) Phagocytosis was blocked by treatment for 1 h before infection with 1 or 50 nM of CytoD, while controls received only the vehicle DMSO (n = 8). (B) Cells were treated with two NFATc inhibitors tacrolimus (FK506, 500 pg/ml) or Cyclosporin (CsA, 50 ng/ml) 1 h before infection by the three rBCG strains. Controls received DMSO (n = 4). (C) For PGE2 production, cells were derived from bone marrow of WT or itgam−/− mice. (D) Translocation of NFATc2 (green) into the nucleus of MPs (blue, DAPI staining) derived from bone marrow of WT or itgam−/− mice was analyzed by confocal microscopy 30 min after infection with rBCG::PGL-I or rBCG::noPGL at MOI of 5. Cells on slides were then fixed, permeabilized and stained with anti-NFATc2 and mounted in medium containing DAPI. Images were acquired with a confocal Leica TCS SP8 microscope, where NFATc2 colocalization with the nucleus appeared in light blue, while NFATc2 remaining in the cytosol appeared in green. Images are from original magnification ×63. (E) After analysis of images (Image J software) the Manders coefficient was determined with the JACoP (26) plugin that allowed to quantify colocalization of NFATc2 with the nucleus (light blue). These coefficients were calculated in 10 fields from 4 slides (obtained from 2 independent experiments). Box and whisker plot shows median ± SEM. ***P < 0.001 (Student's t-test). (F) Cytokine production measured as in (A,B) from WT or itgam−/− cells stimulated with rBCG::PGL-I at MOI of 5 or γ-irradiated M. leprae (strain NHDP, equivalent to MOI of 10). WT cells received treatment with DMSO or Syk inhibitor GS-9973 or CsA to block NFATc translocation, 1 h before contact with bacteria. Data are presented as mean ± SEM. **P < 0.01; ***P < 0.001.