PspA induces PD-L1 expression in a MyD88 dependent pathway.

<p>Mouse bone marrow derived DCs were transfected with siRNAs against indicated molecules for 36h followed by stimulations with 1 μg/ml Pam₃Csk₄ and 15 μg/ml PspA for 24h. PD-L1 levels were monitored by flow cytometry. Dotted line represents unstimulated cells transfected with control siRNAs. Thin lines represent cells transfected with control siRNAs followed by stimulations with 1 μg/ml Pam₃Csk₄ and 15 μg/ml PspA. Bold lines represent cells transfected with specific siRNAs to indicated molecules followed by stimulations with 1 μg/ml Pam₃Csk₄ and 15 μg/ml PspA. Data from one of three independent experiments are shown. In Panel B, PD-L1 expression is represented as bars indicating fold increase in Relative Mean Fluorescence Intensity (MFI) for various groups. Bars represent mean ± S.D. of three independent experiments.</p

PspA regulates apoptosis of DCs through PD-L1.

<p>Mouse bone marrow derived DCs were transfected with siRNAs against PD-L1 for 36h, followed by stimulation with 15 μg/ml PspA for 24h. Cytoplasmic extracts were immunoblotted for indicated molecules. Numbers below the bands represent density of the band relative to unstimulated control. Data from one of two independent experiments are shown.</p

Confocal images of CACNA1S following stimulations with Rv2463 and <i>M</i>. <i>tb</i> on macrophages.

<p>J774 cells were stimulated with 25 μg/ml Rv2463 or 2 MOI <i>M</i>. <i>tb</i> H37Rv for 48h. CACNA1S expression was monitored by confocal imaging (<i>see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124263#sec002" target="_blank">Materials and Methods</a></i>). Blue colour represents staining of nucleus with DAPI while green colour represents CACNA1S staining with streptavidin conjugated FITC bound to biotinylated antibody to CACNA1S. A representative image of 10 fields is shown. P<0.009 for unstimulated v/s 25 μg/ml of Rv2463; P<0.004 for uninfected v/s 2 MOI of <i>M</i>. <i>tb</i> H37Rv infected macrophages. Two-tailed Student’s t-test was employed for P values for 48h time point.</p

PspA upregulates expression of PD-L1 on DCs.

<p>A, mouse bone marrow derived DCs were stimulated with indicated concentrations of PspA for 24h. B, DCs were stimulated with 15μg/ml PspA for indicated times. PD-L1 expression was monitored by flow cytometry. Bold lines represent expression levels in the presence of PspA while thin lines represent unstimulated controls. Data from one of three independent experiments are shown. The MFI values from three independent experiment (expressed as mean ± SD) are summarized as a bar graph in the right panel.</p

Route of calcium entry differentially regulates PspA induced PD-L1 expression.

<p>Mouse bone marrow derived DCs were incubated with bio-pharmacological inhibitors to indicated molecules for 1h followed by stimulation with 15 μg/ml PspA for 24h. PD-L1 levels were monitored by flow cytometry. Dotted lines represent unstimulated cells. Thin lines represent cells stimulated with PspA. Bold lines represent cells treated with inhibitors to indicated molecules followed by stimulation with PspA. One of three independent experiments is shown. PD-L1 expression is represented as bar graph indicating fold increase in Relative Mean Fluorescence Intensity (MFI) for various groups. Bars represent mean ± SD of three independent experiments. For Panel B, mouse bone marrow derived DCs were incubated in the presence or absence of TMB-8 or EGTA for 1h followed by stimulations with 15 μg/ml PspA for 24h. Cells were incubated with phycoerythrin conjugated anti-mouse PD-L1 antibody. Merged images with DAPI (blue) and PD-L1 (red) staining are depicted. For Panel C total RNA was isolated from cells stimulated as indicated and PD-L1 transcript levels were measured by semi-quantitative RT-PCR. One of three independent experiments is shown.</p

<i>S</i>. <i>pneumoniae</i> upregulates PD-L1 on DCs.

<p>For Panel A, mouse bone marrow derived DCs were infected with wild typ<i>e S</i>. <i>pneumoniae</i> strain D39, R6 or JY2008 (<i>see</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133601#sec002" target="_blank">Materials and Methods</a>) and PD-L1 levels were monitored by flow cytometry. Thin lines represent uninfected cells while bold lines represent cells incubated with indicated strain of <i>S</i>. <i>pneumoniae</i>. For Panel B, DCs were incubated with either TMB-8 or EGTA for 1h followed by incubation with D39 and PD-L1 levels were monitored by flow cytometry. Dotted line represents uninfected cells. Thin lines represent cells incubated with D39 while thick lines represent cells treated with indicated inhibitors followed by incubation with D39. Data from one of two independent experiments are shown. Bars in Panel C and D represent fold increase in Relative Mean Fluorescence Intensity (MFI; mean ± SD) for various groups. ns represents non-significant differences between compared groups.</p

Calcium homeostasis regulates Rv2463 and <i>M</i>. <i>tb</i> mediated CACNA1S expression on macrophages.

<p>J774 cells were treated with inhibitors to internal calcium release (TMB8) or external calcium influx (EGTA) for 1h followed by stimulation with 25 μg/ml Rv2463 (panels A and C) or infection with 2 MOI of <i>M</i>. <i>tb</i> H37Rv (panels B and D) for 48h. CACNA1S expression was monitored by flow cytometry. Grey lines represent cells stimulated in the absence of inhibitors while bold lines represent cells stimulated in the presence of inhibitors. Dotted line represents unstimulated or uninfected cells. One of three independent experiments is shown. P<0.033 for macrophages+Rv2463 v/s macrophages+Rv2463+EGTA; P<0.019 for macrophages+Rv2463 v/s macrophages+Rv2463+TMB8. P<0.006 for macrophages+<i>M</i>. <i>tb</i> v/s macrophages+<i>M</i>. <i>tb</i>+EGTA; P<0.038 for macrophages + <i>M</i>. <i>tb</i> v/s macrophages+<i>M</i>. <i>tb</i>+TMB8; Two-tailed Student’s t-test was employed for P values.</p

<i>M</i>. <i>tb</i> H37Rv induces the upregulation of CACNA1S on macrophages.

<p>J774 macrophages were infected with <i>M</i>. <i>tb</i> H37Rv at indicated Multiplicity of Infection (MOI) for indicated times. CACNA1S expression was monitored by flow cytometry. Bold lines represent infections with <i>M</i>. <i>tb</i> H37Rv while dotted lines represent uninfected controls. Data from one of four independent experiments are shown. P<0.011 for uninfected v/s 1 MOI of <i>M</i>. <i>tb</i> infected macrophages; P<0.004 for uninfected v/s 2 MOI of <i>M</i>. <i>tb</i> H37Rv infected macrophages; P<0.007 for uninfected v/s 5 MOI of <i>M</i>. <i>tb</i> infected macrophages. Two-tailed Student’s t-test was employed for P values at the 48h time point.</p

Transcription factors CREB, SOX5 and GATA2 regulate Rv2463 and <i>M</i>. <i>tb</i> mediated CACNA1S expression on macrophages.

<p>For Panel A, J774 cells were transfected with siRNAs against CREB or SOX5 or GATA2 for 36h followed by stimulations with 25 μg/ml Rv2463 or infection with 2 MOI of <i>M</i>. <i>tb</i> H37Rv for 48h. CACNA1S expression was monitored by flow cytometry. Grey lines represent cells transfected with control siRNAs followed by stimulations with Rv2463 or infection with <i>M</i>. <i>tb</i>. Bold lines represent cells transfected with specific siRNA to indicated molecules followed by stimulations with Rv2463 or infection with <i>M</i>. <i>tb</i> H37Rv. Dotted lines represent unstimulated cells transfected with control siRNAs. One of three independent experiments is shown. For Panel B, J774 cells were stimulated with 25 μg/ml Rv2463 or infection with 2 MOI of <i>M</i>. <i>tb</i> H37Rv for indicated times and nuclear extracts were western blotted for indicated molecules. Arrow indicates specific band. Numbers below the bands indicates relative intensities of the blots. One of two independent experiments is shown. For Panel A, P<0.035, control siRNA+Rv2463 v/s Creb+Rv2463; P<0.038 for control siRNA+Rv2463 v/s siSOX5+Rv2463; P<0.036 for control siRNA+Rv2463 v/s siGATA2+Rv2463. Two-tailed Student’s t-test was employed for P values. For Panel B, Creb phosphorylation; P<0.006, Unstimulated v/s Rv2463 stimulation 30 minutes; P<0.004, Unstimulated v/s Rv2463 stimulation 60 minutes; P<0.002, Unstimulated v/s Rv2463 stimulation 120 minutes; P<0.001, Unstimulated v/s <i>M</i>. <i>tb</i> infection 30 minutes P<0.003, Unstimulated v/s <i>M</i>. <i>tb</i> infection 60 minutes; P<0.004, Unstimulated v/s <i>M</i>. <i>tb</i> infection 120 minutes. Two-tailed Student’s t-test was employed for P values.</p

Cross regulation of CACNA1S, ROS and pCREB during Rv2463 stimulation or <i>M</i>. <i>tb</i> infection.

<p>For Panel A, J774 cells were stimulated with 25 μg/ml Rv2463 or infected with 2 MOI <i>M</i>. <i>tb</i> H37Rv for 48h in the presence (bold lines) or absence (dotted lines) of indicated inhibitors to ROS. CACNA1S levels were monitored by flow cytometry. For Panel B, J774 cells were transfected with siRNAs to CACNA1S for 36h followed by stimulation with 25 μg/ml Rv2463 for 48h. ROS levels were monitored by FACS. Grey line represents Rv2463 stimulated cells transfected with control siRNAs, while bold line represents Rv2463 stimulated cells transfected with siRNAs specific to CACNA1S. Dotted line represents unstimulated cells transfected with control siRNAs. For Panel C, J774 cells were stimulated with 25 μg/ml Rv2463 or infected with 2 MOI <i>M</i>. <i>tb</i> H37Rv for indicated times in the presence or absence of indicated inhibitors to ROS and nuclear extracts were probed for pCREB levels. Numbers below the blots indicate relative intensities of the bands. Arrow shows the specific band. Data from one of three experiments is shown. In Panel A, P<0.024 for Rv2463 v/s DPI+Rv2463; P<0.013 for <i>M</i>. <i>tb</i> v/s DPI+<i>M</i>. <i>tb</i>. In Panel B, P<0.006 for siControl+Rv2463 v/s siCACNA1S+Rv2463. In Panel C, at 120 minutes, P<0.024, for <i>M</i>. <i>tb</i> v/s DPI+<i>M</i>. <i>tb</i>. Two-tailed Student’s t-test was employed for P values.</p