The Journal of Immunology
c-Maf–Dependent Growth of Mycobacterium tuberculosis in
a CD14hiSubpopulation of Monocyte-Derived Macrophages
Rohan Dhiman,* Anuradha Bandaru,†Peter F. Barnes,* Sudipto Saha,‡Amy Tvinnereim,*
Ramesh C. Nayak,xPadmaja Paidipally,* Vijaya Lakshmi Valluri,†L. Vijaya Mohan Rao,x
and Ramakrishna Vankayalapati*
Macrophages are a major component of the innate immune response, comprising the first line of defense against various intra-
cellular pathogens, including Mycobacterium tuberculosis. In this report, we studied the factors that regulate growth of M.
tuberculosis H37Rv in subpopulations of human monocyte-derived macrophages (MDMs). In healthy donors, M. tuberculosis
H37Rv grew 5.6-fold more rapidly in CD14hiMDMs compared with that in CD14loCD16+MDMs. Compared with CD14loCD16+
cells, M. tuberculosis H37Rv-stimulated CD14himonocytes produced more IL-10 and had increased mRNA expression for c-Maf,
a transcription factor that upregulates IL-10 gene expression. c-Maf small interfering RNA (siRNA) inhibited IL-10 production
and growth of M. tuberculosis in CD14hicells. Compared with CD14loCD16+monocytes, M. tuberculosis H37Rv-stimulated CD14hi
cells had increased expression of 22 genes whose promoters contained a c-Maf binding site, including hyaluronan synthase 1
(HAS1). c-Maf siRNA inhibited HAS1 expression in M. tuberculosis-stimulated CD14himonocytes, and HAS1 siRNA inhibited
growth of M. tuberculosis in CD14hiMDMs. M. tuberculosis H37Rv upregulated expression of HAS1 protein and its product,
hyaluronan, in CD14hiMDMs. We conclude that M. tuberculosis grows more rapidly in CD14hithan in CD14loCD16+MDMs
because CD14hicells have increased expression of c-Maf, which increases production of two key factors (hyaluronan and IL-10)
that promote growth of M. tuberculosis. The Journal of Immunology, 2011, 186: 1638–1645.
can proliferate and survive in alveolar and tissue macrophages,
favoring establishment and progression of infection, as well as
the capacity to persist and reactivate disease many years later. To
develop new tools to prevent tuberculosis, it is important to iden-
tify the macrophage subpopulations that are most susceptible to
M. tuberculosis infection and to understand the factors that reg-
ulate intracellular growth of M. tuberculosis.
There is increasing evidence that mononuclear phagocytes, in-
cluding monocytes and dendritic cells, are heterogeneous popu-
uberculosis is a leading cause of death from infectious
diseases worldwide, claiming an estimated 1.3 million
lives worldwide annually (1). Mycobacterium tuberculosis
lations that have different effects on T cells (2–5). Based on the
expression of CD14 and CD16 receptors, human blood monocytes
are classified as classical (CD14hiCD162) and nonclassical (CD14lo
CD16+) (3, 5). In various experimental models, it was shown that
CD14loCD16+monocytes produce the proinflammatory cytokine
TNF-a and do not produce IL-10 (3, 4, 6–9). In contrast, CD14hi
CD162monocytes produce the anti-inflammatory cytokine IL-10
(3, 4, 6–9). These monocyte subpopulations also differ in expres-
sion of chemokine receptors (10–12).
In this report, we isolated CD14hiand CD14loCD16+monocytes
from human peripheral blood, matured them to macrophages, and
compared their capacity to restrict growth of virulent M. tuber-
culosis H37Rv and to produce cytokines. We also identified a
transcription factor and a gene that regulates growth of M. tuber-
Materials and Methods
Blood was obtained from 30 healthy donors, 23 of who had negative Quan-
tiFERON TB-Gold test results, and 7 of who had positive QuantiFERON-
TB Gold test results. All studies were approved by the Institutional Re-
view Board of the University of Texas Health Center at Tyler and the Blue
Peter Public Health & Research Center. Written informed consent was
provided by study participants.
For flow cytometry, we used FITC anti-CD14, allophycocyanin anti-CD14,
allophycocyanin anti-CD56, and PE anti-CD16 (all from eBioscience). For
confocal microscopy, we used goat polyclonal anti-hyaluronan synthase 1
(anti-HAS1) as a primary Ab (Santa Cruz Biotechnology) and Alexa Fluor
568 donkey anti-goat IgG as a secondary Ab to stain HAS1. Hyaluronan
staining was performed by using biotinylated hyaluronan binding protein
(US Biological), followed by streptavidin Alexa Fluor 546 (Invitrogen).
*Center for Pulmonary and Infectious Disease Control, University of Texas Health
Science Center, Tyler, TX 75708;
Cherlapally, Hyderabad 501 301, India;‡Center for Proteomics and Bioinformatics,
Case Western Reserve University, Cleveland, OH 44106; andxBiochemistry, Center
for Biomedical Research, University of Texas Health Science Center, Tyler, TX
†Blue Peter Research Center, LEPRA Society,
Received for publication September 22, 2010. Accepted for publication November
This work was supported by grants from the National Institutes of Health (AI054629,
AI073612, and A1063514), the Cain Foundation for Infectious Disease Research, and
the Center for Pulmonary and Infectious Disease Control. P.F.B. holds the Margaret
E. Byers Cain Chair for Tuberculosis Research. A.B. is supported by a postdoctoral
fellowship awarded by the Council of Scientific and Industrial Research, India.
The sequences presented in this article have been submitted to the Gene Expression
Omnibus database under accession number GSE25505.
Address correspondence and reprint requests to Dr. Ramakrishna Vankayalapati, Center
for Pulmonary and Infectious Disease Control, University of Texas Health Center, 11937
US Highway 271, Tyler, TX 75708. E-mail address: email@example.com
Abbreviations used in this article: HAS1, hyaluronan synthase 1; MDM, monocyte-
derived macrophage; siRNA, small interfering RNA.
Isolation of monocytes
PBMCs were isolated by differential centrifugation over Ficoll-Paque
(Amersham Pharmacia Biotech). CD14+monocytes were isolated with
magnetic beads conjugated to anti-CD14 (Miltenyi Biotec), and positively
selected cells were .97% CD14+. Eight percent to 10% of the positively
selected cells were CD16+, as measured by flow cytometry. To isolate
CD16+cells, first we removed CD56+cells from CD14-depleted PBMCs
with magnetic beads conjugated to anti-CD56 (Miltenyi Biotec). From the
CD142CD562fraction, CD16+cells were isolated by positive immuno-
magnetic selection (Miltenyi Biotec). These cells were 95–100% CD14lo
CD16+and 95–97% CD56–, as measured by flow cytometry. The scheme
for cell isolation is shown in Fig. 2A.
Culture of monocytes with gamma-irradiated M. tuberculosis
CD14hior CD14loCD16+cells were cultured in 24-well plates at 106cells/
well in RPMI 1640 containing penicillin (Life Technologies) and 10%
heat-inactivated human serum, with or without gamma-irradiated M. tu-
berculosis H37Rv (10 mg/ml, provided by Dr. J. Belisle, Colorado State
University, Fort Collins, CO) at 37˚C in a humidified 5% CO2atmosphere.
Cell-free supernatants were collected after 24 h, aliquoted, and stored at
270˚C until IL-10 or TNF-a concentrations were measured by ELISA.
Infection of macrophages with M. tuberculosis H37Rv
CD14hior CD14loCD16+monocytes (106/well) were plated in 12-well
plates (BD Biosciences Labware) in 1 ml antibiotic-free RPMI 1640
containing 10% heat-inactivated human serum. Monocytes were incubated
at 37˚C in a humidified 5% CO2atmosphere for 4 d to allow differentiation
into macrophages. Some MDMs were uninfected, and others were infected
with M. tuberculosis H37Rv at a multiplicity of infection of 2.5:1, as
described previously (13). Viability of MDMs was .90% up to 7 d post-
infection. Cells were incubated for 2 h at 37˚C in a humidified 5% CO2
atmosphere, washed to remove extracellular bacilli, and cultured in RPMI
1640 containing 10% heat-inactivated human serum. In some experiments,
rIL-10 (10 ng/ml) was added immediately postinfection.
To quantify intracellular growth of M. tuberculosis H37Rv, infected
macrophages were cultured for 7 d, the supernatant was aspirated, and
macrophages were lysed. The supernatant was centrifuged to pellet bac-
teria, and the pellets were added to the cell lysates. Bacterial suspensions
were ultrasonically dispersed, serially diluted, and plated in triplicate on
7H10 agar. The number of colonies was counted after 3 wk.
Microarray analysis was used to compare gene expression by CD14hiand
CD14loCD16+monocytes. Total RNA was extracted from unstimulated or
gamma-irradiated M. tuberculosis H37Rv-stimulated monocytes after 48 h,
and pooled RNA from five healthy donors was sent to the Phalanx Biotech
Group for microarray analysis. Microarray data are MIAME compliant.
Raw and processed data have been deposited in the Gene Expression
Omnibus database (http://www.ncbi.nlm.nih.gov/geo/) under accession
Real-time PCR for quantification of mRNA
Total RNA was extracted from 106CD14hiand CD14loCD16+monocytes
24 h after culture with medium alone or with gamma-irradiated M. tu-
berculosis H37Rv using TRIzol reagent (Life Technologies) according to
the manufacturer’s instructions. Total RNA was reverse transcribed using
the Clone AMV First-Strand cDNA synthesis kit (Life Technologies).
The forward and reverse primers for c-Maf were 59-TGCACTTCGAC-
GACCGCTTCTC-39 and 59-GGTGGCTAGCTGGAATCGGG-39, respec-
tively. Primers for HAS1 were as follows: forward, 59-GTCATGAGGC-
CCAGGATG-39; reverse, 59-ACCCACTGCGATGAGACAG-39. Primers for
ZIP5 were as follows: forward, 59-CAGCAGAGCAAACTGACGAG-39; re-
verse, 59-CATTGGCTGACCACCTGAAT-39. Primers for TTC22 were as
follows: forward, 59-AAGGCAGGCAGTCTCTTCTG-39; reverse, 59-ACG-
ACAAGGCGCTAGG-39. Primers for GAPDH were as follows: forward, 59-
GCCATCAATGACCCCTTCATT-39; reverse, 59-TTGACGGTGCCATGG-
AAT TT -39. Real-time PCR was performed using the Quantitect SYBR
Green PCR kit (Qiagen) in a sealed 96-well microtiter plate (PE Applied
Biosystems) on a spectrofluorometric thermal cycler (7700 PRISM; PE
Applied Biosystems). PCR reactions were performed in triplicate as follows:
95˚C for 10 min, and 45 cycles of 95˚C for 15 s, 60˚C for 30 s, and 72˚C for
30 s. All samples were normalized to the amount of GAPDH transcript in
Measurement of TNF-a and IL-10 concentrations
Supernatants from gamma-irradiated M. tuberculosis H37Rv-stimulated
CD14hiand CD14loCD16+cells were collected after 24 h and stored at
270˚C until concentrations were measured by ELISA (eBioscience).
Small interfering RNA
Freshly isolated CD14himonocytes or MDMs were transfected with small
interfering RNA (siRNA) for c-Maf, IL-10, HAS1, or TTC22 or with
control siRNA using transfection reagents (all from Santa Cruz Bio-
technology). The efficiency of siRNA knockdown was measured by real-
time PCR of c-Maf or HAS1 or TTC22 mRNA expression. Briefly, 106
CD14himonocytes or MDMs were resuspended in 500 ml transfection
medium and transfected with siRNA (6 pmol). After 6 h, an additional 500
ml 23 RPMI 1640 complete medium was added, and cells were cultured
overnight in a 12-well plate. The next day, monocytes were washed and
either stimulated with gamma-irradiated M. tuberculosis H37Rv or kept in
medium alone as a control. In the case of MDMs, they were infected with
H37Rv, as outlined above, and CFU was measured after 3 d.
Confocal microscopy to detect intracellular HAS1 and
MDMs on chamber slides (Lab Tek) were uninfected or infected with GFP-
expressing M. tuberculosis H37Rv at a multiplicity of infection of 2.5:1 for
2 h, washed thoroughly, and cultured overnight. The next day, cells were
first fixed in 2% paraformaldehyde in PBS (pH 7.2), and then nonspecific
binding was blocked by incubating cell monolayers in blocking buffer.
Cells were then incubated overnight with goat polyclonal anti-HAS1 (4
mg/ml) in blocking buffer. As a control, monocytes were incubated with
either blocking buffer alone or secondary donkey anti-goat 568 Ab (4 mg/
ml). Then, cells were stained with their respective secondary Abs and
DAPI to identify the primary signal and nuclei. The cells were washed
and mounted with aqueous gel mounting media (Biomedia) containing
tuberculosis patients. A, PBMCs were stained with anti-CD14, anti-CD16,
and anti-CD56 to identify monocyte subpopulations expressing different
levels of CD14 and CD16. A representative figure is shown. B, Freshly
isolated PBMCs from seven healthy tuberculin reactors were stained with
Abs to CD14, CD16, and CD56. Flow cytometry was used to measure the
percentages of CD14hiand CD14loCD16+CD562cells. Boxes show the
median and interquartile range, and whiskers show the 5th and 95th per-
Monocyte subpopulations in healthy tuberculin reactors and
The Journal of Immunology1639
antifading agent. Confocal images were obtained, using an LSM 510 Meta
confocal system (Carl Zeiss) equipped with an inverted microscope (Axio
Observer Z1; Carl Zeiss). Immunostained cells were viewed through
a Plan-APOCHROMAT 633/1.4 NA oil objective lens, with 2.53 digital
magnification, to detect green fluorescence (GFP-expressing M. tubercu-
losis H37Rv) and red fluorescence (HAS1). Images were acquired with
Zen 2007 software (Carl Zeiss), and scanned images were exported and
processed using Adobe Photoshop version 7.0 software (Adobe Systems).
Results are shown as the mean 6 SD. For data that were normally dis-
tributed, comparisons between groups were performed by a paired or un-
paired t test, as appropriate. For data that were not normally distributed, the
Wilcoxon rank-sum test was used.
Monocyte heterogeneity in M. tuberculosis H37Rv infection
By staining with anti-CD14 and anti-CD16, we identified pop-
ulations of CD14hiCD162, CD14hiCD16+, and CD14loCD16+
monocytes. In addition, there were CD142CD16+cells that were
CD56+, representing NK cells (Fig. 1A). The distribution of
CD14hiand CD14loCD16+monocytes in freshly isolated PBMCs
from healthy tuberculin reactors is shown in Fig. 1B.
Virulent M. tuberculosis H37Rv grows more rapidly in
macrophages derived from CD14himonocytes
To determine whether monocyte heterogeneity affects the in-
tracellular growth of M. tuberculosis H37Rv in macrophages,
CD14hiand CD14loCD16+monocytes were isolated from the
blood of healthy donors on day 0 (Fig. 2A), and differentiated to
macrophages, as outlined in Materials and Methods. Purified
CD14hicells contained ,10% CD16+cells, and CD14loCD16+
cells contained ,1% CD14hiand ,1% CD56+cells (Fig. 2B). On
day 4, MDMs were infected with H37Rv, and CFU was quantified
after 2 h and 7 d. For five healthy donors, CFU was similar in
CD14hiand CD14loCD16+MDMs 2 h postinfection (Fig. 2C),
suggesting that both macrophage subpopulations were infected
by M. tuberculosis H37Rv with similar efficiency. However, 7 d
postinfection, CFU in CD14hiMDMs was 5-fold higher than that
in CD14loCD16+MDMs (p = 0.003; Fig. 2C).
Cytokine production by CD14hiand CD14loCD16+monocytes
To determine whether CD14hiand CD14loCD16+monocytes differ
in their capacity to produce cytokines in response to M. tubercu-
losis H37Rv, these monocyte subpopulations from 13 healthy
donors were cultured in the presence or absence of gamma-
irradiated M. tuberculosis H37Rv. Unstimulated CD14hicells
produced 130 6 39 pg/ml IL-10, and culture with M. tuberculosis
increased this almost 3-fold to 330 6 61 pg/ml. In contrast,
CD14loCD16+monocytes produced no detectable IL-10 in the
presence or absence of M. tuberculosis (p , 0.001, compared with
stimulated CD14hicells; Fig. 3A). CD14loCD16+monocytes also
produced lower concentrations of TNF-a upon exposure to M.
tuberculosis (2349 6 485 pg/ml versus 4761 6 661 pg/ml for
CD14himonocytes, p , 0.001; Fig. 3B).
CD16+cells is shown. B, Examples are provided of flow cytometry plots of the CD14hiand CD14loCD16+populations that we isolated. C, CD14hiand
CD14loCD16+MDMs from healthy donors were infected with M. tuberculosis H37Rv, and CFU per well was measured after 2 h and 7 d. Mean values and
SDs are shown.
Monocyte heterogeneity affects growth of M. tuberculosis H37Rv. A, A schematic of the procedure for isolation of CD14hiand CD14lo
1640 c-Maf HELPS TUBERCULOSIS
M. tuberculosis induces c-Maf mRNA expression by CD14hibut
Previous studies have shown that c-Maf, a transcription factor,
upregulates IL-10 production by binding to the IL-10 promoter
(14). Because we found that CD14himonocytes produce more IL-
10 in response to M. tuberculosis H37Rv and allow enhanced
bacillary growth compared with CD14loCD16+cells, we com-
pared c-Maf expression by CD14hiand CD14loCD16+monocytes
cultured with or without gamma-irradiated M. tuberculosis
H37Rv. After 48 h, c-Maf mRNA was quantified by real-time
PCR. In eight healthy donors, c-Maf mRNA was upregulated in
response to M. tuberculosis in CD14himonocytes and not in
CD14loCD16+monocytes. c-Maf levels were 4.5-fold higher in M.
tuberculosis-induced CD14himonocytes compared with those in
CD14loCD16+monocytes (3 6 0.8 versus 0.7 6 0.07 arbitrary
units, p = 0.02; Fig. 4).
c-Maf contributes to growth of M. tuberculosis in CD14hi
MDMs, in part through increased IL-10 production
The above findings indicate that M. tuberculosis increases c-Maf
expression in CD14himonocytes, and previous studies showed
that c-Maf upregulates IL-10 production (14). To determine if this
pathway operates in mononuclear phagocytes exposed to M. tu-
berculosis H37Rv, we used c-Maf siRNA to inhibit specifically c-
Maf–mediated gene expression. c-Maf mRNAwas reduced by 65–
75% in siRNA-treated cells, as measured by real-time PCR (data
not shown). In seven healthy donors, c-Maf siRNA reduced IL-10
levels in M. tuberculosis H37Rv-stimulated CD14hicells by 75%
compared with levels in cells treated with scrambled siRNA
(p = 0.02; Fig. 5A). These findings indicate that c-Maf contributes
significantly to M. tuberculosis-induced IL-10 production by
We next determined whether c-Maf siRNA inhibits growth of M.
tuberculosis H37Rv in CD14hiMDMs that were transfected with
c-Maf or scrambled siRNA and infected with M. tuberculosis as
outlined in Materials and Methods. In six healthy donors, c-Maf
siRNA reduced CFU per well in CD14hiMDMs by 75% compared
with that in scrambled siRNA-treated cells (p = 0.0002; Fig. 5B).
These findings demonstrate that c-Maf enhances growth of M.
tuberculosis in CD14himacrophages.
The above results indicate that c-Maf enhances growth of M.
tuberculosis H37Rv in CD14himonocytes and increases IL-10
production. To determine if c-Maf–dependent growth of M. tu-
berculosis is mediated through IL-10, we infected CD14hiand
CD14loCD16+MDMs with M. tuberculosis H37Rv in the presence
monocytes. Freshly isolated CD14hiand CD14loCD16+monocytes from
healthy donors were cultured with gamma-irradiated M. tuberculosis
H37Rv. After 24 h, RNA was isolated, reverse-transcribed to cDNA, and
quantified by real-time PCR. After normalization for GAPDH mRNA
content, c-Maf mRNA values for control monocytes were arbitrarily set as
1.0, and other values were adjusted accordingly. Boxes show the median
and interquartile range, and whiskers show the 5th and 95th percentile
c-Maf mRNA expression by CD14hiand CD14loCD16+
M. tuberculosis H37Rv. A, IL-10 production. Freshly isolated CD14hi
monocytes were mock-transfected, or transfected with c-Maf siRNA or
scrambled siRNA. After 24 h, cells were cultured with gamma-irradiated
M. tuberculosis (M. tb) H37Rv. Some cells were cultured in medium alone
and not transfected. IL-10 levels were measured by ELISA. Boxes show
the median and interquartile range, and whiskers show the 5th and 95th
percentile values. B, Growth of M. tuberculosis H37Rv. CD14hiMDMs
were untreated, mock-transfected, or transfected with siRNA for c-Maf
or scrambled siRNA. After 24 h, cells were infected with M. tuberculosis
(M. tb) H37Rv, as described in Materials and Methods. After 3 d, CFU per
well was measured. Mean values and SDs are shown.
Effect of c-Maf siRNA on IL-10 production and growth of
monocytes in response to M. tuberculosis H37Rv. A, IL-10 production. B,
TNF-a production. Freshly isolated CD14hiand CD14loCD16+monocytes
from healthy donors were cultured with 10 mg/ml gamma-irradiated M.
tuberculosis (M. tb) H37Rv, and supernatants were collected after 24 h. IL-
10 and TNF-a concentrations were measured by ELISA. Boxes show the
median and interquartile range, and whiskers show the 5th and 95th per-
IL-10 and TNF-a production by CD14hiand CD14loCD16+
The Journal of Immunology1641
or absence of rIL-10. CFU per well in CD14loCD16+cells in-
creased 2-5-fold in rIL-10–treated cells (p , 0.01; Fig. 6A). rIL-
10 did not significantly affect CFU in CD14hiMDMs (Fig. 6A).
IL-10 siRNA also reduced CFU in CD14hiMDMs by 54%
(p = 0.04; Fig. 6B). We conclude that c-Maf enhances growth of
M. tuberculosis in CD14hicells, in part through increased pro-
duction of IL-10. However, because growth of M. tuberculosis
was inhibited more markedly by c-Maf siRNA than IL-10 siRNA,
other c-Maf–regulated molecules are likely to contribute to in-
tracellular mycobacterial growth.
Microarray analysis to identify c-Maf–regulated genes in
M. tuberculosis H37Rv-stimulated CD14himonocytes
To identify c-Maf–regulated genes that could affect growth of M.
tuberculosis H37Rv, we used microarray analysis to compare
expression of 40,000 genes by CD14hiand CD14loCD16+mono-
cytes that were unstimulated or cultured with gamma-irradiated
M. tuberculosis H37Rv for 48 h. For each monocyte sub-
population, we computed a ratio for each gene of expression in
stimulated versus unstimulated cells. This strategy may not id-
entify genes for which a small variation in transcription leads to
large differences in protein production. Nevertheless, we identi-
fied 230 genes for which this ratio was at least 2-fold higher in
CD14hithan in CD14loCD16+cells. Of these genes, 22 have c-Maf
binding site in their promoter region. Among these genes, we
selected that encoding HAS1, which was shown to enhance ex-
tracellular growth of M. tuberculosis H37Rv in the lungs (15). We
also selected ZIP5 and TTC22, which are highly expressed in
CD14himonocytes after stimulation with M. tuberculosis H37Rv.
Only TTC22 has a binding site for c-Maf in its promoter region.
To confirm the microarray results for HAS1, ZIP5, and TTC22,
we used real-time PCR. Expression of all three genes was up-
regulated in CD14himonocytes that were stimulated with gamma-
irradiated M. tuberculosis H37Rv: 8- to 9-fold for HAS1 and
TTC22 and 2.5-fold for ZIP5 (Fig. 7). In contrast, gene expression
did not increase in CD14loCD16+monocytes after culture with
c-Maf controls expression of mRNA for HAS1 and TTC22, but
not ZIP5, by CD14hicells
To determine if M. tuberculosis H37Rv-induced expression of
HAS1, ZIP5, and TTC22 by CD14himonocytes is dependent on c-
Maf, we used c-Maf siRNA. In seven healthy donors, c-Maf
siRNA inhibited gamma-irradiated M. tuberculosis H37Rv-in-
duced HAS1 expression by CD14hicells by 75% compared with
that in control siRNA-treated cells (p = 0.02; Fig. 8A). Similarly,
in four healthy donors, c-Maf siRNA inhibited M. tuberculosis-
induced TTC22 expression by CD14hicells by 56% compared
with that in control siRNA-treated cells (p = 0.03; Fig. 8B). In
TTC22 (B), and ZIP5 (C) by CD14hiand CD14lo
CD16+monocytes. Freshly isolated CD14hiand
CD14loCD16+monocytes were cultured, with or
without gamma-irradiated M. tuberculosis (M. tb)
H37Rv. After 24 h, RNAwas isolated, and cDNAwas
quantified by real-time PCR. Values were normalized
to GAPDH mRNA content, and the values for un-
infected (control) monocytes were assigned a value
of 1.0. Boxes show the median and interquartile
range, and whiskers show the 5th and 95th percentile
mRNA expression for HAS1 (A),
CD14hiand CD14loCD16+MDMs. A, Effect of rIL-10. MDMs were
infected with M. tuberculosis H37Rv, as described in Materials and Me-
thods, and some infected macrophages were cultured with rIL-10. After
7 d, CFU per well was measured. Boxes show the median and interquartile
range, and whiskers show the 5th and 95th percentile values. B, Effect of
IL-10 siRNA. MDMs from CD14himonocytes were transfected with IL-10
or scrambled siRNA. After 24 h, cells were infected with H37Rv, and CFU
per well was measured after 3 d. Mean values and SDs are shown.
Effect of IL-10 on growth of M. tuberculosis H37Rv in
1642c-Maf HELPS TUBERCULOSIS
contrast, c-Maf siRNA had no effect on M. tuberculosis-induced
ZIP5 expression (p = 0.3; Fig. 8C).
HAS1 enhances growth of M. tuberculosis H37Rv in CD14hi
and TTC22, but not ZIP5, in M. tuberculosis H37Rv-stimulated
CD14himonocytes. We next used siRNA to ask whether HAS1 and
TTC22 mediate intracellular growth of M. tuberculosis. In six
healthy donors, HAS1 siRNA reduced growth of M. tuberculosis
H37Rv in CD14hiMDMs by 75% (p = 0.001; Fig. 9). In contrast,
TTC22 siRNA had no effect (Fig. 9), indicating that c-Maf–de-
pendent HAS1 expression enhances growth of M. tuberculosis in
Expression of HAS1 and hyaluronan are increased in
M. tuberculosis-infected CD14hiMDMs
To extend the findings regardingHAS1mRNA expression toHAS1
protein, we infected CD14hiand CD14loCD16+MDMs from five
healthy donors with GFP-expressing M. tuberculosis H37Rv and
performed confocal microscopy 24 h later, after staining with anti-
HAS1. M. tuberculosis induced HAS1 expression by CD14hibut
not CD14loCD16+MDMs (Fig. 10A). HAS1 catalyzes production
of hyaluronan, expression of which was also increased in M. tu-
berculosis-infected but not uninfected CD14hiMDMs (Fig. 10B).
Monocyte subpopulations differ in their capacity to differentiate
into macrophages and dendritic cells, produce cytokines and
chemokines, and present Ag to T cells (2–5). However, limited
information is available about differences in their capacity to in-
teract with intracellular pathogens. In the current study, we found
that M. tuberculosis, a bacterium that kills an estimated 1.3 mil-
lion people annually (1), grew 5-fold more rapidly in CD14hithan
in CD14loCD16+human MDMs. Enhanced growth of M. tuber-
culosis H37Rv was mediated through increased production of IL-
10 and HAS1, the latter leading to increased production of hya-
luronan. Gene expression of IL-10 and HAS1 are upregulated by
the transcription factor c-Maf. Our study provides the first evi-
dence, to our knowledge, that a bacterial pathogen upregulates
expression of c-Maf to enhance intracellular growth.
Monocytes were first separated into CD14hiCD162and CD14lo
CD16+subpopulations in 1989 (16), and these subpopulations
differ in their receptor expression and cytokine production (4, 9–
12, 17). CD14loCD16+monocytes are more permissive for infection
with several viruses, including HIV, hepatitis C, and Merkel cell
polyomavirus (18–20), and CD14hiCD162cells more effectively
inhibit conidial germination during infection with Aspergillus fumi-
gatus (21). The current report demonstrates differential susceptibility
transfected, or transfected with c-Maf siRNA or scrambled siRNA. After 24 h, gamma-irradiated M. tuberculosis (M. tb) H37Rv was added to mock-
transfected and transfected cells. After 24 h, RNAwas isolated, and cDNAwas quantified by real-time PCR to measure HAS1 (A), TTC22 (B), and ZIP5 (C)
expression. Values were normalized to GAPDH mRNA content, and value for uninfected (control) monocytes were assigned a value of 1.0. Boxes show the
median and interquartile range, and whiskers show the 5th and 95th percentile values.
Regulation of expression of mRNA for HAS1, TTC22, and ZIP5 by c-Maf. Freshly isolated CD14himonocytes were untreated, mock-
berculosis H37Rv in CD14hiMDMs. CD14hiMDMs were untreated or
transfected with HAS1 or TTC22 siRNA or scrambled siRNA. After 24 h,
cells were infected with M. tuberculosis H37Rv, as described in Materials
and Methods. After 3 d, CFU per well was measured. Mean values and
SDs are shown.
Effect of HAS1 and TTC22 siRNA on growth of M. tu-
The Journal of Immunology1643
of CD14hiand CD14loCD16+cells to an intracellular bacterial
We found that M. tuberculosis H37Rv grows more rapidly in
CD14hithan in CD14loCD16+MDMs and that CD14hicells pro-
duce IL-10 in response to M. tuberculosis H37Rv, whereas CD14lo
CD16+monocytes do not, confirming and extending previous
reports indicating that only CD14hiCD162monocytes produce
IL-10 in response to stimulation with LPS (8). Intracellular growth
of M. tuberculosis H37Rv in CD14hicells was reduced by IL-10
siRNA, and c-Maf siRNA inhibited growth of M. tuberculosis
H37Rv and IL-10 production, suggesting that c-Maf–induced IL-
10 contributed to bacillary replication. These results are consistent
with previous findings that the intracellular growth rates of M.
tuberculosis H37Rv clinical strains correlate strongly with their
capacity to induce IL-10 production rapidly (22) and that IL-10
produced by M. tuberculosis-infected human macrophages blocks
phagosome maturation (23). The effect of IL-10 on multiplication
of M. tuberculosis H37Rv is likely to be relevant in vivo, as
transgenic mice that overproduce IL-10 in macrophages have in-
creased bacillary burdens and increased mortality, despite normal
recruitment and activation of T cells (24).
We found that stimulation with M. tuberculosis H37Rv in-
creased expression of c-Maf in CD14hibut not CD14loCD16+
monocytes. c-Maf is a transcription factor that is associated with
differentiation of monocytes to macrophages (25). Because
CD14himonocytes are less differentiated than CD14loCD162cells
(26), the former are likely to have lower baseline c-Maf levels,
which may be more susceptible to increases during M. tubercu-
losis infection. In the T cell response to infection, c-Maf is a
master regulator of Th2 responses, inducing production of IL-10,
IL-4, and IL-21 by binding to a c-Maf response element in the
promoter regions of their encoding genes (14, 27–31). c-Maf ex-
pression is reduced during HIV infection of CD4+cells, de-
creasing IL-21 expression (32), and bacterial adenylate cyclase
toxins increase c-Maf expression and expansion of Th2 cells (33).
Our findings demonstrate that pathogens can also modulate c-Maf
expression in mononuclear phagocyte subpopulations to enhance
bacterial growth. Additional studies are needed to understand the
differences in signaling pathways between CD14hiand CD14lo
CD16+cells that are exploited by M. tuberculosis H37Rv to in-
crease c-Maf expression.
We found that the effects of c-Maf on intracellular growth of M.
tuberculosis H37Rv were greater than those of IL-10. HAS1 has
a c-Maf binding site in its promoter region, c-Maf siRNA inhibits
HAS1 expression, and HAS1 siRNA reduces growth of M. tu-
berculosis H37Rv in CD14himonocytes, suggesting that c-Maf
regulates intracellular growth of M. tuberculosis in part through
HAS1. The latter is an enzyme that controls production of hya-
luronan, an abundant component of the extracellular matrix that
can be broken down by mycobacterial hyaluronidases and used as
a carbon source to enhance extracellular replication of M. tuber-
culosis (15). Hyaluronan is also present in the cytoplasm (34), and
we found that HAS1 expression was markedly upregulated in
M. tuberculosis H37Rv-infected CD14hicells but not in CD14lo
CD16+monocytes. Similarly, hyaluronan expression was greatly
enhanced after infection of CD14himonocytes. It is intriguing to
speculate that c-Maf increases expression of HAS1, which in turn
stimulates production of intracellular hyaluronan, some of which
may be secreted into the phagosome and provide a carbon sub-
strate for M. tuberculosis, thus enhancing bacillary replication.
Alternatively, hyaluronan fragments can induce formation of im-
munosuppressive macrophages (35) that may allow more rapid
replication of M. tuberculosis. Hyaluronan and HAS1 are both
present in the lungs of M. tuberculosis-infected mice and rhesus
monkeys (15), suggesting that they contribute to the pathogenesis
of tuberculosis in vivo.
and hyaluronan (B) by CD14hiand CD14loCD16+
MDMs. A, CD14hiand CD14loCD16+MDMs from
five healthy donors were cultured in medium alone
or infected with GFP-expressing M. tuberculosis
H37Rv, as outlined in Materials and Methods.
Cells were then fixed in 2% paraformaldehyde,
washed thoroughly, and incubated overnight with
goat polyclonal anti-HAS1 (red) or isotype control
Abs. After overnight incubation, cells were ana-
lyzed by confocal microscopy. Original magnifi-
cation 3157.5. B, CD14hicells from five healthy
donors were treated as in A, except that they were
stained with hyaluronan binding protein (red) to
identify hyaluronan instead of anti-HAS1. A rep-
resentative result is shown for both panels. Original
Expression of HAS1 protein (A)
1644 c-Maf HELPS TUBERCULOSIS
In summary, we found that CD14hiand CD14loCD16+MDMs
differ in their capacity to restrict growth of M. tuberculosis and
that increased intracellular replication is mediated through c-Maf,
which upregulates production of IL-10 and HAS1. Our studies
provide insight into the cellular mechanisms that control growth of
M. tuberculosis in human MDMs.
The authors have no financial conflicts of interest.
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