Acc
epte
d A
rtic
le
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an 'Accepted Article', doi: 10.1111/imm.12164 This article is protected by copyright. All rights reserved.
Received Date : 15-May-2013 Revised Date : 19-Aug-2013 Accepted Date : 20-Aug-2013 Article type : Original Article Mycobacterium tuberculosis infection of human dendritic cells decreases integrin expression,
adhesion, and migration to chemokines
Lawton L. Roberts and Cory M. Robinson*
Department of Pathology, Microbiology, & Immunology, University of South Carolina School of
Medicine, Columbia, SC, 29209, USA.
Summary: Mycobacterium tuberculosis limits integrin surface expression and distribution on
human dendritic cells that impacts adhesion and migration.
Running title: Mycobacteria modulate integrins and migration
*Corresponding author: Cory M. Robinson
6439 Garners Ferry Road, Building 1 room B46, Columbia, SC, 29209
Telephone: 803-216-3421; Fax: 803-216-3413
Email: [email protected]
Keywords: integrins, dendritic cells, CD18, migration, mycobacteria
Acc
epte
d A
rtic
le
This article is protected by copyright. All rights reserved.
Abbreviations: BSA= bovine serum albumin, CCL-19= chemokine (C-C motif) ligand-19,
CCL-21= chemokine (C-C motif) ligand-21, CR= complement receptor, CCR7= chemokine
receptor 7, DC= dendritic cell, DC-SIGN= dendritic cell-specific intercellular adhesion
molecule-3-grabbing non-integrin, GM-CSF= granulocyte-macrophage colony stimulating
factor, HMVLEC= human microvascular lung endothelial cell, ICAM-1= intercellular adhesion
molecule-1, LFA-1= lymphocyte function associated antigen-1, LPS= lipopolysaccharide, Mac-
1= macrophage-1 antigen, MOI= multiplicity of infection, MTB= Mycobacterium tuberculosis,
PBS= phosphate buffered saline, PE= phycoerythrin, PPD= purified protein derivative, TB=
tuberculosis, VCAM-1= vascular cell adhesion molecule-1, VLA-4= very late antigen-4
Abstract
Tuberculosis (TB) remains a major global health problem accounting for millions of deaths
annually. Approximately one-third of the world’s population is infected with the causative agent
Mycobacterium tuberculosis (MTB). The onset of an adaptive immune response to MTB is
delayed compared to other microbial infections. This delay permits bacterial growth and
dissemination. The precise mechanism(s) responsible for this delay have remained obscure. T-
cell activation is preceded by dendritic cell (DC) migration from infected lungs to local lymph
nodes and synapsis with T-cells. We hypothesized that MTB may impede the ability of DCs to
reach lymph nodes and initiate an adaptive immune response. We used primary human DCs to
determine the effect of MTB on expression of heterodimeric integrins involved in cellular
adhesion and migration. We also evaluated the ability of infected DCs to adhere to and migrate
through lung endothelial cells which is necessary to reach lymph nodes. We show by flow
cytometry and confocal microscopy that MTB-infected DCs exhibit a significant reduction in
Acc
epte
d A
rtic
le
This article is protected by copyright. All rights reserved.
surface expression of the β2 (CD18) integrin. β2 integrin distribution is also markedly altered in
MTB-infected DCs. A corresponding reduction in the αL (CD11a) and αM (CD11b) subunits
that associate with β2 was also observed. Consistent with reduced integrin surface expression,
we show a significant reduction in adherence to lung endothelial cell monolayers and migration
towards lymphatic chemokines when DCs are infected with MTB. These findings suggest that
MTB modulates DC adhesion and migration to increase the time required to initiate an adaptive
immune response.
Introduction
Tuberculosis (TB) remains a formidable infectious disease accounting for approximately
2 million deaths worldwide each year.1 Estimates of infection indicate that one-third of the
world’s population has been infected with the causative agent, Mycobacterium tuberculosis
(MTB).1 The host immune response is typically sufficient to contain the infection but seldom
able to eradicate the bacterium. Nearly 5-10% of infections result in active TB while many
result in a latent state, which can later reactivate depending on host immune status.2 In humans,
development of adaptive immunity to MTB is measured as a response to the tuberculin skin test
and evidence shows a 5-6 week period following exposure before acquired immunity develops.3
In mice, approximately 18-20 days are required before antigen specific T-cells arrive in the
infected lungs [reviewed in reference 4]. Compared to other microbial infections this is
substantially longer and allows for expansion of the mycobacterial population.4-12 Detailed
mechanisms that underlie the delayed acquired immune response to MTB have remained
obscure.
Acc
epte
d A
rtic
le
This article is protected by copyright. All rights reserved.
MTB is spread by aerosol route and first encountered by alveolar phagocytes such as
macrophages and dendritic cells (DCs). DCs can internalize MTB and MTB-derived antigens
through numerous cell surface receptors. These include complement receptors (CR), mannose
receptor, and DC-SIGN [reviewed in reference 13]. Although evidence suggests that DC-SIGN
may be the primary receptor for mycobacterial uptake by DCs.14 Subsequently the DC
undergoes maturation and upregulates chemokine receptor 7 (CCR7) expression. This allows for
directed migration to secondary lymphoid organs in response to a gradient of CCL19 and CCL21
chemokines.15, 16 Other coordinated cellular events involve increased expression of T-cell
costimulatory and adhesion molecules.17, 18 The latter aid in adhesion to endothelial cells and
migration through afferent lymphatics to regional lymph nodes.
Integrins are transmembrane heterodimers that consist of a unique alpha subunit and a
shared β subunit, and are indispensable for proper immune function. The β1 family consists of a
diverse group of integrins. Among the most notable adhesion integrin is VLA-4 (CD49d/CD29:
α4/β1), a ligand for VCAM-1 and fibronectin expressed by vascular endothelium. In addition,
the well characterized β2 family consists of 4 different α subunits that share a common β2
(CD18) subunit. Most notable are LFA-1 (CD11a/CD18 or αL/β2), Mac-1/CR3 (CD11b/CD18
or αM/β2) and CR4 (CD11c/CD18 or αX/β2). They are all expressed by DCs and involved in
cell-cell adhesion and T-cell activation.19 In the context of TB, integrins have been found to be
absolutely required for host control of infection. Survival is substantially reduced in mice
deficient for CD11a and CD18.20 Moreover, CD11a knock-out mice are impaired in their ability
to control MTB and exhibit decreased numbers of effector T-cells in the lungs compared to wild
type mice.20
Acc
epte
d A
rtic
le
This article is protected by copyright. All rights reserved.
The process of DC attachment to endothelial cells and subsequent migration is mediated
by members of the β1 and β2 integrin family of adhesion molecules. Blocking antibodies to
CD18, CD11a, CD11b and VLA-4 have been reported to inhibit DC adhesion to and
transmigration through endothelial cells.21, 22 Similarly, blocking antibodies to their cognate
ligands ICAM-1 and VCAM-1 block DC adhesion to and transmigration across lymphatic
endothelium.23 In addition, LFA-1 on DCs is involved in regulating the duration of contact with
T-cells allowing the enhancement of Th1 cell proliferation.24-26
The effect of MTB on primary human DC integrin expression has not been explored.
Given the prominent role of these integrins in DC migration, adhesion to vascular endothelium,
and interactions with T-cells, we investigated the influence of MTB infection on integrin
expression in primary human DCs. Our results demonstrate that DC exposure to MTB results in
a decrease in α and β integrin subunit expression with impaired DC adherence to endothelial
cells and migration toward a chemokine gradient. We further suggest that this could influence
the delayed onset of an adaptive immune response during TB.
Materials and Methods
Cell Culture
Monocytes were isolated from buffy coats purchased from the New York Blood Center,
(New York, NY) by sequential Ficoll (Amersham Bioscienes) and OptiPrep™ (Sigma) density
gradient centrifugations as described previously.27 Eligible donors were 16 years of age or older,
at least 110 pounds, and in good physical health. The donor samples were anonymous and
deidentifed. Monocytes were washed once with phosphate-buffered saline (PBS) and
Acc
epte
d A
rtic
le
This article is protected by copyright. All rights reserved.
resuspended in RPMI (Thermo Scientific) supplemented with 25 mM HEPES and 2 mM L-
glutamine. Monocytes were seeded on 24-well tissue culture plates (1 x 106 cells/well) and
allowed to adhere for 1 hour before washing twice with PBS to remove non-adherent cells.
Adherent monocytes were then immediately cultured in RPMI supplemented with 10% human
serum AB, 25 mM HEPES, 2 mM L-glutamine, 0.1 mM nonessential amino acids, 1 mM sodium
pyruvate, 0.05 mM 2-mercaptoethanol, and recombinant human IL-4 and GM-CSF (Gemini
Bioproducts) were added at 1,600 U and 290 U per 106 cells, respectively. Cells were cultured
for 7 days at 37ºC with 5% CO2. On day 7 cells were considered immature DCs and harvested
with PBS that contained 4 mg/mL lidocaine and 5 mM EDTA for 15 min at 37ºC. Monocytes
cultured in this manner routinely yield a DC population that is heterogenous in size and shape,
MHC IIhigh, CD86high, CD14-, CD80+, CD1b+, CD40high, CD11chigh, and DC-SIGN+ (CD 209;
Figure 1). Harvested DCs were pelleted and washed with PBS. DCs were then cultured in
medium described above without IL-4 and GM-CSF (i.e. infection medium) for downstream
assays.
Primary blood DCs were isolated by immunomagnetic selection from frozen human
PBMC’s with a blood DC isolation kit (Miltenyi Biotech) following the instructions provided by
the manufacturer. Blood DCs were then cultured in infection medium with or without MTB-
infection.
Mycobacterium strains, culture conditions, cell infections and bead treatment
Mycobacterium tuberculosis strain mc27000 was described before and generously
provided by Dr. William Jacobs (Albert Einstein College of Medicine) and grown in
Acc
epte
d A
rtic
le
This article is protected by copyright. All rights reserved.
Middlebrook 7H9 broth supplemented with albumin, dextrose, and pantothenate (24 μg/mL) at
37ºC with 5% CO2.28, 29 For DC infection, MTB were washed with PBS to remove residual
broth, suspended in infection medium (as described above) at the indicated multiplicity of
infection (MOI), and passaged through a 27-guage needle. Determination of the actual MOI was
performed by plating the inoculum on Middlebrook 7H10 agar plates supplemented with oleic
acid, albumin, dextrose, and pantothenate (24 μg/mL) and grown at 37ºC with 5% CO2 for three
weeks. Irradiated Mycobacterium tuberculosis H37Rv was obtained from BEI Resources
(Manassas, VA). For bead treatments, 10 μL of latex beads (Sigma) were suspended in 1 mL of
infection medium followed by two washes with PBS and again suspended in 1 mL infection
medium. This preparation of beads was diluted one thousand-fold in the DC culture to yield a
ratio of 2 beads per cell.
Flow Cytometry
For cell surface protein expression analysis, 2 x 105 DCs were seeded on 24-well low
binding plates (Nalge Nunc) and infected or subjected to 2 μm latex beads (Sigma) at a quantity
comparable to the MOI. DCs were harvested as described above and suspended in 0.1 mL of
flow cytometry staining buffer (R&D Sciences). Fc receptors were blocked with human FcR
blocking reagent (Miltenyi Biotec) according to supplier recommendation for 30 min at room
temperature with gentle shaking. Cells were then incubated with the specific monoclonal
antibody indicated or isotype control for 30 min at room temperature with gentle shaking. All
monoclonal antibodies were purchased from Ebiosciences and used according to manufacturer
recommendation. Monocloncal antibodies used are as follows: mouse anti-CD18 conjugated to
phycoerythrin (PE), mouse anti-CD11a PE-conjugated, mouse anti-CD11b PE-conjugated,
Acc
epte
d A
rtic
le
This article is protected by copyright. All rights reserved.
mouse anti-CD29 PE-conjugated, mouse anti-CD14 FITC-conjugated, mouse anti-CD1b FITC-
conjugated, mouse anti-CD80/86 PE-conjugated, mouse anti-CD40 PE-conjugated, mouse anti-
MHCII PE-conjugated, and mouse anti-CD54 PE-conjugated. DCs were then washed once
with 1 mL of flow cytometry staining buffer, pelleted, and fixed in 0.5 mL of 4%
paraformaldehyde. A minimum of 5,000 cells per sample were analyzed by flow cytometry on a
FC 500 (Beckman Coulter).
RNA isolation and quantitative RT-PCR
RNA was extracted and isolated using PureZOL (Bio-Rad) following the manufacturer’s
instructions. The RNA was reverse transcribed using an iScript cDNA synthesis kit (Bio-Rad).
The resulting cDNA was diluted 20-fold and real-time PCR performed using iQ SYBR Green
Supermix and an iQ5 thermal cycler (Bio-Rad) with 40 cycles of amplification. Samples were
analyzed in duplicate. Gene expression was normalized to that of GAPDH and expressed
relative to untreated controls using the 2-ΔΔCt method. Oligonucleotide primer sets used were as
follows: GAPDH, 5’-CAGCCGCATCTTCTTTTG-3’(forward), 5’-
GCAACAATATCCACTTTACCA-3’(reverse), integrin β2 (itgb2), 5’-
CACCTACGACTCCTTCTGC-3’(forward), 5’-TGACAAACGACTGCTCCTG-3’ (reverse),
integrin αL (itgal), 5’-CCTCTTCCATGTTCAGCCTC-3’ (forward), 5’-
TTCTCATACACCACGTCAACC-3’ (reverse), and integrin αM (itgam), 5’-
CACGGATGGAGAAAAGTTTGG-3’ (forward), 5-TGGATGCGATGGTATTAAGCT-3’
(reverse).
Acc
epte
d A
rtic
le
This article is protected by copyright. All rights reserved.
Confocal Microscopy
DCs (1 x 105) were seeded on glass coverslips in 24-well plates in infection medium and
either left uninfected or infected with a 5 μM Syto-9 (Life Technologies)-stained log-phase
culture of MTB at a MOI of ~2 for 48 h. At that time the medium was removed and cells were
washed with PBS followed by fixation with 350 μL of 4% paraformaldehyde for 30 min at room
temperature. Cells were then washed thrice with 0.5 mL PBS that contained 0.2% BSA (PB
solution) for 30 min. Next, mouse anti-CD18, mouse anti-CD11a or CD11b (Santa Cruz
Biotechnology) or isotype control diluted in 150 μL PB solution was added for 2 h with gentle
shaking in the dark. DCs were then washed thrice with PB solution and 150 μL of goat anti-
mouse Alexaflour 568-conjugated secondary antibody (Invitrogen) was added for 1 h with
shaking in the dark. Following three washes with PBS, coverslips were mounted on microscope
slides with ProLong® Gold mounting solution (Life Technologies). Confocal microscopy was
performed on a Zeiss LSM 510 Meta.
DC/Endothelial cell adhesion assay
Human microvascular lung endothelial cells (HMVLEC) were used and cultured to
confluency following the manufacturer’s instructions (Lonza). HMVLEC were harvested with a
trypsin/EDTA solution (ThermoScientific), washed with PBS, and seeded at a density of 105 per
well in a 96 well plate in 0.2 mL of infection medium. After 24 h, HMVLEC were activated to
express adhesion molecules by 16 h culture with TNF-α (20 ng/mL). All wells were blocked to
reduce nonspecific DC adhesion with HEPES-buffered RPMI that contained 0.5% BSA for 90
min. Untreated, MTB-infected (MOI ~ 2), LPS-treated, or latex bead-treated (as above) DCs
were stained with 1 mL of a 5 μM Syto-61 (Molecular Probes) solution in HEPES-buffered
Acc
epte
d A
rtic
le
This article is protected by copyright. All rights reserved.
saline (HBS) for 30 min with gentle shaking. DCs were then washed twice with 1 mL of HBS,
suspended in HEPES-buffered RPMI, and 1 x 105 were overlaid onto HMVLEC. DCs were
allowed to adhere for 30 min, then all wells were washed thrice with 0.1 mL of room
temperature HEPES-buffered RPMI to remove non-adherent cells. The cultures were then fixed
with 4% paraformaldehyde and fluorescence was measured with a Synergy plate reader (Biotek)
at an excitation of 620 nm and emission of 645 nm. Each treatment was performed in duplicate.
This was repeated with three independent donors for LPS and six for all other DC treatments.
Transendothelial migration assay
DC migration was assayed using the CytoselectTM Leukocyte Transmigration Assay kit
from Cell Biolabs, Inc. following the instructions provided by the manufacturer. Briefly,
HMVLECs (1 x 105) were seeded on 0.3 μm porous inserts for 48 h for monolayer formation.
TNF-α (20 ng/mL) was used to activate HMVLECs to expression adhesion molecules for 16 h.
DCs (2 x 105) that were cultured in medium alone, stimulated with LPS (200 ng/mL) or infected
with MTB (MOI ~ 2) for 48 h were fluorescently stained and 1 x 105 were overlaid. The
lymphatic chemokines CCL19 and CCL21 (100 ng/mL each) were added to the lower chamber
of the well, and cells were incubated for 24 h. Migrated cells were then lysed, and fluorescence
measured with a Synergy plate reader using an excitation of 480 nm and emission of 520 nm.
Each treatment was performed in duplicate. This was repeated with three independent donors.
Statistical Anlaysis
A student’s t-test was used as indicated to determine statistical significance in the 95%
confidence interval.
Acc
epte
d A
rtic
le
This article is protected by copyright. All rights reserved.
Results
Human dendritic cells infected with M. tuberculosis express reduced levels of cell surface
integrins.
Surface expression levels of the β1 and β2 family of integrins have been shown to be
unchanged during maturation of human DCs in response to LPS.30 We wanted to examine the
effect of MTB infection on human DC integrin expression. First, to confirm our DC
differentiation technique, cells were harvested and immunolabeled against the indicated
molecules and analyzed by flow cytometry. The phenotype of the monocyte-derived DCs
cultured as described is demonstrated in Figure 1. Notably, these cells are CD86high, MHC class
IIhigh, CD11chigh, CD1b+ and CD14-. To begin, we first investigated surface levels of the β
chains that are common among a number of distinct heterodimers. DCs were infected with MTB
(MOI ~ 2) for 48 h, immunolabeled for surface integrins, and analyzed by flow cytometry.
CD18 surface expression was significantly decreased when DCs were infected with MTB (Fig.
2A and B). The mean fluorescent intensity (MFI) from eight independent donors was reduced by
approximately two-fold during infection (p= 0.033, Fig. 2A). The expression of CD18 was
distributed in a bimodial manner for some blood donors; this was observed in both infected and
control cultures. To investigate the possibility that reduced surface levels of CD18 were
mediated by a decrease in CD18 (itgb2) transcription upon infection with MTB, quantitative RT-
PCR was performed. We observed an approximate two-fold mean decrease in CD18 transcripts
over four independent donors that was not statistically significant compared to uninfected DCs
(data not shown). We also evaluated CD29 (β1) integrin surface expression. Although there
was a modest decrease in the surface expression of CD29 during infection, the findings did not
reach statistical significance (Fig. 2C and D). To determine if changes in surface protein
Acc
epte
d A
rtic
le
This article is protected by copyright. All rights reserved.
expression were limited to integrins, the expression of ICAM-1 (CD54) was evaluated on
infected DCs. In contrast to CD18 and CD29, ICAM-1 expression was significantly increased
on MTB-infected DCs (p= 0.012, Fig. 2E and F). This is consistent with previous reports.31, 32
Integrin subunits cell surface distribution and expression are altered following M. tuberculosis
infection.
Figure 2 indicated a specific change in surface expression of CD18 on human DCs during
infection by MTB. To further confirm this result and examine cellular distribution upon
infection with MTB, CD18, CD11a, and CD11b surface expression were evaluated by confocal
microscopy. Consistent with flow cytometric data, we observed that uninfected DCs expressed
higher levels of all three integrin subunits (Fig. 3). In addition, we observed a redistribution of
CD18 during infection. Rather than diffuse localization throughout the cell in the uninfected
control, CD18 was located primarily at the cell periphery in MTB-infected DCs (Fig. 3). The
levels of CD11b were also reduced during infection with some redistribution. In contrast,
CD11a surface expression was reduced when DCs were MTB-infected with more intermediate
redistribution compared to CD11b (Fig. 3). CD18 has been shown to be recruited to
podosomes and focal adhesions where an actin-integrin linkage is required to maintain
stability30. In the absence of the ability to regulate actin organization, CD18 has been shown to
be located at the cell periphery with the consequence of a reduced ability to adhere to ICAM-1.30
By comparison, this suggests that the distribution of CD18 is important to its function in
adherence and motility. Importantly in our experiments, mycobacteria induce a redistribution
that is also accompanied by a loss of cell surface protein levels.
Acc
epte
d A
rtic
le
This article is protected by copyright. All rights reserved.
Reduced CD18 expression is mediated by M. tuberculosis-derived antigen and maintained over
time.
To determine if live bacteria are required for the reduction in CD18 levels, we exposed
DCs to irradiated MTB (uviMTB) at a MOI ~ 2 for 48 h. Irradiated MTB mediate a comparable
reduction in cell surface CD18 to live mycobacteria (Fig. 4A). As a control for phagocytosis,
we included 2 μm latex beads in DC cultures for 48 h and evaluated CD18 levels as before.
Latex beads are ideal since the number supplied does not change as would be the case with other
faster-growing bacteria, and they remove inflammatory signaling as a compounding factor.
Treatment with beads did not lead to a reduction of CD18 surface levels that was comparable to
MTB (Fig. 4A). Cumulatively, this suggests that MTB-derived antigens are responsible for the
observed change in CD18 expression; neither bacterial replication nor secretion are required.
Moreover, CD18 is a component of CR3 used for internalization of MTB by phagocytes. Since
CD18 expression was limited 48 h after infection, we considered the possibility that CD18
surface levels may be reduced as a consequence of mycobacterial internalization. CD18 surface
levels were not strongly influenced by increasing numbers of bacteria in a linear fashion that
would be expected if this was purely a result of phagocytosis. To further evaluate the reduction
kinetics and determine if CD18 surface expression returns to levels observed with uninfected
DCs at later time points, CD18 expression was evaluated at 48 h intervals over a period of six
days. Although there is some increase in CD18 over time with uninfected cells, the reduction in
CD18 expression was maintained over six days in the MTB-infected cultures (Fig. 4C).
Cumulatively, these data suggest a MTB-driven event and indicate that internalization of
complement receptors during MTB phagocytosis is not responsible for the observed reduction in
CD18 expression.
Acc
epte
d A
rtic
le
This article is protected by copyright. All rights reserved.
M. tuberculosis infection results in a reduction of cell surface expressed integrins.
Since we observed a reduction in CD18 expression, we also investigated the effect of
MTB infection on the two α subunits that pair with CD18 to form integrin heterodimers LFA-1
and Mac-1 that are commonly involved in DC adhesion and biology. To do this, DCs were
immunolabeled for CD18 in parallel with each α subunit. MTB infection resulted in reduced
surface expression of both α subunits CD11a and CD11b, respectively (Fig. 5). However, these
did not achieve statistical significance. Reduced surface levels of CD11a or CD11b were not the
product of meaningful changes in gene expression observed over three independent donors (data
not shown).
To confirm our findings using primary blood DCs, these cells were isolated from PBMCs
by immunomagnetic selection. Blood DCs were then either left in cell culture medium or
infected with MTB at a MOI ~ 2 for 48 h. They were then immunolabeled for CD18 in parallel
with each α subunit and analyzed by flow cytometry. Similar to monocyte-derived DCs, blood
DCs exhibited reductions in both LFA-1 (Fig 6. A-C) and Mac-1 (D-F) after infection with
MTB, respectively. The decrease was more substantial for Mac-1, which exhibited more than a
two-fold decrease following infection.
M. tuberculosis-infected DCs exhibit a reduced ability to adhere to lung endothelial cells and
migrate toward a chemokine gradient.
Migration of DC from infected lung to local lymph nodes is preceded by adhesion to and
transmigration through lymphatic endothelia. To determine the functional consequence of
limited integrin expression on the surface of infected DCs, we measured DC adherence to
Acc
epte
d A
rtic
le
This article is protected by copyright. All rights reserved.
primary human microvascular lung lymphatic endothelial cells (HMVLECs). DCs were left
untreated, infected with MTB, or treated with LPS or latex beads for 48 h. Latex beads were
used as a control to determine if cells that have phagocytosed a nonstimulating agent are
generally less adherent. The DCs were then harvested, labeled with Syto-61 and cultured with
endothelial cells for 30 min. Non-adherent DCs were removed by washing and the fluorescence
associated with adhered DCs was quantified. Unstimulated DCs bound efficiently to HMVLECs
and this was not altered by uptake of latex beads. LPS-stimulated DCs bound as efficiently to
HMVLECs as bead-treated DCs (Fig. 7A). However, MTB-infected DCs (MOI ~ 2) exhibited
a significant reduction (p< 0.0001) in their ability to adhere to HMVLECs.
Upon firm adhesion to lymphatic endothelial cells, DCs traverse the endothelium and
enter the afferent lymphatic system for migration to local lymph nodes. CCR7 facilitates DC
migration to lymph nodes. We observed a significant increase (p= 0.047) in CCR7 expressing
DCs upon MTB-infection relative to untreated DCs (Fig. 7B). LPS-treated DCs exhibited a
similar increase in CCR7. To test the ability of MTB-infected DCs to migrate through lung ECs,
we used an in vitro migration assay. DCs were treated with LPS as a positive control, latex
beads as negative control, infected with MTB (MOI ~ 2), or left untreated for 48 h. The DCs
were then placed over a monolayer of activated HMVLECs (1 x 105) and allowed to migrate for
24 h toward CCL19 and CCL21 (lymphatic-produced chemokines) placed in the lower chamber
of the well. MTB-infected DCs exhibit a substantial reduction (p= 0.029) in their ability to
migrate through HMVLECs and towards lymphatic chemokines as compared to the LPS positive
control (Fig. 7C). As expected, latex bead treated cells did not migrate.
Acc
epte
d A
rtic
le
This article is protected by copyright. All rights reserved.
Discussion
DCs in peripheral tissues sample the microenvironment for antigens and upon
recognition migrate through afferent lymphatics to regional lymph nodes. DCs can then present
antigens to T cells and in this way serve as the link between innate and adaptive immunity.
Numerous studies in vivo have estimated that migration is typically complete in 24 h.33-35 The
onset of adaptive immunity during TB suggests that this process is prolonged with DCs
following mycobacterial infection, as compared to other pathogens.36-39 In this study we show
that DCs infected with MTB express reduced levels of cell surface integrins that contain CD18.
This impacts their adhesion to lymphatic endothelial cells and the ability to migrate through a
lung endothelial monolayer toward chemokines in vitro.
Surface levels of CD29, CD18, and α chain partners have previously been shown to be
unaffected by DC maturation.30 We wanted to determine if their expression was altered as a
consequence of infection by MTB. To the best of our knowledge there are no reports that have
examined integrin expression on primary human DCs infected by MTB. There have been
numerous studies conducted with macrophages, monocytes, and murine DCs that report
increases in integrin expression during MTB infection. In one study, human monocyte-derived
macrophages exhibited increased expression of LFA-1 that augmented cellular adhesion.40
Similarly, Mac-1 cell surface expression was increased on murine macrophages upon infection
with MTB in vitro.41 Moreover, it was shown that murine lung DCs increased cell surface
expression of both CD11a and CD11b in response to MTB after 24 hours.42 In contrast, our
findings demonstrate that both monocyte-derived and blood DCs infected with MTB exhibit
reduced expression of surface molecules that facilitate adherence to and transmigration through
Acc
epte
d A
rtic
le
This article is protected by copyright. All rights reserved.
endothelial cells. This does not suggest a motile phenotype capable of initiating a prompt
effector T-cell response. The modulation of adhesive and migratory molecules on DCs may
alter/disrupt the trafficking of these cells and contribute to the delayed onset of adaptive
immunity to MTB in vivo.
Flow cytometric analysis of cell surface integrins sometimes revealed a bimodal
expression pattern, although this was not observed with all donors. Similarly, others have
reported bimodal integrin expression on various cell types including DCs.43-45 It is unclear why
this may be in some cases but there does not appear to be a relationship to MTB infection.
Confocal microscopy showed a redistribution of CD18, CD11a and CD11b when DCs are
infected with MTB; this was not as pronounced for CD11a. Rather than demonstrating a
uniform distribution throughout the cell as observed in the unstimulated group, MTB-infected
DCs exhibited a reduced level of all three integrin subunits examined with surface expression
that was localized mainly to the cell periphery for CD18 and CD11a. While still reduced,
CD11b distribution was not as clearly localized to the cell periphery as the above mentioned. In
a previous report by Burns, et al.30 that examined integrin distribution in LPS-matured DCs,
CD18 and CD11b associated with actin-rich podosomes that were shown in a separate report to
assemble and disassemble during DC maturation.46 However, in the absence of the Wiskott-
Aldrich Syndrome protein that contributes to actin organization, CD18 is localized at the cell
periphery in a manner similar to what is reported here in infected DCs.30 This may suggest an
influence of the actin cytoskeleton during MTB infection. Additionally and unique to MTB-
infected DCs is the reduction of integrin surface levels. In contrast, a four-fold increase in
Acc
epte
d A
rtic
le
This article is protected by copyright. All rights reserved.
ICAM-1 surface expression on MTB-infected DCs suggests a specific influence on integrins
involved in interactions with endothelial cells and DC migration.
A reduction in surface-expressed CD18-containing integrins would be expected to impact
adherence and migration. Antibodies specific for CD18 significantly reduce adherence of human
DCs to ICAM-1.30 Therefore, we wanted to evaluate if there was a functional consequence to
limited integrin surface expression during infection of DCs by MTB. We report a significant
reduction in the ability of MTB-infected DCs to adhere to lung endothelial cells. Latex beads
and LPS-stimulation were used in parallel which resulted in an increase in cellular adhesion, as
opposed to decreased adhesion in MTB-infected DCs. This indicates that MTB-infection, and
not phagocytosis or general activation, are responsible for the decrease in CD18 that impacts
adhesiveness of DCs to endothelial cells. Consistent with reduced adhesion to lung endothelial
cells, we report a significant reduction in the ability of MTB-infected DCs to migrate toward the
lymphatic chemokines CCL19/CCL21. DCs responded and migrated when treated with LPS
suggesting that MTB infection of DCs mediates the reduced migratory capacity. Under the same
conditions, we observe a comparable increase in CCR7 expressing DCs between LPS-treated and
MTB-infected. This indicates that they possess the ability to respond to the above chemokines
and migrate accordingly; further highlighting this inhibition is exclusive to MTB and is
suggestive of a mechanism independent of CCR7. This is in contrast to reports of mycobacteria
inhibiting chemotaxis of DCs through a block in the upregulation of CCR7.47, 48 Our data also
suggest a mechanism by which a loss of surface-expressed integrins impacts migration. In line
with this, several studies have shown that specific blocking of integrins resulted in drastic
reductions in DCs ability to adhere and migrate through endothelial cells.21, 22 In further support
Acc
epte
d A
rtic
le
This article is protected by copyright. All rights reserved.
of impaired DC migration during tuberculosis, another study also found that MTB-infected
murine DCs exhibited an impaired ability to migrate using an in vitro model similar to the one
used in this study.49 Given that adhesion to and migration through endothelial cells is a
prerequisite to reach lymph nodes, this could be a mechanism leading to prolonged presence of
MTB-infected DCs at the site of infection. This could contribute to a high rate of bacterial
growth in the lungs and help explain the delayed onset of an adaptive immune response that
characterizes TB. However, in vivo assays will be needed to further substantiate these ideas.
There are several hypotheses that attempt to account for the delayed adaptive immune
response during TB. Evidence in mice indicates that migration of DCs from the infected lung to
lymph nodes takes at least 8-9 days.36 Studies have shown that DCs reach lymph nodes in much
shorter time frames following uptake and activation by other stimuli.12, 33 One hypothesis has
been that there is insufficient antigen to mount an immune response. Wolf and colleagues50
examined this in vivo by increasing the MTB inoculum per mouse and examined proliferation of
adoptively transferred, transgenic T-cells. They found that even the highest inoculum did not
account for earlier T-cell proliferation. This indicates that the delay seen between infection and
CD4+ T-cell activation is not solely attributed to the low number of bacteria, and further implies
that slow bacterial growth does not explain the slow immune response to MTB. T-cell
proliferation is preceded by DC migration/transport of live MTB to draining lymph nodes and
synapsis with T-cells50, 51--further highlighting an inhibition of DC migration to lymph nodes as
a bottleneck in initiation of adaptive responses. Additionally, the authors provide information
that MTB may directly target DC migration and inhibit antigen presentation in vivo.51 The
authors also report that the limiting step in the development of the adaptive immune response is
Acc
epte
d A
rtic
le
This article is protected by copyright. All rights reserved.
attributed to a delay in the initial activation of CD4+ T-cells. In line with this, a recent report
found that mannose-capped lioparabinomannan (a MTB cell wall component) preferentially
inhibited murine and human Th1 cell migration toward the lymph node egress signal
sphingosine-1-phosphate.52 A recent report directly monitored DC migration into the
mediastinal lymph nodes rather activation of T-cells as a measure of DC arrival in mice
receiving intratracheal delivery of either MTB or purified protein derivative (PPD). The authors
observed disparate patterns in the migration timing of DCs to the mediastinal lymph node.53
DCs from PPD-treated mice were detectable and peeked much earlier (7 days) in the mediastinal
lymph node whereas DCs from MTB-infected mice were not detectable early and did not peek
until much later (21 days). The authors suggested that this may be a mechanism whereby MTB
evades an early expansion of effector T-cells. Our data support those observations and provide a
mechanistic explanation. Collectively, this indicates that MTB is capable of interfering with the
migration of multiple subsets of leukocytes to ultimately delay/subvert the onset of a timely
adaptive immune response.
In conclusion, we report for the first time a reduction in cell surface integrin expression
on primary human DCs infected with MTB. It is clear that a robust host T-cell response is
mounted following infection54 as manifested in the granuloma. However, as mentioned
previously, the time taken for DCs to reach T-cell rich zones in the lymph nodes is delayed and
the mechanisms responsible have remained unknown. Our data suggests that the modulation of
adhesion molecules, adherence to lung endothelial cells, and impaired migration by MTB-
infected DCs could account for the delayed activation and presence of T-cells at the site of
pulmonary TB. Measures aimed at combating the MTB-mediated influence on CD18 integrin
Acc
epte
d A
rtic
le
This article is protected by copyright. All rights reserved.
expression may promote an earlier and more efficient immune response with the hope of limiting
early exponential increase in the bacterial burden.
Acknowledgments
This work was supported by institutional funds supplied by the University of South Carolina
School of Medicine and NIH grant HL093300.
Authorship
LLR contributed to the design of the experiments and was responsible for the performance of
experiments, data analysis, and manuscript preparation. CMR conceived the study idea, designed
experiments, and contributed equally to the preparation of the manuscript.
References
1. Global tuberculosis control report, 2011. World Health Organization.
http://www.who.int/tb/publications/global_report/2011/gtbr11_full.pdf
2. Marino S, Pawar S, Fuller C, Reinhart T, Flynn JL, Kirschner DE. Dendritic cell trafficking
and antigen presentation in the human immune response to Mycobacterium tuberculosis. J
Immunol 2004; 173:494-506.
3. Wallgren A. The time-table of tuberculosis. Tubercle 1948; 29:245–51
4. Cooper AM. Cell-mediated immune responses in tuberculosis. Annu Rev Immunol 2009;
27:393-422.
Acc
epte
d A
rtic
le
This article is protected by copyright. All rights reserved.
5. Baron SD, Singh R, Metzger DW. Inactivated Francisella tularensis live vaccine strain
protects against respiratory tularemia by intranasal vaccination in an immunoglobulin A-
dependent fashion. Infect Immun 2007; 75:2152-62.
6. Chakravarty S, Cockburn, IA, Kuk S, Overstreet MG, Zavala F. CD8+ T lymphocytes
protective against malaria liver stages are primed in skin-draining lymph nodes. Nat Med
2007; 13:1035-41.
7. Kursar M, Bonhagen K, Kohler A, Mittrucker HW. Organ-specific CD4+ T cell response
during Listeria monocytogenes infection. J Immmunol 2002; 168:6382-7.
8. Lira R, Doherty M, Modi G, Sacks D. Evolution of lesion formation, parasitic load, immune
response, and reservoir potential in c57bl/6 mice following high and low-dose challenge with
Leishmania major. Infect Immun 2000; 68:5176-82.
9. McSorley SJ, Asch S, Costalonga M, Reinhardt RL, Jenkins MK. Tracking Salmonella-
specific CD4 T cells in vivo reveals a local mucosal response to a disseminated infection.
Immunity 2002; 16:365-77.
10. Moskophidis D, Kioussis D. Contribution of virus-specific CD8+ cytotoxic T cells to virus
clearance or pathologic manifestations of influenza virus infection in a T cell receptor
transgenic mouse model. J Exp Med 1998; 188:223-32.
11. Srinivasan A, Foley J, Ravindran R, McSorley SJ. Low-dose Salmonella infection evades
activation of flagellin-specific CD4 T cells. J Immunol 2004; 173:4091-99.
12. Grayson MH, Ramos MS, Rohlfing MM, Kitchens R, Wang HD, Gould A, Agapov E,
Holtman, MJ. Controls for lung dendritic cell maturation and migration during respiratory
viral infection. J Immunol 2007; 179:1438-48.
Acc
epte
d A
rtic
le
This article is protected by copyright. All rights reserved.
13. Herrmann JL, Lagrange PH. Dendritic cells and Mycobacterium tuberculosis: which is the
Trojan horse? Pathol Biologie 2006; 53:35-40.
14. Tailleux L, Schwartz O, Herrmann JL, Pivert E, Jackson M, Amara A. DC-SIGN is the
major Mycobacterium tuberculosis receptor on human dendritic cells. J Exp Med 2003; 197:121-7.
15. Randolph GJ. Dendritic cell migration to lymph nodes: cytokines, chemokines, and lipid
mediators. Semin Immunol 2001; 13:267-74.
16. Riol-Blanco L, Sanchez N, Torres A, Tejedor A, Narumiya S, Corbi A, Mateos P,
Rodriguez-Fernandez J. The chemokine receptor CCR7 activates in dendritic cells two
signaling molecules that independently regulate chemotaxis and migratory speed. J Immunol
2005; 174:4070-80.
17. Hanekom WA, Mendillo M, Manca C, Haslett P, Siddiqui M., Barry C, Kaplan G.
Mycobacterium tuberculosis inhibits maturation of human monocyte-derived dendritic cells
in vitro. J Infect Dis 2003; 188:257-66.
18. Puig-Kroger A, Sanz-Rodriquez F, Longo N, Sanchez-Mateos P, Botella L, Teixido J,
Bernabeu C, Corbi AL. Maturation-dependent expression and function of the CD49d
integrin on monocyte-derived human dendritic cells. J Immunol 2000; 165:4338-45.
19. Ammon C, Meyer P, Schwarzfischer L, Krause SW, Anderseen R, Kreutz M. Comparative
analysis of integrin expression on monocyte-derived macrophages and monocyte-derived
dendritic cells. Immunol 2000; 100:364-69.
20. Ghosh S, Chackerian AA, Parker CM, Ballantyne CM, Behar SM. The LFA-1 adhesion
molecule is required for protective immunity during pulmonary Mycobacterium tuberculosis
infection. J Immunol 2006; 176:4914-22.
Acc
epte
d A
rtic
le
This article is protected by copyright. All rights reserved.
21. D’Amico G, Bianchi G, Bernasconi S, Bersani L, Piemonti L, Sozzani S, Mantovani A,
Allavena, P. Adhesion, transendothelial migration, and reverse transmigration of in vitro
cultured dendritic cells. Blood 1998; 92:207-14.
22. De la Rosa G, Longo N, Rodriguez-Fernandez J, Puig-Kroger A, Pineda A, Corbi AL,
Sanzhez-Mateos P. Migration of human blood dendritic cells across endothelial cell
monolayers: adhesion molecules and chemokines involved in subset-specific transmigration.
J Leuko Biol 2003; 73:639-49.
23. Johnson LA, Clasper S, Holt AP, Lalor PF, Baban D, Jackson DG. An inflammation-
induced mechanism for leukocyte transmigration across lymphatic vessel endothelium. J Exp
Med 2006; 203:2763-77.
24. Balkow S, Heinz S, Schmidbauer P, Kolanus W, Holzmann B, Grabbe S, Laschinger M.
LFA-1 activity state on dendritic cells regulates contact duration with T-cells and promotes
T-cell priming. Blood 2010; 116:1885-94.
25. Smits HH, de Jong EC, Schuitemaker JHN, Geijenbeek TBH, van Kooyk Y, Kapsenberg
ML, Wierenga EA. Intercellular adhesion molecule-1/LFA-1 ligation favors human Th1
development. J Immunol 2002; 168:1710-16.
26. Varga G, Nippe N, Balkow S, Peters T, Wild M, Seeliger S, Beissert S, Krummen M, Roth J,
Sunderkotter C, Grabbe S. LFA-1 contributes to signal 1 of T-cell activation and to the
production of Th1 cytokines. J Invest. Derm 2010; 130:1005-12.
27. Carlson PE, Carroll JA, O’Dee DM, Nau GJ. Modulation of virulence factors in Francisella
tularensis determines human macrophage responses. Microb Pathog 2007; 42:204-14.
28. Sambandamurthy VK, Derrick SC, Hsu T, Chen B, Larsen MH. Mycobacterium
tuberculosis ΔRD1 ΔpanCD: A safe and limited replicating mutant strain that protects
Acc
epte
d A
rtic
le
This article is protected by copyright. All rights reserved.
immunocompetent and immunocompromised mice against experimental tuberculosis.
Vaccine 2006; 24:6309-20.
29. Sambandamurthy VS, Wang X, Chen B, Russell R, Collins F. A pantothenate auxotroph of
Mycobacterium tuberculosis is highly attenuated and protects mice against tuberculosis. Nat
Med 2002; 8:1171-74.
30. Burns S, Hardy S, Buddle J, Yong K, Jones G, Thrasher A. Maturation of DC is associated
with changes in motile characteristics and adherence. Cell Mot Cytoskel 2004; 57:118-32.
31. Henderson R, Watkins S, Flynn JL. Activation of human dendritic cells following infection
with Mycobacterium tuberculosis. J Immunol 1997; 159:635-43.
32. Mihret A, Mamo G, Tafesse M, Hailu A, Parida S. Dendritic cells activate and mature after
infection with Mycobacterium tuberculosis. BMC Res Note 2011; 4:247-53.
33. Macatonia SE, Knight SC, Edwards AJ, Fryer P. Localization of antigen on lymph node
dendritic cells after exposure to the contact sensitizer fluorescein isothiocyanate. Functional
and morphological studies. J Exp Med 1987; 166:1654-67.
34. Kupiec-Weglinski JW, Austyn JM, Morris JP. Migration patterns of dendritic cells in the
mouse. Traffic from the blood, and T cell-dependent and independent entry to lymphoid
tissues. J Exp Med 1988; 176:632-45.
35. Cumberbatch M, Kimber I. Dermal tumor necrosis factor-alpha induces dendritic migration
to draining lymph nodes, and possibly provides one stimulus for Langerhans’ cell migration.
Immunol 1992; 75:257-63.
36. Reiley WW, Calayag MD, Wittmer ST et al. ESAT-6 specific CD4 T cell responses to
aerosol Mycobacterium tuberculosis infection are initiated in the mediastinal lymph nodes.
Proc Natl Acad Sci 2008; 105:10961-66.
Acc
epte
d A
rtic
le
This article is protected by copyright. All rights reserved.
37. Roman E, Miller E, Harmsen A, Wiley J, von Adrian U, Huston G, Swain S. CD4 Effector T
cell subsets in the response to influenza: heterogeneity, migration, and function. J Exp Med
2004; 196:957-68.
38. Chakerian AA, Alt JM, Perera TV, Behar SM. Dissemination of Mycobacterium
tuberculosis is influenced by host factors and precedes the initiation of T-cell immunity.
Infect Immun 2002; 70:4501-09.
39. Flynn KJ. Virus-specific CD8+ T cells in primary and secondary influenza pneumonia.
Immunity 1998; 8:683-91.
40. Desjardin LE, Kaufman TM, Potts B, Kutzbach B, Yi H, Schlesinger LS. Mycobacterium
tuberculosis-infected human macrophages exhibit enhanced cellular adhesion with increased
expression of LFA-1 and ICAM-1 and reduced expression and/or function of complement
receptors, FcγRII and the mannose receptor. Microbiol 2002; 148:3161-71.
41. Ghosh S, Saxena RK. Early effect of Mycobacterium tuberculosis infection on Mac-1 and
ICAM-1 expression on mouse peritoneal macrophages. Exp Mol Med 2004; 36:387-95.
42. Gonzalez-Juarrero M, Orme IM. Characterization of murine lung dendritic cells infected
with Mycobacterium tuberculosis. Infect Immun 2001; 69:1127-33.
43. Li, A, Simmons, P, Kaur, P. Identification and isolation of candidate human keratinocyte
stem cells based on cell surface phenotype. Proc. Natl. Acad. Sci. 1998; 95:3902-3907.
44. Andreason, S, Thomsen, A, Christensen, J. Expression and functional importance of
collagen-binding integrins α1β1 and α2β1, on virus activated T cells. J. Immunol. 2003;
171:2804-2811.
Acc
epte
d A
rtic
le
This article is protected by copyright. All rights reserved.
45. Tjomsland V, Ellegard R, Kjolhede P, Larsson M. Blocking of integrins inhibits HIV-1
infection of human cervical mucosa immune cells with free and complement-opsonized
virions. Eur. J. Immunol. 2013; 43:1-12.
46. West MA, Wallin RP, Matthews SP, Svensson HG, Zaru R, Ljunggren HG, Prescott AR, Watts C.
Enhanced dendritic cell antigen capture via toll-like receptor-induced acting remodeling. Science 2004; 305:1153-57.
47. Rajashree P, Supriya P, Das SD. Differential migration of human monocyte-derived
dendritic cells after infection with prevalent clinical strains of Mycobacterium tuberculosis.
Immunobiol 2008; 213:567-75.
48. Sanarico N, Colone A, Grassi M, Mariani F. Different transcriptional profiles of human
monocyte-derived dendritic cells infected with distinct strains of Mycobacterium tuberculosis
and Mycobacterium bovis Bacillus Calmette-Guérin. Clin Devel Immunol 2011; 1-14.
49. Blomgran R, Ernst JD. Lung neutrophils facilitate activation of naïve antigen-specific CD4+
T cells during Mycobacterium tuberculosis infection. J Immunol 2011; 186:7110-19.
50. Wolf J, Desvignes L, Linas B, Banaiee N, Tamura T, Takatsu K, Ernst JD. Initiation of the
adaptive immune response to Mycobacterium tuberculosis depends on antigen production in
the local lymph node, not the lungs. J Exp Med 2008; 205:105-15.
51. Wolf AJ, Linas B, Tervejo-Nunez G, Kincaid E, Tumara T, Takatsu K, Ernst JD.
Mycobacterium tuberculosis infects dendritic cells with high frequency and impairs their
function in vivo. J Immunol 2007; 179:2509-19.
52. Richmond J, Lee J, Green D, Kornfeld H, Cruikshank W. Mannose-capped
lipoarabinomannan from Mycobacterium tuberculosis preferentially inhibits sphingosine-1-
phosphate-induced migration of Th1 cells. J Immunol 2012; 189:5886-95.
Acc
epte
d A
rtic
le
This article is protected by copyright. All rights reserved.
53. Garcia-Romo G, Pedroza-Gonzalez, A, Flores-Romo L. Mycobacterium tuberculosis
manipulates pulmonary APCs subverting early immune responses. Immunobiology 2013;
218:393-401.
54. Lopez-Ramierz GM, Rom W, Ciotoli C, Martiniuk F, Cronstein B, Reibman J.
Mycobacterium tuberculosis alters expression of adhesion molecules on monocytic cells.
Infect Immun 1994; 62:2515-20.
FIGURE LEGENDS
Figure 1. Culture of adherent monocytes with IL-4 and GM-CSF results in a characteristic
DC phenotype.
Monocytes were isolated from human peripheral blood by sequential density gradient
centrifugations followed by adhesion in serum free medium. Adherent monocytes were then
washed twice with PBS and cultured in medium containing IL-4 and GM-CSF for 7 days. After
7 days, cells were harvested and immunolabeled with monoclonal antibodies against the
indicated molecule or isotype control and analyzed by flow cytometry. A representative scatter
plot and histogram overlays are shown; grey= isotype control.
Figure 2. M. tuberculosis infection results in a reduction of cell surface integrin expression.
Primary human DCs were cultured in medium alone or infected with MTB (MOI ~ 2), harvested,
immunolabeled for CD18 (A-B), CD29 (C-D), or CD54 (E-F) and analyzed by flow cytometry.
The mean fluorescent intensity (MFI) ± standard error for eight (A) or three independent donors
(C and E) along with a representative histogram overlay (B, D, and F; grey= isotype control;
thin line= infected; bold black line= medium alone) for each molecule is displayed. Statistical
significance in the 95% confidence interval was determined by a student’s t-test.
Acc
epte
d A
rtic
le
This article is protected by copyright. All rights reserved.
Figure 3. Cell surface distribution and expression of integrin subunits is altered by M.
tuberculosis infection.
To visually assess CD18, CD11a and CD11b expression and distribution, primary human DCs
were infected with syto-61 (green)-stained MTB. Integrin subunits were immunolabeled (red)
and analyzed by confocal microscopy. The expression of all three integrin subunits is reduced
upon MTB-infection. CD18 and CD11a are redistributed from a uniform pattern in medium
alone to the cell periphery in MTB-infected cells. CD11b exhibits a uniform cellular distribution
in either condition. A representative of three independent donors demonstrating similar results is
shown.
Figure 4. CD18 expression decrease is mediated by MTB-derived antigen and maintained
over time.
Primary human DCs were treated as indicated and CD18 cell surface expression evaluated by
flow cytometry. (A) A representative experiment of three performed with different donors
displaying the MFI of CD18 in response to fluorescent beads, ultraviolet-inactivated MTB
(uviMTB), and live MTB at a MOI ~ 2. (B) CD18 MFI ± standard error in response to
increasing numbers of MTB (MOI ~ 0.1 to 10) for three combined experiments with separate
donors. (C) CD18 MFI ± standard error for DCs cultured in in medium alone or infected with
MTB (MOI ~ 2) over six days. The data represents three combined experiments done with
different donors.
Acc
epte
d A
rtic
le
This article is protected by copyright. All rights reserved.
Figure 5. M. tuberculosis infection results in a reduction of cell surface expressed integrin
heterodimers and individual alpha subunits.
DCs were cultured in medium alone or infected with MTB (MOI ~ 2), harvested, and either
double- immunolabeled for CD18/CD11a (LFA-1) or CD18/CD11b (Mac-1) and analyzed by
flow cytometry. Representative scatter plots of LFA-1 on medium alone cells (A) or MTB-
infected (B) are shown. Representative scatter plots of Mac-1 on medium alone cells (D) and
MTB-infected (E) are shown. Overlaid histogram representations of the same data sets highlight
the changes in a CD11a (C) or CD11b (F) surface expression. (G) The mean fluorescent
intensity (MFI) ± standard error for CD11a (αL) and CD11b (αM); five combined experiments
done with independent donors are shown. Representative histogram overlays for CD11a (C) and
CD11b (F) are shown; grey= isotype control; thin line= infected; bold black line= medium alone.
Figure 6. M. tuberculosis infected primary blood DCs exhibit reduced cell surface
expression of both LFA-1 and Mac-1.
To confirm our previous findings on blood DCs, these cells were isolated from PBMCs by
immunomagnetic selection. Blood DCs were then either left in cell culture medium or infected
with MTB at a MOI ~ 2 for 48 h. Cells were then immunolabeled for either LFA-1
(CD18/CD11a, A-C) or Mac-1 (CD18/CD11b, D-F) and analyzed by flow cytometry. Shown
are scatter plots of a representative donor, and the MFI ± standard error for two donors.
Statistical significance in the 95% confidence interval was determined by a student’s t-test.
Acc
epte
d A
rtic
le
This article is protected by copyright. All rights reserved.
Figure 7. M. tuberculosis infected DCs exhibit a reduced ability to adhere to lung
endothelial cells and migrate toward lymphatic chemokines.
(A) Human microvascular lung endothelial cells (HMVLECs) were seeded in 96-well plates and
activated to express adhesion molecules with TNF-α (20 ng/mL). DCs were cultured in medium
alone, infected with MTB, treated with LPS (200 ng/mL) or latex beads for 48 h. The DCs were
then harvested, syto-61-stained and placed over the HMVLECs for 30 min. Fluorescence
indicative of DC adherence was measured by spectrophotometry. Data is expressed as average
percent decrease in relative fluorescent units (RFU) ± standard error relative to medium alone.
Data represents three separate donors for LPS and six for all other DC treatments. Each
treatment contained a replicate. Statistical significance in the 95% confidence interval was
determined by a student’s t-test. (B) CCR7 on resting (medium), LPS stimulated (200 ng/mL)
and MTB-infected (MOI ~ 2) DCs was measured by flow cytometry. Shown are percent CCR7
positive cells over four independent blood donors. (C) HMVLECs were seeded on porous
inserts and cultured for 48 h. TNF-α (20 ng/mL) was added to induce expression of adhesion
molecules overnight. DCs (1 x 105) that were cultured in medium alone, LPS-treated (200
ng/mL), or infected with MTB (MOI ~ 2) were fluorescently stained and overlaid. The
lymphatic chemokines CCL19 and CCL21 (100 ng/mL) were added to the lower chamber of the
well. The cultures were incubated for 24 h. Replicate well-readings were taken for each DC
treatment and the data is expressed as the mean percent change in relative fluorescent units
(RFU) ± standard error relative to medium alone. Data represents three experiments done with
separate donors. Statistical significance in the 95% confidence interval was determined by a
student’s t-test; n.s.= not significant.
Acc
epte
d A
rtic
le
This article is protected by copyright. All rights reserved.
Acc
epte
d A
rtic
le
This article is protected by copyright. All rights reserved.
Acc
epte
d A
rtic
le
This article is protected by copyright. All rights reserved.
Acc
epte
d A
rtic
le
This article is protected by copyright. All rights reserved.