mycobacterium tuberculosis infection of human dendritic cells decreases integrin...

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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

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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

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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.

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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

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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

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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

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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,

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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).

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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

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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.

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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

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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.

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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.

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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

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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.

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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

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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

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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

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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

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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

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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.

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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.

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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.

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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.

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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.

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