Originally published online as doi:10.1189/jlb.0206099 on June 29, 2006

Published online before print June 29, 2006

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(Journal of Leukocyte Biology. 2006;80:451-457.)
© 2006 by Society for Leukocyte Biology

TLR4 gene dosage contributes to endotoxin-induced acute respiratory inflammation

Dieudonnée Togbe^*, Silvia Schnyder-Candrian^*, Bruno Schnyder^*,, Isabelle Couillin^*, Isabelle Maillet^*, Franck Bihl^*,1, Danielle Malo, Bernhard Ryffel^*,2 and Valerie F. J. Quesniaux^*

^* CNRS (Centre National de la Recherche Scientifique), Molecular Immunology and Embryology UMR6218, Orléans, France; and
Biomedical Research Foundation, SBF, Matzingen, Switzerland; and
Department of Medicine and Human Genetics, McGill University, Montréal, Québec, Canada

² Correspondence: CNRS, UMR 6218, Molecular Immunology and Embryology Transgenose Institute, 3b rue de la Ferollerie, 45071 Orléans Cédex 2, France. E-mail: bryffel{at}cnrs-orelans.fr

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Toll-like receptor (TLR)4 is critical for endotoxin recognition and cellular responses. Using Tlr4 transgenic mice, we investigated the influence of Tlr4 gene dosage on acute respiratory response to endotoxin. Transgenic mice expressing three, six, or 30 copies of Tlr4, control, and Tlr4-deficient mice received intranasal administration of lipopolysaccharide (LPS; 10 ug), and the airway response was analyzed by plethysmography, lung histology, cell recruitment, cytokine and chemokine secretion and protein leakage into the bronchoalveolar space. We demonstrate that overexpression of Tlr4 augmented a LPS-induced bronchoconstrictive effect, as well as tumor necrosis factor and CXC chemokine ligand 1 (keratinocyte-derived chemokine) production. Neutrophil recruitment, microvascular and alveolar epithelial injury with protein leak in the airways, and damage of the lung microarchitecture were Tlr4 gene dose-dependently increased. Therefore, the TLR4 expression level determines the extent of acute pulmonary response to inhaled endotoxin, and TLR4 may thus be a valuable target for immunointervention in acute lung inflammation as a result of endotoxins.

Key Words: lung • lipopolysaccharide • CXCL1 • KC • Toll-like receptor transgenic mice • vascular leak

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Toll-like receptor (TLR)4 is critical for the recognition of lipopolysaccharide (LPS)/endotoxin from Gram-negative bacteria by different host cells initiating cell activation and the release of proinflammatory cytokines [¹ , ² ]. Systemic LPS administration induces lethal shock including pulmonary neutrophil recruitment, which is TLR4-dependent [³ ], and LPS, given by aerosol, causes bronchoconstriction, recruitment, and extravasation of neutrophils, injury of the alveolar epithelium, and disruption of pulmonary capillary integrity leading to a protein-rich fluid leak in the alveolar space [⁴ ]. LPS has been recognized as a main component in the pulmonary inflammation leading to acute lung injury [⁵ ] or acute respiratory distress syndrome, a leading cause of mortality in humans. Inhaled endotoxins may also play an important role in the development and progression of airway inflammation in asthma [⁶ , ⁷ ]. Although a variety of inflammatory processes and mediators are involved in these responses, LPS recognition by the host cells may be considered as a discrete and critical initial step amenable to therapeutic intervention. The mechanisms underlying LPS recognition are still incompletely understood. The LPS membrane receptor complex comprises accessory molecules such as CD14 and MD2 in addition to TLR4 [⁸ ]. Absence of CD14, MD2, or TLR4 abrogates most LPS responses [⁹ , ¹⁰ ], although CD14-independent effects to high-dose, inhaled LPS has been described recently [¹⁰ ].

We have shown previously that higher expression of Tlr4 gene copies augments the membrane expression of TLR4 on macrophages, amplifies the proliferative response of splenic B cells, and increases the acute lethal effect in response to systemic LPS [¹¹ ]. For these studies, Tlr4 transgenic mice were established on the C57BL/10ScNCr background [¹¹ ], which has complete deletion of the Tlr4 gene [¹² , ¹³ ].

Here, we asked whether the pulmonary response to endotoxin may be modulated by the level of Tlr4 expression using the Tlr4 transgenic mice. We demonstrate that overexpression of Tlr4 augmented the bronchoconstrictive effect of inhaled endotoxin, tumor necrosis factor (TNF), and keratinocyte-derived chemokine [KC; CXC chemokine ligand 1 (CXCL1)] production, lung epithelial and endothelial cell damage, and recruitment of neutrophils in the lungs. Therefore, the data indicate that the level of TLR4 expression may be a limiting factor in the pulmonary response to endotoxin.

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Mice
C57BL/10ScNCr mice, carrying a deletion of the Tlr4 gene (C57BL/10ScNJ, The Jackson Laboratory, Bar Harbor, ME) and the transgenic lines 388, 390, and 394 containing three, six, and 30 copies of the Tlr4 transgene under its natural promoter, respectively, on the C57BL/10ScNCr genetic background, were described previously [^{11 12 13} ]. The C57BL/10J mice were used as wild-type control (B10J, The Jackson Laboratory). The animals were kept in the animal facilities of the Transgenose Institute (CNRS, Orleans, France). All animal experiments complied with the French government’s ethical and animal experiment regulations.

In vivo experimental protocol
Groups of four mice were given a low dose of ketamine/xylazine by the intravenous route immediately prior to LPS intranasal administration (10 µg in a volume of 40 µl Escherichia coli, Serotype O111:B4 LPS, Sigma Chemical Co., St. Louis, MO). Twenty-four hours after LPS administration, the mice were given a high dose of ketamine/xylazine intraperitoneally (i.p.) and bled out. The bronchi were lavaged via a canula inserted into the trachea. The bronchoalveolar lavage fluid (BALF) was analyzed for cell composition and cytokine content as described below. After BAL, the lung was perfused via cardiac puncture with ISOTON® II acid-free balanced electrolyte solution (Beckman Coulter, Krefeld, Gemany). Half of the lung was stored at –20°C for myeloperoxydase (MPO) assay, and the other half was fixed for histological analysis.

Airways resistance
The airways resistance was evaluated by whole-body plethysmography [¹⁴ , ¹⁵ ] over a period of 3–6 h after LPS administration (10 µg LPS by the intranasal route). Unrestrained, conscious mice were placed in whole-body plethysmography chambers (EMKA Technologies, Paris, France). Mean airway bronchoconstriction was estimated by the Enhanced Respiratory Pause (Penh) index, which can be conceptualized as the phase-shift of the thoracic flow and the nasal flow curves; increased phase-shift correlates with increased respiratory system resistance. Penh is calculated by the formula Penh = (Te/RT-1) x PEF/PIF, where Te is expiratory time, RT is relaxation time, PEF is peak expiratory flow, and PIF is peak inspiratory flow as described [⁷ ]. Data were analyzed using "Datanalyst" software (EMKA Technologies) and expressed as mean ± SD of Penh of individual mice per group.

BAL
BALFs were prepared by washing the lungs four times with 0.5 ml ice-cold phosphate-buffered saline (PBS). The cells were obtained by centrifugation at 400 g for 10 min at 4°C. The supernatant of the first lavage was used for cytokine and protein analysis. The cell pellets were resuspended, pooled, and counted. Cytospin preparations were stained with Diff-Quik (Merz and Dade A.G., Dudingen, Switzerland), and differential cell counts were evaluated by counting at least 200 cells for the determination of the relative percentage of each cell type present in the BAL.

Lung histology
The organs were fixed in 4%-buffered formaldehyde overnight and embedded in paraffin as described [¹⁶ ]. Lung sections (3 µm) were stained with haematoxylin and eosin (H&E). The sections were examined with a Leica microscope. Neutrophil and erythrocyte accumulation in alveoli, disruption of alveolar septae, and activation of alveolar epithelial cells (with cuboidal-shaped appearance and basophilic cytoplasm) were quantified using a semiquantitative score with increasing severity of changes (0–4) by two independent observers including a trained pathologist (B. Ryffel) [¹⁷ , ¹⁸ ]. Groups of four to eight mice and 10 randomly selected high-power fields (400x magnifications) per animal were analyzed.

Cytokine quantification
TNF and KC (CXCL1) protein contents in cell culture supernatants and BALF were evaluated by enzyme-linked immunosorbent assay (ELISA) according to the instructions from the kit manufacturer (Duoset, R&D Systems, Minneapolis, MN).

MPO assay
MPO activity in the lung tissue was evaluated as described [¹⁴ ]. In brief, frozen lung was homogenized for 30 s in 1 ml ice-cold PBS with a polytron mixer. The extract was centrifuged, and the pellet was resuspended in 1 ml PBS containing 0.5% hexadecyl-trimethyl ammonium bromide (HTAB; Sigma Chemical Co.) and 1 mM EDTA (PBS-HTAB-EDTA), homogenized for 30 s, and centrifuged. To 100 µl of the supernatant were added 2 ml Hanks’ balanced salt solution (w/Ca²⁺ and Mg²⁺, Gibco-BRL, Grand Island, NY), 200 µl PBS-HTAB-EDTA, 100 µl o-dianisidine (1.25 mg/ml H₂O, Sigma Chemical Co.), and 100 µl H₂O₂. After 15 min incubation at 37°C under agitation, the reaction was stopped by transfer on ice and addition of 100 µl NaN₃ (1%, Sigma Chemical Co.). The MPO activity was quantified as absorbance at 460 nm.

Protein determination
BAL protein content was determined by the Bradford method according to the manufacturer’s (Bio-Rad, Hercules, CA) instructions using ovalbumin as a standard. Protein solutions were assayed in duplicate. Absorbance was measured at 595 nm after 30 min.

Statistical analysis
Data are presented as means and standard deviation (SD) indicated by error bars. Statistical evaluation of differences between the experimental groups was determined by Mann-Witney "U" test for data from plethysmography experiments and Student’s t-test for other data using Prism software. P values of <0.05 were considered statistically significant.

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Levels of TLR4 expression determine the airway response to LPS
TLR4 is essential to LPS-induced, heightened airway responsiveness to methacholine [¹⁹ ]. In addition, inhaled LPS alone induces acute bronchoconstriction, which is TLR4- and myeloid differentiation primary-response protein 88 (MyD88)-dependent, as demonstrated in TLR4- and MyD88-deficient mice [²⁰ ]. We first confirmed the critical role of TLR4 in endotoxin-induced bronchoconstriction using mutant C57BL/10ScNCr mice, which have no functional TLR4 and are unresponsive to inhaled LPS (Fig. 1 ), and the TLR4-positive controls, C57BL/10J mice, had a clear response to LPS. Using the TLR4 transgenic mouse lines 388, 390, and 394, we then investigated whether TLR4 expression levels affect bronchoconstriction in response to endotoxin. Tlr4 transgenic 388, 390, and 394 mice carry three, six, and 30 copies of Tlr4, respectively, on the Tlr4-deficient C57BL/10ScNCr background [¹¹ ]. The two Tlr4 transgenic lines were shown to express different levels of the Tlr4 gene, and a gradient of expression was well-correlated with the number of integrated copies [¹¹ ]. Compared with mice carrying two normal copies of Tlr4, 388, 390, and 394 mice presented a level of TLR4/MD2 protein expression on the cell surface of thioglycolate-elicited peritoneal macrophages well-correlated with the number of Tlr4-integrated copies in the genome and the level of Tlr4 transcription [¹¹ ]. As shown in Figure 1A and 1B , inhaled endotoxin induced a slight but significant bronchoconstriction in Tlr4 transgenic 388 mice expressing three copies over the effect seen in C57BL/10J wild-type control mice. The effect was increased dramatically in 390 and 394 transgenic mice overexpressing six and 30 Tlr4 copies, and the Tlr4 null mutant C57BL/10ScNCr mice did not respond to LPS.

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Figure 1. LPS-induced bronchoconstriction depends on the number of Tlr4 gene copies. Wild-type B10J mice, Tlr4-deficient B10ScNCr, and 388, 390, and 394 transgenic mice expressing, respectively, three, six, and 30 copies of the Tlr4 gene were challenged by intranasal administration of 10 µg LPS. The bronchoconstriction was recorded over 6 h using whole body plethysmography. (A) Penh values are given as the mean ± SD (n=4–7 mice per group). (B) The mean arbitrary units (AUC) of Penh are represented. Saline control induced no increase in Penh (*, P<0.05; **, P<0.01).

The dose-dependence of LPS response was determined (data not shown). Consistent bronchoconstriction was obtained at an intranasal dose of 10 µg LPS, and higher doses up to 50 µg LPS did not augment the response. Therefore, LPS was used at 10 µg/mouse in the rest of this study. Of note, intranasal LPS administration had no effect on TNF levels in plasma or on peripheral blood cell counts, unlike systemic injection (data not shown). Therefore, the results demonstrate that the airway response to inhaled endotoxin is Tlr4 dose-dependent.

Cytokine production and protein leak in the airways are TLR4 dose-dependent
As LPS induces the rapid production of proinflammatory cytokines and chemokines, we investigated the production of TNF and KC (CXCL1) in the BAL upon LPS exposure. TNF was not detected in the BALF of Tlr4 mutant C57BL/10ScNCr mice, and a strong TNF production was detected in the 394 transgenic mice expressing 30 copies of Tlr4 (data not shown). To further assess the Tlr4 dose-dependency of this response, TNF concentrations were determined in the BALF of mice expressing zero, two, three, or six copies of Tlr4 (Fig. 2A ). TNF production was clearly augmented in 388 and 390 mice expressing three and six copies of Tlr4, respective to wild-type controls.

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Figure 2. TLR4-dependent TNF and KC (CXCL1) release and protein leak into the alveolar space. Mice were treated as in Figure 1 , and BALs were obtained at 24 h after intranasal LPS application. TNF (A) and KC (B) concentrations were quantified by ELISA. Protein concentration (C) in the BAL was determined at 24 h. The results represent the mean ± SD (four mice per group; *, P<0.05; **, P<0.01). ns, Not significant.

We then analyzed the production of the chemokine CXCL1, which is critical for the recruitment of neutrophils into the BAL. CXCL1 levels were high in 394 mice expressing 30 copies of Tlr4 upon LPS administration (data not shown), and this effect was clearly gene dose-dependent in 388 and 390 Tlr4 transgenic mice expressing three and six Tlr4 copies as compared with C57BL/10J wild-type mice (Fig. 2B) . Tlr4 mutant C57BL/10ScNCr mice did not produce any detectable CXCL1 upon LPS administration.

LPS induces epithelial and endothelial cell damage directly or through the release of host factors such as TNF or reactive oxygen species, which results in the exudation of plasma proteins. We investigated the extent of protein leakage after LPS administration in the Tlr4 transgenic mice. BAL protein levels were increased in a Tlr4 dose-dependent manner; 394 mice showed the highest levels, and the protein levels in the Tlr4 mutant C57BL/10ScNCr mice were similar to the saline controls (Fig. 2C) .

From this set of experiments, we can conclude that TLR4 is essential for LPS-induced production of TNF, the neutrophil chemokine CXCL1, and protein extravasation into the BAL, and these parameters are increased when the Tlr4 gene is overexpressed.

Inflammatory cell response in the airways of Tlr4-expressing mice
We then asked whether TLR4 overexpression affects inflammatory cell recruitment in the airways after LPS exposure, consisting most prominently of neutrophils. The neutrophil counts present in BAL were slightly augmented in mice expressing three or six copies of the Tlr4 gene and further increased in the 394 transgenic mice expressing 30 copies of Tlr4 as compared with wild-type controls, and C57BL/10ScNCr mutant mice were unresponsive (Fig. 3A ). The recruitment of neutrophils in the lung tissue, as assessed by MPO activity, reflected the data obtained in the BAL (Fig. 3B) . Therefore, the extent of the LPS-induced inflammatory cell recruitment in the airways correlated with the level of TLR4 expression.

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Figure 3. TLR4-dependent neutrophil recruitment in alveolar space and lung tissue. Wild-type B10J mice, Tlr4-deficient B10ScNCr, and 388, 390, and 394 transgenic mice expressing, respectively, three, six, and 30 copies of the Tlr4 gene were challenged by intranasal administration of 10 µg LPS and analyzed at 24 h. Neutrophil counts (A) were determined in the BALF. Neutrophil MPO activity in the respective lung tissue [optical density of 460 nm (OD 460 nm)] is shown (B). Results are the mean ± SD (four mice per group) and are representative of two independent experiments (*, P<0.05; **, P<0.01).

Lung damage exacerbated upon Tlr4 overexpression
The LPS-induced inflammation and lung damage after LPS administration were investigated microscopically. Neutrophils accumulated in the alveolar space in wild-type mice, and this was clearly more pronounced in 394 transgenic mice overexpressing 30 copies of Tlr4, and TLR4 mutant mice were unresponsive (Fig. 4 ). As a sign of endothelial/epithelial damage, erythrocytes were associated with neutrophils. The lung damage reflected by epithelial cell activation with a more columnar appearance and basophilic cytoplasm and focal necrosis, disruption of alveolar septae, and focal edema was found in wild-type mice and more pronounced in 394 transgenic mice but absent in Tlr4 mutant C57BL/10ScNCr mice (Fig. 4) . Neutrophil infiltration, presence of erythrocytes, and alveolar damage were scored on the microscopic sections. Lungs from saline-treated, control mice exhibited neither disruption of alveolar septae nor neutrophil or erythrocytes in alveoli, excluding artifacts from lavage, perfusion, or fixation. In addition to neutrophil recruitment into the alveolar space, the epithelial cell activation and disruption of alveolar septae were found in wild-type mice but more prominent in 390 and 394 transgenic mice expressing six or 30 copies of Tlr4 (Fig. 5 ). The neutrophil infiltration observed microscopically was in good agreement with the increased MPO activities in the lung (Fig. 3B) . Therefore, Tlr4 gene overexpression was associated with increased pulmonary neutrophil recruitment and lung damage induced by local LPS exposure.

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Figure 4. Tlr4 gene overexpression exacerbates pulmonary neutrophil infiltration and pathology induced by endotoxin. The lungs of Tlr4-deficient B10ScNCr, wild-type B10J mice, and 394 transgenic mice expressing 30 copies of the Tlr4 gene were fixed, stained by H&E, and analyzed by microscopy 24 h after LPS administration (10 µg intranasally). Neutrophil infiltration, disruption of microarchitecture, and activation of alveolar epithelia observed in wild-type mice were clearly exacerbated in 394 mice overexpressing Tlr4, and neither cellular infiltration nor lung damage was detected in Tlr4-deficient B10ScNCr mutant mice after LPS treatment. Representative lung sections are shown (A and B) at original magnifications x400 and 1000, respectively.

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Figure 5. Quantification of pulmonary neutrophil infiltration and pathology induced by endotoxin. Tlr4-deficient B10ScNCr, wild-type B10J, and 388, 390, and 394 transgenic mice expressing, respectively, three, six, and 30 copies of the Tlr4 gene were given intranasally 10 µg LPS and analyzed after 24 h. Disruption of alvolar septae (A), activation of alveolar epithelium (B), neutrophils infiltration (C), and erythrocytes presence in alveoli (D) were evaluated based on histological sections. Results are expressed as mean lesion score ± SD (n4–7 mice per group; *, P<0.05; **, P<0.01).

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Here, we demonstrate for the first time that the extent of the acute lung inflammatory response and alveolar epithelial cell damage to inhaled endotoxin depends on the level of TLR4 expression. LPS-induced bronchoconstriction, TNF, and CXCL1 (KC) production, neutrophil recruitment in the airways, and lung tissue damage are absent in mutant Tlr4-deficient mice and are pronounced in Tlr4-overexpressing mice.

Overexpression from three, six, to 30 tlr4 gene copies increased airway responsiveness and neutrophil recruitment in the airways and lung, protein exudation and CXCL1, TNF production, and protein leakage to LPS. The inflammatory response was absent in TLR4-deficient mice and correlated with the tlr4 gene overexpression.

Exposure of LPS via the airways seems to be necessary for establishing the chemokine and cytokine network responsible for bronchoconstriction and inflammatory cell transmigration. Indeed, i.p. administration of 50 µg LPS did not cause bronchoconstriction or neutrophil recruitment in BALF, and neutrophils were only sequestrated in the lung vasculature without any endothelial transmigration into the alveolar space [³ ].

Bronchial hyper-reactivity to methacholine was not affected by the Tlr4 expression levels [¹⁹ ], data that we confirmed (not shown). Hollingsworth et al. [¹⁹ ] further demonstrated that the methacholine response of TLR4-deficient mice was reduced after LPS exposure, and we investigated here a direct LPS-induced bronchoconstriction in the absence of methacholine.

The LPS receptor complex is composed of several components, which include phosphatidylinositol-anchored CD14, TLR4, MD2 and MD1, and RP105. The LPS-binding protein (LBP) enhanced binding to the LPS receptor in serum [¹ ]. Absence of CD14 [²¹ ], MD2 [⁹ , ²² ], or LBP [²³ ] abrogates endotoxin responses, similar to what is seen in loss-of-function mutants of Tlr4. However, some CD14-independent, acute lung injury responses to high-dose LPS have been described recently [¹⁰ ]. MD2 was shown to be essential for the correct intracellular distribution and efficient LPS recognition of TLR4 [⁹ ]. We showed before that Tlr4 expression correlated with TLR4-MD2 expression using the flow cytometric analysis of MTS510 monoclonal antibody-labeled macrophages [¹¹ ]. In contrast, the TLR homologue RP105, together with MD1, originally described on B cells, was recently shown to negatively regulate TLR4 signaling in myeloid cells [²⁴ ].

The mechanisms leading to LPS recognition are not yet fully understood. In particular, the relative abundance of each component in the receptor complex is unclear. TLR4 is recognized as a main switch of the inflammatory response, and part of this may be ascribed to its unique use of all four TLR adaptor molecules known to date: MyD88, Toll-interleukin-1 receptor translation initiation region (TIR) domain-containing adaptor protein (TIRAP), TIR domain-containing adaptor-inducing interferon-ß (TRIF), and TRIF-related adaptor molecule (TRAM). TLR4 and the adapters are proposed to associate in platforms to which kinases from the different signaling pathways may be recruited [²⁵ ]. The higher the complexity of the models proposed, the more relevant it becomes to appreciate whether limitation in some of these shared molecules, coreceptors, or adaptors is likely to introduce a new level of regulation. Using Tlr4 transgenic mice [¹¹ ], we show that response to inhaled LPS can be augmented by the Tlr4 gene dose. The level of the membrane-expressed TLR4/MD2 complex was shown to correlate with the number of Tlr4-integrated copies in the genome and the level of Tlr4 transcription in these mice [¹¹ ]. Here, our data suggest that MD2 may not be limiting functional overexpression of TLR4. Moreover, the data suggest that TLR4 expression is limiting the endotoxin response on the lung and the adaptor proteins such as MyD88, TIRAP, TRAM, or TRIF [²⁶ ], and the enzymes involved in downstream signaling are apparently in excess, capable of accommodating the signaling of additional TLR4 molecules. Conversely, TLR4 overexpression might exceed the availability of the negative regulator RP105, which might also contribute to the strongly increased biological responses in the transgenic mice carrying multiple copies of Tlr4 [¹¹ , ²⁴ ].

In conclusion, the level of TLR4 expression is a limiting factor for the pulmonary inflammatory response to endotoxin, supporting the notion that the pulmonary response to environmental LPS might be regulated at the level of the TLR4, which has been recognized as an interesting target, and TLR4 antagonists might have a therapeutic potential [²⁷ ]. Alternatively, attenuation of LPS responses may be achieved by modulating TLR4 expression, as shown for the cytokine migration inhibitory factor [²⁸ ]. Our data confirm the potential of intervening on TLR4 expression or function and validate the fact that modulating TLR4 expression or function can directly influence lung inflammatory response to endotoxin. Small molecular weight compounds inhibiting Tlr4 expression locally may offer new ways of therapeutic intervention for acute pulmonary inflammation.

 
This work was supported by CNRS and FRM. S. S-C. and B. S. received grant support by the Foundation Le Studium, Orléans, and FRM (Fondation de la Recherche Medicale), France. D. M. is a Dawson McGill scholar and a scholar of Canadian Institutes of Health Research and an International Research Scholar of the Howard Hughes Medical Institute.

 
¹ Current address: CNRS UMR 6097, Institut de Pharmacologie Moléculaire et Cellulaire, Sophia-Antipolis, Valbonne, France.

Received February 14, 2006; revised May 12, 2006; accepted May 18, 2006.

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