Suppression of the NF-κB Pathway by Diesel
Exhaust Particles Impairs Human
Antimycobacterial Immunity
This information is current as
of June 16, 2017.
Srijata Sarkar, Youngmia Song, Somak Sarkar, Howard M.
Kipen, Robert J. Laumbach, Junfeng Zhang, Pamela A.
Ohman Strickland, Carol R. Gardner and Stephan
Schwander
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Copyright © 2012 by The American Association of
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Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2012; 188:2778-2793; Prepublished online 15
February 2012;
doi: 10.4049/jimmunol.1101380
http://www.jimmunol.org/content/188/6/2778
The Journal of Immunology
Suppression of the NF-kB Pathway by Diesel Exhaust
Particles Impairs Human Antimycobacterial Immunity
Srijata Sarkar,* Youngmia Song,* Somak Sarkar,* Howard M. Kipen,†
Robert J. Laumbach,† Junfeng Zhang,*,‡,x Pamela A. Ohman Strickland,{
Carol R. Gardner,‖ and Stephan Schwander*,†,‡
A
ir pollution and tuberculosis (TB) each contribute significantly to global burden of disease. The World Health
Organization estimates that air pollution, which is linked
to a variety of illnesses including cardiopulmonary diseases and
*Department of Environmental and Occupational Health, University of Medicine
and Dentistry of New Jersey-School of Public Health, Piscataway, NJ 08854;
†
Environmental and Occupational Health Sciences Institute, University of Medicine
and Dentistry of New Jersey-Robert Wood Johnson Medical School, Piscataway, NJ
08854; ‡Center for Global Public Health, University of Medicine and Dentistry of
New Jersey-School of Public Health, Piscataway, NJ 08854; xKeck School of Medicine, University of Southern California, Los Angeles, CA 90033; {Department of
Biostatistics, University of Medicine and Dentistry of New Jersey-School of Public
Health, Piscataway, NJ 08854; and ‖Department of Pharmacology and Toxicology,
Rutgers University, Piscataway, NJ 08854
Received for publication May 13, 2011. Accepted for publication January 8, 2012.
This work was supported by National Institute of Environmental Health Sciences
Grants 5R21ES16928-2 (to S. Schwander), U19ES019536 (to J.Z.), and K08
ES013520 (to R.J.L.) and National Institute of Environmental Health Sciences-sponsored University of Medicine and Dentistry of New Jersey Center for Environmental
Exposures and Disease Grant P30ES005022.
Address correspondence and reprint requests to Prof. Stephan Schwander, Department of Environmental and Occupational Health and Center for Global Public
Health, University of Medicine and Dentistry of New Jersey-School of Public Health,
683 Hoes Lane West, Room 305, Piscataway, NJ 08854. E-mail address: schwansk@
umdnj.edu
The online version of this article contains supplemental material.
Abbreviations used in this article: BCG, bacillus Calmette-Guérin; CB, carbon black;
DEP, diesel exhaust particle; IRAK, IL-1R–associated kinase; IRF, IFN regulatory
factor; LAL, Limulus amebocyte lysate; MOI, multiplicity of infection; PAH, polycyclic aromatic hydrocarbon; PI, propidium iodide; PM2.5, particulate matter ,2.5
mm in aerodynamic diameter; PPD, purified protein derivative; qRT-PCR, quantitative RT-PCR; TB, tuberculosis; TEM, transmission electron microscopy; UMDNJ,
University of Medicine and Dentistry of New Jersey.
Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1101380
cancer, causes ∼2 million premature deaths worldwide per year
(1). According to the World Health Organization’s 2009 Global
Health Risk report (2), indoor air pollution (from biomass fuel and
coal combustion) and urban outdoor air pollution rank 10th and
14th, respectively, among 19 leading risk factors for mortality in
low- and middle-income countries. Exposure to ambient-air fine
particulate matter ,2.5 mm in aerodynamic diameter (PM2.5) is
estimated to cause ∼1% of mortality from acute respiratory infections in children .5 y worldwide (3).
TB was estimated to afflict ∼10 million people and cause 2
million deaths in 2010 alone (4). Epidemiologic associations have
been established between the incidence of TB and, respectively,
cigarette smoking (5–11), occupational exposure to aerosolized
silica (12–14), and indoor air pollution (10, 11, 15). Despite these
associations, to the best of our knowledge, no studies have been
conducted to assess the mechanisms that underlie associations
between air pollution and TB development in primary human cells.
Human exposure to diesel exhaust particles (DEP), a major
component of urban PM2.5 in most industrialized urban areas (16),
typically occurs by inhalation. Due to their small size, DEP remain airborne for prolonged time periods and deposit in the lungs
upon inhalation. Likewise, infection with Mycobacterium tuberculosis typically occurs by inhalation of aerosolized M. tuberculosis-containing droplet nuclei (1–5 mm) that are released from
patients with active TB during respiratory maneuvers (17–20).
Thus, concurrent respiratory exposure to DEP and M. tuberculosis
can be expected to occur under real-life conditions and may
subsequently alter host immune responses.
Protective antimycobacterial human host immunity involves
innate and adaptive immune mechanisms that are primarily cell-
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
Epidemiological studies suggest that chronic exposure to air pollution increases susceptibility to respiratory infections, including
tuberculosis in humans. A possible link between particulate air pollutant exposure and antimycobacterial immunity has not been
explored in human primary immune cells. We hypothesized that exposure to diesel exhaust particles (DEP), a major component of
urban fine particulate matter, suppresses antimycobacterial human immune effector cell functions by modulating TLR-signaling
pathways and NF-kB activation. We show that DEP and H37Ra, an avirulent laboratory strain of Mycobacterium tuberculosis,
were both taken up by the same peripheral human blood monocytes. To examine the effects of DEP on M. tuberculosis-induced
production of cytokines, PBMC were stimulated with DEP and M. tuberculosis or purified protein derivative. The production of
M. tuberculosis and purified protein derivative-induced IFN-g, TNF-a, IL-1b, and IL-6 was reduced in a DEP dose-dependent
manner. In contrast, the production of anti-inflammatory IL-10 remained unchanged. Furthermore, DEP stimulation prior to M.
tuberculosis infection altered the expression of TLR3, -4, -7, and -10 mRNAs and of a subset of M. tuberculosis-induced host genes
including inhibition of expression of many NF-kB (e.g., CSF3, IFNG, IFNA, IFNB, IL1A, IL6, and NFKBIA) and IFN regulatory
factor (e.g., IFNG, IFNA1, IFNB1, and CXCL10) pathway target genes. We propose that DEP downregulate M. tuberculosisinduced host gene expression via MyD88-dependent (IL6, IL1A, and PTGS2) as well as MyD88-independent (IFNA, IFNB)
pathways. Prestimulation of PBMC with DEP suppressed the expression of proinflammatory mediators upon M. tuberculosis
infection, inducing a hyporesponsive cellular state. Therefore, DEP alters crucial components of antimycobacterial host immune
responses, providing a possible mechanism by which air pollutants alter antimicrobial immunity. The Journal of Immunology,
2012, 188: 2778–2793.
The Journal of Immunology
Materials and Methods
Approval to perform this study, collect personal health information, and
perform venipunctures was given by the Institutional Review Boards of the
University of Medicine and Dentistry of New Jersey (UMDNJ) in Newark and New Brunswick (Institutional Review Board protocol number
0120060235).
Human subjects
A total of 20 healthy individuals (13 male, 7 female, mean age 33.5 y [range
20–52 y]) were recruited from students and staff of the UMDNJ-School of
Public Health, Rutgers University, the Environmental and Occupational
Health Sciences Institute, and volunteers at the Chandler Clinic in New
Brunswick, NJ. Inclusion and exclusion criteria were as follows: inclusion
criteria: healthy women and men, 18–65 y of age; exclusion criteria:
concurrent infections, use of immunosuppressive medications (steroidal
and/or nonsteroidal anti-inflammatory medications, TNF-a inhibitors, antineoplastic chemotherapy), illnesses affecting host immunity (diabetes,
HIV-1 infection, chronic liver or kidney diseases, and malignancies),
smoking, and illicit drug use. A total of 80 ml heparinized venous whole
blood was obtained from each individual from a cubital vein by venipuncture. Not all subjects were examined in all experiments. Subject
numbers for each experiment are shown in the figures or figure legends. All
study subjects provided written informed consent before personal health
information was acquired and whole heparinized peripheral blood obtained
by venipuncture.
Reagents
Reagents were obtained from the following sources: ELISPOT assays:
capture and biotinylated detection Abs for IL-1b and IL-6 (Cell Sciences,
Canton, MA), IFN-g, TNF-a (Endogen Pierce, Rockford, IL), IL-10, IL-4
(BD Pharmingen, San Diego, CA), streptavidin-peroxidase (Sigma-Aldrich,
St. Louis, MO), chromogen 1% 3-amino-9-ethylcarbazole (Pierce, Rockford, IL), and MultiscreenHTS high protein binding 96-well plates (Millipore, Bedford, MA); PBMC isolation, culture, and stimulation: PHA
(Sigma-Aldrich), LPS (Escherichia coli 026:B6; Sigma-Aldrich), PPD
(Statens Serum Institute, Kopenhagen, Denmark), RPMI 1640 (BioWhittaker, Walkersville, MD), L-glutamine (Cellgro, Manassas, VA), pooled
human AB serum (Gemini Bioproducts, Woodland, CA), and Ficoll-Paque
(GE Healthcare Biosciences, Pittsburgh, PA); RNA extraction and quantitative RT-PCR (qRT-PCR): RNeasy mini kit (Qiagen, Germantown, MD),
RNase-free DNase set (Qiagen), RT2 First Strand kit and RT2 qPCR Master
Mix (SuperArray Bioscience, Frederick, MD), Power SYBR Green PCR
Master Mix (Applied Biosystems, Foster City, CA), TaqMan reverse transcription reagents (Applied Biosystems); pathway-specific gene expression
arrays: human Th1-Th2-Th3 (PAHS-34E) and TLR (PAHS-18E; SuperArray Bioscience); M. tuberculosis culture: H37Ra (ATCC no. 25177;
American Type Culture Collection, Manassas, VA), Middlebrook 7H9 broth
medium (Difco Laboratories, Detroit, MI) supplemented with 10% albumin
dextrose catalase (Difco Laboratories), 7H10 solid agar plates (BD Biosciences, San Jose, CA); flow cytometry: Annexin V/propidium iodide (PI)
apoptosis detection assay (BD Biosciences), PE-conjugated CD14 (BioLegend, San Diego, CA), and allophycocyanin-conjugated CD3 (eBioscience, San Diego, CA) mAbs; and DEP and carbon black (CB): DEP was
a gift from Dr. Sagai (Tokyo, Japan), generated by a diesel-powered automobile, collected in a condensation trap, and stored at 280˚C (65), and CB
(Printex 90, primary particle diameter 16 nm) was from Degussa (Frankfurt,
Germany).
Preparation of DEP and CB suspensions
DEP and CB stock suspensions (10 mg/ml) were prepared by 15-min
sonication in PBS containing 0.05% Tween, aliquoted, and kept frozen at
220˚C until use. Prior to addition to cell cultures, frozen aliquots of DEP
or CB were thawed and vortexed for 5 min (a period determined to provide
reliable dispersion of DEP in preliminary studies). DEP were then diluted
in complete cell-culture medium to generate desired final concentrations
(0.1, 1, 10, 50, and 100 mg/ml).
Determination of endotoxin content and sterility of DEP
Presence of any endotoxin in complete culture fluid with and without DEP
(100 mg/ml) was assessed using a commercial endotoxin detection service
(Lonza Walkersville, Walkersville, MD) that employed the quantitative
kinetic chromogenic Limulus amebocyte lysate (LAL) method (KQCL).
The assay met all validation requirements per U.S. Food and Drug
Administration-established guidelines for LAL testing (“Guideline on
Validation of the Limulus Amebocyte Lysate Test as an End-Product Endotoxin Test for Human and Animal Parenteral Drugs, Biological Products, and Medical Devices”). The assays were conducted in adherence
to USP ,85. Bacterial Endotoxin Test and Lonza’s Quality System
requirements. Testing was performed according to standard operating procedure 162.6.
To rule out the possibility of bacterial or fungal contamination of DEP,
complete culture media (RPMI 1640 + 10% pooled human serum + L-Gln)
and complete culture media containing DEP (100 mg/ml) were examined
on blood brain heart infusion and inhibitory mode agar plates as well as on
sheep blood and chocolate agar plates (all from Voigt Global Distribution,
Lawrence, KS) at the Department of Microbiology at Robert Wood
Johnson University Hospital in New Brunswick, NJ.
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mediated, involving monocytes/macrophages, dendritic cells, CD4+
and CD8+ T cells, NK, and gdT cells. M. tuberculosis Ag-specific
immunity is characterized predominantly by a Th1 response (IL-2,
IFN-g, and TNF-a) (21) that is strongly enhanced and compartmentalized at the infection sites, most commonly the lungs (22–
24). IFN-g, secreted from activated T cells and NK cells, is known
to activate macrophages and promote bacterial killing (25) by
permitting phagosomal maturation and production of antimicrobial
inducible NO synthase and reactive oxygen intermediates (26).
IFN-g has an essential role in the control of mycobacterial infections both in the murine model (27, 28) and in humans (29–33).
Likewise, TNF-a plays an important role in killing of intracellular
M. tuberculosis (34, 35) and granuloma formation. As evidenced
by the increased risk of reactivation TB during TNF-a inhibitor
treatment (36, 37), TNF-a is required in the maintenance of latent
M. tuberculosis infection. IL-6 and IL-10 are important regulatory
cytokines during the early phase of the mycobacterial infection of
macrophages and in the inflammatory responses to M. tuberculosis
and the regulation of IFN-g, respectively (22).
Innate host resistance against M. tuberculosis depends to a large
extent on the engagement of TLRs (38–43) and nucleotide oligomerization domain-like receptors (44). TLR2, TLR4, and TLR9
are activated by M. tuberculosis-derived ligands such as lipoarabinomannan and mycobacterial 19-kDa protein (39, 40, 45),
M. tuberculosis heat shock proteins 65 and 71 (38), and mycobacterial DNA (42), respectively. Stimulation of these TLRs (38,
41–43) on monocytes, alveolar macrophages, or dendritic cells
(46–49) activates signal transduction pathways that culminate in
the activation of MAPK, transcription factor NF-kB, and the IFN
regulatory factor (IRF) family (47), leading to the release of antimicrobial effector molecules (50), proinflammatory cytokines
(51–57), and chemokines (58).
DEP exposure has been shown to shift Th1 to Th2 cytokine
production by T, monocyte-derived dendritic, and spleen cells (59,
60), decrease secretion of IFN-g, and increase secretion of IL-10
in murine bone marrow-derived dendritic cells (61). A study of the
direct effects of DEP on M. tuberculosis immune responses
showed that DEP increases the pulmonary M. tuberculosis burden
concomitant with decreased production of proinflammatory
cytokines (e.g., IL-1b, IL-12p40, and IFN-g) in experimentally
infected mice (62). DEP exposure also decreases the phagocytosis
and bactericidal activity of rat alveolar macrophages exposed to
Listeria monocytogenes infection (63, 64).
The current study was undertaken to examine the effects of DEP
on human host immune responses to M. tuberculosis. We show that
DEP significantly alter live M. tuberculosis and purified protein
derivative (PPD)-induced cytokine production in primary human
PBMC. Furthermore, DEP modulate M. tuberculosis-induced host
gene expression by inhibiting TLR-mediated signaling including
NF-kB and IRF pathways crucial for M. tuberculosis-induced host
immunity. Suppression of M. tuberculosis-induced activation of
these two pathways by DEP provides insights into the possible
mechanisms by which particulate air pollution may modify
pathogen-induced human host immune responses.
2779
2780
Preparation of PBMC
PBMC were isolated from whole heparinized venous blood by Ficoll
gradient centrifugation (66). Briefly, whole blood was diluted 1:1 with Lglutamine supplemented RPMI 1640 medium and subjected to gradient
density centrifugation (1200 rpm, 45 min, 21˚C) over Ficoll-Paque. Following removal from the interface, PBMC were washed three times in
RPMI 1640, resuspended in complete culture medium, counted, and adjusted at required concentrations. Viability of PBMC was 98–100% by
trypan blue exclusion in all experiments.
Preparation of M. tuberculosis for in vitro infection
Preparation of enriched CD14+CD32 blood monocytes for
microscopy
Enriched CD14+CD32 peripheral blood monocytes were generated by
negative selection through immunomagnetic depletion of nonmonocytes
using a mixture of magnetic bead-coupled biotin-conjugated mAbs against
CD3, CD7, CD16, CD19, CD56, CD123, and Glycophorin A (Miltenyi
Biotec, Auburn, CA) from whole heparinized blood of an adult male
healthy donor. The purity of the enriched monocytes was assessed by flow
cytometry using a Cytomics FC500 Cytometer (Beckman Coulter, Miami,
FL) and CXP software (Beckman Coulter). Monocytes were incubated on
ice for 30 min with CD14-PE and CD3- allophycocyanin mAbs, washed
with PBS, and acquired immediately without fixation for analysis. Cells
were then gated in a forward scatter and side scatter dot plot to exclude
debris, an FL2 and FL4 dot plot was drawn from the monocyte gate, and
20,000 cells were acquired for analysis. The CD14+CD32 monocyte
population had a purity of 83.5%, which matched the manufacturerpredicted range (Miltenyi Biotec). CD14+CD32 monocytes were subsequently stimulated with DEP (10 mg/ml) and M. tuberculosis (MOI 10)
in vitro and then incubated at 37˚C in 5% CO2 for 24 h in 15-ml roundbottom polypropylene tubes. This cell-culture incubation period reflected
the duration of the IFN-g, IL-6, and IL-1b ELISPOT assays and that of the
gene expression studies and provided ample time for DEP and M. tuberculosis uptake. CD14+CD32 monocytes were used for cytospin preparations and transmission electron microscopy (TEM) studies.
series of acetone and embedded in EMbed 812 resin (Electron Microscopy
Sciences, Hatfield, PA), and 90-nm thin sections were cut on a Leica EM
UC6 ultramicrotome (Leica Microsystems). Sectioned grids were stained
with a saturated solution of uranyl acetate and lead citrate. Images were
captured with an Advanced Microscopy Techniques XR111 digital camera
(Advanced Microscopy Techniques) at 80 Kv.
Cell viability and apoptosis assays
DEP effects on apoptosis and necrosis in PBMC were assessed by flow
cytometry (FACSCalibur; BD Biosciences, San Jose, CA) using an Annexin
V/PI apoptosis detection kit as described by the manufacturer (BD Biosciences). Proportions of mononuclear cells that were simultaneously undergoing apoptosis (Annexin V positive) and necrosis (PI positive) were
determined. DEP were added to PBMC at final concentrations of 0.1, 1, 10,
and 100 mg/ml for 2, 24, and 72 h.
ELISPOT assays
ELISPOT assays are highly sensitive tools for quantitation of frequencies of
cytokine-producing cells and the semiquantitative assessment of their cytokine output (spot size) (67). In the current study, ELISPOT assays were
performed to enumerate frequencies of IL-1b–, IL-4–, IL-6–, IL-10–, IFNg–, and TNF-a–producing cells. PBMC were stimulated simultaneously
with DEP (0, 1, 10, 50, and 100 mg/ml) and M. tuberculosis at MOI 1 or
MOI 10, or PPD (10 mg/ml), or LPS (100 ng/ml), or complete culture
medium (control) in duplicate wells and incubated at 37˚C in 5% CO2.
PBMC and stimuli were cultured in a total volume of 200 ml/well in high
protein-binding 96-well plates coated with appropriate anti-human primary
capture Abs. Optimal densities of PBMC per well for the detection of the
various cytokines were as follows: 20,000 for IL-6 and TNF-a, 100,000 for
IL-1b, and 200,000 for IL-4, IL-10, and IFN-g. Optimal short-term cell
culture incubation periods for the ELISPOT assays were 4 h for TNF-a, 20 h
for IL-1b, 24 h for IL-6 and IFN-g, and 48 h for IL-4 and IL-10. These
incubation periods were chosen on the basis of published studies (22, 23,
67, 68) and the manufacturer’s recommendations. Plates were then washed
three times with PBS and PBS-Tween 0.05% followed by the addition of
appropriate detection Abs. Plates were incubated for 1 h at 37˚C for IL-1b
and overnight at 4˚C for all other cytokines. Following washing, cytokine
spots were visualized with peroxidase-conjugated streptavidin, HRP, and
chromogen 1% 3-amino-9-ethylcarbazole. After washing, plates were
dried and scanned, images acquired, and frequencies of cytokine-produc-
Cytospin preparations and microscopy
Uptake of DEP and M. tuberculosis by CD14+CD32 blood monocytes was
assessed by histochemistry on thin-layer cytospin preparations of 150,000
cells per glass slide (cytoslides; Thermo Shandon, Pittsburgh, PA) using
a cytocentrifuge (Shandon CytoSpin 4; Thermo Fisher Scientific, Waltham,
MA). Cytospins were stained with Kinyoun stain (Alpha-Tec Systems,
Vancouver, WA) and methyl blue counterstain (Thermo Shandon) following the manufacturer’s protocol to visualize M. tuberculosis and DEP
particles. Photomicrographs were taken at original magnification 31000
(oil immersion) using a BX51 microscope equipped with UPlanSApo
lenses and a DP71 camera with DP image manager software (both from
Olympus, Tokyo, Japan).
Transmission electron microscopy
DEP and M. tuberculosis uptake within CD14+CD32 monocytes was
further corroborated by TEM using a Philips CM12 transmission electron
microscope (Philips). Cells were fixed in 2.5% glutaraldehyde/4% paraformaldehyde in 0.1 M cacodylate buffer and then postfixed in buffered 1%
osmium tetroxide. Samples were subsequently dehydrated in a graded
FIGURE 1. DEP and M. tuberculosis uptake in human blood monocytes.
Magnetic bead-enriched CD14+CD32peripheral blood monocytes were
stained with Kinyoun acid-fast stain on cytospins following overnight
incubation with DEP (10 mg/ml) and M. tuberculosis (MOI 10). Five
randomly selected monocytes that have incorporated both DEP and
M. tuberculosis are shown. Arrows indicate the internalized DEP and
Kinyoun-stained M. tuberculosis visible as black spots and red rods, respectively, at original magnification 31000. Scale bars, 10 mM.
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Suspensions of M. tuberculosis (avirulent M. tuberculosis strain H37Ra)
were prepared in Middlebrook 7H9 broth medium supplemented with 10%
albumin dextrose catalase and 0.2% glycerol. After a 21-d incubation
period at 37˚C on an orbital shaker, M. tuberculosis stock suspensions were
harvested, aliquoted, and stored at 286˚C until use. The concentrations of
the M. tuberculosis stock suspensions were confirmed by assessing CFU
numbers from serial culture dilutions after 21 d of incubation on 7H10
solid agar plates. For PBMC infection experiments, single-cell suspensions
of M. tuberculosis were prepared as follows: frozen M. tuberculosis stock
was thawed, centrifuged for 5 min at 6000 3 g, and resuspended in complete culture medium. To generate single-bacterial-cell suspensions, M.
tuberculosis stock suspensions were declumped by vortexing for 5 min in
the presence of five sterile 3-mm glass beads. Remaining M. tuberculosis
clumps were removed with additional centrifugation at 350 3 g for 5 min.
Appropriate volumes of the supernatant M. tuberculosis suspensions were
used for in vitro infections to obtain multiplicities of infection (MOIs) of 1
and 10. MOIs represent the ratio of M. tuberculosis to monocytes (bacteria/
monocytes), and monocytes were assumed to constitute 10% of PBMC.
Concentrations of frozen M. tuberculosis stock suspensions were confirmed after thawing of aliquots in each experiment by CFU assays.
DEP SUPPRESS HUMAN ANTIMYCOBACTERIAL IMMUNITY
The Journal of Immunology
2781
FIGURE 2. TEM of DEP and M. tuberculosis uptake in human peripheral blood monocytes. The micrographs [(A and B) overviews
of two independent cells; (C) details of the area
surrounded by rectangle in (A); (D) intracellular compartment containing both DEP and
three M. tuberculosis] show uptake of DEP
(asterisks) and M. tuberculosis (arrows) within
the same transects of monocytes, providing
evidence of presence of DEP and M. tuberculosis within the same cell. Enriched peripheral
blood CD14+CD32 monocytes were cultured
with DEP (10 mg/ml) and M. tuberculosis
(MOI 10) for 24 h and processed as described
in Materials and Methods. Scale bars and direct magnifications are included in the figure.
hexamer, 0.4 U/ml RNAse inhibitor, 1.25 U/ml Multiscribe Reverse
Transcriptase, 0.4 M forward primer, and 0.4 M reverse primer). Thermal
cycling parameters of the reverse transcription reaction were 25˚C for 10
min, 48˚C for 10 min, and 95˚C for 5 min. Real-time PCR was performed
with Power SYBR Green PCR master mix in an ABI 7900HT (Applied
Biosystems). A two-step cycling program followed by dissociation curve
as described in the section above was used.
Preparation of RNA from PBMC and generation of cDNA
Statistical analysis
PBMC cultures were incubated at 37˚C in 5% CO2 and stimulated with
DEP, M. tuberculosis, M. tuberculosis plus DEP simultaneously, M. tuberculosis following a 20-h DEP prestimulation, and M. tuberculosis following a 20-h LPS prestimulation or left untreated (control) in six-well
plates (Falcon; BD Biosciences) at concentrations of 1.5 3 106 cells/ml for
24 h. Total RNA was extracted from unstimulated (in complete culture
medium only) or stimulated PBMC using the RNeasy mini kit (Qiagen),
and DNA was removed from RNA samples by RNase-free DNase treatment (Qiagen). Concentration and purity of RNA were determined using
a Nanodrop spectrophotometer (Nanodrop Technologies, Wilmington,
DE). Total RNA (400 ng) was reverse transcribed into cDNA with RT2
First Strand kit (SuperArray Bioscience) according to the manufacturer’s
protocol.
We performed statistical analyses to test the following two hypotheses: the
first was whether there was an effect for DEP within each stimulant; the
second was whether each DEP dose affected M. tuberculosis MOI 1, MOI
10, and PPD stimulation effects differentially. Histograms of the values
were examined for normality and other model assumptions. Linear mixedeffects models were employed with cytokine counts measured as a response and DEP dose as the predictor. Because all DEP doses were applied
to each PBMC sample, a random effect for subject was included to account
for the repeated measures. Also, based on observed heterogeneity, the
variance of the cytokine counts was allowed to vary by DEP dose. The
analyses were then stratified by each stimulant (M. tuberculosis MOI 1, M.
tuberculosis MOI 10, PPD, and LPS). All the statistical analyses were
conducted using the SAS 9.1 for Windows (SAS Institute).
Pathway-specific qRT-PCR arrays
Human Th1-Th2-Th3 and TLR pathway-specific qRT-PCR arrays were
used to assess mRNA expression in stimulated and unstimulated PBMC
samples. First-strand cDNA diluted in water (102 ml) was mixed with 23
RT2 qPCR master mix (550 ml; SuperArray Bioscience), and the total
reaction volume was adjusted to 1100 ml with RNAse-free water. Sample
cocktails (10 ml/well) were loaded onto 384-well plates. A two-step cycling program consisting of 1 cycle of 95˚C for 10 min, 40 cycles of 95˚C
for 15 s, and 60˚C for 1 min was performed with an ABI 7900HT (Applied
Biosystems, Foster City, CA) instrument. A dissociation curve specific to
this instrument was used to assure primer quality. Levels of cDNA were
calculated by the relative quantitation method (DDCt method) from the
PCR array data using the analysis software accessed at http://sabiosciences.
com/pcrarraydataanalysis.php. Statistical differences in fold mRNA expression levels between stimulated and unstimulated PBMC were calculated using the same software.
Real-time PCR
Gene expression data obtained by qRT-PCR arrays were validated by realtime PCR with gene-specific forward and reverse primer pairs. Forward and
reverse primers were designed using Oligoperfect software (Invitrogen),
and cDNA was generated from RNA from PBMC using TaqMan reverse
transcription reagents (Applied Biosystems) according to the manufacturer’s protocols. Briefly, 400 ng total RNA was used for the generation
of cDNA in a 25-ml reaction (13 reverse transcription buffer, 5.5 mM
MgCl2, 500 mM 29-deoxynucleoside 59-triphosphate, 2.5 mM random
Results
Characterization of DEP
DEP used in this study consist of a carbonaceous core to which
organic chemicals such as polycyclic aromatic hydrocarbons (PAH),
nitro derivatives of PAH, oxygenated derivatives of PAH (ketones,
quinines, and diones), heterocyclic compounds, aldehydes, and
aliphatic hydrocarbons are adsorbed (65, 69–71). To determine any
potential endotoxin contamination of the DEP used in this study,
Table I.
Induction of apoptosis and necrosis by DEP in PBMC
Annexin V FITC/Propidium Iodide
Positive (Mean %)
DEP Concentration
(mg/ml)
2h
24 h
72 h
0
0.1
1
10
100
3.2
4.3
4.9
5.4
8.5
6.3
7.2
6.1
6.8
31.4
23.6
24.0
22.4
24.9
40.6
Mean proportions of PBMC from healthy subjects (n = 5) showing positivity for
Annexin V and PI by flowcytometry.
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ing cells (cytokine spots, spot-forming units) enumerated with an automated ELISPOT reader (Immunospot series 5 analyzer, software version
5.0; Cellular Technology, Cleveland, OH). Frequencies were calculated by
averaging spot numbers from duplicate wells per stimulant and exposure
condition. Frequencies of cytokine-producing cells (y-axes) were plotted as
a function of DEP concentration in micrograms per milliliter (x-axes,
shown in the bottom IL-1b panel only).
2782
a quantitative kinetic chromogenic LAL method (KQCL) was
employed (see Materials and Methods). Endotoxin content of
culture media without and with DEP (100 mg/ml) was ,0.05 EU/
ml, indicating that no additional endotoxin was introduced into the
PBMC cultures as a consequence of stimulation with DEP. To
further rule out microbial contaminants in the DEP, complete
culture media without and with DEP (100 mg/ml) were cultured
for bacterial and fungal growth. No bacterial or fungal growth was
detected in either of the samples after 1 wk and 1 mo, respectively.
DEP and M. tuberculosis uptake by CD14+CD32 peripheral
blood monocytes
M. tuberculosis is an intracellular bacterium that is taken up in
monocytes, macrophages, and dendritic cells by phagocytosis, a
prerequisite for the initiation of innate and subsequent adaptive
host immune responses. Because human mononuclear phagocytes
have been demonstrated to take up DEP (72), uptake of DEP and
M. tuberculosis was assessed in enriched peripheral CD14+CD32
monocytes. Cytospin analysis of monocytes stimulated with DEP
and M. tuberculosis (Fig. 1) provided initial evidence for a concurrent cellular uptake of DEP and M. tuberculosis into the boundaries
of the cells. Subsequent studies by TEM confirmed presence of DEP
and M. tuberculosis within the same transect of the monocytes (Fig.
2), implying the possibility of functional interactions of DEP and
M. tuberculosis within the same cells.
Induction of apoptosis and necrosis by DEP
To examine the extent of DEP-induced toxicity in PBMC, we first
assessed apoptosis and necrosis using a flow cytometry-based
Annexin V/PI apoptosis detection assay. PBMC from healthy
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FIGURE 3. Simultaneous stimulation with DEP alters pathogen-induced cytokine production. Frequencies of cytokine-producing PBMC
(IFN-g [n = 20 subjects], TNF-a
[n = 18 subjects], IL-6 [n = 15 subjects], IL-10 [n = 20 subjects, data
for DEP 50 mg/ml from n = 18 only],
and IL-1b [n = 5 subjects]) stimulated with DEP alone (0, 1, 10, 50,
and 100 mg/ml) or the stimuli M.
tuberculosis (M.tb) MOI 1, MOI 10,
and PPD (10 mg/ml) in presence
(1, 10, 50, and 100 mg/ml) or absence (0 mg/ml) of DEP, were measured by ELISPOT assay. Cytokine
data are arranged horizontally for
each of the stimuli, which are
arranged vertically. Frequencies of
cytokine-producing cells (y-axes) are
plotted as a function of DEP concentration in micrograms per milliliter (x-axes shown below the IL-1b
panel only). Data are presented in
box plots showing from top to bottom: the maximum value (black dot),
the 95th (whisker), 75th (top of box
plot), 50th (median, dotted line),
mean (solid line in middle of box
plot), 25th (solid line bottom of box
plot), and 5th (whisker) percentiles
and the minimum value (black dot).
The p values are shown on top of
box plots in which differences in
cytokine expression between no
DEP and different doses of DEP
were regarded to be statistically
significant (p # 0.05). Additional
statistical comparisons are shown in
Supplemental Table I.
DEP SUPPRESS HUMAN ANTIMYCOBACTERIAL IMMUNITY
The Journal of Immunology
2783
subjects (n = 5) were exposed to DEP for 2, 24, and 72 h at final
concentrations of 0.1–100 mg/ml and proportions of mononuclear
cells undergoing combined apoptosis (Annexin V positive) and
necrosis (PI positive) determined. Stimulation of PBMC with DEP
for 2 h did not result in significant toxicity (combined apoptosis
and necrosis) at any of the concentrations of DEP (0–100 mg/ml)
tested. Furthermore, no significant cell toxicity with DEP stimulation in the dose range from 0.1–10 mg/ml relative to unstimulated cells was observed at any of the time points examined.
Combined apoptosis and necrosis levels were increased only by
20–25% above the spontaneous level in PBMC exposed to DEP at
a concentration of 100 mg/ml (Table I). It is worth noting that even
at the highest concentration of DEP examined, ∼90, 70, and 60%
of cells were viable at 2, 24, and 72 h, respectively. Based on these
observations, we chose the DEP concentrations and PBMC stimulation durations for the subsequent ELISPOT assay and mRNA
expression studies.
ELISPOT assays were used to assess frequencies of cytokineproducing PBMC as a measure of cytokine protein production. Representative images of cytokine-producing PBMC in developed
wells are shown in Supplemental Fig. 1. To assess whether DEP alter
antimycobacterial host immune responses, PBMC were stimulated
with DEP alone or M. tuberculosis MOI 1, MOI 10, and PPD in
presence of DEP at 0 (no DEP control), 1, 10, 50, and 100 mg/ml.
Stimulation of PBMC with DEP alone induced negligible
amounts of IFN-g (Fig. 3, leftmost column). Frequencies of IFN-g–
producing cells in PBMC stimulated with M. tuberculosis MOI 1
and MOI 10 were reduced significantly at DEP concentrations of
$10 mg/ml relative to DEP 0 mg/ml. As expected, frequencies of
PBMC producing all cytokines tested (IFN-g, TNF-a, IL-1b, IL-6,
and IL-10) were greater upon stimulation with M. tuberculosis MOI
10 than with MOI 1 (DEP 0) (Fig. 3, second and third columns from
the left). Following PPD stimulation, IFN-g production was significantly decreased at DEP concentrations of $50 mg/ml (Fig. 3, IFNg). Interestingly, suppressive effects of DEP were also observed on
the per-cell IFN-g output, as the size of the IFN-g spots (diameters)
decreased with increasing DEP concentrations (data not shown).
TNF-a production was barely detectable upon stimulation of
PBMC with DEP alone (Fig. 3, leftmost column). TNF-a production by PBMC stimulated with DEP at a concentration of $50
mg/ml was significantly lower (p # 0.05) compared with that of
unstimulated PBMC (culture media). TNF-a production upon
stimulation with M. tuberculosis (MOI 1 and MOI 10) and PPD
was significantly decreased at DEP concentrations of $50 and
$10 mg/ml, respectively (Fig. 3, TNF-a panel).
As shown for IFN-g and TNF-a, M. tuberculosis and PPDinduced IL-1b and IL-6 production was significantly decreased
by DEP in a dose-dependent manner (Fig. 3, IL-1b and IL-6
panels). Unlike IFN-g and TNF-a, however, stimulation of
PBMC with DEP alone induced significant levels of cytokine
production at concentrations of 1 and 10 mg/ml for IL-6 and 10
mg/ml for IL-1b compared with PBMC in culture media (Fig. 3,
leftmost column). It is worth noting that the DEP-mediated suppression of cytokine production was most striking for IFN-g and
IL-1b, both of which are crucial for M. tuberculosis-induced host
immune responses (21, 28–30, 73).
We also investigated the effect of DEP on the production of the
immunoregulatory cytokines IL-4 and IL-10, which have suppressive effects on innate and acquired immune responses to intracellular pathogens including M. tuberculosis (74–77). In contrast to the findings with IFN-g, TNF-a, IL-1b, and IL-6, IL-10
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Simultaneous exposure to DEP and stimuli alters
M. tuberculosis-induced cytokine production
FIGURE 4. Simultaneous stimulation with DEP alters LPS-induced
cytokine production. Frequencies of IFN-g–, TNF-a–, IL-6–, IL-10–, and
IL-1b–producing PBMC (y-axis) represent spot frequencies following
stimulation with LPS in presence or absence of indicated amounts of DEP
(0, 1, 10, 50, and 100 mg/ml) were measured by ELISPOT assay. Subject
numbers, incubation time, and cell numbers for each cytokine were
identical to that stated in legend to Fig. 3. Presentation of data in the box
plots is described in Fig. 3. Additional statistical comparisons are shown in
Supplemental Table I.
2784
DEP SUPPRESS HUMAN ANTIMYCOBACTERIAL IMMUNITY
production in response to M. tuberculosis or PPD stimulation remained unchanged in presence of DEP concentrations up to 50
mg/ml relative to unstimulated PBMC (DEP 0) (Fig. 3, IL-10). No
significant IL-10 production was detected when PBMC were
stimulated with DEP alone (Fig. 3, leftmost column). No IL-4
production by PBMC was detected following stimulation with
DEP, M. tuberculosis, or PPD (data not shown).
To examine whether DEP effects on M. tuberculosis-induced
cytokine expression were similar to those induced by LPS, a TLR4
agonist, we determined frequencies of IFN-g–, TNF-a–, IL-1b–,
FIGURE 6. M. tuberculosis (M.tb)-induced mRNA expression. Upregulation (A, C) and downregulation (B, D) of mRNA levels in M. tuberculosis MOI
10-stimulated PBMC relative to that of untreated PBMC from healthy subjects using Th1-Th2-Th3 (n = 11) and TLR signaling (n = 8) pathway-specific
qRT-PCR arrays are shown. y-axes represent statistically significant (p # 0.01) mean fold changes ($2-fold) 6 SEM in the mRNA expression level of M.
tuberculosis-induced genes compared with that in untreated PBMC. No M. tuberculosis-induced fold change relative to untreated (defined by ,2-fold; p .
0.01) was set as 1 in (A) and (C) and as 0 in (B) and (D), in which logarithmic and linear scales were used, respectively.
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FIGURE 5. Comparison of DEP and CB effects on M. tuberculosis- (M.tb) and PPD-induced cytokine production. PBMC from healthy subjects (n = 3)
were stimulated with M. tuberculosis at MOI 10 and PPD (10 mg/ml) in presence of varying doses of DEP or CB. DEP or CB was added at final concentrations of 0 (DEP0, CB0, negative control), 1, 10, and 50 mg/ml. Numbers of IFN-g–, IL-10–, and TNF-a–producing PBMC were enumerated by
ELISPOT assay, and frequencies of cytokine-producing cells (y-axes) are plotted as a function of DEP concentration in micrograms per milliliter (x-axes
shown below the TNF-a panel only). Mean frequencies 6 SEM are shown.
The Journal of Immunology
To determine whether the observed effects of DEP were due to its
particle nature or the adsorbed surface chemical species (65, 69–
71), we assessed the effects of CB as a chemically inert particle
control on M. tuberculosis-induced production of IFN-g, TNF-a,
and IL-10, in parallel with DEP. CB represents the chemically
inert inner carbon core of DEP (78) and is devoid of surface
chemical species. In contrast to the findings following DEP exposure, M. tuberculosis- or PPD-induced production of IFN-g,
TNF-a, and IL-10 did not change with increasing CB concentrations (0, 1, 10, and 50 mg/ml) (Fig. 5). Taken together, these
results suggest that the alterations in cytokine production observed
FIGURE 7. DEP effects on M. tuberculosis (M.tb)-induced Th1-Th2-Th3
pathway-specific mRNA expression.
Depicted on y-axes are statistically
significant (p # 0.01) mean fold
changes ($2-fold) 6 SEM in mRNA
expression levels of in vitro-stimulated
PBMC from healthy subjects (n = 11).
(A) The DEP-mediated (10 mg/ml)
mRNA expression in PBMC relative
to mRNA expression levels of unstimulated PBMC in culture medium (set
as 0-line). (B) mRNA expression levels from simultaneous DEP (10 mg/
ml) and M. tuberculosis (MOI 10)
stimulation of PBMC compared with
mRNA expression levels from PBMC
stimulated with M. tuberculosis alone
(set as 0-line). (C) The effect of DEP
prestimulation on M. tuberculosis-induced mRNA expression. PBMC were
prestimulated with DEP (10 mg/ml)
for 20 h and subsequently stimulated
with M. tuberculosis at MOI 10 for an
additional 24 h. The mRNA expression levels of DEP prestimulated and
subsequently M. tuberculosis-stimulated PBMC were compared with that
of PBMC stimulated with M. tuberculosis alone (set as 0-line).
in PBMC stimulated with DEP (Figs. 3, 4) are in large part due to
adsorbed surface chemical species (see Characterization of DEP)
rather than the inert carbon core of DEP.
DEP alters M. tuberculosis-induced mRNA expression profiles
A broad spectrum of gene targets was studied using Th1-Th2-Th3
and TLR pathway-specific gene expression qRT-PCR arrays to
determine the effect of DEP on M. tuberculosis-induced host gene
expression.
M. tuberculosis-induced mRNA expression. In a first step, mRNA
expression profiles of PBMC stimulated with M. tuberculosis MOI
10 were compared with that of unstimulated PBMC (cultured
in complete medium). Levels of M. tuberculosis-induced mRNAs
(increased or decreased by $2-fold; p # 0.01) relative to unstimulated cells were evaluated with Th1-Th2-Th3 (Fig. 6A, 6B) and
TLR pathway-specific (Fig. 6C, 6D) qRT-PCR arrays. Using human
Th1-Th2-Th3 qRT-PCR arrays, a significant increase (100–1000fold) of mRNAs encoding Th1 cytokines IFNG, IL12B, and CSF2
relative to untreated controls was observed (Fig. 6A). In addition,
the level of mRNA encoding Th1 cytokines and related genes
IL12RB2, IL2RA, IRF1, IL18R1, SOCS1, STAT4, TBX21, and TNFA
was increased by 2–50-fold upon M. tuberculosis stimulation (Fig.
6A). The expression of a smaller number of Th2 cytokines and
related genes (e.g., CCL7, IL10, IL13, IL5, ICOS, and NFATC2)
was increased by 2–50-fold in M. tuberculosis-stimulated cells
relative to unstimulated PBMC. Increased expression (2- to
.2,000-fold) of genes encoding CD4+ T cell markers CD40LG,
CTLA4, FASG, IL6, IL7, LAG3, TNFRSF8, TNFRSF9, and
TNFRSF4 was observed in M. tuberculosis-stimulated PBMC (Fig.
6A), whereas IL18, TLR4, IL13RA1, IL1R2, CD4, and CD86 genes
were downregulated by 2–30-fold following M. tuberculosis stimulation (Fig. 6B).
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IL-6–, and IL-10–producing cells in PBMC stimulated with LPS in
presence and absence of DEP. Effects of DEP on frequencies of
IFN-g–, TNF-a–, IL-1b–, IL-6–, and IL-10–producing PBMC
following stimulation with LPS were comparable to effects of DEP
following stimulation with M. tuberculosis or PPD (Fig. 4). Production of all cytokines was completely abrogated at the DEP concentration 100 mg/ml (Figs. 3, 4). It is worth noting that DEPmediated apoptosis and necrosis of PBMC cannot account for the
observed inhibition of cytokine production because 60–90% of
PBMC were viable even after 72 h of incubation with DEP (Table I).
Taken together, these results indicate that stimulation of PBMC
with DEP inhibited M. tuberculosis, PPD, and LPS-induced production of IFN-g, TNF-a, IL-1b, and IL-6 in a concentrationdependent manner. On the contrary, IL-10 production remained
unchanged at all but the highest concentration of DEP examined.
The observed suppression of M. tuberculosis-induced production
of proinflammatory cytokines together with the concomitantly
unaltered IL-10 production strongly suggest that DEP interfere
with protective host antimycobacterial immunity.
Particle versus chemical effects of DEP
2785
2786
a ratio of the mRNA levels of M. tuberculosis plus DEP and M.
tuberculosis alone. Thus, all DEP-mediated mRNA expression
changes are represented relative to the mRNA expression levels in
PBMC stimulated with M. tuberculosis alone (set as baseline).
IFNG, TLR4, and CXCL10 mRNAs decreased to comparable
levels during both the simultaneous stimulation (Figs. 7B, 8B,
Table II) and the 20-h prestimulation with DEP (Figs. 7C, 8C,
Table II). DEP prestimulation followed by M. tuberculosis infection, however, led to the downregulation of a larger number of
M. tuberculosis-induced genes with known involvement in antimycobacterial immunity such as costimulatory molecules, type I
and type II IFNs, proinflammatory cytokines, chemokines, chemokine receptors, and TLRs 3, 4, 7, and 10. In addition to the
downregulation of the genes mentioned above, stimulation of
PBMC with DEP upregulated the expression of several M. tuberculosis-induced genes including CSF2, IL1R1 during simultaneous, and CD86, CEBPB, IGSF6, IL13RA1, and IL18 during
prestimulation by 2–4-fold (p # 0.01) compared with PBMC
stimulated with M. tuberculosis alone (Figs. 7B, 7C, 8B, 8C, Table
II). Thus, prestimulation with DEP followed by M. tuberculosis
stimulation alters M. tuberculosis-induced host responses more
profoundly than simultaneous stimulation with DEP and M. tuberculosis as evidenced by the alteration of 4 and 5 mRNAs in
Figs. 7B and 8B, respectively, compared with 23 and 17 mRNAs
in Figs. 7C and 8C, respectively. Stimulation of PBMC with DEP
alone altered the expression of only a small number of mRNAs
(Figs. 7A, 8A). The expression of proinflammatory mediators
FIGURE 8. DEP Effects on TLR pathway-specific mRNA expression. Depicted in y-axes are statistically significant (p # 0.01) mean fold changes ($2fold) 6 SEM in mRNA expression levels of in vitro-stimulated PBMC from healthy subjects (n = 8). (A) DEP-mediated alteration of mRNA expression in
PBMC. DEP-induced mRNA expression levels were compared with that of unstimulated PBMC in culture medium (set as 0-line). (B) The effect of DEP
(10 mg/ml final concentration) on M. tuberculosis (M.tb) MOI 10-induced mRNA expression during simultaneous combined stimulation compared with
mRNA expression levels from PBMC that were stimulated with M. tuberculosis MOI 10 alone (set as 0-line). (C) The effect of DEP prestimulation on M.
tuberculosis MOI 10-induced mRNA expression. PBMC were prestimulated with DEP (10 mg/ml) for 20 h and subsequently stimulated with M. tuberculosis at MOI 10 for an additional 24 h. The mRNA expression levels of DEP prestimulated and subsequently M. tuberculosis-stimulated PBMC were
compared with that of PBMC stimulated with M. tuberculosis alone (set as 0-line).
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Using human TLR signaling pathway-specific qRT-PCR arrays,
upregulation of NF-kB target genes CCL2, CSF2, CSF3, IFNA1,
IFNB1, IFNG, IL1A, IL1B, IL6, IL8, IL10, TNF, NFKB1, and
NFKB1A and of genes downstream of IRF pathway, CXCL10,
IFNA1, IFNB1, IFNG, and IRF1, were observed in M. tuberculosis-stimulated PBMC relative to unstimulated PBMC (Fig. 6C).
In addition to M. tuberculosis-mediated upregulation (Fig. 6C),
downregulation of several genes related to TLR-mediated signal
transduction pathway by M. tuberculosis was observed (Fig. 6D).
DEP-mediated modification of M. tuberculosis-induced mRNA
expression. Engagement of TLRs by M. tuberculosis ligands initiates signaling pathways that ultimately activate transcription
factor NF-kB, leading to subsequent release of cytokines and
chemokines (38, 41, 42, 73). The effects of DEP were studied
upon simultaneous stimulation of PBMC with DEP and M. tuberculosis and upon a 20-h prestimulation of PBMC with DEP
followed by stimulation with M. tuberculosis. The prestimulation
with DEP simulated the sequential exposures to DEP and M. tuberculosis that may occur in real life. Thus, PBMC were stimulated with DEP, M. tuberculosis plus DEP (simultaneous), M.
tuberculosis following DEP (prestimulation), and M. tuberculosis
alone or left unstimulated. The mRNA expression profile of
PBMC that had been stimulated with M. tuberculosis following
DEP pre-exposure was considerably different from that of PBMC
stimulated with M. tuberculosis plus DEP simultaneously (compare Fig. 7B, 7C and Fig. 8B, 8C, Table II). The mRNA fold
changes plotted on y-axes in Figs. 7B, 7C, 8B, and 8C represent
DEP SUPPRESS HUMAN ANTIMYCOBACTERIAL IMMUNITY
The Journal of Immunology
2787
Table II. Effect of DEP exposure on M. tuberculosis-induced host gene expression
Functional Group
Cytokines and cytokine receptors
Chemokines and receptors
TLR
Costimulatory molecules, T cell activation
IFN-responsive effector
Others
DEP Stimulation Condition
Fold Change Relative to
M. tuberculosis
IL5
IL6
IFNG
IFNA1
IFNB1
IL1A
CSF3
IL18
CSF2
IL7
IL2RA
IL18R1
IL1R1
IL13RA
CXCL10
CXCR3
CCL5
CCL7
CCR4
NFKB1A
SOCS1
FOS
GATA3
CEBPB
TLR7
TLA4
TLR3
TLR10
TLR5
CD86
CD80
LAG3
CTLA4
ICOS
TNFSF4
EIF2AK2
SPP1
FASLG
PTGS2
IGSF6
Pre
Pre
Simultaneous and Pre
Pre
Pre
Pre
Pre
Pre
Simultaneous
Pre
Pre
Pre
Simultaneous
Pre
Simultaneous and Pre
Pre
Pre
Simultaneous
Pre
Pre
Pre
Pre
Pre
Pre
Pre
Simultaneous and Pre
Pre
Pre
Simultaneous
Pre
Pre
Pre
Pre
Pre
Pre
Pre
Pre
Pre
Pre
Pre
↓9
↓ 8.9-9.5
↓ 5–5.4 and 6.1–7.3
↓ 6.4
↓ 4.7
↓4
↓ 3.5
↑ 3.2
↑ 2.4–2.7
↓ 2.2
↓ 2.3
↓ 2.2
↑ 2.2
↑ 2.1
↓ 3.48 and 5.3
↓3
↓ 2.4
↓ 2.3
↓ 2.1
↓ 9.6
↓ 2.6
↑ 2.6
↓2
↑2
↓ 4.7
↓ 2.3 and 3.8
↓ 3.1
↓ 2.4
↑ 2.1
↑ 2.8–3.5
↓ 2.4–2.7
↓ 2.7
↓ 2.6
↓ 2.3
↓ 2.2
↓ 5.6
↑ 2.1
↓ 3.0
↓ 3.6
↑ 3.9
The mRNA levels of PBMC stimulated simultaneously with M. tuberculosis and DEP (simultaneous) and with M. tuberculosis following DEP
prestimulation (Pre) were compared with that of PBMC stimulated with M. tuberculosis alone. mRNA expression levels that were increased or decreased
by $2-fold (p # 0.01) relative to the mRNA expression levels of PBMC stimulated with M. tuberculosis alone are shown.
including IL1R1, IL8, PTGS2, and TNF was increased, whereas
the expression of the receptors CD4 and CD80 (B7-1) was decreased (Figs. 7A, 8A).
The gene expression analyses with qRT-PCR arrays were validated by real-time PCR of cDNAs generated from a subset of RNA
samples in the qRT-PCR arrays using primers listed in Fig. 9. The
expression of eight genes altered by simultaneous and/or prestimulation with DEP is shown in Fig. 9. In general, real-time PCR
data were consistent with the data obtained from the qRT-PCR
arrays.
Simultaneous and prestimulation with DEP were found to
downregulate the expression of a large number of genes including
that of TLRs (Fig. 8C). Considering the importance of TLRmediated antimycobacterial immunity, DEP-induced suppression
of TLR pathways could potentially inhibit robust host immune
responses against microbial infection.
Comparison of DEP and LPS prestimulation
DEP prestimulation modified M. tuberculosis-induced cytokine
responses in a manner comparable to that shown in other studies
following repeated LPS stimulation of cells (40, 79–81). Such LPS
stimulation induces a state of cellular tolerance of monocytes/
macrophages to subsequent secondary stimulation, which is char-
acterized by impairment of IFN-g, TNF-a, IL-1b, and IL-6 and
preservation of anti-inflammatory IL-10 production (80). Stimulation with LPS, a potent ligand of TLR4 and CD14, leads to activation of the NF-ĸB pathway via MyD88-dependent and MyD88independent signaling (47, 82). To examine whether LPS prestimulation suppresses M. tuberculosis-induced gene expression, the
gene expression profile of PBMC infected with M. tuberculosis
following prestimulation with LPS was compared with that of
M. tuberculosis-infected PBMC without LPS prestimulation using
Th1-Th2-Th3 (Fig. 10A) and TLR pathway-specific (Fig. 10B)
qRT-PCR arrays. Prestimulation with LPS, like with DEP, suppressed several M. tuberculosis-induced genes encoding host immune effector proteins including IFN-g and CXCL10, which are
crucial for protective antimycobacterial immunity. However, unlike with DEP, LPS prestimulation inhibited the M. tuberculosisinduced expression of IL12B, IL12RB2, and IRF4 (Fig. 10, Table
III). As seen in Table III, prestimulation with DEP suppressed
a broader spectrum of M. tuberculosis-induced host immune genes
in PBMC than prestimulation with LPS. This is not surprising
considering that LPS prestimulation suppresses predominantly
TLR4 signaling, whereas DEP may potentially suppress signaling
through other TLRs involved in M. tuberculosis-induced host
responses. Thus, prestimulation with DEP, like with LPS, induced
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Transcription factors and inhibitors
Genes
2788
a state of cellular hyporesponsiveness toward subsequent stimulation with M. tuberculosis.
It is worth noting that the mRNA expression profile of the M.
tuberculosis-induced host immune response in presence of DEP
FIGURE 10. Effect of LPS prestimulation on
M. tuberculosis (M.tb)-induced mRNA expression.
Depicted in y-axes are statistically significant (p #
0.01) mean fold changes ($2-fold) 6 SEM in
mRNA expression levels of in vitro-stimulated
PBMC from healthy subjects (n = 11 for Th1-Th2Th3, n = 8 for TLR pathway-specific qRT-PCR
arrays). PBMC were prestimulated with LPS (100
ng/ml) for 20 h and subsequently stimulated with
M. tuberculosis at MOI 10 for an additional 24
h. Following RNA extraction and generation of
cDNA, gene expression was assessed by qRT-PCR
arrays. mRNA expression levels were compared
with that from PBMC stimulated with M. tuberculosis alone (set as 0-line). (A) Effects of LPS
prestimulation on M. tuberculosis-induced Th1Th2-Th3 pathway-specific mRNA expression. (B)
Effects of LPS prestimulation on M. tuberculosisinduced TLR pathway-specific mRNA expression.
stimulation compared with that in the presence of M. tuberculosis
alone was consistent with the reduced IFN-g, IL-1b, and IL-6 and
increased or unchanged IL-10 protein expression in the ELISPOT
assays (compare Figs. 7B, 7C, 8B, 8C with Fig. 3). DEP had no
effect on M. tuberculosis-induced TNF-a mRNA expression
(measured 24 h after stimulation), whereas DEP decreased M. tuberculosis-induced TNF-a protein expression (Fig. 3). This discrepancy in gene and protein expression is likely due to the transient
and early expression of mRNA encoding TNF-a. Taken together,
these cytokine production and mRNA expression data suggest that
DEP exposure modulates and interferes (see hypothetical model in
Fig. 11) with antimycobacterial immune responses.
Discussion
TB remains a major global public health concern that is aggravated
by the HIV pandemic and emergence of drug-resistant M. tuberculosis strains. Deteriorating air quality from rapid industrial
growth and urban traffic coincide with high prevalence and incidence rates of endemic TB in densely populated regions of lowand middle-income countries. Although epidemiological evidence
suggests that exposure to air pollutants (e.g., silica, indoor pollution from fossil fuel combustion, cigarette smoke) increase the
incidence of TB, only a few studies have assessed mechanisms
that may underlie the effects of urban air PM2.5 on innate and
adaptive human immune responses to mycobacteria (62, 72, 83).
Because concomitant aerosol exposure to DEP and M. tuberculosis occur frequently in real-life situations, we hypothesized
that DEP may alter human antimycobacterial host immunity. This
study is the first, to our knowledge, to assess the effects of DEP,
a major component of urban air pollution, on human primary
immune cell responses to M. tuberculosis. We focused on the
effects of DEP on M. tuberculosis-specific cytokine production
and gene expression in PBMC, which, unlike studies in isolated
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FIGURE 9. Validation of qRT-PCR array results. Total RNA (400 ng)
from PBMC of a subset of donors (n = 4–6) was reverse transcribed to
generate cDNA. Real-time PCR was performed and mRNA fold change in
DEP-prestimulated and M. tuberculosis (M.tb) MOI 10-exposed PBMC
relative to M. tuberculosis was evaluated. Means 6 SEM are shown. IFNG:
forward primer, 59-GCATCCAAAAGAGTGTGGAGAC-39, reverse primer,
59-TACTGGGATGCTCTTCGACCT-39; CXCL10: forward primer, 59-TGGCATTCAAGGAGTACCTCTC-39, reverse primer, 59-CTTGATGGCCTTCGATTCTG-39; CD86: forward primer, 59-GGTGCTGCTCCTCTGAAGATT-39, reverse primer, 59-TGA AGTCTCAGGGTCCAACTG-39; IL13RA:
forward primer, 59-GTCCCAGTGTAGCACCAATGA-39, reverse primer,
59-CCAGGCTTCTGTGCCAATAGT-39; IL6: forward primer, 59-AGACAGCCACTCACCTCTTCA-39, reverse primer, 59-CACCAGGCAAGTCTCCTCATT-39; CSF3: forward primer, 59-ATAGCGGCCTTTTCCT CTACC-39, reverse primer, 59-GCCATTCCCAGTTCTTCCAT-39; PTGS2: forward primer, 59-GGTGATGAGCAGTTGTTCCAG-39, reverse primer, 59GAAGGGGATGCCAGTGATAGA-39; and LAG3: forward primer, 59-TCCTGGTGACTGGAGACAATG-39, reverse primer, 59-TGGAGTCACCTCACAAAGCAG-39.
DEP SUPPRESS HUMAN ANTIMYCOBACTERIAL IMMUNITY
The Journal of Immunology
2789
Table III. Alteration of M. tuberculosis-induced genes by DEP and LPS
M. tuberculosis-Induced Genes
Altered by LPS Prestimulation
(Relative to M. tuberculosis)
M. tuberculosis-Induced Genes
Not Altered by DEP Prestimulation
(Relative to M. tuberculosis)
M. tuberculosis-Induced Genes
Not Altered by LPS Prestimulation
(Relative to M. tuberculosis)
CCL5 ↓
CCR4 ↓
CD86 ↑
CEBPB ↑
CSF3 ↓
CTLA4 ↓
CXCR3 ↓
FOS ↑
GATA3 ↓
ICOS ↓
IFNA1 ↓
IGSF6 ↑
IL13RA ↑
IL18 ↑
IL1A ↓
IL5 ↓
IL6 ↓
IL7 ↓
NFkB1A ↓
PTGS2 ↓
SOCS1 ↓
SSP1 ↑
TLR10 ↓
TLR4 ↓
TLR7 ↓
TNFSF4 ↓
CD80 ↓
CXCL10 ↓
EIF2AK2 ↓
FASLG ↓
IFNB1 ↓
IFNG ↓
IL18R1 ↓
IL2RA ↓
LAG3 ↓
TLR3 ↓
CCL7 ↓
CLEC4E ↑
CSF2 ↓
HAVCR2 ↓
IL1R1 ↑
IL12B ↓
IL12RB2 ↓
INHBA ↓
IRF4 ↓
TBX21 ↓
TNF ↓
TNFRSF9 ↓
CD80 ↓
CXCL10 ↓
EIF2AK2 ↓
FASLG ↓
IFNB1 ↓
IFNG ↓
IL18R1 ↓
IL2RA ↓
LAG3 ↓
TLR3 ↓
CCL2
CCL7
CD180
CD40LG
CLEC4E
CSF2
ELK1
HAVCR2
IL10
IL12RB2
IL13
IL1R2
IL23A
IL8
INHBA
IRAK2
IRF1
IRF4
JUN
LTA
MAP4K4
NFKB1
RIPK2
SIGIRR
SOCS2
STAT4
TBX21
TLR1
TLR5
TLR8
IL12B
TNF
TNFRSF8
TNFRSF9
UBE2V1
CCL2
CCL5
CCR4
CD180
CD40LG
CD86
CEBPB
CSF3
CTLA4
CXCR3
ELK1
FOS
GATA3
ICOS
IFNA1
IGSF6
IL10
IL13
IL13RA
IL18
IL1A
IL23A
IL6
IL7
IL8
IRAK2
IRF1
JUN
LTA
MAP4K4
NFKB1
NFKB1A
PTGS2
RIPK2
SIGIRR
SOCS1
SOCS2
SSP1
STAT4
TLR1
TLR10
TLR4
TLR5
TLR7
TLR8
IL5
TNFRSF8
TNFSF4
UBE2V1
The mRNA levels of PBMC stimulated with M. tuberculosis following DEP or LPS prestimulation were compared with that of PBMC stimulated with M. tuberculosis alone.
Increased or decreased ($2-fold; p # 0.01) mRNA expression levels relative to mRNA expression levels of M. tuberculosis-stimulated PBMC (set as 0-value) are shown.
mRNAs altered by both DEP and LPS (columns 1 and 2) are in boldface.
primary cell populations or cell lines (61, 84–91), permit evaluation of immunological interactions between APCs and lymphocytes in a quasi-physiological context. The current study demonstrates that DEP and M. tuberculosis localize within the same
monocytes (Figs 1, 2), a finding that is consistent with a report of
combined uptake of bacillus Calmette-Guérin (BCG) and DEP by
human monocyte-derived macrophages (72). Because combined
uptake of M. tuberculosis and DEP in host immune cells suggests
the possibility of subsequent alterations of cellular functions, we
studied the DEP effects on TLR-mediated proinflammatory immune responses and adaptive immunity (92).
Our studies show that DEP suppress M. tuberculosis, PPD, and
LPS-induced IFN-g, IL-6, IL-1b, and TNF-a protein production
as determined by a dose-dependent reduction of the frequencies of
cytokine-producing PBMC, whereas the frequencies of IL-10–
producing PBMC remain unaltered except at the highest DEP dose
(Figs. 3, 4). To gain further insights into the mechanisms of these
immunosuppressive effects of DEP, we studied a broad spectrum
of genes corresponding to TLRs and their associated pathways as
well as Th1-Th2-Th3 cytokines, chemokines, and their receptors.
The central finding of these studies is that DEP prestimulation
impairs M. tuberculosis-induced effector mechanisms by suppressing NF-kB (IL6, IFNG, NFKNA1, IFNB1, IFNA1, IL1A, and
CSF3) and IRF (IFNG, IFNA1, IFNB1, and CXCL10) pathway
target genes (Fig. 8C, Table II), thus modulating the production of
IFN-g, IL-6, IL-1b, and TNF-a. It has been shown that M. tuberculosis-mediated TLR activation leads to the recruitment of
various adaptor proteins and the initiation of TLR signaling, such
as the activation of adaptor protein MyD88-dependent and -independent pathways and the activation of various transcription fac-
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M. tuberculosis-Induced Genes
Altered by DEP Prestimulation
(Relative to M. tuberculosis)
2790
prestimulation of PBMC suppresses both MyD88-dependent and
-independent M. tuberculosis-induced signaling pathways, leading
to a hyporesponsive state upon subsequent exposure to M. tuberculosis. Such a DEP-induced hyporesponsive state is reminiscent of
the effects of tobacco smoke on alveolar macrophages, which have
been shown to become refractory to challenge with TLR2 and TLR4
agonists compared with alveolar macrophages of nonsmokers (97).
The DEP-induced hyporesponsive state toward M. tuberculosisstimulation described in this study also shows similarities to LPSinduced tolerance (40, 79–81), a state characterized by a transitory
hyporesponsiveness to secondary stimulation following repeated
preceding LPS stimulation. It is interesting in this context that in
contrast to our DEP prestimulation experiments, simultaneous
stimulation of PBMC with DEP and M. tuberculosis affected the
expression of fewer M. tuberculosis-induced genes (Figs. 7B, 8B).
DEP prestimulation thus appears to be a prerequisite for the observed
induction of the cellular hyporesponsiveness to subsequent M. tuberculosis-induced cellular responses. These studies are of relevance,
as periods of respiratory exposure to air pollutants may precede
subsequent M. tuberculosis infection events frequently in real-life
situations.
DEP stimulation has also been linked to a potent Th2 adjuvant
activity and induction of allergic conditions (98–102) and increased Th2 cytokine responses (103). These observations are
FIGURE 11. A hypothetical representation of DEP-mediated suppression of M. tuberculosis-induced TLR signaling and effector functions. M. tuberculosis-derived ligands (lipoarabinomannan [LAM], 19-kDa protein, heat shock proteins 65 and 71, and M. tuberculosis DNA) bind to TLR2, -4, and -9 and
recruit adaptor protein MyD88 to the TLR receptor complex, which associates with IL-1R–associated kinase (IRAK) 1 and 4. TNFR-associated factor 6
(TRAF6) is also recruited to the receptor complex following phosphorylation of IRAK1 by IRAK4. Association of TRAF6–IRAK complex with TGF-b–
activated kinase (TAK) and TAK-binding protein (TAB) 1 and 2 induces activation of TAK1 that phosphorylates IkB-kinase (IKK) complex consisting of
three subunits: IKKa, b, and g. IKK complex phosphorylates IkB, leading to its dissociation from NF-kB complex followed by translocation of NF-kB into
the nucleus, where NF-kB binds and activates target genes. In addition to MyD88, ligand binding to TLR4 recruits adaptor protein Toll/IL-1R domaincontaining adaptor protein inducing IFN-b (TRIF), which associates with TANK-binding kinase 1 (TBK1), leading to the phosphorylation of IRF3.
Phosphorylated IRF3 translocates to the nucleus and induces production of type I IFNs (47–49). DEP suppress M. tuberculosis-induced expression of target
genes IL6, IL1A, and PTGS2 via MyD88-dependent and type I IFNs via MyD88-independent pathways. DEP-mediated downregulation of expression of
TLR4, TLR7, and NF-kB target genes and type I IFNs is shown by downward arrows.
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
tors including NF-kB and IRFs (38, 41–43, 46, 47). Our data
suggest that DEP prestimulation suppresses these MyD88dependent signaling pathways, as indicated by the downregulation of several genes such as IL1A, IL6, and PTGS2 (Fig. 8B). In
addition, DEP may also affect MyD88-independent pathways (91),
as indicated by the inhibition of the expression of M. tuberculosisinduced type I IFN mRNAs (IFNA1 and IFNB1) following DEP
prestimulation. Curiously, the observed increase in expression of
proinflammatory mediators IL8, PTGS2, and TNF and decrease in
expression of costimulatory receptors CTLA4 and CD80 (B7-1) in
PBMC stimulated with DEP alone (Fig. 8A) coincide with that in
monocyte-derived immature dendritic cells following in vitro exposure to ambient PM (93). Further, our finding of suppressed
expression of M. tuberculosis-induced TLR3, TLR4, TLR7, and
TLR10 mRNA following DEP prestimulation (Fig. 8C) is consistent with the reported suppression of expression and function of
TLRs (TLR2 and TLR4) in monocyte-derived dendritic cells exposed to ambient PM (93, 94). In addition, suppression of TLR
signaling may also result from the reduced expression and function
of signaling intermediates downstream of the TLRs, as indicated
by recent studies (80, 95, 96).
Based on the presented findings and published evidence, we
propose a hypothetical model of DEP-mediated suppression of
M. tuberculosis-induced TLR signaling (Fig. 11) in which DEP
DEP SUPPRESS HUMAN ANTIMYCOBACTERIAL IMMUNITY
The Journal of Immunology
Acknowledgments
We thank the study volunteers for participation in this study. We also thank
Drs. S. Levine and S. Marcella at the Chandler Clinic in New Brunswick and
Kathie Kelly McNeill at the Environmental and Occupational Health Sciences Institute for invaluable help with the study subject recruitment and
administrative work, Megan Rockafellow (UMDNJ-School of Public
Health) for help with cytospin preparations, Raj Patel (Electron Micros-
copy Core Imaging Lab, Department of Pathology, UMDNJ) for expert
TEM, and Drs. Martha Torres (National Institute for Respiratory Diseases,
Mexico City, Mexico) and Andrew Gow (Rutgers University, Piscataway,
NJ) for reviewing the manuscript.
Disclosures
The authors have no financial conflicts of interest.
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