Article Figures & Data
Figures
- FIG 1
Activation of innate immune cells by vitamin A metabolites. Primary human monocytes were treated with either vehicle alone (CTRL), retinol (10−8 M), retinaldehyde (ATRH) (10−8 M), or all-trans retinoic acid (ATRA) (10−8 M) for 18 h and the mRNA expression levels of NPC2 (A) and CYP27A1 (B) were measured via qPCR. Data shown are the average fold change (FC) versus CTRL ± standard error of the mean (SEM) (n = 3 to 7). P values by one-way ANOVA. *, P < 0.05; ***, P < 0.001. (C) Black dots represent the serum retinol levels of TB household contact (TB contacts) or active TB patients. The red line indicates the average ± SEM retinol serum levels in each group. P value by Student's t test. ***, P < 0.001.
- FIG 2
Expression of the retinol metabolic pathway in DCs. (A) Primary human monocytes were stimulated with GM-CSF for 0, 3, and 12 h, and then gene expression was profiled using microarrays. Expression data of vitamin A pathway genes, DHRS3, DHRS9, RDH10, ALDH1A1, ALDH1A2, and ALDH1A3, are displayed as mean expression in arbitrary units (AU) from three independent donors ± SEM. Primary human monocytes stimulated with a titration of GM-CSF or IL-15 for 18 h and mRNA expression of DHRS9 (n = 3 to 5) (B) and ALDH1A2 (n = 3 to 6) (C) were measured by qPCR. Data shown are the average fold change (FC) versus CTRL ± SEM. (D) Induction of CYP27B1 mRNA expression in monocytes by IL-15 was measured by qPCR. Data shown are the average fold change (FC) versus CTRL ± SEM (n = 3). P values by one-way ANOVA. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
- FIG 3
Transcriptional profiling of retinol metabolism in TB infected lung. Expression of vitamin A metabolism (ALDH1A2 and DHRS9) and activation (NPC2 and CYP27A1) genes as well as DC (CD1B) and macrophage (CD163) markers in human caseous TB versus normal lung tissue (A) and rabbit lung 2 and 16 weeks after M. tuberculosis infection (B) as measured by gene microarray. P values by one-way ANOVA. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
- FIG 4
CD1B and ALDH1A2 expression in TB lung. TB patient lung and normal lung tissue protein expression of ALDH1A2 (A), CD163 (B), and CD1B (C) by immunohistochemistry. Data shown are representative images (TB lung [TB], n = 8 to 10; normal lung [NL], n = 4 to 5). Scale bar, 40 μm. Expression of ALDH1A2 (D), CD163 (E), and CD1B (F) in TB versus normal lung was quantified per nucleated cell using Immunoratio. Data shown are the average percentage of positive cells/nucleus + SEM. P value by Student’s t test. *, P < 0.05; ***, P < 0.001.
- FIG 5
Activation of monocytes and macrophages with DC-produced ATRA. (A) GM-CSF-derived DCs were treated with the indicated amounts of retinol for 6 h under serum-free conditions, and the amounts of ATRA in the cellular (Cell) and supernatant (Supe) fractions were analyzed via HPLC. Data represent the means ± SEMs of the average area of ATRA peak from HPLC plots (n = 4). Data shown are the average fold change versus control ± SEM (n = 3 to 7). P values by one-way ANOVA. *, P < 0.05; ***, P < 0.001. Expression levels of NPC2 (B) and CYP27A1 (C) were measured by qPCR in primary human monocytes cultured with CM from DCs with or without retinol. From the same experiments, expression levels of NPC2 (D) and CYP27A1 (E) were measured in primary human monocytes cultured with CM from DCs pretreated with DEAB for 20 min prior to addition of retinol. Primary human monocytes were also pretreated with RARi and treated with CM from DC with or without retinol, and mRNA expression levels of NPC2 (F) and CYP27A1 (G) were measured by qPCR. All experimental conditions (panels B to G) were performed in parallel. Data shown are the average fold change versus control ± SEM (n = 3 to 7).
Supplemental Material
FIG S1
Ingenuity Pathway Analysis predicts GM-DCs are cellular source of retinol metabolism. Analysis of the RAR activation gene signature in GM-CSF, IL-10, IL-15, and IL-4 stimulated primary human monocytes. Download FIG S1, PDF file, 0.01 MB.
Copyright © 2019 Kim et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
FIG S2
GM-CSF induces DC phenotype. Primary human monocytes were differentiated into DCs by stimulation with GM-CSF for 48 h. DC surface markers CD206, CD86, and CD1B were assessed by flow cytometry using monoclonal antibodies (mAB) and their corresponding isotype controls (Iso). Data shown are averages of the mean fluorescence intensity (MFI) ± SEM (n = 4). P value by Student’s t test. **, P < 0.01; ***, P < 0.001. Download FIG S2, PDF file, 0.06 MB.
Copyright © 2019 Kim et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
FIG S3
Representative isotype control stains for normal and TB lung. Corresponding isotype controls for immunohistochemistry comparing TB lung tissue versus normal lung. (A) Rb IgG isotype control antibody for ALDH1A2; (B) mouse IgG1 isotype control antibody for CD163 and CD1B. Scale bars, 40 μm. Download FIG S3, PDF file, 0.04 MB.
Copyright © 2019 Kim et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
FIG S4
RARi blocked ATRA-induced genes but not DEAB. Primary human monocytes were pretreated with DEAB (A) or RARi (B) at the indicated concentrations and then stimulated with 10−8 M ATRA for 18 h. Expression of NPC2 and CYP27A1 was measured by qPCR. Data shown are the average fold change (FC) ± SEM (n = 4). P values by one-way ANOVA. **, P < 0.01; ***, P < 0.001. Download FIG S4, PDF file, 0.01 MB.
Copyright © 2019 Kim et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
