The Endogenous Retinoic Acid Receptor Pathway Is Exploited by Mycobacterium tuberculosis during Infection, Both In Vitro and In Vivo

Abstract

Retinoic acid (RA) is a fundamental vitamin A metabolite involved in regulating immune responses through the nuclear RA receptor (RAR) and retinoid X receptor. While performing experiments using THP-1 cells as a model for Mycobacterium tuberculosis infection, we observed that serum-supplemented cultures displayed high levels of baseline RAR activation in the presence of live, but not heat-killed, bacteria, suggesting that M. tuberculosis robustly induces the endogenous RAR pathway. Using in vitro and in vivo models, we have further explored the role of endogenous RAR activity in M. tuberculosis infection through pharmacological inhibition of RARs. We found that M. tuberculosis induces classical RA response element genes such as CD38 and DHRS3 in both THP-1 cells and human primary CD14+ monocytes via a RAR-dependent pathway. M. tuberculosis-stimulated RAR activation was observed with conditioned media and required nonproteinaceous factor(s) present in FBS. Importantly, RAR blockade by (4-[(E)-2-[5,5-dimethyl-8-(2-phenylethynyl)-6H-naphthalen-2-yl]ethenyl]benzoic acid), a specific pan-RAR inverse agonist, in a low-dose murine model of tuberculosis significantly reduced SIGLEC-F+CD64+CD11c+high alveolar macrophages in the lungs, which correlated with 2× reduction in tissue mycobacterial burden. These results suggest that the endogenous RAR activation axis contributes to M. tuberculosis infection both in vitro and in vivo and reveal an opportunity for further investigation of new antituberculosis therapies.

FIGURE 1.

Increased baseline levels of RAR-mediated CD38 expression in FBS-supplemented THP-1 cell cultures. (A) Representative scheme showing the experimental approach used to investigate the effects of FBS on RAR activation, created with BioRender.com. THP-1 cells were expanded in RPMI-1640 medium containing 10% FBS. Cells were cultured in RPMI containing either 10% FBS or 1% BSA. By day 4, CD38 expression was evaluated by flow cytometry and (B) represents a typical histogram of CD38 MFI in cells grown either in 1% BSA or 10% FBS. (C and D) Frequency of CD38⁺ (C) and expression of CD38 (MFI) (D) on viable cells. (E) CD38 MFI on viable serum-free THP-1 cells stimulated with various concentrations of FBS for 48 h. (F) CD38 expression (MFI) on viable serum-free THP-1 cells stimulated with various concentrations of ROL for 48 h. (G) Metabolic pathway of vitamin A for regulation of RARE gene expression. (H) CD38 expression (MFI) on live serum-free THP-1 cells stimulated with various concentrations of ROL in the presence or absence of DEAB (100 μM) and BMS493 (3 μM) for 48 h. (I) CD38 expression (MFI) on live serum-free THP-1 cells stimulated with various concentrations of RA in the presence or absence of BMS493 (3 μM) for 48 h. (J and K) Frequency (J) and MFI (K) of CD38 on viable cells grown in either 10% FBS or 10% FBS containing BMS493 (3 M) for 4 d. The data are shown as mean ± SEM. The results in (C) and (D) are pooled from 13 independent experiments performed in duplicate or triplicate, and (E)–(K) are from two independent experiments performed in triplicate. Statistical analyses were performed using a Mann–Whitney U test (C, D, J, and K), Kruskal–Wallis with Dunn’s multiple comparisons test (E), and ordinary one-way ANOVA with Dunnett’s multiple comparisons test (F, H, and I). p value is shown.

FIGURE 2.

M. tuberculosis activates endogenous RA/RAR pathway in primary human monocytes and THP-1 cells in vitro. PBMCs from healthy donors were exposed to M. tuberculosis (H37Rv) at MOI 3 in the presence of 1% autologous serum. By 72 h postinfection, RAR activation was evaluated by measuring CD38 expression in CD14⁺ monocytes, CD3⁺ lymphocytes, CD19⁺ B cells, CD56⁺ NK cells, and HLA-DR⁺CD11c⁺ DCs by flow cytometry. (A) CD38 MFI on viable CD14⁺ cells exposed or not to M. tuberculosis (MOI 3) in the presence or absence of BMS493 (3 μM). (B–E) CD38 MFI on viable CD3⁺ lymphocytes, CD19⁺ B cells, CD56⁺ NK cells, and HLA-DR⁺CD11c⁺ DCs. (F) THP-1 exposed to M. tuberculosis (H37Rv) at MOI 3 in the presence of 10% FBS. CD38 expression was measured 48 h postinfection. Data are shown as fold increase calculated over uninfected cells. (G and H) THP-1 cells were treated or not with BMS493 (3 μM) and exposed to M. tuberculosis (MOI 3). mRNA expression was evaluated 24 h postinfection. (I) CD38 expression on viable THP-1 cells stimulated by the combination of LPS (10 ng/ml) and IFN-γ (500 U/ml) for 48 h in the presence or absence of BMS493 (3 μM). The data are shown as mean ± SEM. The results shown in (A) are pooled from three independent experiments (n = 11 healthy donors), results in (B)–(E) are from two independent experiments (n = 6 healthy donors), and results in (F)–(H) are from two independent experiments performed in duplicate or triplicate. Results shown in (I) are from two independent experiments performed in duplicate or triplicate. Statistical analyses were performed using an ordinary one-way ANOVA with Dunnett’s multiple comparisons test (A, D, and F–I), and Kruskal–Wallis with Dunn’s multiple comparisons test (B, C, and E). p value is shown.

FIGURE 3.

CM from M. tuberculosis cultures activates RAR-mediated CD38 expression in THP-1 cells. (A) Representative scheme showing the experimental approach used to produce CM from M. tuberculosis cultures, created with BioRender.com. (B) CD38 MFI on live serum-free THP-1 cells exposed to RA (10 nM), CM from M. tuberculosis cultures, and CM-control (CM-Ø, without bacteria). CD38 fold increase was calculated over unstimulated cells. (C) CD38 MFI on live serum-free THP-1 cells stimulated with CM-Ø, CM from M. tuberculosis cultures, or CM from M. tuberculosis cultures HK in the presence or absence of BMS493 (3 μM) for 48 h. (D) THP-1 cells were exposed to high-dose M. tuberculosis H37Rv total lipids (10 μg/ml) in the presence or absence of BMS493 (3 μM) for 48 h. (E) CD38 MFI fold increase on live serum-free THP-1 cells stimulated with CM-Ø, CM–M. bovis BCG, CM–M. smegmatis, and CM–E. coli 48 h poststimuli. (F) CD38 MFI fold increase on live serum-free THP-1 cells stimulated with CM-control, CM from M. tuberculosis cultures in the presence or absence of DEAB (100 μM) (ALDH inhibitor) 48 h poststimuli. (G) CD38 MFI fold increase on live serum-free THP-1 cells exposed to either CM from M. tuberculosis cultures–treated or proteinase K 48 h poststimuli. Fold increase was calculated relative to unstimulated cells. The data are shown as mean ± SEM. The results shown in (B) are from one representative experiment performed in triplicate. (C–G) Data are pooled from six independent experiments performed in triplicate (C), are from one representative experiment performed in triplicate (D), are pooled from two to three independent experiments performed in duplicate or triplicate (E), and are pooled from two independent experiments performed in duplicate or triplicate (F and G). Statistical analysis was performed using a two-way ANOVA with Tukey’s multiple comparisons test (B), ordinary one-way ANOVA with Tukey’s multiple comparisons test (C–F), and Kruskal–Wallis with Dunn’s multiple comparisons test (D). p value is shown.

FIGURE 4.

BMS493 treatment reduces lung M. tuberculosis growth and CD38 expression by AMs. (A) Study timeline, created with BioRender.com. (B) Live CD45⁺ absolute cell counts. (C) Live AMs (Siglec-F⁺CD11c⁺CD64⁺) and (D) IMs (Siglec-F⁻CD11c⁻CD64⁺CD11b⁺) absolute cell counts. (E and F) Live CD4⁺ (E) and CD8⁺ (F) absolute T cell counts. (G and H) CD38 MFI on live AMs (G) and IMs (H). (I and J) Lung CFUs (I) and CFU fold change (J). Each symbol represents an individual mouse. Data are pooled from two independent experiments (n = 5–8 mice/group). The data are shown as mean ± SEM. Statistical analysis was performed using an ordinary one-way ANOVA with Tukey’s multiple comparisons test (B, D, and F–I), Kruskal–Wallis with Dunn’s multiple comparisons test (E), and unpaired t test (J and K). p value is shown.