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Lipopolysaccharides from Bordetella

pertussis and Bordetella parapertussis

Differently Modulate Human Dendritic Cell

Functions Resulting in Divergent Prevalence

of Th17-Polarized Responses

Giorgio Fedele, Maria Nasso, Fabiana Spensieri, Raffaella

Palazzo, Loredana Frasca, Mineo Watanabe and Clara M.

Ausiello

J Immunol 2008; 181:208-216; ;

http://www.jimmunol.org/content/181/1/208

This article cites 49 articles,

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Copyright © 2008 by The American Association of

Immunologists All rights reserved.

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Lipopolysaccharides from Bordetella pertussis and Bordetella

parapertussis Differently Modulate Human Dendritic Cell

Functions Resulting in Divergent Prevalence of

Th17-Polarized Responses 1

Giorgio Fedele,* Maria Nasso,* Fabiana Spensieri,* Raffaella Palazzo,* Loredana Frasca,*

Mineo Watanabe, 2† and Clara M. Ausiello 3 *

Bordetella pertussis and B. parapertussis are the etiological agents of pertussis, yet the former has a higher incidence and is the

cause of a more severe disease, in part due to pertussis toxin. To identify other factors contributing to the different pathogenicity

of the two species, we analyzed the capacity of structurally different lipooligosaccharide (LOS) from B. pertussis and LPS from B.

parapertussis to influence immune functions regulated by dendritic cells. Either B. pertussis LOS and B. parapertussis LPS triggered

TLR4 signaling and induced phenotypic maturation and IL-10, IL-12p40, IL-23, IL-6, and IL-1 production in human

monocyte-derived dendritic cells (MDDC). B. parapertussis LPS was a stronger inducer of all these activities as compared with B.

pertussis LOS, with the notable exception of IL-1, which was equally produced. Only B. parapertussis LPS was able to induce

IL-27 expression. In addition, although MDDC activation induced by B. parapertussis LPS was greatly dependent on soluble CD14,

B. pertussis LOS activity was CD14-independent. The analysis of the intracellular pathways showed that B. parapertussis LPS and

B. pertussis LOS equally induced IB and p38 MAPK phosphorylation, but B. pertussis LOS triggered ERK1/2 phosphorylation

more rapidly and at higher levels than B. parapertussis LPS. Furthermore, B. pertussis LOS was unable to induce MyD88independent

gene induction, which was instead activated by B. parapertussis LPS, witnessed by STAT1 phosphorylation and

induction of the IFN-dependent genes, IFN regulatory factor-1 and IFN-inducible protein-10. These differences resulted in a

divergent regulation of Th cell responses, B. pertussis LOS MDDC driving a predominant Th17 polarization. Overall, the data

observed reflect the different structure of the two LPS and the higher Th17 response induced by B. pertussis LOS may contribute

to the severity of pertussis in humans. The Journal of Immunology, 2008, 181: 208–216.

Pertussis or whooping cough is an acute respiratory disease

caused by Bordetella pertussis or B. parapertussis, two

strictly human pathogens (1). These two bacteria possess

overlapping subsets of virulence factors, with the notable exception

of pertussis toxin, which is not expressed by B. parapertussis

(2). Whooping cough in neonates infected with B. pertussis develops

huge bacterial loads and massive lymphocytosis, and

may be lethal. In contrast, severe disease was never observed

following B. parapertussis infection (3–5). Although pertussis

toxin is considered to play a critical role for disease occurrence

*Department of Infectious, Parasitic and Immune-mediated Diseases, Anti-infectious

Immunity Unit, Istituto Superiore di Sanità, Rome, Italy; and † Department of Molecular

Genetics, Biochemistry and Microbiology, University of Cincinnati, Cincinnati,

OH 45267-0524

Received for publication March 3, 2008. Accepted for publication April 30, 2008.

The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordance

with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by Grants 5303 and 28C6 from Istituto Superiore di

Sanità-National Institutes of Health (USA) Scientific Cooperation Agreement, by

Grant 6ACF/6 from the Italian Ministry of Health, Istituto Superiore di Sanità, Sixth

AIDS Project and l’Agenzia Italiana del Farmaco Project (to C.M.A.), and Contract

LSHP-CT-2003-503240 from the Commission of the European Communities, Sixth

Framework Program, “Mucosal Vaccines for Poverty-Related Diseases.”

2 Current address: Graduate School of Infection Control Sciences, Kitasato University

5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan.

3 Address correspondence and reprint requests to Dr. Clara M. Ausiello, Department

of Infectious, Parasitic and Immune-mediated Diseases, Anti-infectious Immunity

Unit, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy. E-mail

address: clara.ausiello@iss.it

www.jimmunol.org

The Journal of Immunology

and severity (6, 7), it has also been suggested that pertussis

toxin may not play a decisive role in causing the typical symptoms

of whooping cough (8).

Another major difference between the two species is represented

by the structure of their LPS. These molecules share a degree of

common architecture, both having a penta-acylated lipid A and a

branched-chain core oligosaccharide, yet also structural differences.

B. pertussis lipooligosaccharide (LOS) 4 lacks an O-side

chain, having in its place a nonrepeating trisaccharide, whereas B.

parapertussis LPS has an O-Ag structure consisting of a homopolymer

of 2,3-dideoxy-2,3-di-N-acetylgalactosaminuronic

acid (9–11).

LPS is a critical molecular pattern, exerting a major role in the

host-bacteria relationship, particularly driving a number of activation

processes in professional and nonprofessional APC such as

macrophages and dendritic cells (DC). Thus, differences in LPS

structures may fundamentally affect differences in pathogenesis

(12).

To evaluate whether structural differences in LPS between the

two species may in some way explain different pathogenicity, the

impact of B. pertussis LOS and B. parapertussis LPS was studied

on human monocyte-derived DC (MDDC), an ex vivo model that

4 Abbreviations used in this paper: LOS, lipooligosaccharide; IP, IFN-inducible protein;

IRF, IFN regulatory factor; HEK, human epithelial kidney; EBI, EBV-induced

gene; DC, dendritic cell; MDDC, monocyte-derived DC; SEAP, secreted alkaline

phosphatase.

Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00

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The Journal of Immunology

fold increase

10

8

6

4

2

0


0 0.01 0.1 1

allows the evaluation of aspects of the regulation of immune

response.

DC are professional APC distributed in lymphoid and nonlymphoid

organs with the ability to translate innate into adaptive immunity

(13). They express a wide array of receptors specialized in

pathogen recognition, permitting detection and identification of

pathogen-associated molecular patterns (14, 15). TLR4 is required

for detection of LPS (15, 16), together with other proteins directly

involved in ligand-binding such as MD-2, which mediates subsequent

receptor activation, and CD14, which controls ligand presentation

to the TLR4/MD-2 complex (17). The interactions between

LPS and the TLR4 receptorial complex crucially influence

the amplitude of cellular responses (12, 17).

We previously showed that B. pertussis LOS induces partially

mature MDDC not expressing IL-12p70. Such committed MDDC

are able to induce Th2 polarization in vitro (18). In this study, to

propose a differential pathogenetic role of LPS molecules from B.

parapertussis and B. pertussis, we adopted a strategy based on

comparative analysis of the ability of B. pertussis LOS and B.

parapertussis LPS to induce TLR4-mediated signaling and the

functions that are specifically acquired by MDDC upon maturation.

Escherichia coli LPS served as control. The consequent approach

was the identification of MDDC intracellular pathways potentially

helping in the transduction of activation signals. The last

approach was the exploration of the regulatory role exerted by B.

pertussis LOS and B. parapertussis LPS on MDDC in the polarization

of T cells, which may have a crucial role in Bordetella

pathogenicity.

Materials and Methods

Reagents

*

µg/ml



*

*° ‡

none

BpLOS

BppLPS

EcLPS

FIGURE 1. Triggering of TLR4 receptorial complex in transfected

HEK 293 cells. HEK/TLR4/pNifty2-SEAP cells were either untreated

(none) or treated with B. pertussis LOS (BpLOS), B. parapertussis LPS

(BppLPS), or E. coli LPS (EcLPS) at the indicated doses for 16 h. SEAP

activity in supernatants of cell cultures was then measured. Data are reported

as the fold induction of SEAP activity over untreated activity. Results

represent mean SE of four independent experiments. , p 0.05

vs untreated cells; °, p 0.05 vs B. pertussis LOS-treated cells; and ‡, p

0.05 vs B. parapertussis LPS-treated cells.

Ultrapure E. coli LPS from O111:B4 strain, Pam 2CSK 4 (synthetic bacterial

lipoprotein S-[2,3-bis(palmitoloxy)-(2RS)-propyl]-[cysteinyl-[S]-lysil-[S]lysine

x 3 CF3COOH), p44/42 MAPK (ERK1/2) inhibitor PD98059, and

p38 MAPK inhibitor SB203580 were purchased from InvivoGen. Human

recombinant GM-CSF and recombinant IL-4 were from R&D Systems.

Culture supernatants (4%, v/v) of IL-4–62 cell line were also used as a

source of IL-4 (19). Recombinant IL-2 was obtained from Roche. Fluorochrome-conjugated

anti-human CD1a, CD14, CD38, CD80, CD83, and

CD86 mAbs and purified anti-human IFN--inducible protein (IP)-10 mAb

were from BD Biosciences. Anti-human CD14-neutralizing mAb was from

*

*

MFI

% positive cells

R&D Systems. Rabbit polyclonal IgG anti-phospho-STAT1 (Tyr 701 ), antiphospho-ERK1/2

(Thr 202 /Tyr 204 ), anti-phospho-p38 MAPK (Thr 180 /

Tyr 182 ), and anti-phospho-IB (Ser 32 ) were from Cell Signaling Technology.

Mouse anti--tubulin and brefeldin A were from Sigma-Aldrich.

Mouse anti-STAT1 was from Transduction Laboratories.

B. parapertussis and B. pertussis LPS molecule purification

B. pertussis LOS from BP338 strain and B. parapertussis LPS from

CN8234 strain were purified and characterized as described (20). By SDS-

PAGE characterization, purified B. pertussis LOS revealed distinct band A

and band B, whereas B. parapertussis LPS revealed ladder-like bands

showing that the B. parapertussis LPS has an O-Ag polysaccharide; the

parallel gel that was stained to check protein contamination revealed no

bands (20). Protein content checked by Bradford assay (Bio-Rad) was below

detection limit.

Cell lines

160

120

80

40

100

0

75

50

25

*

*



CD80 CD86


*° ‡

*° ‡

none

BpLOS

BppLPS

EcLPS

0

CD83 CD38

FIGURE 2. Induction of human MDDC maturation. MDDC either untreated

(none) or treated with B. pertussis LOS (BpLOS) (1 g/ml), B.

parapertussis LPS (BppLPS) (1 g/ml), or E. coli LPS (EcLPS) (1 g/ml)

for 48 h and analyzed for indicated surface markers associated with mature

phenotype. Fluorescence data are reported as median fluorescence intensity

(MFI) (top) when treatment increased the expression of the marker in cells

that were already positive (CD80, CD86); otherwise, the percentage of

positive cells (CD83, CD38) was used (bottom). Mean expression SE of

eight independent experiments is indicated. , p 0.05 vs untreated cells;

°, p 0.05 vs B. pertussis LOS-treated cells; and ‡, p 0.05 vs B.

parapertussis LPS-treated cells.

Human epithelial kidney (HEK) 293 cells stably transfected with human

TLR4, MD-2 and CD14 (HEK/TLR4) or with human TLR2 (HEK/TLR2)

were purchased from InvivoGen. The HEK/TLR clones were grown in

DMEM (Life Technologies), supplemented with heat-inactivated 10%

LPS-screened FCS (Limulus amoebocyte lysate 1 ng/ml; HyClone Laboratories)

supplemented with 1 mM sodium pyruvate, 0.1 mM nonessential

amino acids, 2 mM L-glutamine (all from HyClone Laboratories), and Normocin

(100 g/ml; InvivoGen). HEK/TLR4 culture medium was supplemented

with 0.2 mM blasticidin (InvivoGen) and HygroGold (50 g/ml;

InvivoGen). HEK/TLR2 culture medium was supplemented with 0.6 mM

G418 sulfate (InvivoGen).

Stable transfection with NF-B-inducible reporter plasmid and

TLR signaling assay

HEK/TLR cells were transfected with a plasmid encoding secreted alkaline

phosphatase (SEAP, pNifty2-SEAP; InvivoGen) as previously described

*

*



*° ‡

none

BpLOS

BppLPS

EcLPS

209

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210 Bordetella LOS OR LPS DRIVE DIFFERENTIAL Th17 POLARIZATION

FIGURE 3. Analysis of cytokine

expression by human MDDC. A,

MDDC were either untreated (none)

or treated with B. pertussis LOS

(BpLOS) (1 g/ml), B. parapertussis

LPS (BppLPS) (1 g/ml), or E. coli

LPS (EcLPS) (1 g/ml) for 48 h. IL-

12p70, IL-10, IL-12p40, IL-6, IL-23,

and IL-1 release in culture medium

was assessed by ELISA. Values are

expressed as mean SE from eight

(for IL-12 p70 and IL-10), five (for

IL-12p40 and IL-1), and three (for

IL-6 and IL-23) independent experiments

and expressed as picograms per

milliliter of cytokine released. B,

MDDC were treated as in A, and total

RNA extracted at the indicated time

points. Kinetics of mRNA expression

for EBI3 and p28 were evaluated

by real-time quantitative RT-PCR.

mRNA transcript levels were expressed

as fold increase over those in

unstimulated MDDC at 5 h. Results of

three independent experiments are expressed

as mean SE. , p 0.05 vs

untreated cells; °, p 0.05 vs B. pertussis

LOS-treated cells; and ‡, p

0.05 vs B. parapertussis LPS-treated

cells.

(18). HEK/TLR/pNifty2-SEAP cells were either untreated or treated for

16 h with B. pertussis LOS, B. parapertussis LPS, ultrapure E. coli LPS,

or Pam 2CSK 4 at a dose range from 0.01 to 1 g/ml. Supernatants were then

incubated with QUANTI-Blue (InvivoGen), and SEAP activity was measured

by reading OD at 655 nm with a 3550 UV Microplate Reader

(Bio-Rad).

Purification and culture of MDDC

CD14 monocytes were purified and cultured as previously described (19),

At day 7, MDDC were analyzed for CD1a and CD14 expression by use of

a FACScan flow cytometer (BD Biosciences). MDDC were further cultured

for 48 h in the presence of 1 g/ml B. pertussis LOS, B. parapertussis

LPS, or E. coli LPS to induce maturation. When indicated, MDDC were

pretreated, using a predetermined optimal dose, with neutralizing anti-

CD14 mAb (10 g/ml), or ERK1/2 inhibitor PD98059 (100 M) or p38

inhibitor SB203580 (20 M) for 1 h and further stimulated.

Polarization of T lymphocytes

To evaluate the potential of MDDC to polarize T lymphocytes, experiments

were performed using allogeneic T cells purified from PBMC by

negative sorting with magnetic beads (pan-T cell kit; Miltenyi Biotec) (21).

MDDC (0.5 10 5 /ml) and T cells (0.5 10 6 /ml) were cultured in complete

medium in 24-well plates (Corning). On day 5, IL-2 (50 U/ml) was

added. On day 12, supernatants were harvested for cytokine measurement.

Immunophenotypic analysis

MDDC were incubated with specific fluorochrome-conjugated mAbs for

immunophenotypic analysis. Isotype-matched Abs (BD Biosciences) were

A

pg/ml

pg/ml

pg/ml

B

Fold increase

800

600

400

200

0

60000

45000

30000

15000

0

4000

3000

2000

1000

0

1600

1200

800

400

0

IL-12p70

none BpLOS BppLPS EcLPS

IL-12p40

*

none BpLOS BppLPS EcLPS

*

IL-23




*° ‡


none BpLOS BppLPS EcLPS

IL-10


4000

*°‡

pg/ml

pg/ml

pg/ml

5 h

20 h

3000

2000

1000

used as negative control. Cells were analyzed with a FACScan (BD Biosciences).

Fluorescence data are reported as a percentage of positive cells

when treatment induces the expression of the marker in cells that were

negative; median fluorescence intensity was used when treatment increased

the expression of the marker in cells that were already positive.

Cytokine measurement by ELISA

MDDC culture supernatants were collected, and cytokines assayed by

ELISA specific for IL-1, IL-6, IL-10, IL-12p40, IL-12p70 (Quantikine;

R&D Systems), and IL-23 (Bender MedSystems). The lower detection limits

were 1.0 pg/ml for IL-1, 0.7 pg/ml for IL-6, 3.9 pg/ml for IL-10, 15.0

pg/ml for IL-12p40, 5.0 pg/ml for IL-12p70, and 20.0 pg/ml for IL-23. OD

was read at 450 nm with a 3550 UV Microplate Reader (Bio-Rad).

Cytokines in the supernatants from polarized T cells were assayed by

ELISA specific for IFN-, IL-5, and IL-17 (Quantikine; R&D Systems).

The lower detection limits were 8.0 pg/ml for IFN-, 3.0 pg/ml for IL-5,

and 15.0 pg/ml for IL-17.

IP-10 intracellular staining

0

12000

10000

8000

6000

4000

2000

0

250

200

150

100

50

EBI3 p28

50000

none BpLOS BppLPS EcLPS

0

Fold increase

none BpLOS BppLPS EcLPS

40000

30000

20000

10000

0

IL-6

none BpLOS BppLPS EcLPS

IL-1

none BpLOS BppLPS EcLPS

none BpLOS BppLPS EcLPS

MDDC were incubated with different stimuli. After 1 h, brefeldin A, a

compound that blocks proteins in the endoplasmic reticulum, was added

(10 g/ml) and cells further cultured for 5 h. MDDC were then fixed and

permeabilized using Cytofix/Cytoperm and Perm/Wash protocols (BD Biosciences)

and then stained with pretitrated anti-IP-10 mAb or an appropriate

isotype control, followed by incubation with FITC-conjugated goat

anti-mouse Ig (DakoCytomation). Cells were analyzed by FACScan (BD

Biosciences).

*

*

*



*


*

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The Journal of Immunology

mRNA cytokine expression by TaqMan real-time RT-PCR

analysis

To measure cytokine mRNA expression, TaqMan RT-PCR analysis was

used (Applied Biosystems). Total RNA was extracted from MDDC at different

time points and reverse transcription was conducted as previously

described (21, 22). PCR was performed, amplifying the target cDNA transcripts

and the -actin cDNA as endogenous control. Specific primers and

probes were obtained from Applied Biosystems. mRNA transcript levels

were expressed as fold increase compared with basal condition.

Western blot analysis

MDDC were starved by culturing overnight in culture medium supplemented

with 1% LPS-screened FCS. Cells were then stimulated and lysed

at the indicated time points as previously described (23). Proteins were

separated by 12% SDS-PAGE and transferred onto a nitrocellulose membrane

(GE-Healthcare) and immunoreactive proteins were detected by incubating

blots (23). Specific phosphorylation levels were measured by Image

Station 2000R (Kodak), and data were expressed in pixel intensity

arbitrary units.

Statistical analysis

All data were recorded in a computerized database. Results are reported as

mean SEM. Statistical analyses were conducted using the SPSS 13.0

software. Differences between mean values were assessed by Student’s t

test. The statistical significance was set at p 0.05.

Results

B. parapertussis and B. pertussis LPS molecules differentially

trigger TLR4 receptorial complex

To identify the ability of B. parapertussis LPS and B. pertussis

LOS to engage TLR4 receptorial complex, we used HEK 293 cells

expressing TLR4, MD-2 and CD14, and stably transfected with

pNifty2-SEAP, a reporter gene under the control of a NF-B-dependent

promoter (18). Penta-acylated B. parapertussis LPS and

B. pertussis LOS triggered TLR4/MD-2/CD14-dependent signaling

at each dose tested. B. parapertussis LPS triggered signaling

more efficiently than B. pertussis LOS, particularly at lower doses

(Fig. 1). However, Bordetella LPS induced lower reporter gene

expression compared with hexa-acylated E. coli LPS. To evaluate

whether LPS from Bordetella may activate TLR2, as described for

other LPS molecules (24), a similar experiment was performed

using pNifty2-SEAP transfected HEK/TLR2 cells (18). Both types

of LPS were unable to trigger TLR2-dependent signal transduction

at any dose tested, whereas Pam2CSK4, a synthetic TLR2 ligand,

efficiently induced the expression of the reporter gene (data not

shown).

Overall this set of experiments documented that TLR4 receptorial

complex is triggered at different intensity by structurally different

LPS and underlined a hierarchy that correlated with the

degree of lipid A acylation and the presence of a distal O-chain, E.

coli LPS being a stronger stimulus compared with B. parapertussis

LPS, which in turn proved more potent than B. pertussis LOS.

B. parapertussis and B. pertussis LPS molecules differentially

promote MDDC maturation and function

The acquisition of a mature phenotype by MDDC in response to B.

parapertussis LPS and B. pertussis LOS was studied. As shown in

Fig. 2, upon stimulation, immature cells shifted to a mature phenotype,

witnessed by enhanced expression of maturation (CD83,

CD38) and costimulatory (CD80, CD86) molecules. The degree of

phenotypic maturation was dependent on the stimulus used, showing

the same hierarchy observed upon stimulation of TLR4 receptorial

complex. Indeed, maximal maturation was induced by E. coli

LPS, whereas B. parapertussis LPS was a more efficient inducer

compared with B. pertussis LOS, achieving statistical significance

relative to all markers studied.

MFI

% positive cells

pg/ml

100

80

60

40

20

0

90

75

60

45

30

15

0

1200

900

600

300

0

CD80

none Bp LOS BppLPS EcLPS

CD83

none BpLOS BppLPS EcLPS

IL -10

none BpLOS BppLPS EcLPS

ctr mAb

anti-CD14

ctr mAb

anti-CD14

ctr mAb

anti-CD14

FIGURE 4. Neutralization of soluble CD14. MDDC were either untreated

(none) or treated with B. pertussis LOS (BpLOS) (1 g/ml), B.

parapertussis LPS (BppLPS) (1 g/ml), or E. coli LPS (EcLPS) (1 g/ml)

for 48 h either in the absence or presence of a neutralizing anti-CD14 mAb

(10 g/ml) or irrelevant control mAb (ctr), as indicated. Expression is

reported as median fluorescence intensity (MFI) when treatment increased

the expression of the marker in cells that were already positive (CD80);

otherwise, the percentage of positive cells (CD83) was used as described in

Fig. 2. IL-10 release in culture medium was assessed by ELISA and expressed

as picograms per milliliter of cytokine released as described in Fig.

3. Results of three independent experiments are expressed as mean SE.

, p 0.05 anti-CD14 mAb vs control mAb.

B. parapertussis LPS- and B. pertussis LOS-induced cytokine

production by MDDC was then analyzed (Fig. 3A). Both Bordetella

LPS were inducers of a set of immunoregulatory cytokines

including IL-10, IL-12p40, IL-23, IL-6, and IL-1. E. coli LPS

was the only stimulus able to drive IL-12p70 secretion. In fact,

neither B. pertussis LOS, as already described (18), nor B. parapertussis

LPS were inducers of this cytokine. In addition, E. coli

LPS was a more potent cytokine inducer than B. parapertussis

LPS, which in turn promoted statistically significant higher levels

of cytokine production compared with B. pertussis LOS, with the

notable exception of IL-1 (Fig. 3A).

Fig. 3B showed gene expression analysis of EBV-induced

gene (EBI)3 and p28 as subunits of heterodimeric IL-27, which

is a cytokine belonging to the IL-12 family endowed with important

immunoregulatory functions and implicated in the

down-regulation of Th17 cells (25, 26). Data demonstrated a

differential behavior of Bordetella LPS because B. parapertussis

LPS induced EBI3 and p28 subunit transcription of IL-27,

whereas B. pertussis LOS was able to induce only the EBI3

subunit (Fig. 3B). E. coli LPS induced the expression of both

IL-27 subunits (Fig. 3B).

*

*

*

*

*

*

211

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212 Bordetella LOS OR LPS DRIVE DIFFERENTIAL Th17 POLARIZATION

A

β-tubulin

B

5’ 15’ 30’ 5’ 15’ 30’ 5’ 15’ 30’ 5’ 15’ 30’ 5’ 15’ 30’ 5’ 15’ 30’ 5’ 15’ 30’ 5’ 15’ 30’ 5’ 15 ’ 30’ 5’ 15’ 30’ 5’ 15’ 30’ 5’ 15’ 30’

pIκBα pp38 pERK1/2

Intensity

MFI

300

150

50

25

0

100

80

60

40

20

0

none BpLOS BppLPS EcLPS

*

*

CD80

° * *

*

*

*

*

B. parapertussis and B. pertussis LPS molecules differentially

trigger CD14-mediated signaling

It has been proposed that CD14 contributes to TLR4 signaling via

binding to the LPS distal O-chain (27). Although MDDC are

CD14-negative, soluble CD14 present in the serum added to the

cell culture medium may take part into LPS signaling in this ex

vivo model (28). To evaluate the involvement of CD14 in B. pertussis

LOS- and B. parapertussis LPS-induced signaling, MDDC

were stimulated in the presence of a neutralizing anti-CD14 mAb

to prevent the interactions of LPS with soluble CD14. CD80,

CD83, and IL-10 induction were then measured as a readout for

MDDC maturation and activation. The results obtained showed

that the activity of E. coli LPS and B. parapertussis LPS rely on

soluble CD14 binding. Indeed, the addition of the anti-CD14 mAb

significantly reduced the expression of CD80, CD83, and IL-10

release (Fig. 4). Remarkably, signals delivered by B. pertussis

LOS were shown to be CD14-independent.

B. parapertussis and B. pertussis LPS molecules induce

signaling pathways involved in MDDC functions

The intracellular signal transduction events triggered by LPS from

Bordetella in MDDC were then investigated. It is known that exposure

to E. coli LPS results in DC activation through the receptor

complex TLR4/MD-2/CD14 and subsequent recruitment of Toll/

IL-1R domain-containing adaptor molecules to the cytoplasmic

domain of the receptor. These adaptors include MyD88 (29). Recruitment

of the MyD88 adaptor initiates a MyD88-dependent

pathway that culminates in the early activation of NF-B, which

plays an important role in DC maturation and function (30). In

% positive cells

β-tubulin β-tubulin

60

45

30

15

Intensity

80

60

40

20

0

*

Intensity

0

none BpLOS BppLPS EcLPS none BpLOS BppLPS EcLPS

CD83

*

0

0

none BpLOS BppLPS EcLPS none BpLOS BppLPS EcLPS none BpLOS BppLPS EcLPS

*

*

pg/ml

3000

2500

2000

1500

1000

500

60

45

30

15

*

°

IL-10

*

*

*

*

ERK1/2 inhibitor

P38 inhibitor

FIGURE 5. Analysis of intracellular signaling in human MDDC. A, MDDC were either untreated (none) or treated with B. pertussis LOS (BpLOS) (1

g/ml), B. parapertussis LPS (BppLPS) (1 g/ml), or E. coli LPS (EcLPS) (1 g/ml). Phosphorylation of IB, p38, and ERK1/2 was determined at the

indicated time points by Western blot. Results are reported as both blots and phosphorylated protein (p) level measured by densitometric analysis. Data

are shown as pixel intensity (for ERK1/2 the sum of p42 plus p44 is reported) from one of four independent experiments. B, MDDC were treated as in

A, either in the absence or presence of ERK1/2 inhibitor PD98059 or p38 inhibitor SB203580 for 48 h. Expression is reported as median fluorescence

intensity (MFI) when treatment increased the expression of the marker in cells that were already positive (CD80); otherwise, the percentage of positive cells

(CD83) was used as described in Fig. 2. IL-10 release in culture medium was assessed by ELISA and expressed as picograms per milliliter of cytokine

released as described in Fig. 3. Results of four independent experiments are expressed as mean SE. , p 0.05 inhibitors vs control treatment; and °,

p 0.05 vs p38 inhibitor.

parallel, a MyD88-independent pathway results in a late-phase activation

of NF-B (29).

To evaluate the Bordetella LPS signaling, we first evaluated

MyD88-dependent pathway by the analysis of NF-B and the

MAPK pathways induction. To follow the NF-B activation, we

focused on IB phosphorylation because this process is necessary

to induce nuclear translocation of NF-B. LPS from Bordetella

promoted IB phosphorylation, notwithstanding E. coli LPS

being the stronger inducer of IB-phosphorylated protein especially

at later time points (Fig. 5A).

Concerning MAPK family members, we measured p38 and p44/

p42 (ERK1/2), which have been described as master regulators of

DC maturation and inducers of cytokine transcription (31). B. pertussis

LOS and B. parapertussis LPS both induced phosphorylation

of p38 and ERK1/2. However, B. pertussis LOS showed ability

to induce a more pronounced ERK1/2 phosphorylation,

especially at early time points (Fig. 5A).

This specific induction of signaling pathways was confirmed using

inhibitors of ERK1/2 (PD98059) and p38 (SB203580), measuring

CD80, CD83, and IL-10 induction as readout of cell maturation and

activation, respectively (Fig. 5B). When cells were treated with B.

pertussis LOS, CD80 expression and IL-10 production were significantly

reduced by both inhibitors, and remarkably, inhibition of

ERK1/2 was significantly more potent compared with p38 inhibition.

When B. parapertussis LPS and E. coli LPS were used,

CD80 and IL-10 were significantly reduced by both inhibitors,

with no statistically significant difference. CD83 expression

was inhibited only by p38 inhibitor independently of the stimulus

used. Overall these data confirmed a predominant role for

ctr

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The Journal of Immunology

A

B

C

pSTAT1

STAT1

300

240

180

120

60

0

IItensity

fold increase

% positive cells

150

120

90

60

30

80

60

40

20

0

0

none BpLOS BppLPS EcLPS

IRF1

none BpLOS BppLPS EcLPS

IP10

none BpLOS BppLPS EcLPS

5 h

20 h

ERK1/2-mediated signaling when MDDC are stimulated with

B. pertussis LOS, whereas B. parapertussis LPS appears to act

through the activation of both p38 and ERK1/2.


*° ‡

FIGURE 6. Analysis of TLR4-dependent MyD88-independent gene induction.

A, MDDC were either untreated (none) or treated with B. pertussis

LOS (BpLOS) (1 g/ml), B. parapertussis LPS (BppLPS) (1 g/ml), or E. coli

LPS (EcLPS) (1 g/ml) for 2 h. Phosphorylation of STAT1 was determined by

Western blot. Data are reported as both blots and phosphorylated protein (p)

level measured by densitometric analysis and shown as pixel intensity. Data

are from one representative experiment of four independent experiments. B,

MDDC were treated as in A and total RNA extracted at the indicated time

points. Kinetics of mRNA expression for IRF-1 was evaluated by real-time

quantitative RT-PCR. mRNA transcript levels were expressed as fold increase

over those in unstimulated MDDC at 5 h. One representative experiment is

shown of three performed. C, MDDC were treated as in A for 1 h, and brefeldin

A was added. After further incubation for 5 h, intracellular staining for

IP-10 was performed. Results of three independent experiments are expressed

as mean percentage SE of positive cells. , p 0.05 vs untreated cells; °,

p 0.05 vs B. pertussis LOS-treated cells; and ‡, p 0.05 vs B. parapertussis

LPS-treated cells.

pg/ml

5000

4000

3000

2000

1000

0


B. parapertussis LPS activates TLR4-dependent

MyD88-independent gene induction

MyD88-independent signaling is responsible for the activation of

intracellular pathways that lead to the induction of IFN- and IFNinducible

genes (29, 32). To study this aspect in more depth, we

analyzed by Western blot the phosphorylation of the transcription

factor STAT1, pivotal in the regulation of this pathway (29). Fig.

6A, shows that B. pertussis LOS induced only a weak STAT1

phosphorylation, with a modest increase with respect to untreated

cells, whereas B. parapertussis LPS promoted substantial levels of

phosphorylated protein. E. coli LPS stimulation induced high levels

of phosphorylated STAT1, confirming other studies in human

MDDC (32). The expression of the IFN-inducible gene IFN regulatory

factor (IRF)-1 was then studied. As shown in Fig. 6B, both

B. parapertussis LPS and E. coli LPS stimulation induced high

levels of IRF-1, and in contrast, B. pertussis LOS was unable to

promote consistent mRNA transcription. IP-10 expression, which

is a marker of MyD88-independent pathway (33), was then assessed

(Fig. 6C). B. parapertussis LPS increased the percentage of

MDDC positive for IP-10 staining, reaching statistical significance

in comparison to B. pertussis LOS and to untreated cells. B. pertussis

LOS did not enhance the number of IP-10-positive cells with

respect to untreated MDDC. E. coli LPS promoted high levels of

IP-10-expressing cells.

Overall these data indicate that B. parapertussis LPS, differently

from B. pertussis LOS, activates the MyD88-independent pathway

in MDDC, although not to its full potential, as witnessed by intermediate

phospho-STAT1 and IP-10 level induction.

Bordetella LPS-matured MDDC drive the expansion of

polarized Th2/Th17 effectors

In a previous study we demonstrated that B. pertussis LOS-matured

MDDC skewed Th cell polarization toward a Th2 phenotype

(18). In this study, we have shown that both B. pertussis LOS and

B. parapertussis LPS induce in MDDC IL-23, IL-6, and IL-1 (but

not IL-12p70), a cytokine profile compatible with the induction of

Th17 lymphocytes (34–36). To verify this point, polarization experiments

were performed by coculturing purified allogeneic T

lymphocytes with MDDC stimulated with the different LPS molecules

(Fig. 7). Both B. pertussis LOS- and B. parapertussis LPStreated

MDDC induced statistically significant higher levels of

IL-5 and IL-17 as compared with untreated and E. coli LPS-treated

MDDC. However, IL-17 was induced to significantly greater

amounts by B. pertussis LOS as compared with B. parapertussis

LPS, suggesting a predominant Th17 polarization.

Noteworthy, MDDC maturated by B. parapertussis LPS were

more prone to drive expansion of IFN--producing Th1 cells, as

IFN-γ IL-5

IL-17

*

1200

1000

800

‡ *

*

1000

750 *° ‡


*

pg /ml

600

400

200

0

0

none BpLOS BppLPS EcLPS none BpLOS BppLPS EcLPS none BpLOS BppLPS EcLPS

FIGURE 7. T lymphocyte polarization. MDDC either untreated (none) or treated with B. pertussis LOS (BpLOS) (1 g/ml), B. parapertussis LPS (BppLPS)

(1 g/ml), or E. coli LPS (EcLPS) (1 g/ml) for 48 h were cocultured with purified T cells as described in Materials and Methods. On day 12, supernatants were

collected, and secreted cytokines were measured by ELISA. Results are mean SE of four independent experiments. Data are expressed as picograms per milliliter

of cytokine released. , p 0.05 vs untreated cells; °, p 0.05 vs B. parapertussis LPS-treated cells; and ‡, p 0.05 vs E. coli LPS-treated cells.

*

pg /ml

500

250

* ‡

213

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214 Bordetella LOS OR LPS DRIVE DIFFERENTIAL Th17 POLARIZATION

shown by the IFN- levels measured in coculture supernatants,

which were significantly higher compared with IFN- cells induced

by untreated MDDC and intermediate compared with B.

pertussis LOS- or E. coli LPS-treated MDDC. E. coli LPS stimulation

drove the expansion of Th1 effectors producing high levels

of IFN- and low levels of IL-5 and IL-17, confirming results

reported in our study and other studies (18, 36).

Discussion

Several earlier studies indicated that B. pertussis infection promotes

a Th1 immune response, based largely on IFN- production

(37–39). We have previously demonstrated that incubation of human

MDDC with B. pertussis induced a Th1 predominant polarization

and expression of IL-23, involved in Th17 cell expansion,

in the absence of the Th1-inducing cytokine IL-12p70 (21, 22).

Furthermore, evidence is accumulating to show that Bordetella

infection might bias the host response toward a Th17 response (40,

41). In this study, novel data support this view.

Bordetella LPS triggered TLR4 signaling, but their signaling

potency was reduced in comparison to E. coli LPS because we

found that E. coli LPS, bearing a hexa-acylated lipid A, is a stronger

activator of the TLR4 receptorial complex and inducer of DC

responses compared with penta-acylated lipid A present in B.

parapertussis LPS and B. pertussis LOS.

However, the number of acylated lipid A chains is not the only

factor influencing the amplitude of LPS-induced response because

B. parapertussis LPS was significantly more potent than B. pertussis

LOS in determining the intensity of TLR4 receptor signaling.

It was recently reported that the presence of a distal O-chain

is required for LPS binding to CD14, and that the intensity of LPS

activation parallels the presence of the polysaccharide portion and

consequently triggers the induction of MyD88-independent signaling

and maximal TLR4 response (12, 27). Because B. pertussis

LOS lacks an O-side chain and B. parapertussis LPS has an O-Ag

structure, to understand the structural basis for the differences observed,

we inhibited the interactions that may have taken place

with soluble CD14 molecules present in cell culture medium. The

results were consistent with our study hypothesis; in fact, E. coli

LPS and B. parapertussis LPS activities were dramatically inhibited

by the presence of the neutralizing anti-CD14 mAb, whereas

B. pertussis LOS stimulation was unaltered. Differences in CD14

usage by Bordetella LPS may explain the apparent contrast between

our findings and the results published by Mann and colleagues

(42), who have reported that B. pertussis LOS is a more

efficacious stimulator of TLR4-transfected HEK 293 cells compared

with B. parapertussis LPS. However, the cell line used by

Mann et al. (42) did not express CD14, thus nullifying the contribution

of B. parapertussis LPS distal O-chain to TLR4 signaling.

Differences in structure of B. pertussis LOS and B. parapertussis

LPS are at the basis of quantitative and qualitative differences

observed in intracellular pathways activated by Bordetella LPS. B.

pertussis LOS triggered ERK1/2 phosphorylation more rapidly

and at higher levels than B. parapertussis LPS, and a preferential

role for ERK1/2 in B. pertussis LOS-mediated MDDC activation

was confirmed by using specific MAPK inhibitors. Furthermore, B.

pertussis LOS was unable to induce MyD88-independent gene induction,

which was instead activated by B. parapertussis LPS,

although at low levels. This pathway leads to the induction of

IFN-, which in turn, activates STAT1 phosphorylation, leading to

the induction of several IFN-inducible genes, including IP-10 (32,

33). TLR4-induced type I IFN was shown to be involved in the

expression of the p35 subunit of IL-12p70, probably through the

induction of IRF family members such as IRF-1 and IRF-8 (32,

43). In this study, we showed that B. parapertussis LPS, although

unable to induce IL-12p35 as verified by quantitative RT-PCR

(data not shown) and inferred by the detection of IL-12p40 but not

IL-12p70 dimeric protein, promoted STAT1 phosphorylation and

the expression of IRF-1 and IP-10. These findings provide evidence

in support of the activation of the TLR4-MyD88-independent

pathway (29, 32, 33).

Differences in intracellular pathways triggered by Bordetella

LPS in MDDC reflect the diverse ability of B. pertussis LOS and

B. parapertussis LPS to induce MDDC activation. B. parapertussis

LPS was a significantly more potent activator compared with B.

pertussis LOS in all MDDC functions examined in this study, including

up-regulation of maturation markers and expression of IL-

10, IL-12p40, IL-23, and IL-6 cytokines. The notable exception

was IL-1, recently described as a pivotal cytokine in Th17 polarization

(36), equally induced by B. pertussis LOS and B. parapertussis

LPS. Another principal difference between B. pertussis

LOS- and B. parapertussis LPS-induced MDDC activation was the

ability of B. parapertussis LPS to promote the transcription of the

IL-27 subunits, EBI3 and p28. The capacity of B. parapertussis

LPS to induce IL-27p28 expression again supports its ability to

activate the TLR4-MyD88-independent pathway, as reported by

recent studies showing the involvement of the MyD88-independent

pathway and IRF-1 in up-regulation IL-27p28 subunit

(44, 45).

The different ability shown by B. pertussis LOS and B. parapertussis

LPS to activate intracellular pathways and cytokine induction

in MDDC might determine differences in the regulation of

Th cell responses during infection by Bordetella. The induction

and expansion of Th17 cells in humans has been described recently

to be dependent on the production of IL-1, IL-6, and IL-23,

whereas IL-12p70 possesses an inhibitory activity. Furthermore, it

was demonstrated that IL-1 is fundamental in Th17 differentiation,

whereas IL-6 and IL-23 enhance Th17 cell expansion (36).

MDDC treated with LPS from Bordetella produced high levels of

IL-6, IL-1, and IL-23 in the absence of IL-12p70. Of interest, B.

pertussis LOS induced a relatively higher amount of IL-1, giving

support for a possible major involvement in Th17 differentiation.

Indeed, we found that purified T cells cocultured with B. pertussis

LOS-driven or B. parapertussis LPS-driven MDDC expressed IL-

17. Interestingly, the capacity of B. parapertussis LPS to induce

IL-27 expression in MDDC may be linked to the reduced IL-17

and enhanced IFN- levels measured in T cell B. parapertussis

LPS-MDDC cocultures as compared with T cell B. pertussis LOS-

MDDC cocultures. IL-27, initially characterized as a proinflammatory

cytokine with Th1-inducing activity (25) was subsequently

attributed important immunoregulatory functions in vivo, specifically

down-regulating Th17 cells (26, 46, 47).

B. pertussis LOS- or B. parapertussis LPS-matured MDDC

drove the expansion of IL-5-producing Th2 cells, as previously

demonstrated for B. pertussis LOS (18). It is conceivable that Th2

polarization occurs by default, due to absence of IL-12p70 and

production of IL-10. However, it is not possible to rule out that a

specific Th2-polarizing signal is induced by Bordetella LPS, and

further studies are needed to identify which surface molecules or

cytokines are involved.

Overall these results suggest that B. pertussis infection biases

the host immune system toward a Th17 response through the action

exerted by B. pertussis LOS, in accordance with the recent

hypothesis that Th17 may be one major cause of cough pathology

in pertussis (41). These data are also in agreement with that obtained

in mice, in which vaccination with a whole cell pertussis

vaccine, known to contain LOS in high concentration, promotes

the expansion of IL-17-producing T cells dependent on TLR4 signaling,

as well as expansion of Th1 cells (48). It has also been

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The Journal of Immunology

shown that B. bronchiseptica induces a strong Th17 immune response

both in vitro and in vivo (40). IL-17-producing T cells may

determine in patients a sustained inflammation of the airways causing

cough, as reported for other respiratory infections (49). In contrast,

B. parapertussis LPS is a stronger activator of innate immune

response compared with B. pertussis LOS in terms of DC maturation

and cytokine production, and mediates a less robust Th17

polarization with enhanced levels of Th1 cells.

In conclusion, we provide in this study evidences that the different

immunomodulatory properties are related to the diverse

structures of Bordetella LPS molecules and may contribute to explaining

differences in the natural history of diseases caused by B.

pertussis and B. parapertussis; however, these data should be confirmed

in infected patients. Furthermore, differences in LPS structures

might not be the only factors involved in the differential

balance of Th cell polarization in Bordetella infections; other virulence

factors may indeed be relevant in vivo.

Acknowledgments

The editorial assistance of Adam Nixon is gratefully acknowledged. We

thank Antonio Cassone for constructive discussions, support, and critical

reading of the manuscript and to Alison Weiss for critical reading of the

manuscript.

Disclosures

The authors have no financial conflict of interest.

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