TLR4 is involved in mediating fatal murine pneumonia due to <i>Burkholderia cenocepacia</i>

doi:10.4236/oji.2011.13012

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Vol.1, No.3, 97-102 (201

doi:10.4236/oji.2011.13012

1) Open Journal of Immunology

TLR4 is involved in mediating fatal murine pneumonia

due to Burkholderia cenocepacia

Viviane Balloy1,2#, Heidi Nagel1,2#, Reuben Ramphal3, Mustapha Si-Tahar1,2#,

Michel Chignard1,2#*

1Unité de Défense Innée et Inflammation, Institut Pasteur, Paris, France;

2INSERM U874, Paris, France; *Corresponding Author: [email protected]

3Department of Medicine, University of Florida, Gainesville, Florida, USA.

Received 14 April 2011; revised 15 July 2011; accepted 11 August 2011.

ABSTRACT

Background: We previously showed that MyD88

knocked out mice were protected from death

due to B. cenocepacia pneumonia implying that

a toll-like receptor(s) (TLR) was involved in me-

diating death. The aim of the present study w as

to determine which TLR(s) was involved in trig-

gering the inflammatory response responsible

for the pathogenesis. We specifically focus on

the TLRs 4 a nd 5, as th e s e two recept ors are t he

main ones involved in the recognition of P. ae-

ruginosa, a flagellated Gram-bacterium similar

to B. cenocepacia. Methods: Mice were inf-

ected intratracheally with a suspension of B. ce-

nocepacia. Animals were then observed daily

for signs of morbidity. Alternatively, bronchoal-

veolar lavages (BAL) w ere collected at different

time points to further determine cytokine con-

centrations and the number of CFU of B. ceno-

cepacia. Results: The data clearly indicate that

the innate immune response of the host to B.

cenocepacia lung infection was due to TLR4

that senses the pathogen while TLR5 does not

do so in vivo. As with the MyD88-/- strain, TLR4-/-

mice were protected from death and cytokine

and chemokine synthesis to infection were re-

duced. The only paradoxical observation was the

reduced pathogen burden in the case of TLR4-/-

mice compared to the enhanced (but transient)

pathogen burden observed with MyD88-/- mice,

suggesting that another TLR was involved in

bacterial clearance. Conclusion: The dat a clearly

demonstrate a deleterious implication of TLR4

in the host to B. cenocepacia lung infection.

Keywords: Rodent; Bacterial; Inflammation;

Innate Immunity; Lung

1. INTRODUCTION

Burkholderia cepacia complex (Bcc) strains have

emerged as problematic opportunistic pathogens causing

severe infections in patients with immune suppression,

chronic granulomatous disease and cystic fibrosis [1-3].

Infections due to Bcc are specially a serious concern to

CF patients due to their inherent antibiotic resistance and

high potential for patient-to-patient transmission [4]. At

least 17 Bcc species have been recovered from respira-

tory secretions of CF patients in many countries [5-6],

but B. cenocepacia is the most common species isolated

[5,7-8]. The clinical outcomes of Bcc infections range

from asymptomatic carriage to a fulminant and fatal

pneumonia, the so-called “cepacia” syndrome [9]. There

is increasing evidence that the acute pulmonary deterio-

ration associated with B. cenocepacia infection is the con-

esquence of a marked inflammatory host response [10].

This could be due to lipopolysaccharide (LPS) as it has

been shown that LPS purified from Bcc species exhibits

potent proinflammatory activity mediated through Toll-

like receptor (TLR) 4 [11], and may account, at least in

part, for the sepsis-like cepacia syndrome [10-12].

TLR recognize conserved pathogen-associated mo-

lecular patterns and trigger the activation of genes in-

volved in innate defense. Four main TLRs are involved

in sensing bacteria, TLR2 in tandem with TLR1 or 6

detects the presence of lipopeptides and peptidoglycan,

TLR4 in association with CD14 and MD2 recognizes

LPS, TLR5 detects the presence of flagellin, and TLR9

recognizes hypomethylated DNA [13-15].

Numerous groups have established the importance of

TLRs in pulmonary host defense and this laboratory has

demonstrated their involvement during influenza A [16],

Aspergillus fumigatus [17,18], and Pseudomonas aeru-

ginosa [19-21] infections. Recently, we showed that

# These authors contributed equally to the work.

V. Balloy et al. / Open Journal of Immunology 1 (2011) **-**

MyD88, a key downstream adapter for most of the TLRs,

was involved the death due to B. cenocepacia pneu mo-

nia suggesting a role of TLRs in the pathogenesis [22].

Thus, MyD88 knocked out mice (MyD88-/-) were pro-

tected from death following B. cenocepacia lung infec-

tion. We demonstrated that the pathogenesis was due to a

hyperinflammatory host response mediated by TNF-



The aim of the present study was to determine which

TLR(s) is playing a role in the recognition of B. cenoce-

pacia and triggering of hyperinflammation. We specifi-

cally focus on the TLR4 and 5, knowing that these two

receptors are the main actors in the recognition of P.

aeruginosa, a flagellated Gram- bacterium which like B.

cenocepacia is a highly problematic pathogen for indi-

vidual with CF [19,20,23-25].

2. METHODS

2.1. Bacterial Strain and Growth Conditions

B. cenocepacia of the epidemic ET12 lineage (strain

J2315) was provided by the Pasteur Institute microor-

ganisms depository. Bacteria were grown on tryptic soy

agar (TSA) at 33˚C for 48 h. Single colonies removed

from the plate were grown in 5 mL of tryptic soy broth

at 33˚C with shaking for 16 h - 18 h, corresponding to

midlog phase. Bacteria were harvested by centrifugation

(3,000 ×g for 15 min), resuspended in saline and optical

densities of the suspensions were adjusted to give the

desired bacterial concentration which was verified by

serial dilutions and plating on TSA.

2.2. Mouse Strains

TLR4-/- and TLR5-/- mice were obtained from S.

Akira (Osaka University, Osaka, Japan). All mice

were backcrossed at least eight times with C57BL/6

to ensure similar genetic backgrounds. Double knock-

ed-out TLR4,5-/- mice were generated by breeding

TLR4-/- mice and TLR5-/- mice. C57/BL6 mice from

which these mice were derived were used as the control

mice. These latter mice were supplied by the Centre

d’Elevage R. Janvier, Le Genest Saint-Isle, France and

used at about 8 weeks of age. Upon arrival for experi-

mentation mice were fed normal mouse chow and

water ad libitum and were housed under standard

conditions with air filtration. Mice were cared for in

accordance with Pasteur Institute guidelines in com-

pliance with the European animal welfare regulation.

2.3. Immunosuppressive Treatment and

Infection

As mice are relatively resistant to B. cenocepacia and

generally do not die from pulmonary infection unless

encased in agar beads, we developed a lethal model in

the neutropenic mouse [22], where wild type mice die

after an intratracheal challenge with about 4 - 5 × 107 cfu.

Chemotherapy-induced neutropenia was achieved by the

intravenous administration of 5 mg/kg of the antineo-

plastic drug vinblastine (Cell Pharm GmbH, Germany)

66 h before infection. Under these conditions, the hae-

matological profile of the mice after vinblastine and

during the infection displayed total polymorphonuclear

cell depletion from day 0 (the day of infection) until day

2 for both wild-type and TLR-/- mice. Of note, in sepa-

rate investigations, we previously demonstrated that with

this vinblastine regimen, mouse survival and cytokine

production are similar to those obtained when neutro-

penia is induced with the antigranulocyte monoclonal

antibody RB6-8C5, [26]. Animals were infected intra-

tracheally under general anesthesia achieved with a

mixture of ketamine (40 mg/kg) and xylazine (8 mg/kg)

administered via the intramuscular route and infected as

previously described [19] with a 50-μl suspension of B.

cenocepacia suspension (4 × 107

cfu/mouse). Mice were

then observed daily for signs of morbidity. Alternatively,

mice were killed at different time points (6 h or 24 h) by

intraperitoneal injection of 300 mg/kg sodium pentobar-

bital. Airways were washed twice with 1 ml saline, and

the bronchoalveolar lavage (BAL) was collected [27] to

further determine cytokine concentrations using DuoSet

ELISA kits (R&D Systems). One hundred microliters of

the BAL were diluted and plated on TSA plates to deter-

mine the number of CFU of B. cenocepacia.

2.4. Statistics

Survival of wild-type and TLR-/- animals was com-

pared using Kaplan-Meier analysis log-rank test. In-

flammatory mediators levels and bacterial counts were

expressed as the mean ± SEM. Differences between

groups were assessed for statistical significance using the

Kruskal-Wallis ANOVA test, followed by the Mann-

Whitney U test. A value of p < 0.05 was considered sta-

tistically significant.

3. RESULTS

3.1. Effect of TLR4 and TLR5 Deletion on

Mouse Survival

The survival of WT, TLR4-/-, TLR5-/- and TLR4,5

double knockout neutropenic mice was followed for 10

days following intratracheal challenge with B. cenoce-

pacia. The survival curves are shown in Figure 1. It was

noted that only 22% of WT mice survived during the 10

day observation whereas 84% of the mice that had TLR4

deleted (p < 0.0001 compared to WT), and 75% of the

V. Balloy et al. / Open Journal of Immunology 1 (2011) **-**

9999

Wild-type, TLR5-/-, TLR4-/- and TLR4,5-/- mice treated with vinblastine

and infected by 4 × 107 cfumouse B. cenocepacia. Survivals were checked

every 24 h. Logrank (Mantel-Cox) test for comparisons of Kaplan-Meier

survival curves indicated a significant difference in the survival of

TLR4-deficiente mice (n = 19 for TLR4-/-; p < 0.0001; and n = 24 for TLR

4,5-/-; p < 0.0005) and no significant difference in the survival of TLR5-/-

mice (n = 34 for TLR5-/-; p > 0.05) compared to that of wild-type animals

(n = 36).

Figure 1. Mice survival upon infection by B. cenocepacia as a

function of TLR4 or 5 expression.

mice that had both TLR4 and TLR5 deleted (p < 0.0005

compared to WT) survived. Mortality among TLR5-/-

mice was non significantly different from that of WT

mice, both in terms of percentage mortality and time to

death. Collectively, these data suggested that the TLR4

response to B. cenocepacia was indeed deleterious as

has been noted for the response to LPS challenge [10,12]

and that the TLR5 response did not play a significant

role either in protecting the mice or inducing a deleteri-

ous inflammatory response.

3.2. Role of TLR in the Clearance of B.

Cenocepacia from the Airw ays

In order to examine whether there was a correlation

between bacterial clearance, survival and TLR function,

we examined bacterial clearance from the airways of

WT and mutant mice by culturing of BAL fluid (Figure

2). At 24 h hours post-infection, both WT and TLR5-/-

mice had retained similar amounts of the challenge dose.

Thus, clearance of this bacterium did not appear to be

affected by the absence of a TLR5 response, which was

not surprising as the TLR4 arm of the innate immune

response was intact. However, the number of bacteria

remaining in the airways of TLR4-/- (p < 0.01) and

TLR4,5-/- (p < 0.01) was significantly lower than that

seen in WT mice despite the latter mice not having the

two major TLRs that are involved in defense against

flagellated gram negative bacteria. Thus the response

mediated through TLR4 appears to both lead to death

Wild-type, TLR5-/-, TLR4-/- and TLR4,5-/- mice were treated with vin-

blastine and infected by 4 × 107 cfumouse B. cenocepacia. BAL were per-

formed at 24 h p.i. for the measurements of bacterial loads determined by

serial dilutions and plating on TSA. Data are the mean ± SEM values ob-

tained from four animals. **, p < 0.01 when compared with the corre-

sponding WT values. ns = non significant.

Figure 2. Pathogen burden of the lung upon infection by B.

cenocepacia as a function of TLR4 and 5 expression.

and to inhibit bacterial clearance, since in its absence

there is significantly better survival and bacterial clear-

ance from the airways compared to WT mice. This find-

ing is consistent with enhanced survival seen with

MyD88-/- mice [22] where multiple MyD88-/- depend-

ent TLRs are not functional. One salient difference

however is that the bacterial burden in MyD88-/- mice at

24 h was much greater than that seen in WT mice [22],

suggesting that another MyD88 dependent factor, active

in TLR4, 5-/- mice, functions to assist in the early clear-

ance of B. cenocepacia.

3.3. Role of TLR in the Induction of

Cytokine/ Chemokine Secretion during

B. Cenocepacia Lung Infection

Using the BAL fluids collected to examine bacterial

clearance from the airways, we measured several cyto-

kines/chemokines responses of the mice to explain the

sometimes paradoxical data obtained for survival and

bacterial clearance. TNF



, which we had previously

shown to be involved in the death of mice infected with

this bacterium [22], was significantly lower in TLR4-/-

mice than in WT mice but just as high in TLR5-/- mice

as in WT mice (Figure 3), confirmaing a significant role

for this cytokine in LPS induced inflammation. Similarly

all other cytokines/chemokines measured showed low

levels in the absence of TLR4 and similar levels in WT

and TLR5-/- mice (Figure 4).

4. DISCUSSION

The innate immune response of the host to B. ceno-

cepacia lung infection clearly indicates that TLR4

senses the pathogen while TLR5 does not do so in vivo.

In the absence of the former, the host response in terms

V. Balloy et al. / Open Journal of Immunology 1 (2011) **-**

100

Wild-type, TLR5-/-, TLR4-/- and TLR4,5-/- mice were treated with vin-

blastine and infected by 4 × 107 cfumouse B. cenocepacia. BAL were per-

formed at 6 and 24 h p.i. for the measurements of TNF- concentrations.

Data are the mean ± SEM values obtained from eight animals. **, p < 0.01

when compared with the corresponding WT values. ns = non significant.

Figure 3. Induction of TNF-



synthesis upon infection by B.

cenocepacia as a function of TLR4 and 5 expressions.

of cytokine and chemokine synthesis to infection is re-

duced, although not totally ablated, indicating the par-

ticipation of other signaling pathway(s). Moreover, as

with the MyD88-/- strain, TLR4-/- mice were protected

from death. We thus deduce that activation of TLR4 by

B. cenocepacia triggers a hyperinflammatory state that is

responsible for the observed pathogenesis [22]. The only

paradoxical observation is the reduced pathogen burden

in the case of TLR4-/- mice compared to the enhanced

(but transient) pathogen burden observed with MyD88-/-

mice. How TLR4 inhibits bacterial clearance was not

ascertainable, but the observation appears to be consis-

tent for the two different groups of mice with the TLR4-/-

deletion. It is also of note that the in vivo data do not fit

with our previous in vitro data [28]. Using respiratory

epithelial cells isolated from TLR4-/- mice or cells over-

expressing a functional form of TLR5, we established

that TLR5, but not TLR4, mediated B. cenocepacia-

induced lung epithelial inflammatory response. Such a

discrepancy between in vivo and in vitro results suggests

that the role of respiratory epithelial cells in lethal B.

cenocepacia pneumonia is limited. We have however

previously noted differences between in vivo and in vitro

data. Thus, with P. aeruginosa, we observed the lack of

involvement of TLR2 and 4 in the induction of IL-6

synthesis in vivo [19] while the absence of expression of

the very same TLRs by respiratory epithelial cells in-

fected in vitro reduced the synthesis of IL-6 considerably

[21]. Altogether, it is clear that examination of epithelial

cells in tissue culture is prone to variations and may not

always be reflective of infection.

The observations reported here also do not concur with

those of Urban et al. [29] who used an agar bead model of

infection to examine the role of flagella in B. cenocepacia

chronic lung infection. They observed reduced mortality

Wild-type, TLR5-/-, TLR4-/- and TLR4,5-/- mice were treated with vin-

blastine and infected by 4 × 107 cfumouse B. cenocepacia. BAL were

performed at 6 and 24 h p.i. for the measurements of IL-6, KC and G-CSF

concentrations. Data are the mean ± SEM values obtained from at least

eight animals. *, p < 0.05 and **, p < 0.01 when compared with the corre-

sponding WT values. ns = non significant.

Figure 4. Induction of IL-6, KC and G-CSF synthesis upon

infection by B. cenocepacia as a function of TLR4 and 5 ex-

pressions.

and noted significant reductions of KC, a murine ho-

molog of IL-8 both in BAL fluid and serum,when wild

type mice were challenged with a flagellin mutant, indi-

cating that there is normally a TLR5 mediated response

in that model of infection. However, their data in vitro

showing that flagellin does stimulate a response in cells

through TLR5 is similar to ours [28]. Rather than dis-

miss a role for TLR5, this discordance raises the ques-

V. Balloy et al. / Open Journal of Immunology 1 (2011) **-**

101101

tion whether B. cenocepacia expresses flagellin in vivo

during an acute infection versus the agar bead model of

infection. This possibility is not with precedent, as

members of the genus Bord etella, which are responsible

for a variety of acute bronchial infections repress flagel-

lum production upon entry into the airways [30]. Lastly,

while we have chosen to examine the role of LPS medi-

ated inflammation in death, it is likely that other viru-

lence factors may also be involved, as some TLR4-/-

mice do still die. This organism is known to have at least

four protein secretion systems, any or all of which may

also be involved in death. Analysis of these factors is

however beyond the scope of this study.

It is concluded that B. cenocepacia induces a hyperin-

flammatory state mediated through its very potent LPS

[10,12]. However, suppression of the LPS-TLR4 inter-

action and the consequent down-regulation of lung in-

flammation still leaves an effective innate immune re-

sponse that is capable of controlling bacterial growth.

5. ACKNOWLEDGEMENTS

This work is supported by Institut Pasteur, Inserm and Legs Poix.

REFERENCES

[1] Mahenthiralingam, E., Baldwin A. and Vandamme, P.

(2002) Burkholderia cepacia complex infection in pa-

tients with cystic fibrosis. Journal of Medical Microbi-

ology, 51, 533-538.

[2] Coenye, T. and Vandamme, P. (2003) Diversity and sig-

nificance of Burkholderia species occupying diverse

ecological niches. Environmental Microbiology, 5, 719-

729. doi:10.1046/j.1462-2920.2003.00471.x

[3] Vandamme, P., Holmes, B., Coenye, T., Goris, J., Ma-

henthiralingam, E., LiPuma, J.J. and Govan J.R. (2003)

Burkholderia cenocepacia sp. nov.—a new twist to an

old story. Research in Microbiology, 154, 91-96.

doi:10.1016/S0923-2508(03)00026-3

[4] Mahenthiralingam, E. and Vandamme, P. (2005) Taxon-

omy and pathogenesis of the Burkholderia cepacia com-

plex. Chronic Respiratory Disease, 2, 209-217.

doi:10.1191/1479972305cd053ra

[5] Reik, R., Spilker. T. and Lipuma, J.J. (2005) Distribution

of Burkholderia cepacia complex species among isolates

recovered from persons with or without cystic fibrosis.

Journal of Clinical Microbiology, 43, 2926-2928.

doi:10.1128/JCM.43.6.2926-2928.2005

[6] Vanlaere, E., Baldwin, A., Gevers, D., Henry, D., De

Brandt E., LiPuma, J.J., Mahenthiralingam, E., Speert,

D.P., Dowson, C., Vandamme, P. and Taxon, K. (2009) A

complex within the Burkholderia cepacia complex, com-

prises at least two novel species, Burkholderia contami-

nans sp. nov. and Burkholderia lata sp. nov. rnational

Journal of Systematic and Evolutionary Microbiology, 59,

102-111.

[7] LiPuma, J.J., Spilker, T., Gill, L.H., Campbell, P.W. 3rd,

Liu, L. and Mahenthiralingam, E. (2001) Disproportion-

ate distribution of Burkholderia cepacia complex species

and transmissibility markers in cystic fibrosis. American

Journal of Respiratory and Critical Care Medicine, 164,

92-96.

[8] Speert, D.P., Henry, D., Vandamme, P., Corey, M. and

Mahenthiralingam E. (2002) Epidemiology of Burk-

holderia cepacia complex in patients with cystic fibrosis,

Canada. Emerging Infectious Diseases, 8, 181-187.

[9] Mahenthiralingam, E., Urban, T.A. and Goldberg, J.B.

(2005) The multifarious, multireplicon Burkholderia ce-

pacia complex. Nature Reviews Microbiology, 3, 144-

156. doi:10.1038/nrmicro1085

[10] De Soyza, A., Ellis, C.D., Khan, C.M., Corris, P.A. and

Demarco de Hormaeche, R. (2004) Burkholderia ceno-

cepacia lipopolysaccharide, lipid A, and proinflamma-

tory activity. American Journal of Respiratory and Cri-

tical Care Medicine, 170, 70-77.

doi:10.1164/rccm.200304-592OC

[11] Bamford, S., Ryley, H. and Jackson S. (2007) Highly

purified lipopolysaccharides from Burkholderia cepacia

complex clinical isolates induce inflammatory cytokine

responses via TLR4-mediated MAPK signalling path-

ways and activation of NFkappaB. Cellular Microbiology,

9, 532-543. doi:10.1111/j.1462-5822.2006.00808.x

[12] Zughaier, S.M., Ryley, H.C. and Jackson, S.K. (1999)

Lipopolysaccharide (LPS) from Burkholderia cepacia is

more active than LPS from Pseudomonas aeruginosa and

Stenotrophomonas maltophilia in stimulating tumor ne-

crosis factor alpha from human monocytes. Infection and

Immunity, 67, 1505-1507.

[13] Iwamura, C. and Nakayama, T. (2008) Toll-like receptors

in the respiratory system: their roles in inflammation.

Current Allergy and Asthma Reports, 8, 7-13.

doi:10.1007/s11882-008-0003-0

[14] Bauer, S., Müller, T. and Hamm, S. (2009) Pattern recog-

nition by Toll-like receptors. Advances in Experimental

Medicine and Biology, 653, 15-34.

doi:10.1007/978-1-4419-0901-5_2

[15] Kumar, H., Kawai, T and Akira, S. (2009) Toll-like re-

ceptors and innate immunity. Biochemical and Biophysi-

cal Research Communications, 388, 621-625.

doi:10.1016/j.bbrc.2009.08.062

[16] Le Goffic, R., Balloy, V., Lagranderie, M., Alexopoulou,

L., Escriou, N., Flavell, R., Chignard, M. and Si-Tahar,

M. (2006) Detrimental contribution of the Toll-like re-

ceptor (TLR)3 to influenza A virus-induced acute pneu-

monia. PLoS Pathogens, 2, e53.

doi:10.1371/journal.ppat.0020053

[17] Balloy, V., Si-Tahar, M., Takeuchi, O., Philippe, B., Na-

hori, M.A., Tanguy, M., Huerre, M., Akira, S., Latgé, J.P.

and Chignard, M. (2005) Involvement of toll-like recep-

tor 2 in experimental invasive pulmonary aspergillosis.

Infection and Immunity, 73, 5420-5425.

doi:10.1128/IAI.73.9.5420-5425.2005

[18] Balloy, V. and Chignard, M. (2009) The innate immune

response to Aspergillus fumigatus. Microbes and Infec-

tion, 11, 919-927. doi:10.1016/j.micinf.2009.07.002

[19] Ramphal, R., Balloy, V., Huerre, M., Si-Tahar, M. and

Chignard, M. (2005) TLRs 2 and 4 are not involved in

hypersusceptibility to acute Pseudomonas aeruginosa

lung infections. Journal of Immunology, 175, 3927-3934.

[20] Ramphal, R., Balloy, V., Jyot, J., Verma, A., Si-Tahar, M.

V. Balloy et al. / Open Journal of Immunology 1 (2011) **-**

102

and Chignard, M. (2008) Control of Pseudomonas aeru-

ginosa in the lung requires the recognition of either lipo-

polysaccharide or flagellin. Journal of Immunology, 181,

586-592.

[21] Raoust, E., Balloy, V., Garcia-Verdugo, I., Touqui, L.,

Ramphal, R. and Chignard, M. (2009) Pseudomonas ae-

ruginosa LPS or flagellin are sufficient to activate

TLR-dependent signaling in murine alveolar macro-

phages and airway epithelial cells. PLoS One, 4, e7259.

doi:10.1371/journal.pone.0007259

[22] Ventura, G.M., Balloy, V., Ramphal, R., Khun, H., Huerre,

M., Ryffel, B., Plotkowski, M.C., Chignard, M. and

Si-Tahar, M. (2009) Lack of MyD88 protects the immu-

nodeficient host against fatal lung inflammation triggered

by the opportunistic bacteria Burkholderia cenocepacia.

Journal of Immunology, 183, 670-676.

doi:10.4049/jimmunol.0801497

[23] Feuillet, V., Medjane, S., Mondor, I., Demaria, O., Pagni,

P.P., Galán, J.E., Flavell, R.A. and Alexopoulou, L. (2006)

Involvement of Toll-like receptor 5 in the recognition of

flagellated bacteria. Proceedings of the National Acad-

emy of Sciences of the United States of America, 103,

12487-12492. doi:10.1073/pnas.0605200103

[24] Skerrett, S.J., Wilson, C.B., Liggitt, H.D. and Hajjar,

A.M. (2007) Redundant Toll-like receptor signaling in

the pulmonary host response to Pseudomonas aeruginosa.

American Journal of Physiology—Lung Cellular and

Molecular Physiology, 292, L312-L322.

doi:10.1152/ajplung.00250.2006

[25] Morris, A.E., Liggitt, H.D., Hawn, T.R. and Skerrett, S.J.

(2009) Role of Toll-like receptor 5 in the innate immune

response to acute P. aeruginosa pneumonia. American

Journal of Physiology—Lung Cellular and Molecular

Physiology, 297, L1112-L1119.

doi:10.1152/ajplung.00155.2009

[26] Balloy, V., Sallenave, J.M., Crestani, B., Dehoux, M. and

Chignard, M. (2003) Neutrophil DNA contributes to the

antielastase barrier during acute lung inflammation.

American Journal of Respiratory Cell and Molecular Bi-

ology, 28, 746-753. doi:10.1165/rcmb.2002-0119OC

[27] Gonçalves de Moraes, V.L., Singer, M., Vargaftig, B.B.

and Chignard, M. (1998) Effects of rolipram on cyclic

AMP levels in alveolar macrophages and lipopolysac-

charide-induced inflammation in mouse lung. British

Journal of Pharmacology, 123, 631-636.

doi:10.1038/sj.bjp.0701649

[28] de C Ventura, G.M., Le Goffic, R., Balloy, V., Plotkowski,

M.C., Chignard, M. and Si-Tahar, M. (2008) TLR 5, but

neither TLR2 nor TLR4, is involved in lung epithelial

cell response to Burkholderia cenocepacia. FEMS Im-

munology & Medical Microbiology, 54, 37-44.

doi:10.1111/j.1574-695X.2008.00453.x

[29] Urban, T.A., Griffith, A., Torok, A.M., Smolkin, M.E.,

Burns, J.L. and Goldberg, J.B. (2004) Contribution of

Burkholderia cenocepacia flagella to infectivity and in-

flammation. Infection and Immunity, 72, 5126-5134.

doi:10.1128/IAI.72.9.5126-5134.2004

[30] Akerley, B.J., Cotter, P.A. and Miller, JF. (1995) Ectopic

expression of the flagellar regulon alters development of

Bordetella-host interaction. Cell, 80, 611-620.

doi:10.1016/0092-8674(95)90515-4

LIST OF ABBREVIATIONS USED

BAL, bronchoalveolar lavage;

CF, cystic fibrosis;

MyD, myeloid-differentiation;

p.i., post-infection;

TLR, Toll-like receptor.