Vitamin d rescues impaired Mycobacterium tuberculosis-mediated tnf release in hiv+
M. Kirby, C. Saltini, and R. G. Crystal
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572 Figure 1. 1,25D3 rescues MTb-mediated TNF-release in HIV+ human macrophages. 573
Differentiated U937(a) and HIV+U1(b,c) macrophages were incubated with irradiated virulent 574
MTb (MOI 10:1 for 24 hours) in the presence and absence of 1,25D3 pre-treatment (24 hours). 575
TNF was measured in cell culture supernatant by ELISA (R&D). Figures are representative of 576
individual experiments with similar results, n=6) . Quantitative data represent mean+SEM * 577
p<0.05 578
Figure 2. 1,25D3 enhances TNF transcription in HIV+ human macrophages. (a) RT-PCR and 579
real-time PCR for VDR was performed on total RNA from differentiated U937 and HIV+U1 580
macrophages (n=4). (b,c) Differentiated U937 and HIV+U1 cells were incubated with MTb or 581
BCG (M.bovis) (MOI 10:1) for 3 hours in the presence and absence of 1,25D3 pre-treatment (24 582
hours). TNF mRNA was measured by real-time PCR. (n=4). Quantitative data represent 583
mean+SEM *p<0.05, **p<0.01 584
Figure 3. 1,25D3 increased TLR signaling but not TLR expression in HIV+U1 585
macrophages . (a,b) Differentiated human U937 and HIV+U1 macrophages were incubated with 586
TLR ligands Lipid A (LA-TLR4), Pam3CCSK4 (PamCys-TLR2/1) and 19kDa lipoprotein from 587
MTb (19kDa MTb-TLR2/1) [1μg/ml] for 24 hours in the presence and absence of 1,25D3 588
pretreatment. Cell-free culture supernatants were assayed for TNF by ELISA (n= 3). (c) 589
Differentiated U937 and HIV+U1 macrophages were incubated for 24h in the presence or 590
absence of 1,25D3 pretreatment and stained with PE-labeled anti-TLR antibodies or isotype 591
control. Surface expression was measured by flow cytometry. Left panels show isotype control 592
(grey line) and TLR-labeled cells (black line); right panels show TLR-labeled cells (grey line) 593
and 1,25D3 treated TLR-labeled cells (black line). Representative histograms of independent 594
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595 real-time PCR (n=3). Quantitative data represent mean+SEM.* p<0.05 596
597
U937 and HIV+U1 macrophages were incubated with MTb (MOI 10:1) for 0-120 minutes in the 598
presence and absence of 1,25D3 pretreatment and PDTC. Cell lysates were resolved by Western 599
blot using specific antibody to IKBα. Representative blot of three independent experiments with 600
similar results. Quantitative densitometric analysis of IKBα bands are displayed directly 601
beneath the blot. (b) NF-kB nuclear translocation in nuclear extracts was measured by ELISA. 602
(n=3) Quantitative data represent mean+SEM.* p<0.05 603
Figure 5. 1,25D3 induces CD14 expression in human macrophages. (a) Differentiated U937 604
and HIV+U1 macrophages were incubated for 24 hours in the presence or absence of 1,25D3 605
pretreatment and stained with PE-labeled anti-CD14 antibody or isotype control. Surface 606
expression was measured by flow cytometry. Left panels show isotype control (grey line) and 607
CD14-labeled cells (black line); right panels show CD14-labeled cells (grey line) and VD treated 608
CD14-labeled cells (black line). Representative histograms from individual experiments with 609
similar results (n=3). (b,c) Differentiated U937 and HIV+U1 macrophages were treated with 610
MTb (MOI 10:1) or LPS (1μg/ml) in the presence and absence of Vit D pretreatment (24 hours) 611
and indicated antibodies. TNF was measured in cell culture supernatants by ELISA (R&D). 612
(Representative figure from individual experiments with similar results, n=3). Quantitative data 613
represent mean+SEM *p<0.05 614
Figure 6. 1,25D3 rescues MTb-mediated TNF release in human alveolar macrophages from 615
HIV+ persons. (a) Healthy (n=6) and HIV+ (n=2) AM were incubated with MTb or BCG (MOI 616
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617 measured in cell culture supernatant by ELISA (R&D). (b) Specific TLR2 and TLR4 mRNA was 618 detected by real-time PCR (n=3). (c) AM were incubated for 24h in the presence or absence of 619 1,25D3 pretreatment and stained with PE-labeled anti-TLR or isotype control antibodies. Surface 620 expression was measured by flow cytometry. Left panels show isotype control (grey line) and 621 receptor-labeled cells (black line); right panels show receptor-labeled cells (grey line) and 622 1,25D3- treated receptor-labeled cells (black line). Representative histograms from individual 623 experiments with similar results (Healthy n=3, HIV+ n=2). Quantitative data represent 624 mean+SEM.* p<0.05, **p<0.01 625 Figure 7. 1,25D3 rescue of MTb-mediated TNF release in human alveolar macrophages 626
from HIV+ persons is dependent on CD14 (a) AM were incubated for 24h in the presence or 627
absence of 1,25D3 pretreatment and stained with PE-labeled anti-CD14 or isotype control 628
antibodies. Surface expression was measured by flow cytometry. Left panels show isotype 629
control (grey line) and receptor-labeled cells (black line); right panels show receptor-labeled 630
cells (grey line) and 1,25D3- treated receptor-labeled cells (black line) (n=3). (b) HIV+AM were 631
treated with MTb (0.25:1), BCG (10:1), or LPS (1μg/ml) in the presence and absence of 1,25D3 632
pretreatment (24 hours) and indicated antibodies. TNF was measured in cell culture supernatants 633
by ELISA (n=2). (c) 25D3 levels were measured in the cell-free BALF of healthy and HIV- 634
infected Indian patients with and without active MTb infection [HIV-MTb- (n=38), HIV-MTb+ 635
(n=35), HIV+MTb- (n=12), HIV+MTb+ (n=17)] by ELISA. Quantitative data represent 636
mean+SEM.* p<0.05 637
638
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