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Am J Physiol Lung Cell Mol Physiol 292: L1422-L1431, 2007. First published February 23, 2007; doi:10.1152/ajplung.00337.2006
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Omega-3 polyunsaturated fatty acids improve host response in chronic Pseudomonas aeruginosa lung infection in mice

Maud Pierre,1,2 Marie-Odile Husson,2 Rozenn Le Berre,1 Jean-Luc Desseyn,3 Claude Galabert,4 Laurent Béghin,2 Christopher Beermann,5 André Dagenais,6 Yves Berthiaume,6 Bruno Cardinaud,7,8 Pascal Barbry,7,8 Frédéric Gottrand,2 and Benoît P. Guery1

1Faculté de Médecine and 2Clinique de Pédiatrie, Hôpital Jeanne de Flandre, Université de Lille and CHRU de Lille, France; 3JPARC Research Centre, Institut National de la Santé et de la Recherche Médicale, IMPRT, Place de Verdun, Lille, France; 4CERM, Hôpital Renée Sabran, Giens, Hyères, CHRU Lyon, France; 5Lipids Research Division, Numico Research, Friedrichsdorf, Germany; 6Centre de Recherche, Centre Hospitalier de l'Université de Montréal/Hôtel Dieu, Département de Médecine, Université de Montréal, Montréal, Québec, Canada; 7Centre National de la Recherche Scientifique and 8Université de Nice Sophia Antipolis, Institut de Pharmacologie Moléculaire et Cellulaire, Sophia Antipolis, France

Submitted 30 August 2006 ; accepted in final form 18 February 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Pseudomonas aeruginosa is a gram-negative bacilli frequently encountered in human pathology. This pathogen is involved in a large number of nosocomial infections and chronic diseases. Herein we investigated the effects of polyunsaturated fatty acids (PUFA) in chronic Pseudomonas aeruginosa lung infection. C57BL/6 mice were fed for 5 wk with specifically designed diets with high contents in either omega-3 ({omega}-3) or {omega}-6 PUFA and compared to a control diet. P. aeruginosa included in agarose beads was then instilled intratracheally, and the animals were studied for 7 days. On the 4th day, the mice fed with the {omega}-3 diet had a higher lean body mass gain and a lower {omega}-6:{omega}-3 ratio of fatty acids extracted from the lung tissue compared with the other groups (P < 0.05). The {omega}-3 group had the lowest mortality. Distal alveolar fluid clearance (DAFC) as well as the inflammatory response and the cellular recruitment were higher in the {omega}-3 group on the 4th day. The effect on DAFC was independent of {alpha}-epithelial Na+ channels ({alpha}-ENaC), beta-ENaC, and {alpha}1-Na-K-ATPase mRNA expressions, which were not altered by the different diets. In conclusion, a diet enriched in {omega}-3 PUFA can change lung membrane composition and improve survival in chronic pneumonia. This effect on survival is probably multifactorial involving the increased DAFC capacity as well as the optimization of the initial inflammatory response. This work suggests that a better control of the {omega}-6/{omega}-3 PUFA balance may represent an interesting target in the prevention and/or control of P. aeruginosa infection in patients.

alveolar liquid clearance

DIETARY SUPPLEMENTATION with fish oil, a source of highly polyunsaturated n-3 fatty acids, has been demonstrated to exert beneficial effects in a number of diseases (26, 30, 39). Among the fatty acids, the omega-3 ({omega}-3) polyunsaturated fatty acids (PUFA) possess the most potent immunomodulatory activities (4). Some of the effects of omega-3 PUFA are related to the types of eicosanoids; eicosapentaenoic acid (EPA) can be converted to the 3-series prostaglandins and 5-series leukotrienes (25). These mediators are considered less biologically active and less inflammatory than the arachidonic acid (AA)-derived 2-series prostaglandins and 4-series leukotrienes (17, 27). Animal experiments as well as clinical studies have already shown that omega-3 fatty acids have anti-inflammatory properties, and, therefore, might be useful in the management of inflammatory and autoimmune diseases (9).

Another potential effect of the PUFA could be related to the influence on membrane transport: Hulbert and Else (22) have proposed a general paradigm where biological membranes could act as "pacemakers" for overall metabolic activities. This paradigm depends in some way on the relative metabolic cost to maintain a sodium gradient across the plasma membrane, i.e., it can be particularly valid in tissues characterized by an active ion transport, such as the brain, the kidney, or the lung epithelium. Interestingly, the authors noticed that membrane polyunsaturation increases the molecular activity of many membrane-bound proteins and particularly some specific membrane leak-pump cycles and cellular metabolic activity. Crossover experiments have thus shown that the functional activity of the sodium pump was linked to the membrane composition (13, 14, 47, 48).

Pseudomonas aeruginosa is a gram-negative bacilli frequently encountered in human pathology. This pathogen is involved in a large number of nosocomial infections such as urinary tract infections and pneumonia (8, 16). In chronic infections, Pseudomonas is commonly found in patients with chronic obstructive pulmonary disease and cystic fibrosis. P. aeruginosa produces a large number of secreted and cell-associated virulence factors that have been implicated in the pathogenesis of infection. It is well known that in chronic lung infection with this pathogen, distal alveolar fluid clearance (DAFC) is decreased and stays unresponsive to stimulation (5). Dagenais et al. (11) explained these observations by a decreased expression of {alpha}-epithelial Na+ channel ({alpha}-ENaC) mRNA at days 3, 7, and 14 after infection by P. aeruginosa.

From these data, we hypothesized that oral lipid supplementation with PUFA could influence the host response in chronic P. aeruginosa-induced lung injury. We used a murine model of P. aeruginosa infection combined with the design of isocaloric fatty acid mixtures. A control diet was compared with AA ({omega}-6)-enriched or EPA/docosahexaenoic acid (DHA) ({omega}-3)-enriched diets. We first evaluated the consequences of each diet on body composition and membrane incorporation. We then assessed the host response in terms of mortality, DAFC, lung permeability, and cytokine production. We finally studied by Northern blotting the expression of {alpha}-ENaC, beta-ENaC, and {alpha}1-Na-K-ATPase mRNA in infected lungs.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals and food. Male C57BL/6 mice (5 wk of age) purchased from Charles River Laboratories (Domaine des oncins, L'Arbresle, France) were fed during 5 wk with three different diets produced by Numico Research. Mice were maintained in a protected unit. All experiments were performed with the approval of the Lille Institutional Animal Care and Use Committee.

The diets were adapted from the AIN-93 (American Institute of Nutrition) rodent diet (Table 1). We substituted lipids (soybean oil) with three different isocaloric fatty acid mixtures: {omega}-3 PUFA, {omega}-6 PUFA, or a control diet (Table 2). Mice were randomized in one of the three groups during the 5 wk preceding P. aeruginosa infection and until the end of the experiment. All experiments were performed blindly. The number of animals used in each group for all the specific protocols is detailed in Table 3.


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  		Table 1. Diet composition in gram per kilogram of diet

 

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  		Table 2. Fatty acid repartition in grams per 100 grams of lipids

 

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  		Table 3. Number of animals in each group for the different protocol performed in the study

 
Preparation of the bacterial inoculum. P. aeruginosa (PAO1 strain) was grown at 37°C in a rotating, shaking water bath in a tryptic soy broth (Biomerieux Laboratories, Lyon, France) for 8 h. The inoculum was calibrated at 109 colony-forming units (cfu)/ml by spectroscopy. The pathogen was then entrapped in agar beads as previously described (5). A control group was performed with sterile beads: 1 ml of sterile water was mixed with agarose.

Mice were anesthetized with sevoflurane inhalation (Abbott, UK) and placed in dorsal recumbency. Transtracheal insertion of a 24-G animal feeding needle was used to instillate 60 µl of a 1:4 dilution of the agarose beads (2 x 105 cfu/mouse).

Quantitative bacteriology. The lungs were excised aseptically at the 1st, the 4th, and the 7th day after the inoculation. These lungs were then homogenized in saline buffer, and the samples were cultured quantitatively by serial dilution on BCP agar plates (Biomerieux Laboratories). The plates were incubated at 37°C and inspected for P. aeruginosa colonies after at least 24 h.

Body composition. Mice were anesthetized (100 mg/kg ketamine, 10 mg/kg chlorpromazine) and scanned using a Lunar PIXImus densitometer (GE Lunar, Madison, WI) for body composition measurements before and after infection. The total body percentage of fat and lean was measured (34).

Lung tissue composition. Lipid extraction from homogenized lungs was performed with chloroform/methanol according to the method of Bligh and Dyer (3). Approximately 100 mg of tissue was used for each analysis. The organic phase obtained after the extraction was concentrated, and individual lipid classes within the extract were separated by bidimentional high-performance thin-layer chromatography (HPTLC) as previously described (21). Briefly, HPTLC plate was developed with chloroform/methanol/16.5 N aqueous ammonia, vol/vol/vol 65:25:5, in the first dimension, followed by chloroform/acetone/methanol/acetic acid/water, vol/vol/vol/vol/vol 3:4:1:1:0.5, in the second dimension. Isolated phospholipid subclasses phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were scraped from the HPTLC plate and transesterified in 14% boron trifluoride-methanol (Alltech, code 18017) in a sealed vial under a nitrogen atmosphere at 100°C for 45 min (33). The resulting fatty acid methylesters (FAME) were extracted with pentane, desiccated over sodium sulfate decahydrated (Na2SO4, 10H2O), evaporated in a vacuum concentrator, and prepared for gas chromatography (GC) by sealing the pentane extract under nitrogen.

FAME were separated and quantified by capillary GC using a gas chromatograph (model 5890; Hewlett-Packard, Wilmington, DE) equipped with a 60-m/0.32-mm DB-23 capillary column (J&W Scientific, Folsom, CA) and a flame-ionization detector.

Fatty acid analysis data were calculated as mol% data. Mol% data were calculated as the percentage that each individual fatty acid represented relative to the sum of the concentrations of all fatty acids within the lipid (phospholipids) class.

DAFC. DAFC was measured in the absence of blood flow and ventilation using a previously described in situ model (18). DAFC was determined by measuring the increase in protein tracer activity over 30 min following intratracheal instillation of a 5% albumin solution containing 0.1 µCi of 125I-labeled albumin (41). Five minutes after instillation, a baseline sample (time 0) (0.1 ml) was aspirated from the distal airways using PE50 tubing (Becton Dickinson, Sparks, MD). This allowed for the determination of tracer dilution by edema fluid present in the lungs. An infrared lamp, placed 30 cm above the body, was used to maintain body temperature at 37°C. Body temperature was monitored by placing a temperature probe into the abdominal cavity. Thirty minutes later, another sample was collected from the distal airways. DAFC, expressed as the percent of alveolar fluid volume cleared in 30 min, was determined using the following: DAFC = [1 – (PI/PF)] x 100, where PI is the initial 125I-albumin activity/g at time 0, and PF is the 125I-albumin activity/g 30 min later.

Permeability and extravascular lung water. To evaluate the alveolar capillary barrier permeability, 0.1 µCi of 125I-labeled albumin in 0.1 ml of saline was administered intraperitoneally (41). After 2 h, a blood sample was obtained by heart puncture, and the lungs were removed, homogenized (PT 1600 E; Polytron, Fisher Bioblock Scientific), and centrifuged. The supernatant hemoglobin content was measured.

Permeability was calculated as extravascular plasma equivalent (EVPE) in µl: EVPE = ({radioactivity in lung – [Qb x (1-hematocrite) x radioactivity in plasma]}/[radioactivity in plasma]) x 1,000, where Qb is the lung blood volume.

The lung wet-to-dry weight ratio (W/D) was determined by removing the lungs at the end of the experiment and recording the wet weight. The lungs were then placed in a 37°C incubator for 7 days, at which time the dry weight was recorded. The W/D weight ratio was then calculated for each pair of lungs (1, 5).

Bronchoalveolar lavage. Bronchoalveolar lavage (BAL) was performed by cannulating the trachea. Lungs from each experimental group were lavaged three times with 0.5 ml of normal saline solution. BAL fluid samples were filtered and immediately frozen at –80°C. A cell count was performed directly. Cellular monolayers were prepared with a cytocentrifuge and stained with Wright-Giemsa stain. Cellular morphotype differential was obtained by counting 200 cells/sample and expressing each type of cell as a percentage of the total number counted.

Measurement of cytokines. BAL cytokine levels were quantified using the BD Cytometric Bead Array technology (Becton-Dickinson), which employs a series of particles with discrete fluorescence intensities to simultaneously detect multiple soluble analytes in a small volume sample. The BD CBA Mouse Inflammation kit was used as recommended by the supplier to quantitatively measure IL-6 and TNF-{alpha} protein levels in the different samples. Briefly, each concentrated BAL sample was incubated for 2 h at room temperature in the presence of mixed antibodies-coupled beads (50 µl) and Phycoerythrin Detection Reagent (50 µl), followed by two washes. Subsequently, bead fluorescence was analyzed using the BD FACS Array Bioanalyzer. The median relative fluorescent intensity values were converted into cytokine concentrations by using nine-point calibration curves created by serial dilution cytokine standard.

Northern blotting. Total RNA from mouse lung was extracted in TRIzol (Invitrogen) following homogenization of the samples with Ultraturrax (IKA, Janke & Kunkel). For Northern blotting, 7.5 µg of total RNA were electrophoresed on 1% agarose-formaldehyde gel and transferred to Amersham Hybond N+ membrane (GE Healthcare) after overnight blotting with 10x SSC. Hybridization was performed, as reported previously, in Church buffer [0.5 M Na phosphate, pH 7.2, 7% SDS (wt/vol), 1 mM EDTA, pH 8] (12). The nylon membranes were hybridized successively with different cDNA probes ({alpha}-ENaC, beta-ENaC, {alpha}1-Na-K-ATPase). {alpha}-ENaC mRNA was detected with a 1,886-bp EcoRI-BamHI rat {alpha}-ENaC cDNA probe (Glu76 to the stop codon). beta-ENaC mRNA was detected with a 1,458-bp BamHI rat beta-ENaC cDNA probe (ATG to Trp469). The rat {alpha}-ENaC and beta-ENaC cDNA were gifts from Dr. B. C. Rossier (Institut de Pharmacologie et de Toxicologie, Univ. de Lausanne, Lausanne, Switzerland). The {alpha}1-Na-K-ATPase probe was a gift from Dr. J. Orlowski (Physiology Dept., McGill Univ., Montreal, Quebec, Canada) and consisted of a 3,626-bp fragment that encompasses the whole cDNA. The RNA loaded on each lane was normalized to 18S rRNA, detected by fluorescence scanning of the gels after staining with SYBR green II (Molecular Probes, Eugene, OR) before blotting RNA on nylon membrane. The SYBR green-stained gels as well as the Northern blots were scanned and analyzed with a Typhoon PhosphorImager (Molecular Dynamics, Sunnyvale, CA).

Statistical analysis. Results are presented as means ± SE. Data were analyzed using the Kruskal-Wallis test. If the overall P value was less than 0.05, a Dunn posttest comparing all groups was performed with a 95% confidence interval. P values less than 0.05 were regarded as statistically significant. A Bonferroni correction was used for multiple analysis. GraphPad Prism 4.02 software was used. Cumulative survival rates were compared by using a log rank test. The criterion for statistical significance was P ≤ 0.05.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Diets do not influence the lung bacterial load. The influence of diet on the lung bacterial load was studied on the 1st, 4th, and 7th day of infection in 3 groups of 50 mice fed with each specific diet during 5 wk before infection. Figure 1 shows a significant decrease in the number of bacteria over time in the three groups with the persistence of bacteria on the 7th day. There was no significant difference in the lung bacterial load of the study between the three groups over time.


Figure 1
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  		Fig. 1. Lung bacterial load was assessed as the ratio between the number of bacteria recovered in each mouse and the number of bacteria injected. This was measured on days 1, 4, and 7 for the 3 diets. The lungs of infected mice were excised, homogenized, and cultured quantitatively by incubation on BCP agar plate. ***P < 0.001 vs. day 1.

 
The weight loss is not affected by the diets. The weight was measured in each group before the infection and on the 1st, 4th, and 7th day postinfection. Sterile beads induced a weight loss only after the 1st day after inoculation in the three diets (Fig. 2). After the infection, a significant weight loss was observed on the 4th day compared with the sterile bead group (P < 0.001). Even if the weight of the animals increased from the 4th to the 7th day, it remained significantly lower than in the sterile bead group (P < 0.05). This result was comparable in the three groups of animals.


Figure 2
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  		Fig. 2. Evolution of the mice weight. The mice were weighed before and after the injection on days 1, 4, and 7. °P < 0.05 and °°°P < 0.001 vs. sterile beads. For infected mice, §§§P < 0.001 vs. day 0, **P < 0.01 vs. day 4. For sterile beads, {Delta}{Delta}{Delta}P < 0.001 day 0 vs. day 1.

 
The gain of lean body mass was higher for the infected animals fed with the {omega}-3 diet. The mice were fed for 5 wk, and the percentage of fat was measured before and after the infection for each diet. Before the infection, the {omega}-6 diet (16.5 ± 0.1%) had a statistically lower percentage of fat compared with both the control (18.2 ± 0.1%, P < 0.001) and the {omega}-3 groups (18.5 ± 0.1, P < 0.001). After the infection, the percentage of lean body mass gain was evaluated for each diet (Fig. 3). On the 4th day, the mice fed with the {omega}-3 diet had a higher lean body mass gain (2.35 ± 0.23%) compared with the control (0.9 ± 0.4%) and the {omega}-6 diets (0.8 ± 0.3%) (P < 0.05). On the 7th day, all groups presented an equivalent gain of lean body mass ({omega}-3: 2.9 ± 0.5%, control: 2.8 ± 0.6%, {omega}-6: 1.8 ± 0.5%).


Figure 3
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  		Fig. 3. Percentage of lean body mass gain of mice fed with the {omega}-3 diet, the control diet, and the {omega}-6 diet measured with the PIXImus densitometer during the course of infection. *P < 0.05 vs. the 2 other diets.

 
The fatty acid composition of the lung tissue was influenced by the diet. As infection itself had no influence on the membrane lipid composition (data not shown), the results shown correspond, for each diet, to the pooling of data collected over the time course of the experiment. PC and PE were extracted and assessed for their content in AA, EPA, and DHA (Table 4). DHA was increased in the {omega}-3 and control diets compared with the {omega}-6 group. Consistent with these data, AA was significantly increased in the {omega}-6 group (Table 4). When PC and PE data were analyzed according to the {omega}-6:{omega}-3 ratio, ratios were statistically higher in the {omega}-6 group than in the control or the {omega}-3 diets. The {omega}-6:{omega}-3 ratio was significantly lower in the {omega}-3 group than in the control and {omega}-6 groups (Table 4).


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  		Table 4. Fatty acid composition of lung phospholipids

 
The mice fed with the {omega}-3 diet had a better survival than {omega}-6 and control groups. No mortality was observed during the first 24 h after instillation of the pathogen. On the 4th and 7th days, the percentage of survival was statistically higher for the mice fed with the {omega}-3 (64%) compared with both the {omega}-6 (51%) and control (50%) diets. None of the animals instilled with the sterile beads died during the experiment. The mice died between the 2nd and 3rd day after the infection, and no mortality was observed afterward (Fig. 4). All the mice were still infected with Pseudomonas on the 7th day (Fig. 1).


Figure 4
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  		Fig. 4. Survival of mice infected with Pseudomonas aeruginosa or sterile beads (n = 126) fed with the {omega}-3 diet, the control diet, and the {omega}-6 diet. *P < 0.001 vs. the sterile beads, control, and {omega}-6 diets, **P < 0.001 vs. sterile beads and {omega}-3 diet.

 
The {omega}-3 diet significantly improved DAFC on the 4th day after infection. On the 1st day after the infection, there was a slight decrease of the DAFC compared with the animals that received only the sterile beads (not significant). No difference was observed between the three groups (Fig. 5, A and B). Interestingly, on the 4th day, DAFC remained elevated for the animals fed with the {omega}-3 diet (13.8 ± 1.7%), statistically higher than the two other groups (control: 2.9 ± 1.7%, {omega}-6: 5.7 ± 1.5%) (Fig. 5A). On the 7th day after the infection, DAFC increased in the control group to levels comparable to the {omega}-3 diet (18.4 ± 0.9% vs. 15 ± 0.9%). This parameter remained lower in the {omega}-6 group (7.2 ± 2.3%). In all the groups that received sterile beads, DAFC was comparable between the groups and over time (Fig. 5B).


Figure 5
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  		Fig. 5. Evaluation of the distal alveolar fluid clearance (DAFC) on the 1st, 4th, and 7th days in mice infected with P. aeruginosa (A) or sterile beads (B). The mice were fed with the {omega}-3 diet, the control diet, and the {omega}-6 diet during 5 wk. *P < 0.001 vs. the other groups on the same day.

 
The diets did not influence P. aeruginosa-induced increase in permeability and extravascular lung water. The alveolar-capillary barrier permeability was significantly increased after P. aeruginosa instillation, and this parameter was not influenced by the diets. We did not observe major changes for the lung W/D weight ratio, which was not statistically different between the experimental groups (Table 5).


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  		Table 5. Evaluation of the alveolar barrier permeability and extravascular lung water

 
The number of polymorphonuclear cells in the BAL was increased in the {omega}-3 group the 1st day after infection. On the 1st day after the infection, the mice fed with the {omega}-3 diet had a higher number of polymorphonuclear cells compared with the two other diets (Table 6). On the 4th day, the number in the {omega}-3 group decreased to reach values comparable to the control group. The {omega}-6 group had a number of cells that was lower than both of the other groups. The number of cells recovered for the animals instilled with sterile beads remained low and stable over time with no difference between the diets (Table 6).


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  		Table 6. Number of white blood cells and percentage of PMN in the bronchoalveolar lavage

 
TNF-{alpha} was increased during the early period of the infection, and IL-6 remained elevated until the 4th day. Measurement of BAL TNF-{alpha} level did not show any influence of the diet at any time of the study. The values were only elevated on the 1st day after the infection (Fig. 6A). There was a trend for a lower value in the {omega}-3 group that did not reach a statistical significance. IL-6 level was elevated on the 1st and 4th days in all the infected groups compared with the sterile bead-instilled animals (Fig. 6B). The values of IL-6 reached on the 1st day after infection in the {omega}-3 group were, however, significantly lower than in the two other experimental groups.


Figure 6
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  		Fig. 6. Level of TNF-{alpha} (A) and IL-6 (B) (pg/ml) in the bronchoalveolar lavage on the 1st (D1), 4th (D4), and 7th (D7) days in mice infected with P. aeruginosa (P) or instilled with sterile beads (SB). *P < 0.01 vs. SB, °P < 0.05 vs. other diets with P. aeruginosa on the same day.

 
Modulation of ENaC and Na-K-ATPase expression following lung chronic infection with P. aeruginosa. Diet had an impact on the expression of {alpha}-ENaC and beta-ENaC, two pore-forming subunits of the amiloride-sensitive Na+ channel, in the absence of any infection. {alpha}-ENaC expression was significantly higher in the control group compared with the {omega}-6 and {omega}-3 groups (Fig. 7). A comparable pattern was observed with the beta-ENaC subunit. After infection, we did not observe any significant changes over time for the {alpha}-ENaC subunit expression. Interestingly, for beta-ENaC, the value observed in the control diet was significantly higher before infection than at any other time point (Fig. 7). The expression of beta-ENaC mRNA was significantly decreased on day 4 and day 7 of infection for the {omega}-3 diet and the control diet (Fig. 7). Analysis of the {alpha}1-Na-K-ATPase mRNA showed a comparable increase in the control diet before infection. No difference was observed between the diets during the course of the infection (Fig. 8). After the infection, we could not find any significant differences in the modulation of {alpha}-ENaC and beta-ENaC mRNA and {alpha}1-Na-K-ATPase mRNA expression among the three different diets.


Figure 7
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  		Fig. 7. Analysis of the modulation of {alpha}-epithelial Na+ channels ({alpha}-ENaC) and beta-ENaC mRNA before infection (day 0) and on the 1st, 4th, and 7th day after P. aeruginosa instillation. The modulation of this mRNA by Northern blot hybridization was subjected to a densitometric analysis. The expression of mRNA was calculated at each time point as the % of expression relative to an untreated control. §P < 0.05 vs. day 4, *P < 0.05 vs. other days, °P < 0.05 vs. other diets, °°P < 0.05 vs. day 0.

 

Figure 8
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  		Fig. 8. Analysis of the modulation of {alpha}1-Na-K-ATPase mRNA before infection (day 0) and on the 1st, 4th, and 7th day after P. aeruginosa instillation. The modulation of this mRNA by Northern blot hybridization was subjected to a densitometric analysis. The expression of mRNA was calculated at each time point as the % of expression relative to an untreated control. °P < 0.05 vs. other diets.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
P. aeruginosa is a pathogen frequently involved in human diseases. It is associated with acute as well chronic infections such as ventilator-associated pneumonia, chronic obstructive pulmonary disease, and cystic fibrosis (10, 16). In a chronic murine model of P. aeruginosa infection, our results showed that {omega}-3 or {omega}-6 fatty acids can, after 5 wk, modulate lung phospholipids and body composition. Survival of animals with P. aeruginosa infection was improved by the {omega}-3 diet, compared with the control and the {omega}-6 diets. This improvement in survival is probably multifactorial. We have shown that alveolar liquid clearance is preserved, there is a greater neutrophil influx in the first 24 h and potentially a decrease in the IL-6 response, and finally, there is a change in body composition and the percentage of lean body mass.

The first part of our study was to evaluate the consequences, on the host, of a 5-wk diet associated with different levels of {omega}-3 and {omega}-6 PUFA. The diets were specifically designed from the reference mixture AIN-93. We could therefore obtain two derived diets with either a high content in EPA and DHA or AA (Table 2). In this study, we used an animal model of chronic infection by a mouse intratracheal inoculation with the P. aeruginosa agar beads. Consistent with van Heeckeren et al. (43), mice infected with P. aeruginosa lost weight on the 4th day after inoculation, and no difference was observed between the three diets. However, on the 4th day, the mice fed with the {omega}-3 diet had a higher lean body mass gain compared with the control and the {omega}-6 diets (P < 0.05). To further characterize the changes induced in our model, we studied the fatty acid composition of the lung, focusing on the phospholipids that are an essential component of the biological membranes. PE and PC represent, with sphingomyelin, the most abundant lipids in these membranes. PC is located in the outer leaflet of cell membranes, and, in the lung, originates also from the alveolar surfactant. On the contrary, PE is located in the inner leaflet of cell membranes, reflecting more accurately the membrane lipid environment. The analysis of both of these fractions showed, in our study, that in 5 wk, we could modulate the lung membrane composition: the {omega}-6:{omega}-3 ratio was significantly lower in the {omega}-3 diet compared with the control and {omega}-6 groups. These results are in general accordance with the literature. Several studies demonstrated that oral administration of DHA could, in mice and humans, correct this imbalance and reverse the pathological manifestations (20, 24, 29). The first part of the current study shows that oral administration of realistic amounts of {omega}-3 or {omega}-6 PUFA in the diets is indeed able to modulate the body composition and the fatty acid ratio in phospholipid fractions from the lung. Based on these initial observations, we therefore decided to evaluate the benefits of each diet on the host response. These benefits were evaluated in a chronic model of P. aeruginosa pneumonia.

An initial survival analysis showed a significant improvement of the survival in the {omega}-3 diet-fed group. All animals instilled with sterile beads survived. All deaths in the {omega}-3 and {omega}-6 diet-fed groups occurred on the 4th day after infection. Recently, the effect of PUFA on the survival of mice challenged with P. aeruginosa was analyzed by van Heeckeren et al. (42). These authors showed that cumulative survival rates between Cftr knockout mice and wild-type mice did not differ when animal diets were supplemented with DHA. In this work, the authors used a different strain of mice, and DHA was administered as a supplementation; moreover, the strain of Pseudomonas is not clearly stated. All of these differences may explain the absence of effect on survival even if the bacterial load is in the same range. This difference in mortality observed in our work is probably multifactorial. We tried to explore several pathways.

We initially focused on the lung bacterial load. In fact, a lower inoculum or a better clearance of the pathogen could be associated with the improvement in survival. We show that the decrease in lung bacterial load is strictly comparable among the three diets (Fig. 1), therefore ruling out this hypothesis.

In acute lung injury, DAFC represents a major parameter correlated to survival (31, 44). In our study, we did not find any difference in DAFC on the 1st day after infection. However, on the 4th day, we observed a major decrease for the control group and the {omega}-6-fed animals compared with the {omega}-3 diet. On the 7th day, only the {omega}-6 group remained with a low DAFC, suggesting a deleterious effect of the {omega}-6 PUFA on the recovery process. Interestingly, Rezaiguia et al. (36) showed a major increase in DAFC only 24 h after Pseudomonas instillation. They proposed that the effect was mostly due to a TNF-{alpha}-dependent mechanism. In our model, DAFC increase was observed later, i.e., at a time when TNF-{alpha} levels were nearly back to normal. Several experimental differences can explain these apparently contradictory results: we used a chronic model with lower inocula per animal, and experiments were performed in mice rather than in rats. In our model of chronic lung infection adapted from Cash et al. (7) in rats, we have previously shown that DAFC remained unresponsive to stimulation on the 5th day after chronic Pseudomonas infection (5). It is likely that the major difference between these two studies is due to differences in the bacterial load. In rats, there was an initial increase in alveolar permeability that returned to baseline 5 days after instillation (5). The adaptation of the model to mice showed different results with a persistent increase of permeability throughout the 7 days of experiment.

On the 4th day, with a similar increase in permeability and extravascular lung water, the {omega}-3-fed animals kept a normal DAFC, whereas it decreased more than twofold in the other two groups. We did not observe in our study any difference in permeability and extravascular lung water. A potential bias could be related that most of the mortality occurs on the 4th day of infection; we therefore only studied the animals that survived at this time point. It is possible that both permeability and extravascular lung water were, in the animals that died within those 4 days, a lot higher than in the survivors. Concerning the alveolar response, the membrane composition could be involved. Besides the well-documented impact of dietary fatty acids on the inflammatory response (26, 40), several authors attribute a major influence to the lipid environment to tune up or down the sodium transport. Experiments of delipidation and reconstitution of the sodium pump lipidic environment from cattle or crocodile showed a major role of the lipid type on the pump activity (48). {omega}-3 significantly improved this activity. In the first part of our study, analysis of lung tissue composition revealed that each diet could modulate the {omega}-6:{omega}-3 ratio in both PC and PE fractions. One likely explanation of our results would be that the improvement in the {omega}-3 group is due to a direct modulation of the transepithelial transport of fluids, caused by a {omega}-3 PUFA-dependent effect on one or several of the molecular entities associated with this transport. So even if there is no difference in permeability or extravascular lung water, DAFC is improved through PUFA incorporation in the membrane. Interestingly, the influence of the nutrition on alveolar clearance has already been suggested: in rats, Sakuma et al. (38) showed that malnutrition could impair amiloride-insensitive and dibutyryl-cGMP-sensitive alveolar fluid clearance. The specific analysis of the membrane composition was, however, not studied, but DAFC was restored after refeeding or sodium glutamate supply.

Vectorial sodium transport in the lung is the main force driving water out of the alveoli (2, 37). This transport relies on sodium absorption at the apical side of pneumocyte by the ENaC and at the basolateral side by its extrusion by Na-K-ATPase activity. The {alpha}-ENaC and beta-ENaC mRNA expression has been found to be modulated in the lung in a chronic model of Pseudomonas infection (11). In our model, {alpha}-ENaC mRNA was not significantly modulated over time during infection, although a decrease was observed at day 4 and day 7 of infection, especially for the {omega}-3 group (45% of uninfected control at day 4). The expression of beta-ENaC mRNA was significantly decreased on day 4 and day 7 of infection for the {omega}-3 and control diets. No modulation of {alpha}1-Na-K-ATPase mRNA could be found. These results are in general agreement with the findings reported by Dagenais et al. (11). We could not find any significant differences in the modulation of {alpha}-ENaC and beta-ENaC mRNA expression among the three different diets that could explain the protective effect of the {omega}-3 diet on DAFC at day 4 postinfection. Because the expression profile of {alpha}-ENaC and beta-ENaC mRNA shows a similar pattern for the three diets, it suggests that besides ENaC mRNA expression, the {omega}-3 diet could modulate ENaC activity. Membrane composition of {omega}-3-fed animals shows a significant decrease in AA compared with the control and {omega}-6 diet. This could have a consequence on ENaC since AA and AA derivatives have been found to decrease ENaC activity. AA production driven by cytosolic phospholipase A2 has been shown to decrease ENaC open probability in A6 kidney cells (46) and kidney CCD cells (45), whereas AA or arachidonic analog decreases ENaC activity and surface expression in Xenopus oocyte (6). Lung liquid clearance depends on ENaC activity but also on amiloride-insensitive transport that contributes to roughly 50% of the lung liquid clearance (23, 35). The {omega}-3 diet could therefore also modulate amiloride-insensitive channel. Our study failed to show a link between the diets and sodium transport, but we did not study the membrane protein expression and function of ENaC and Na-K-ATPase.

The better prognosis of the animals fed with the {omega}-3 diet could also be related to the modulation of the inflammatory response (15, 32). Several hypotheses may explain this phenomenon. The most interesting is based on the biochemical properties of EPA. EPA can be converted to the 3-series prostaglandins and 5-series leukotrienes (25); these mediators are considered less biologically active and less inflammatory than the AA-derived 2-series prostaglandins and 4-series leukotrienes (17, 27). Although it did not reach a statistical significance for TNF-{alpha}, we observed on the 1st day after Pseudomonas instillation a lower level of TNF-{alpha} and IL-6 in the {omega}-3 group compared with the two other groups. These data are in accordance with the literature (15, 32). With regard to the inflammatory cells found in the BAL, we observed a higher number of polymorphonuclear cells in the {omega}-3 group on the 1st day after instillation. We did not find any difference in chemokine production in the BAL (data not shown).

It is important to note that this study has several limitations. We cannot conclude a direct link between the improvement in survival and the DAFC. We observed several changes in the host response (cytokine production, cellular response, body composition), all of which can participate in the decrease in mortality. The analysis of mRNA expression for ENaC and the Na-K-ATPase failed to show a statistical difference between the diets, but an analysis of the membrane protein expression and function would be necessary to completely study this hypothesis.

In conclusion, our study shows that a 5-wk diet with {omega}-3 PUFA can improve lung and host response to a bacterial challenge. The survival can be related to the improvement of the DAFC, the recruitment of the inflammatory cells, and the inflammatory response, but also to the change in body composition and the percentage of lean body mass. The clinical consequences of these data could be interesting. In fact, it has been shown in cystic fibrosis that these patients often have fatty acid deficiencies (19, 20, 28). We propose that modulation of the {omega}-6:{omega}-3 balance can represent an interesting target in diseases where Pseudomonas aeruginosa is frequently observed such as chronic obstructive pulmonary disease or cystic fibrosis.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
We are grateful to the association Vaincre la Mucoviscidose (B. P. Guery and P. Barbry), Numico Laboratories Research (B. P. Guery and F. Gottrand), and La Société Francaise de Nutrition (Nutricia grant, M. Pierre) for financial support.


   ACKNOWLEDGMENTS
 
The expert technical assistance of Julie Cazareth is greatly appreciated. We are grateful to the Institut de Médecine Prédictive et de Recherche Thérapeutique for the PIXImus acquisition.


   FOOTNOTES
 

Address for reprint requests and other correspondence: B. P. Guery, EA 2689, Faculté de Médecine, Physiologie, 1 Place de Verdun, F-59045 Lille, France (e-mail: bguery{at}invivo.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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