The Journal of Immunology
All-Trans Retinoic Acid–Triggered Antimicrobial Activity
against Mycobacterium tuberculosis Is Dependent on NPC2
Matthew Wheelwright,*,1Elliot W. Kim,*,1Megan S. Inkeles,†Avelino De Leon,‡
Matteo Pellegrini,†Stephan R. Krutzik,* and Philip T. Liu*,‡
A role for vitamin A in host defense against Mycobacterium tuberculosis has been suggested through epidemiological and in vitro
studies; however, the mechanism is unclear. In this study, we demonstrate that vitamin A–triggered antimicrobial activity against
M. tuberculosis requires expression of NPC2. Comparison of monocytes stimulated with all-trans retinoic acid (ATRA) or 1,25-
dihydroxyvitamin D3 (1,25D3), the biologically active forms of vitamin A and vitamin D, respectively, indicates that ATRA and
1,25D3 induce mechanistically distinct antimicrobial activities. Stimulation of primary human monocytes with ATRA did not
result in expression of the antimicrobial peptide cathelicidin, which is required for 1,25D3 antimicrobial activity. In contrast,
ATRA triggered a reduction in the total cellular cholesterol concentration, whereas 1,25D3 did not. Blocking ATRA-induced
cellular cholesterol reduction inhibits antimicrobial activity as well. Bioinformatic analysis of ATRA- and 1,25D3-induced gene
profiles suggests that NPC2 is a key gene in ATRA-induced cholesterol regulation. Knockdown experiments demonstrate that
ATRA-mediated decrease in total cellular cholesterol content and increase in lysosomal acidification are both dependent upon
expression of NPC2. Expression of NPC2 was lower in caseous tuberculosis granulomas and M. tuberculosis–infected monocytes
compared with normal lung and uninfected cells, respectively. Loss of NPC2 expression ablated ATRA-induced antimicrobial
activity. Taken together, these results suggest that the vitamin A–mediated antimicrobial mechanism against M. tuberculosis
requires NPC2-dependent expression and function, indicating a key role for cellular cholesterol regulation in the innate immune
response.The Journal of Immunology, 2014, 192: 2280–2290.
Mycobacterium tuberculosis, the causative agent of tuberculosis,
micronutrients have proven to be critical as part of a successful
antimicrobial response. Epidemiological evidence demonstrates
an association between vitamin A and tuberculosis: serum vitamin
A levels are significantly higher in healthy household contacts
compared with tuberculosis patients (1, 2). In the laboratory, the
biologically active form of vitamin A, all-trans retinoic acid (ATRA),
was shown to inhibit the growth of virulent M. tuberculosis in
macrophages (3, 4). However, the molecular mechanisms and
ne key function of the innate immune system is the rapid
recognition and destruction of invading pathogens via
the activation of antimicrobial pathways. In the case of
cellular processes induced by ATRA that lead to this antimi-
crobial activity are unclear.
Vitamin D and vitamin A share similar molecular and biochem-
ical characteristics: both are fat-soluble secosteroids that are rec-
ognized by and effect changes in cells by binding to the vitamin D
receptor and the retinoic acid receptor (RAR), respectively (5).
RAR and vitamin D receptor are members of the nuclear hormone
receptor family and heterodimerize with the retinoid X receptor
(5). In relation to tuberculosis, deficient serum vitamin D levels
are associated with tuberculosis (6,7), and treatment of M. tu-
berculosis–infected cells in vitro with the active 1,25-dihydroxy-
vitamin D3 (1,25D3) form of vitamin D triggers antimicrobial
activity (8, 9), which is comparable to the epidemiological and
biochemical properties of vitamin A. Based on these similarities,
we compared the 1,25D3-triggered antimicrobial response, which
is dependent on production of the antimicrobial peptide cath-
elicidin (10, 11), with the ATRA-triggered response to elucidate
the vitamin A–mediated antimicrobial mechanism.
Materials and Methods
Comparisons between two different conditions were analyzed using the
Student t test. All experiments with three or more measurements were
analyzed using one-way ANOVA or the Kruskal–Wallis one-way ANOVA
on Ranks, as appropriate, with Student–Newman–Keuls method for pair-
wise analyses. Error bars represent the SEM.
ATRA was purchased from Sigma-Aldrich (St. Louis, MO), dissolved in
DMSO, and stored at 280˚C in small aliquots protected from light. 1,25D3
was purchased from Enzo Life Sciences (Farmingdale, NY), dissolved in
ethanol, and stored at 280˚C in small aliquots protected from light. Both
ATRA and 1,25D3 were used at 1028M. LysoSensor Green DND 189
(Life Technologies) was used at 1:2000 dilution (0.5 nM), as recom-
mended by the manufacturer. Oregon Green 488–dextran m.w. 10,000 and
*Division of Dermatology, Department of Medicine, David Geffen School of Medicine,
University of California, Los Angeles, Los Angeles, CA 90095;†Department of Mo-
lecular, Cell, and Developmental Biology, University of California, Los Angeles, Los
Angeles, CA 90095; and
Hospital Department of Orthopaedic Surgery and the Orthopaedic Hospital Research
Center, Los Angeles, CA 90095
‡University of California, Los Angeles and Orthopaedic
1M.W. and E.W.K. contributed equally to this work.
Received for publication June 27, 2013. Accepted for publication January 5, 2014.
This work was supported by National Institutes of Health K22 Career Development
Award AI 85025 (to P.T.L.).
Address correspondence and reprint requests to Dr. Philip T. Liu, University of
California, Los Angeles, 615 Charles E. Young Drive East, Room 410, Los Angeles,
CA 90095. E-mail address: firstname.lastname@example.org
The online version of this article contains supplemental material.
Abbreviations used in this article: ATRA, all-trans retinoic acid; 1,25D3, 1,25-dihy-
droxyvitamin D3; IPA, Ingeniuty Pathways Analysis; kME, module eigengene–based
connectivity; MDM, monocyte-derived macrophage; MFI, mean fluorescence inten-
sity; MOI, multiplicity of infection; Nel, nelfinavir; qPCR, quantitative real-time
RT-PCR; RAR, retinoic acid receptor; Rit, ritonavir; siCTRL, small interfering RNA
oligonucleotide control; siNPC2, small interfering RNA targeting NPC2; siRNA, small
interfering RNA; UCLA, University of California, Los Angeles; WGCNA, weighted
gene coexpression network analysis.
Alexa Fluor 647–dextran m.w. 10,000 were purchased from Life Tech-
nologies and used at 250 and 30 mg/ml, respectively. Nelfinavir (Nel) and
ritonavir (Rit) were obtained through the AIDS Research and Reference
Reagent Program, Division of AIDS, National Institute of Allergy and
This study was conducted according to the principles expressed in the Dec-
laration of Helsinki and was approved by the Institutional Review Board of
the University of California, Los Angeles (UCLA). All donors provided
written informed consent for the collection of peripheral blood and sub-
sequentanalysis. We obtainedwhole blood from healthy donorsthroughthe
UCLA Center for AIDS Research Virology Core with informed consent.
Mononuclear cells (PBMCs) were isolated from peripheral blood of healthy
donors using Ficoll-Paque, as previously described (12,13). Monocytes
were purified using two methods: plastic adherence and negative selec-
tion. For plastic adherence, PBMCs were cultured for 2 h in RPMI 1640
medium (Life Technologies) supplemented with 1% FCS (Οmega Sci-
entific, Tarzana, CA). The cultures were washed vigorously, and the
remaining adherent cells were cultured in RPMI 1640 with 10% FCS. For
negatively selected monocytes, we used an EasySep Human Monocyte
Enrichment Kit (STEMCELL Technologies, Vancouver, BC, Canada),
according to the manufacturer’s recommended protocol. Monocyte-derived
macrophages (MDMs) were produced as previously described with M-CSF
M. tuberculosis H37Ra and H37Rv were plated on 7H11 agar plates from
frozen stocks. All experiments involving H37Rv were conducted at Bio-
safety Level 3. Following 3–4 wk of incubation at 37˚C in a water-jacketed
incubator with 5% CO2, the solid colonies were scraped off the agar plate
and placed in 13 PBS. The bacterial suspension was gently separated with
a sonicating water bath (Branton 2510) for 30 s and then centrifuged at
735 3 g for 4 min to create a single-cell suspension. To enumerate the
bacteria, the supernatant was separated from the pellet, and the absorbance
at 600 nm was measured using spectrophotometry. Normal monocytes and
MDMs were infected at a multiplicity of infection (MOI) of 1 and trans-
fected monocytes were infected at an MOI of 0.5 overnight, and the cells
were vigorously washed three times with fresh RPMI 1640 media to re-
move extracellular bacteria.
To assess M. tuberculosis viability from infected monocytes, we used the
real-time PCR–based method, as previously described (15, 16), which
compares 16S RNA levels with genomic DNA (IS6110) levels as an in-
dicator of bacterial viability. Monocytes were purified and infected with
M. tuberculosis and stimulated as indicated for 3 d. For H37Ra-infected
monocytes, the cells were harvested and divided following the incubation.
Half of the cells were lysed by boiling at 100˚C for 5 min and then snap-
frozen at 280˚C. Total RNA was isolated from the remaining half using
TRIzol reagent (Life Technologies) via phenol-chloroform extraction,
followed by RNA cleanup and on-column DNase digestion using an
RNeasy Miniprep Kit (QIAGEN, Valencia, CA). cDNA was synthesized
from the total RNA using the iScript cDNA Synthesis kit (Bio-Rad, Her-
cules, CA), according to the manufacturer’s recommended protocol. The
bacterial 16S rRNA and genomic element DNA levels were assessed from
the cDNA and cellular lysate, respectively, using real-time PCR using iQ
SYBR Green (Bio-Rad). Comparison of the bacterial DNA with the
mammalian genomic 36B4 levels was used to monitor infectivity between
all of the conditions in the assay, as well as PCR quality. The relative 16S
values were calculated using DDCT analysis, with the IS6110 value
serving as the “housekeeping gene.” The following IS6110 genomic ele-
ment and 16S primer sequences were used: 16S Forward 59-GGT GCG
AGC GTT GTC CGG AA-39, 16S Reverse 59-CGC CCG CAC GCT CAC
AGT TA-39 and IS6110 Forward 59-GGA AGC TCC TAT GAC AAT GCA
CTA G-39, IS6110 Reverse 59-TCT TGTATA GGC CGT TGATCG TCT-
39. For H37Rv-infected cells, the DNA was isolated from the interphase
and phenol-chloroform phase using the back-extraction protocol, as de-
scribed by the manufacturer.
Quantitative real-time RT-PCR for mRNA
Total RNA was extracted from cells using TRIzol reagent (Life Technol-
ogies), and mRNA was reverse transcribed using iScript (Bio-Rad). Gene
expression of CAMP, CYP27A1, NPC2, and IL6 was analyzed by quan-
titative real-time RT-PCR (qPCR), as described above, with 36B4 as the
housekeeping gene. The following primers were used: CAMP Forward
59-GGA CCC AGA CAC GCC AAA-39, CAMP Reverse 59-GCA CAC
TGT CTC CTT CAC TGT GA-39; CYP27A1 Forward 59-GCT ATG CCC
TGC AAC TGC ACC A-39, CYP27A Reverse 59-TCC TTC CGT GGT
GAA CGG CCC ATA G-39; NPC2 Forward 59-TAT CCC TCT ATA AAA
CTG GTG GTG-39, NPC2 Reverse 59-CCA GAT GCA CCG AAC TCA
AT-39; and IL6 Forward 59-GCC CAC CGG GAA CGA AAG AGA-39,
IL6 Reverse 59-GAC CGA AGG CGC TTG TGG AGA AG-39.
Cellular cholesterol measurement
Monocytes were cultured and stimulated as indicated for 18 h, collected,
and enumerated. The lipid fraction was extracted using 3:2 hexane:iso-
propyl alcohol at room temperature for 30 min. Following 10 min of
centrifugation, the supernatant was collected and dried in glass test tubes
using nitrogen gas. Cholesterol levels were assessed with the Amplex Red
Cholesterol Assay Kit (Life Technologies), using the recommended pro-
tocol, and expressed as total cholesterol/cell.
Total RNAwas isolated from monocytes treated as indicated in the Results.
The total RNA samples were processed and analyzed by the UCLA Clinical
Microarray Core Facility using the Affymetrix U133 GeneChip. Cluster
diagrams were generated using the Cluster and TreeView software programs
from the Eisen Lab (http://rana.lbl.gov/) (17). Biological functions and
cholesterol-related functions were identified using Ingenuity Pathways
Analysis (http://www.ingenuity.com/). For the caseous tuberculosis gran-
uloma and weighted gene coexpression network analysis (WGCNA) val-
idation microarray analysis, data files were obtained from the Gene
Expression Omnibus database (http://www.ncbi.nlm.nih.gov/geo/; acces-
sion numbers GSE20050, GSE23073, GSE13762, and GSE28995) (18).
NPC2 and IL6 levels were normalized to G3PDH. Because there are
multiple G3PDH probes represented on the microarray, the NPC2 and IL6
probe values were normalized to every G3PDH probe and averaged.
We performed WGCNA, as previously described, using the R package
“WGCNA.” (19). A signed weighted correlation network was constructed
using the blockwiseModules() command with a soft thresholding power
b = 9. Using an adjacency matrix calculated from pairwise correlations
between all pairs of genes across all samples raised to the power b, the
topological overlap was calculated as a measure of network interconnec-
tedness. Module eigengenes were calculated for each resulting module and
were correlated to ATRA or 1,25D3 treatment using a binary vector rep-
resentation of treatment status.
WGCNA module preservation
WGCNA can assess whether individual modules are preserved between
two data sets (19). To validate our microarray results, we examined the
preservation of ATRA- and 1,25D3-specific modules against published
and publically available microarray data (20–22). The R function “mod-
ulePreservation” in the WGCNA R package was applied to our data and
the published data, and the Zsummary value was calculated. Zsummary
scores . 10 are interpreted as “strongly preserved,” Zsummary scores
between 2 and 10 are interpreted as “weak to moderately preserved,” and
Zsummary scores , 2 are “not preserved.” Significance of the Zsummary
scores was calculated by permutation analysis (23).
Identification of hub genes
Genes with the highest module membership values, or module eigengene–
based connectivity (kME), are referred to as intramodular “hub” genes,
which are genes that have the highest number of connections within the
module. kME was calculated for each gene in each module using the
signedKME() command. For each module, genes were sorted by kME, and
VisANT was used to visualize the gene connections among the top 50 hub
genes, as ranked by kME (24).
Two methods were used to determine monocyte viability: trypan blue
exclusion and TUNEL assay. Following infection for 16 h with H37Ra,
monocytes were harvested, and viability was assessed. For trypan blue
exclusion, the harvested cells were incubated with a final concentration of
0.04% trypan blue (Bio-Rad) and enumerated for the number of blue-
labeled cells compared with total cells using an automated cell counter
(Bio-Rad TC10). The data are shown as a percentage of viable cells. For
The Journal of Immunology2281
TUNEL assay, the APO-BrdU TUNEL Assay Kit (Life Technologies) was
used according to the manufacturer’s recommended protocol.
Transfection of monocytes
Purified monocytes were transfected with pooled small interfering RNA
(siRNA) oligonucleotides using the Lonza Nucleofector 4D system with the
Human Monocyte Nucleofector kit (Lonza, Allendale, NJ), according to the
manufacturer’s recommended protocol. To knockdown the expression of
NPC2, the predesigned and validated ON-TARGETplus siRNA targeting
NPC2 (siNPC2) oligonucleotide pool was used in conjunction with the ON-
TARGETplus Non-targeting Control Pool siRNA oligonucleotide control
(siCTRL). The siRNA oligonucleotides were purchased from Thermo
Fisher (Rockford, IL) and stored as recommended by the manufacturer.
Lysosomal acidification was measured using two dyes, LysoSensor and
Oregon Green (both from Life Technologies), as previously described (25).
Monocytes were purified and transfected, as described above, with siCTRL
or siNPC2 and then stimulated with ATRA for 18 h. LysoSensor (1:2000)
was added to each well, rocked gently for 5 s to mix, and incubated for
30 min. After incubation, the cells were fixed in 1% paraformaldehyde,
and fluorescence was acquired using flow cytometry. The mean fluorescent
intensity (MFI) was determined on the monocytes in the samples, which
were the only cellular population in the samples given that they were
magnetically separated as indicated in the “Transfection of monocytes”
section above. For Oregon Green labeling, the transfected and stimulated
monocytes were harvested, washed, and incubated with Oregon Green 488
(250 mg/ml) and Alexa Fluor 647 (Life Technologies; 30 mg/ml) for 30
min. The cells were washed and analyzed by flow cytometry. Both Oregon
Green 488 and Alexa Fluor 647 were conjugated to 10,000 m.w. dextran
for targeting into the lysosome. Data shown are the ratio of the fluores-
cence detected for Oregon Green 488/Alexa Fluor 647.
ATRA- and 1,25D3-induced monocyte function
Primary human monocytes were infected overnight with M. tube-
rculosis H37Ra, an avirulent strain used to model mycobacterial
infection (10, 15), and then treated with carrier control, ATRA, or
1,25D3 for 3 d. Following treatment, the ratio of bacterial 16S
RNA/IS6110 genomic repeat element was determined as an indi-
cator of bacterial viability, as we previously described (15). Both
ATRA and 1,25D3 treatment resulted in a decrease in bacterial
viability compared with carrier control (representative figure,
Fig. 1A). On average, M. tuberculosis viability, measured as
log10(16S/IS6110) in monocytes treated with ATRA or 1,25D3,
was significantly reduced (Fig. 1B) and of comparable magni-
tude at these concentrations (1028M) to previously published studies
using macrophages and the CFU assay (3, 8–10).
Given that the 1,25D3-mediated antimicrobial activity is de-
pendent on expression of the antimicrobial peptide cathelicidin
(10,11), we determined whether ATRA could induce cathelicidin
(CAMP) mRNA in primary human monocytes. Although 1,25D3
was able to significantly induce expression of CAMP mRNA,
ATRA did not (Fig. 1C). In contrast, CYP27A1 mRNA, a known
ATRA response gene (26), was induced by ATRA but not 1,25D3
(Fig. 1D), indicating that the cells were responding to ATRA.
Because cholesterol is a key factor in the interactions between
innate immune cells and M. tuberculosis (27–29), and ATRA is
known to induce cholesterol efflux (26, 30, 31), we hypothesized
that the ability of ATRA to regulate cellular cholesterol content is
linked to antimicrobial activity. The cellular cholesterol concen-
tration (total cholesterol/cell) in monocytes treated with carrier
control, ATRA, or 1,25D3 was measured (representative figure,
Fig. 1E). ATRA treatment resulted in a significant reduction
(234 6 7% versus control, p , 0.001) in total cellular cholesterol
content, whereas 1,25D3 had no effect (Fig. 1F). These results
suggest that ATRA and 1,25D3 induce distinct intracellular path-
ways and likely use different antimicrobial mechanisms.
Role of cholesterol regulation in ATRA-induced antimicrobial
To determine whether an ATRA-induced reduction in cellular
cholesterol plays a role in ATRA-mediated antimicrobial activity,
we used Nel and Rit, two compounds previously described to
inhibit cholesterol efflux from human macrophages and mac-
rophage-derived foam cells (32,33). Monocytes were infected
with M. tuberculosis H37Ra for 18 h, washed to remove extra-
cellular bacterium, preincubated for 10 min with Nel at 10 mM
and Rit at 30 mM (concentrations previously described to inhibit
cholesterol efflux) (32,33), and stimulated with ATRA for 3 d.
ATRA induced a decrease in cellular cholesterol compared with
carrier control, which was significantly inhibited by the presence
of Nel or Rit (Fig. 2A). There were no significant effects of Nel
or Rit on baseline total cellular cholesterol levels (Supplemental
viability of M. tuberculosis H37Ra–infected primary monocytes treated
with carrier control (CTRL), ATRA, or 1,25D3 for 3 d, displayed as
a representative experiment (A) or log10(mean bacterial viability versus
CTRL 6 SEM) (B) (n = 4). mRNA expression levels assessed by qPCR of
cathelicidin (CAMP) (C) and CYP27A1 (D) in primary human monocytes
stimulated with CTRL, ATRA, or 1,25D3 for 18 h, displayed as mean fold
change versus CTRL 6 SEM (n = 7). Cellular cholesterol levels/cell of
monocytes treated with CTRL, ATRA, or 1,25D3 for 18 h shown as a
representative experiment (E) or mean percentage change versus CTRL 6
SEM (F) (n = 5).
ATRA- and 1,25D3-induced cellular responses. Bacterial
2282RETINOIC ACID ANTIMICROBIAL ACTIVITY IS DEPENDENT ON NPC2
Fig. 1A). Importantly, ATRA-induced antimicrobial activity was sig-
nificantly inhibited in M. tuberculosis H37Ra–infected monocytes
by Nel (from 21.3 to 20.05 6 0.17, p = 0.0002, versus ATRA
only) and Rit (from 21.3 to 20.13 6 0.22, p = 0.002, versus
ATRA only) (Fig. 2B).
Because macrophages are the natural cellular host type for
M. tuberculosis infection, we used MDMs, a cellular model for
antimicrobial activity against M. tuberculosis in macrophages
(14). MDMs were infected with the virulent M. tuberculosis H37Rv
strain at an MOI of 1 for 18 h, washed to remove extracellular
bacteria, preincubated for 10 min with Nel and Rit, and stimulated
with ATRA for 3 d. Pretreatment of M. tuberculosis H37Rv–
infected MDMs with Nel or Rit inhibited ATRA-induced antimi-
crobial activity (Nel: from 20.9 to 20.05 6 0.16, p = 0.05, versus
ATRA only; Rit: from 20.85 to 20.28 6 0.15, p = 0.05, versus
ATRA only) (Fig. 2C). These data suggest that regulation of cellular
cholesterol may play an important role in ATRA-induced antimi-
crobial activity in both monocytes and macrophages.
ATRA- versus 1,25D3-induced gene-expression profiles
To identify specific genes driving the ATRA-induced cholesterol
regulation, we compared the monocyte gene-expression profiles
induced by ATRA (1028M) or 1,25D3 (1028M) treatment for
18 h from four independent donors using microarrays. Analysis of
genes significantly upregulated (1.2-fold versus control, p , 0.05)
by either ATRA or 1,25D3 revealed three gene groups: induced
by ATRA only, induced by 1,25D3 only, and induced by both
(Fig. 3A). A total of 868 genes was represented in the ATRA-only
group, 2591 genes were in the 1,25D3-only group, and 205 genes
were induced by both. As expected, CYP27A1 was significantly
upregulated (2.6 6 0.3-fold versus control, p , 0.001) and was
present in the ATRA-only group (Supplemental Fig. 1B).
To confirm the differential gene-expression signatures induced
by ATRA or 1,25D3, we applied WGCNA to the microarray data
(34). WGCNA identified transcripts that organize into distinct
modules of coexpressed genes (Fig. 3B). In particular, the
“salmon” module eigengene was significantly correlated with
ATRA stimulation (r = +0.95, p = 2 3 1026), whereas the “cyan”
module eigengene was correlated with 1,25D3 stimulation
(r = +0.91, p = 4 3 1025) (Fig. 3C). To validate these ATRA- and
1,25D3-induced gene-expression profiles, we determined the pre-
servation of the cyan and salmon modules in published and publi-
cally available microarray studies that examined CD14+monocytes
differentiating into dendritic cells treated with 1,25D3 (1028M) for
18 h (20), human primary monocytes treated with 1,25D3 (1028M)
for 12 h (21), and the THP-1 monocytic cell line treated with ATRA
(2 3 1028M) for 2, 6, or 16 h (22). These studies were chosen for
comparison with our current study because they were conducted on
myeloid immune cells, used similar doses of ATRA and 1,25D3,
and used similar incubation times. Because of the low number of
THP-1 samples at 2 h (n = 2) and 6 h (n = 2) for the ATRA study,
the data from these two time points were combined to provide the
necessary resolution for the module-preservation test. The cyan
module demonstrated a strong preservation in the 1,25D3 studies
(Fig. 3D), whereas the salmon module was only preserved in the
ATRA study (Fig. 3E). These results suggest that the gene-expression
profiles we obtained in ATRA or 1,25D3 stimulated primary human
monocytes are representative of the core gene signatures induced
by ATRA or 1,25D3.
Identification of candidate genes
To determine the relationship between the gene-expression pro-
files and cellular function, the upregulated genes were analyzed
by Ingeniuty Pathways Analysis (IPA), a knowledge-guided bio-
or 1,25D3 (analysis scheme displayed in Supplemental Fig. 2).
Based on our hypothesis that control of cellular lipids is a key
element of the ATRA-induced antimicrobial response, the cate-
gories “lipid metabolism,” “molecular transport,” and “small
molecule biochemistry” (the second-, third-, and fourth-ranked
for 18 h. The total cellular cholesterol level/cell was determined using Amplex Red. Data shown are the mean percentage change (6 SEM) versus control
(CTRL)-treated cells (n = 5). Bacterial viability of M. tuberculosis H37Ra (B) in monocytes or M. tuberculosis H37Rv in MDMs (C) treated with ATRA and
with or without Nel or Rit for 3 d. Data shown are log10(bacterial viability versus CTRL) for each individual donor monocytes/MDMs tested. Gray line
indicates the mean.
Effects of Nel and Rit on ATRA-induced responses. (A) Monocytes were treated with Nel and Rit for 20 min and then stimulated with ATRA
The Journal of Immunology2283
functions, respectively) were examined further (Fig. 4A). The
same three categories (“lipid metabolism,” “molecular transport,”
and “small molecule biochemistry”) were the 27th-, 23rd-, and
2nd-ranked 1,25D3-induced categories, respectively (Fig. 4B).
Comparing the ATRA- and 1,25D3-induced genes in the “small
molecule biochemistry” category reveals 14 genes in common,
which represents 6.2% of the total genes in the category induced
by either ATRA or 1,25D3 (Supplemental Fig. 2). Taken together,
these analyses suggest that ATRA induces a lipid metabolism and
intracellular molecular transport gene profile that is not present in
The comparison of the ATRA-induced genes in the “lipid me-
tabolism,” “molecular transport,” and “small molecule biochem-
istry” categories revealed a high degree of similarity among the
categories, with 44 genes in common (Fig. 4C). A total of 11 of
the 44 common genes also were identified as hub genes by
WGCNA (Fig. 4D). Of the 44 common genes, 16 genes were
annotated by IPA with functions related to regulation of cellular
cholesterol (functions: “accumulation of cholesterol,” “concen-
tration of cholesterol,” and “efflux of cholesterol”), three of which
(NPC2, CYP27A1, and LAMP2) were also hub genes. Only one
gene, NPC2, was annotated with all three cholesterol-related func-
tions and was induced by ATRA (Fig. 4E) but not by 1,25D3
(Fig. 4F). The kME value for NPC2 was 0.98, which was the
highest ranked hub gene identified by WGCNA in the salmon
module. These data suggest that NPC2 may play a central role in
the ATRA-induced gene signature that mediates the intracellular
regulation of cholesterol content.
Induction of NPC2 by ATRA
Monocytes were stimulated with ATRA or 1,25D3, and NPC2
mRNA levels were measured by qPCR. Confirming the microarray
analysis, the qPCR results demonstrated specific induction of NPC2
mRNA in ATRA-stimulated monocytes compared with 1,25D3
stimulation (Fig. 5A). To better characterize the induction of
NPC2, monocytes were stimulated with ATRA at 1028M for 1, 4,
16, or 24 h. Total RNA was harvested, and NPC2 mRNA levels
were measured by qPCR. NPC2 was significantly induced (3.6-
fold versus control, p = 0.026) at the 16-h time point in monocytes
(Fig. 5B). MDMs stimulated with ATRA also showed a signif-
icant induction (3.4-fold versus control, p = 0.008) of NPC2
mRNA at 16 h (Fig. 5C). An RAR response element is present
1312 bp upstream of the NPC2 mRNA start site (Supplemental
Fig. 3A). These results demonstrate that NPC2, which has an
RAR response element, can be induced by ATRA stimulation of
pression profiles. (A) Hierarchical clustering of genes
induced (1.2-fold over control [CTRL], p , 0.05) in
primary human monocytes by ATRA or 1,25D3 after
18 h. (B) Identification of coexpression modules by
WGCNA. The dendrogram was obtained by average
linkage hierarchical clustering of ATRA and 1,25D3
gene-expression profiles. Height on the dendrogram
represents kME, with more connected genes located
toward the bottom of the tree. The corresponding
module colors for each gene are indicated in the color
bar. (C) Correlation of module eigengenes versus stim-
ulus displayed in a heat map. The r values (p value) are
indicated in the map for each module. Preservation
(Zscore) of the salmon (D) and cyan (E) modules in
published microarray studies on ATRA- or 1,25D3-
stimulated immune cells. The p values are indicated by
line graph overlay. * and ** denote modules most highly
and significantly correlated with ATRA or 1,25D3 treat-
ATRA- and 1,25D3-induced gene-ex-
2284 RETINOIC ACID ANTIMICROBIAL ACTIVITY IS DEPENDENT ON NPC2
Role of NPC2 in ATRA-induced regulation of cellular
Mutations in NPC1 or NPC2 are associated with Niemann–Pick
disease, a lysosomal storage disorder that is characterized by ab-
normally high cholesterol accumulation in cells. NPC1 and NPC2
have common (35) and nonredundant functions related to lyso-
somal lipid transport (36,37). Stimulation of monocytes with ATRA
resulted in expression of NPC2, but not NPC1, detected by qPCR
correlating with the microarray results, suggesting a specific role for
NPC2 as a lipid transporter in ATRA-stimulated cells (Supplemental
Fig. 3B). To ascertain the role of NPC2 in ATRA-mediated regu-
lation of cellular cholesterol concentration, we transfected mono-
cytes with siNPC2 or siCTRL and then stimulated the cells with
carrier control or ATRA for 18 h. siNPC2 transfection resulted in
a significant decrease in NPC2 mRNA levels in both resting and
ATRA-stimulated monocytes (Fig. 5D). Neither siNPC2 nor
siCTRL had an effect on the CYP27A1 mRNA levels of resting
and ATRA-stimulated monocytes (Fig. 5E), indicating that the
siNPC2 knockdown was specific. Correlating with the expression
of NPC2, siCTRL-transfected monocytes stimulated with ATRA
demonstrated a reduction (242 6 15% versus control treated, p ,
0.05) in total cellular cholesterol content (Fig. 5F). In marked
contrast, ATRA stimulation of siNPC2-transfected monocytes re-
sulted in an increase (126 6 69% versus control treated, p , 0.05)
in total cellular cholesterol content (Fig. 4F). There was no sig-
nificant difference in baseline cellular cholesterol concentration
the ATRA-induced genes in the “lipid metabolism,” “molecular transport,” and “small molecule biochemistry” categories. (D) Network connection between
the top 50 ranked hub genes identified by WGCNA. The overlap between the 44 common genes and the hub genes are indicated. (E) Annotation of the 44
common genes with IPA functional categories (Fx) related to cholesterol. Intensity of red indicates level of significance (p , 0.05, Fisher exact test), gray
indicates not significant for ATRA- or 1,25D3-induced expression. Asterisk indicates WGCNA-identified hub genes.
Regulation of cellular cholesterol by ATRA. IPA of the top biological functions induced by ATRA (A) or 1,25D3 (B). (C) Venn diagram of
The Journal of Immunology2285
between untreated siCTRL- and siNPC2-transfected cells (Sup-
plemental Fig. 3C).
Role of NPC2 in lysosomal acidification
Blocking cholesterol egress from lysosomes prevents acidifica-
tion (38), which is a key process in antimicrobial activity against
M. tuberculosis (39). We sought to address the role of NPC2 in
ATRA-induced lysosomal acidification using two dyes: Lyso-
Sensor, which accumulates and increases fluorescence intensity
in acidic organelles, and Oregon Green 488, a pH-sensitive fluo-
rescent dye (25). siCTRL- and siNPC2-transfected monocytes
were treated with ATRA for 18 h and labeled using LysoSensor.
Stimulation with ATRA resulted in increased LysoSensor labeling in
siCTRL-transfected, but not siNPC2-transfected, monocytes (Fig. 6A).
The change in MFI in siCTRL cells treated with ATRA (11.1%,
p = 0.012) was significantly higher (p = 0.009) compared with
siNPC2 cells (1.2%) treated with ATRA (Fig. 6B).
Oregon Green 488 is pH sensitive and exhibits a decrease in
fluorescence when exposed to acidic environments, whereas Alexa
Fluor 647 is pH resistant, and the fluorescence remains constant.
Therefore, a decrease in the Oregon Green 488/Alexa Fluor 647
ratio (OG:A647) indicates an increase in acidification. siCTRL-
and siNPC2-transfected monocytes were treated with ATRA for
18 h and then colabeled with Oregon Green 488 and Alexa Fluor
647, both conjugated to dextran for lysosomal targeting. Stimu-
lation with ATRA resulted in a decreased OG:A647 in siCTRL-
transfected, but not siNPC2-transfected, monocytes (Fig. 6C). The
decreased OG:A647 in siCTRL-transfected cells treated with ATRA
(16.6%, p , 0.001) was significantly higher (p , 0.001) compared
with siNPC2-transfected cells (1.4%) treated with ATRA (Fig. 6D).
These data suggest that ATRA induces lysosomal acidification that
is dependent upon the expression of NPC2.
Role of NPC2 during M. tuberculosis infection
To determine whether M. tuberculosis infection regulates NPC2
expression in situ, we analyzed a previously published gene-
microarray experiment that compared uninvolved lung tissue
with caseous tuberculosis granulomas (40). The relative expression
of NPC2 and IL6 was compared with G3PDH in the same samples
to account for differences between the sample types. Based on this
analysis, NPC2 levels trended lower (0.46-fold, p = 0.07), whereas
IL6 levels, which is inducible by M. tuberculosis (41), were sig-
(CTRL), ATRA, or 1,25D3 for 18 h (mean fold change [FC] 6 SEM versus CTRL, n = 4) (A), monocytes stimulated with ATRA over a time course (mean
FC 6 SEM versus CTRL, n = 4/time point) (B), and human MDMs stimulated with ATRA for 18 h (mean FC 6 SEM versus CTRL, n = 5) (C). mRNA
levels of NPC2 (D) and CYP27A1 (E) in monocytes transfected with siCTRL or siNPC2 and then stimulated with CTRL or ATRA. Data shown are mean
FC 6 SEM versus CTRL (n = 3–4). (F) Percentage change in total cholesterol/cell of monocytes transfected with siCTRL or siNPC2 and then stimulated
with ATRA for 18 h. Data shown are mean percentage change 6 SEM versus CTRL (n = 6).
Regulation of NPC2 by ATRA. mRNA levels of NPC2 assessed by qPCR in primary human monocytes stimulated with carrier control
2286RETINOIC ACID ANTIMICROBIAL ACTIVITY IS DEPENDENT ON NPC2
nificantly higher (2.7-fold, p = 0.0004) in caseous tuberculosis
granulomas compared with uninvolved lung tissue (Fig. 7A). Be-
cause lung biopsies contain multiple cell types, including nonim-
mune cells, the effects of M. tuberculosis infection on the NPC2
signal may be confounded. Therefore, we addressed the effects of
M. tuberculosis infection on NPC2 expression directly in mono-
cytes. In monocytes infected with M. tuberculosis H37Ra, we
observed a significant decrease (0.4 6 0.1-fold versus uninfected,
p = 0.002) in NPC2 mRNA after 18 h (Fig. 7B) and an increase in
IL6 mRNA (Fig. 7C) correlating with the microarray data. There
was no change in monocyte viability following infection, as de-
termined by trypan blue exclusion and TUNEL assay (Fig. 7D).
Treatment of infected monocytes with ATRA for 3 d, which
parallels the antimicrobial assay time course, resulted in a signif-
icant increase in NPC2 mRNA levels in monocytes (Fig. 7E)
and MDMs (Fig. 7F). Knockdown of NPC2 mRNA ablated the
ATRA-induced antimicrobial activity (0.29 6 0.22 versus siNPC2
control stimulated), whereas transfection of siCTRL had no effect
(20.30 6 0.08 versus siCTRL control stimulated, p , 0.05)
(Fig. 7G). In contrast, knockdown of NPC2 did not affect the
ability of 1,25D3 to induce antimicrobial activity (Fig. 7G), which
is expected based on the fact that NPC2 was not induced by
1,25D3 and that 1,25D3 uses a cathelicidin-dependent pathway
(10). Comparison of siCTRL- and siNPC2-transfected monocytes
treated with vehicle control showed no significant difference in
bacterial viability during the course of the antimicrobial activity
experiment (Supplemental Fig. 3D). Taken together, these findings
indicate an important role for NPC2 in the ATRA-triggered anti-
microbial response against M. tuberculosis infection.
Although vitamin A has been associated with host protection
against M. tuberculosis both in vivo (1,2) and in vitro (3,4), the
precise vitamin A–induced antimicrobial mechanism remained
unclear. In this study, we sought to explore the mechanism(s)
driving the vitamin A–triggered antimicrobial response by com-
paring vitamin A (ATRA)-induced and vitamin D (1,25D3)-
induced cellular and genomic responses, given that both are
known to induce antimicrobial activity in M. tuberculosis–infected
monocytes and macrophages (3,8,9). Previously, we demonstrated
that 1,25D3-induced antimicrobial activity was dependent on ex-
pression of the antimicrobial peptide cathelicidin (10,11); how-
ever, our current study found that ATRA did not induce cathelicidin
expression. In contrast, ATRA, but not 1,25D3, stimulation resulted
in the reduction of cellular cholesterol content. Blocking cholesterol
egress inhibited ATRA-mediated antimicrobial activity in mono-
cytes infected with M. tuberculosis H37Ra, as well as MDMs
infected with M. tuberculosis H37Rv. Bioinformatic analysis
combining WGCNA and IPA revealed NPC2, a lysosomal to en-
doplasmic reticulum lipid transporter, as a potential mediator of
ATRA-induced regulation of cellular cholesterol content. NPC2
was required for the ATRA-mediated decrease in cholesterol con-
tent, as well as the increase in lysosomal acidification. In the context
of disease, NPC2 expression is decreased in caseous tuberculosis
granulomas and infected monocytes compared with normal lung
tissue and uninfected cells, respectively. Stimulation of M. tuber-
culosis H37Ra–infected monocytes or M. tuberculosis H37Rv–
infected MDMs with ATRA recovered NPC2 expression levels, and
knockdown of NPC2 expression ablated the ATRA-induced anti-
transfected with siCTRL or siNPC2 and stimulated with
control (CTRL) or ATRA for 18 h, and lysosomal
acidification was measured using LysoSensor or Oregon
Green. LysoSensor data are shown as representative
graphs of the flow cytometry data (A) and average
change (ATRA versus CTRL) in MFI (DMFI) 6 SEM
(n = 4) (B). Detection of lysosomal acidification by
Oregon Green is calculated as the fluorescence ratio
of Oregon Green 488–dextran (OG)/Alexa Fluor 647–
dextran (647). The Oregon Green data are shown as a
representative experiment (C) and average change (ATRA
versus CTRL) in OG:647 ratio 6 SEM (n = 4) (D).
Lysosomal acidification. Monocytes were
The Journal of Immunology2287
microbial activity, suggesting that NPC2 plays a pivotal role in
vitamin A–mediated host defense. These results demonstrate that
vitamin A–induced immune defense against M. tuberculosis is de-
pendent on the expression and function of NPC2.
Regulation of cholesterol is an important facet of the host–
pathogen interaction between immune cells and M. tuberculosis.
In a caseous tuberculosis granuloma there is an increased ex-
pression of genes involved in lipid sequestration and metabolism,
as well as an accumulation of cholesterol, cholesteryl esters, tri-
acylglycerols, and lactosylceramide (40). The presence of foamy
macrophages in the granuloma (42) and host hypercholesterolemia
both correlate with loss of protection against M. tuberculosis (43).
Studies also demonstrated that phagocytosis of M. tuberculosis
(27) and bacterial persistence within the macrophages are de-
pendent on cholesterol (28). M. tuberculosis can accumulate and
use cholesterol as a source of nutrition (29, 44, 45), as well as
exploit host-derived lipids to reduce metabolic stress (46), which
could be a determinant of pathogen virulence and immunogenicity
(47). Accumulation of lipids within lysosomes alters the pH of the
vesicle to favor bacterial survival (38), and fusion of lysosomes
with phagosomes harboring M. tuberculosis is a critical host de-
fense process against the infection (39,48,49).
Our data demonstrate that the ATRA-mediated decrease in
cellular cholesterol requires the expression and function of NPC2.
Mutations in the NPC2 gene have been well defined to be re-
sponsible for Niemann–Pick disease type C2, a lysosomal storage
disease characterized by abnormally high cholesterol accumula-
tion in cells. Our experiments show that loss of NPC2 expression
ablated the ATRA-mediated reduction in cellular cholesterol
content, and it resulted in a significant accumulation of cellular
cholesterol, paralleling the cellular etiology of Niemann–Pick
disease. Important to macrophage antimicrobial defense against
M. tuberculosis, knockdown of NPC2 expression also ablated
ATRA-induced lysosomal acidification, which is required for an-
timicrobial activity against the infection (38,39,48,49). Thus, de-
creasing NPC2 expression levels during infection can inhibit
(cTB) versus normal lung tissue normalized to G3PDH levels by microarray analysis. Expression of NPC2 (B) and IL6 (C) mRNA in M. tuberculosis
H37Ra–infected monocytes treated with control (CTRL) or ATRA for 3 d. Data shown are mean fold change (FC) 6 SEM versus CTRL (n = 5). (D)
Monocyte viability following infection assessed with trypan blue exclusion and TUNEL assay; data shown are average percentage viability 6 SEM (n = 4).
Expression of NPC2 mRNA in M. tuberculosis H37Ra–infected monocytes (n = 5) (E) or M. tuberculosis H37Rv–infected MDMs (n = 13) (F) treated with
CTRL or ATRA for 3 d. Data shown are mean FC 6 SEM versus CTRL. (G) Bacterial viability of M. tuberculosis H37Ra in monocytes transfected with
siCTRL or siNPC2 and then stimulated with ATRA or 1,25D3 for 3 d. Data shown are log10(mean bacterial viability 6 SEM versus siCTRL) (n = 3–4).
Role of NPC2 in host defense against M. tuberculosis infection. (A) Expression of NPC2 and IL6 mRNA in caseous tuberculosis granuloma
2288RETINOIC ACID ANTIMICROBIAL ACTIVITY IS DEPENDENT ON NPC2
important macrophage defense mechanisms, as well as increase
a nutrient source within the cell, which favors bacterial survival.
These data suggest that regulation of cellular cholesterol through
proteins, such as NPC2, may be a key part of the innate immune
response, and further studies investigating the consequence of cel-
lular cholesterol modulation on bacterial viability are warranted.
Although vitamin A deficiency is associated with tuberculosis,
vitamin A supplementation has not proven effective as a treatment
(50,51). This is likely because vitamin A status is determined by
serum retinol levels, and vitamin A supplementation similarly
modulates retinol levels. In contrast, the ability to induce an-
timicrobial activity through vitamin A metabolites, such as in this
study and previous studies (3,4) use the active ATRA form. This
suggests that, although activation of infected macrophages with
ATRA results in antimicrobial activity against intracellular M. tuber-
culosis infection, the intricate pathways that regulate retinol me-
tabolism, especially in tuberculosis patients, are still unclear. If
retinol metabolism were inhibited by infection, then the effects
of vitamin A supplementation will be effectively negated. Further
studies are needed to understand how vitamin A is metabolized
during the immune response to M. tuberculosis infection.
In summary, our data demonstrate a novel role for NPC2 in
the ATRA-mediated innate immune response against M. tuber-
culosis, which suggests that regulation of intracellular cholesterol
may be an important facet of defense against infection. Under-
standing how vitamin A–mediated functions are regulated during
infection in the host will be an important step in determining how
this micronutrient can influence the outcome of disease.
We thank Dr. J.S. Adams, Dr. B.R. Bloom, and Dr. R.L. Modlin for critical
reading of the manuscript. Nel and Rit were obtained through the AIDS
Research and Reference Reagent Program, Division of AIDS, National In-
stitute of Allergy and Infectious Diseases, National Institutes of Health.
Experiments using virulent M. tuberculosis were performed in the UCLA
Department of Microbiology, Immunology and Molecular Genetics Select
Agent Biosafety Level 3 Facility with approval and support from the
Director of UCLA High Containment Facilities and the Office of the Vice
Chancellor for Research.
The authors have no financial conflicts of interest.
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