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J. Nutr. First published December 3, 2008; doi:10.3945/jn.108.096115
 Journal of Nutrition, doi:10.3945/jn.108.096115
Vol. 139, No. 1, 82-89, January 2009
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© 2009 American Society for Nutrition

Nutrition and Disease

Dietary (n-3) Polyunsaturated Fatty Acids Affect the Kinetics of Pro- and Antiinflammatory Responses in Mice with Pseudomonas aeruginosa Lung Infection1–3,

Hélène Tiesset4–6, Maud Pierre4–6, Jean-Luc Desseyn5–7, Benoît Guéry5,6,8, Christopher Beermann9, Claude Galabert10, Frédéric Gottrand4–6 and Marie-Odile Husson4–6*

4 EA 3925, Faculty of Medicine, 59045 Lille, France; 5 IMPRT/IFR 114, 59045 Lille, France; 6 University of Lille 2, 59045 Lille, France; 7 INSERM U837-JPARC, 59045 Lille cedex, France; 8 EA 2689, Faculty of de Medicine, 59045 Lille, France; 9 Danone Research, Center of Specialized Nutrition, D-61276 Friedrichsdorf, Germany and Food Technology, University of Applied Science Fulda, D-36039 Fulda, Germany; and 10 CERM, Hospital Renée-Sabran, CHRU Lyon, 83406 Giens-Hyères, France

* To whom correspondence should be addressed. E-mail: mohusson{at}chru-lille.fr.


   ABSTRACT
TOP
 ABSTRACT
Introduction
 Materials and Methods
 Results
 Discussion
 LITERATURE CITED
 
The underlying mechanisms by which eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) affect host resistance to Pseudomonas aeruginosa are unclear. The aim of this study was to determine their role on the kinetic of pro- and antiinflammatory response in lung infection. Mice fed either a control diet or a diet enriched with EPA and DHA were infected intratracheally and we studied lung expression of proinflammatory markers [CXCL1, interleukin (IL)-6, tumor necrosis factor-{alpha}], antiinflammatory markers (IL-10, A20, and I{kappa}B{alpha}), and PPAR{alpha} and PPAR{gamma}. The inflammatory response was assessed using recruitment of neutrophils and macrophages into bronchoalveolar lavage fluid, bacterial clearance from the lung, pulmonary injury, and 7-d survival rate. Compared with the control group, EPA and DHA delayed the expression of proinflammatory markers during the first 2 h (P < 0.05), upregulated proinflammatory marker expression (P < 0.05), and induced overexpression of antiinflammatory markers at 8 h (P < 0.05), enhanced recruitment of neutrophils at 16 h (P < 0.05), and induced PPAR{alpha} and PPAR{gamma} overexpression at 4 and 8 h (P < 0.01), respectively. Pulmonary bacterial load decreased and pulmonary injury and mortality were reduced during the first 24 h (P < 0.05). In conclusion, EPA and DHA modulate the balance between pro- and antiinflammatory cytokines, alter the early response of the host to P. aeruginosa infection, and affect the early outcome of infection.


   Introduction
TOP
 ABSTRACT
 Introduction
Materials and Methods
 Results
 Discussion
 LITERATURE CITED
 
Dietary supplementation with (n-3) long-chain PUFA (LCPUFA),11 such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) improves the health of patients with chronic inflammatory diseases and various autoimmune disorders (1–4). (n-3) LCPUFA modulate lipid synthesis (5–7), cytokine release (8), leukocyte activity (9), endothelial activation (10), and immune function, particularly T cell proliferation and signaling in animals and humans (11). Their antiinflammatory properties are partially mediated by interaction with PPAR, which results in suppression of leukocyte responses (12–14). During the past decade, several authors have suggested that (n-3) LCPUFA also improve resistance to infectious disease (15). In a previous study, we observed that consumption of an (n-3) LCPUFA-enriched diet for 5 wk by mice with chronic Pseudomonas aeruginosa lung infection decreased mortality, enhanced distal alveolar fluid clearance (16), and exerted a suppressive effect on mucin overexpression (17). However, the underlying mechanisms by which (n-3) LCPUFA affect the host response to P. aeruginosa infection are unclear.

P. aeruginosa is a Gram-negative bacterium and an opportunistic pathogen that causes pneumonia in immunocompromised humans and severe pulmonary damage in cystic fibrosis patients (18). It initiates an intense inflammatory response, which progressively destroys pulmonary tissue and is associated with a high mortality rate. Because of P. aeruginosa resistance to antibiotics and the severity of infections, there is currently much interest in improving host resistance to P. aeruginosa, especially by nutritional means (19).

Host defenses in response to infection are normally orchestrated by an early nonspecific inflammatory response. This defense is mediated by a combination of chemical inflammatory mediators (antibacterial peptides, nitric oxide, and complement factors) and cells such as dendritic cells, macrophages, neutrophils, natural killer cells, and T cells. These cells play a major role in the shift to an adaptive response by secreting proinflammatory markers that coordinate the recruitment of immune cells. In acute bacterial lung infection, the rapid release of proinflammatory chemokines such as CXCL1, CXCL2, and CXCL3 and cytokines such as tumor necrosis factor-{alpha} (TNF{alpha}), interleukin (IL)-6, and IL-8 promotes the recruitment and activation of macrophages and neutrophils. Although these cells play an important role in bacterial clearance, they may also cause local tissue damage, because they secrete proteolytic enzymes, free radicals, and reactive oxygen species (20,21). The immune response is normally regulated by negative feedback loops to protect host tissue from damage and to enable immune cells to return to an inactive state after the infection cases. To regulate the production of potent inflammatory agents, activation of the host response triggers an antiinflammatory response. This response is mediated in part by chemical mediators such as IL-10, which inhibits nuclear factor-{kappa}B (NF-{kappa}B)-dependent cytokines (22), the I{kappa}B{alpha} subunit, which facilitates retention of NF-{kappa}B subunits in the cytoplasm, and A20 (also known as TNFAIP3), which inhibits the transcription of NF-{kappa}B subunit genes (23–26). The timing and magnitude of both pro- and antiinflammatory responses are crucial determinants of the severity of infectious diseases (27,28).

The aim of this study was to determine the mechanisms by which an EPA and DHA-enriched diet affects host response during the first 24 h of P. aeruginosa lung infection. Mice were fed either a control diet (control group) or an EPA and DHA-enriched diet (EPA + DHA group) for 5 wk and were then infected intratracheally with a 5 x 1010 colony-forming units (CFU)/L suspension of P. aeruginosa.


   Materials and Methods
TOP
 ABSTRACT
 Introduction
 Materials and Methods
Results
 Discussion
 LITERATURE CITED
 
    Mice and nutrition. Five-wk-old male C57BL/6 mice were purchased from Harlan and housed as recently described (16). The mice were randomly allocated to 1 of 2 groups and fed either a control diet or a diet supplemented with EPA and DHA (Danone Research) for 5 wk before P. aeruginosa infection as previously described (16,17). The 2 formulations used were identical to the AIN-93G diet (29) in the contents of energy, proteins, minerals, micronutrients, and vitamins. In the EPA+ DHA diet, soybean oil was replaced with a combination of 2 isocaloric fatty acid mixtures equal in energy and fat content to soybean oil (Table 1). In the EPA + DHA diet, EPA constituted 11.4% of the total fat content and DHA constituted 4.7% of the total fat content; the control diet contained neither EPA nor DHA (Table 1). All procedures were in accordance with the French Guide for the Care and Use of Laboratory Animals and the Guidelines of the European Union. This study was approved by the local Laboratory Animal Facility Executive Committee.


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  		TABLE 1 Fatty acid composition of experimental diet1

 
    Lung infection with P. aeruginosa and collection of lung and bronchoalveolar lavage fluid. P. aeruginosa strain PAO1 was grown at 37°C in Luria Broth (Sigma) and incubated in a rotating incubator for 12 h. The inoculum was calibrated to a concentration of 1012 CFU/L using spectroscopy. Before the mice were infected, they were anesthetized with sevorane (Abbott) and placed in dorsal recumbency. A 24-gauge animal feeding needle was inserted transtracheally and used to administer 50 µL of P. aeruginosa suspension (5 x 107 CFU per mouse). Mice were killed by intraperitoneal injection of 0.3 mL sodium pentobarbital (Ceva) 0.5, 1, 2, 4, 8, or 16 h after inoculation. In each case, the lungs were excised aseptically. A portion of the lung tissue was homogenized in 1 mL of RNase-free water and stored in lysis solution for real-time quantitative assays. Proteins were extracted from the 2nd portion of lung tissue in 100 µL of PBS containing 1% Nonidet P-40, 0.05% sodium deoxycholate, 0.1% SDS, 100 mmol/L sodium p-nitrophenyl phosphate, and 1 protease inhibitor tablet (Roche) and separated by Western blotting. For bacteria counts, 100-µL volumes of serial dilutions of lung were placed on agar and incubated for 24–48 h at 37°C. Bronchoalveolar lavage (BAL) was performed as previously described (16).

    RT-PCR. An RT-PCR assay was performed using the ABI PRISM 7000 sequence detection system. Total RNA was isolated from the lung tissue using the Invisorb Spin Tissue RNA Mini kit (Laboratory Eurobio). RT was performed using a High-capacity cDNA Archive kit (Applied Biosystems) according to the manufacturer's protocol. cDNA samples were stored at –20°C. All primers (Supplemental Table 1) were used with SYBR green as fluorescent intercalant. RT-PCR was performed according to the manufacturer's protocol. Briefly, the reaction mixture contained 5 µL cDNA, 12.5 µL SYBR green PCR Master mix (Applied Biosystems), and either 300 nmol/L of the CXCL1, A20, or I{kappa}B{alpha} primers, 900 nmol/L of the Il-6 or TNF{alpha} primers, or 200 nmol/L of the β-actin primer in a final volume of 25 µL. RT reactions were carried out in duplicate and were normalized against the expression of β-actin, the endogenous control. Data are expressed as fold changes in expression of infected compared with uninfected mice at various times after infection by using the formula: 2–{Delta}{Delta}Ct.

    Measurement of cytokines in BAL fluid. Levels of chemokine CXCL1 were quantified using an ELISA kit from R&D Systems. IL-6, TNF{alpha}, and IL-10 were quantified using an ELISA kit from CliniSciences. Absorbance at 450 nm was determined using a microplate reader.

    Western blotting and antibodies. Samples containing 20 µg of total protein were electrophoresed by SDS-PAGE and transferred to nitrocellulose membranes by electroblotting. Nonspecific binding was blocked by incubating the membrane in PBS containing 5% low-fat milk for 1 h. Membranes were probed with protein A20, phosphorylated I{kappa}B{alpha}, and nonphosphorylated antibodies (dilution 1:200) (Tebu-bio) for 1 h at room temperature. After incubation with horseradish peroxidase-conjugated secondary antibodies, signals were quantified using a chemiluminescence system (Las 3000, Fuji). Each blot was stripped and probed for β-actin to confirm equal loading.

    Permeability and extravascular lung water. Permeability and extravascular lung water were determined as described previously (16). Permeability was calculated using the following formula: permeability (%) = {[radioactivity in lung – (Qb x radioactivity in plasma)] / radioactivity in plasma} x 0.07 x animal weight x 100, where Qb is the lung blood volume calculated as follows: Qb = (weight lung + blood x hemoglobin concentration supernatant x water ratio homogenate x 1.039) / (hemoglobin concentration blood x water ratio blood).

    Time points of the study. Expression of the proinflammatory molecules CXCL1/KC, TNF{alpha}, and IL-6, the antiinflammatory molecules IL-10, A20, and I{kappa}B{alpha}, and PPAR{alpha} and PPAR{gamma} in the lung and recruitment of neutrophils and macrophages in BAL fluid (BALF) were monitored during the first 16 h of infection. We assessed the effects of the EPA + DHA diet on the inflammatory response from clearance of bacteria from the lung, pulmonary injury, and survival during d 1 of infection.

    Statistical analysis. Results are expressed as the means ± SEM. Expression of pro- and antiinflammatory genes, cytokine concentration, neutrophil and macrophage numbers, lung wet:dry weight ratio, and bacterial load were analyzed in samples obtained from mice killed at different times and in each diet group. Because these 2 factors (time and diet) and the groups were independent, we used a 2-way ANOVA test. When effects (P < 0.05) were detected, we performed post hoc testing using Bonferroni's test. Differences were considered significant at P < 0.05. Results of protein marker concentration, alveolar capillarity, and extravascular water were analyzed using a 1-way ANOVA followed by the Mann-Whitney U test. Cumulative survival rates were compared using the log-rank test.


   Results
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
Discussion
 LITERATURE CITED
 
    Kinetics of gene expression during lung infection according to diets. Diet did not influence the basal expression of genes, because mRNA levels measured in mice fed the control and EPA+DHA diets were similar. P. aeruginosa induced expression of proinflammatory genes (CXCL1, IL-6, and TNF{alpha}) and antiinflammatory genes (IL-10, A20, and I{kappa}B{alpha}) genes in the 2 groups of mice and expression was affected by diet (Table 2). In the control group, expression of CXCL1, IL-6, and TNF{alpha} peaked at 0.5 h and then decreased over the course of the infection. In contrast, in the group fed EPA + DHA, CXCL1 and IL-6 mRNA levels were low for 1 h of infection, increased significantly 2 h after infection, and peaked 8 h after infection (11- to 40-fold higher than the value of mRNA obtained in the control group). In this group, the temporal pattern of TNF{alpha} expression differed from that of CXCL1 and IL-6 mRNA and was similar to that of the control group; it was elevated 0.5 h after infection, moderately increased for 2 h of infection, and then decreased over time.


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  		TABLE 2 mRNA levels of proinflammatory genes (CXCL1, IL-6, TNF{alpha}), antiinflammatory genes (A20, I{kappa}B{alpha}, IL-10), and PPAR{alpha} and PPAR{gamma} at various time during P. aeruginosa lung infection in mice fed the control or EPA + DHA diet for 5 wk12

 
The highest mRNA levels of the antiinflammatory marker A20 were observed 1 h after infection in the control group and 8 h after infection in the EPA + DHA group. The effect of diet on A20 mRNA was similar to the effect of the diet on IL-6 and CXCL1 expression. In contrast, mRNA levels of the antiinflammatory makers I{kappa}B{alpha} and IL-10 were low during the first 4 h of infection in both groups of mice and peaked 8 and 16 h after infection, respectively. mRNA levels of these markers in the EPA + DHA group were 61- and 457-fold higher than the basal group (uninfected mice) and 2- to 9-fold higher than the mRNA measured in infected mice fed the control diet.

The kinetics of PPAR{gamma} mRNA expression were similar to those of the antiinflammatory genes IL-10 and I{kappa}B{alpha}. PPAR{gamma} gene expression was very low during the course of infection in both groups, but PPAR{gamma} was overexpressed 8 h after infection in the EPA + DHA group. Expression of PPAR{alpha} increased earlier than that of PPAR{gamma} with their peak at 0.5 h after infection in the control group and at 4 h after infection in the EPA + DHA group.

    Kinetics of cytokine secretion in BALF and concentration of protein markers in lungs according to diet. Secretion of CXCL1, IL-6, TNF{alpha}, and IL-10 increased progressively over time in both groups (Table 3). However, concentrations of IL-6 and TNF{alpha} 8 h after infection and concentrations of CXCL1, IL-6, and IL-10 16 h after infection were higher in the EPA + DHA group than in the control group.


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  		TABLE 3 CXCL1, IL-6, TNF{alpha}, and IL-10 protein concentrations at various time after infection with P. aeruginosa in the BALF of mice fed a control or EPA + DHA diet for 5 wk1

 
Protein concentrations of A20, I{kappa}B{alpha}, PPAR{alpha}, and PPAR{gamma} in the lungs were measured 8, 12, and 16 h after infection (Fig. 1). Protein concentrations of PPAR{alpha}, PPAR{gamma}, and A20 were significantly elevated in the EPA + DHA groups at 4, 12, and 16 h after infection compared with the control group at the same times of infection. Concentrations of the phosphorylated form of I{kappa}B{alpha} were measured to assess NF-{kappa}B activation pathway activity. The concentration of the phosphorylated form of I{kappa}B{alpha} was significantly lower in the EPA + DHA group than in control mice 12 h after infection.


Figure 1
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  		FIGURE 1  Relative concentration of protein markers A20 (A), phosphorylated I{kappa}B{alpha} (B), PPAR{alpha} (C), and PPAR{gamma} (D) in the lung of mice fed a control diet or EPA + DHA diet 8, 12, and 16 h after P. aeruginosa infection. Data are expressed in arbitrary units corresponding to the ratio of peroxydase-labeled markers:peroxydase-labeled β-actin as quantified using a chemiluminescence system and values are expressed as the mean ± SEM, n = 5. Asterisks indicate differences between the control and EPA + DHA diets within sample times, *P < 0.05; **P < 0.01; ***P < 0.001 (1-way ANOVA followed by the Mann-Whitney U test).

 
These data confirm the results of previous studies on lung cytokine secretion even if the kinetics of proteins did not strictly parallel mRNA levels. The time required for protein synthesis and the probable secretion of cytokines by immune cells that migrated early in BAL could partially explain this difference for the first 2 h. The highest rate of proteins at 8 or 16 h confirmed the postponed secretion of cytokines in the EPA + DHA group.

    Effect of EPA + DHA on the recruitment of macrophages and neutrophils. The recruitment of macrophages was not affected by diet during the first 8 h of infection. Macrophages were the predominant cell population in BALF during the first 2 h of infection. The number of macrophages decreased until 8 h after infection and increased again 16 h after infection. The neutrophils began to migrate into BALF 2 h after infection and the influx was significantly greater for the EPA + DHA group than the control diet group 16 h after infection. (Fig. 2; Supplemental Table 2).


Figure 2
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  		FIGURE 2  Numbers of macrophages (A) and neutrophils (B) 0.5, 1, 2, 4, 8, and 16 h after P. aeruginosa infection in the BALF of mice fed the control or EPA + DHA diet for 5 wk. Values are mean ± SEM of x 108 cells/L, n = 5. Asterisks indicate differences between the control diet and the EPA + DHA diet for each day of the experiment, *P < 0.05 (2-way ANOVA test followed by Bonferroni correction).

 
    Effect of EPA + DHA on the pulmonary injury 8 and 16 h after infection. P. aeruginosa infection significantly increased the alveolar-capillary barrier permeability in both the control group (2.6 vs. 13.9%) and the EPA + DHA group (2.9 vs. 11%) (Fig. 3). Alveolar-capillary barrier permeability was significantly higher in the control group than in the EPA + DHA group 8 h after infection and was still significantly elevated 16 h after infection in the control group, whereas it had normalized by that time in the EPA + DHA group.


Figure 3
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  		FIGURE 3  Percentage alveolar-barrier capillarity permeability 8 and 16 h after P. aeruginosa infection in mice fed control or EPA + DHA diets for 5 wk. Data are expressed as percentage permeability, values are expressed as the mean ± SEM, n = 5. Asterisks indicate differences. Asterisks indicate different from the control at that time: *P < 0.05; **P < 0.01; ***P < 0.001. Within a group, means in a row with superscripts without a common letter differ, P < 0.05 (1-way ANOVA followed by Mann-Whitney U test). Un., control permeability values obtained from uninfected mice 8 h after an endotracheal injection of saline water. Values are mean ± SEM, n = 5.

 
The extravascular water content of the lung was assessed from the lung wet:dry weight ratio. This ratio was higher in infected mice (4.9 ± 0.1) than in uninfected mice (3.6 ± 0.2) at 8 h (P < 0.001) for the control group and 4.6 ± 0.1 vs. 3.9 ± 0.1 (P < 0.001) for the EPA + DHA group and at 16 h (5.1 ± 0.1 vs. 3.6 ± 0.2 (P < 0.1) for the control group, and 4.9 ± 0.3 vs. 3.9 ± 0.1 (P < 0.05) for the EPA + DHA group. However, diet did not affect this ratio (data not shown).

    Effect of EPA + DHA on survival rate. Mice died between d 1 and d 3 of infection and survived thereafter. The EPA + DHA group had a higher survival rate during the first 24 h (71.4%) compared with the control group (42.9%), but the groups did not differ thereafter (Fig. 4).


Figure 4
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  		FIGURE 4  Survival rates after infection with P. aeruginosa in mice fed control or EPA + DHA diets. Data represent the number of dead mice, n = 20. Asterisks indicate differences between the control diet and the EPA + DHA diet for each day of the experiment, *P < 0.05 (log-rank test).

 
    Effect of EPA + DHA on bacterial clearance. Diets did not affect the number of viable bacteria (Fig. 5). This result indicates that group differences in proinflammatory response, antiinflammatory response, and lung injury during the first 8 h of infection were not caused by differences in lung bacterial load. However, the number of viable bacteria in the lung decreased significantly between 8 and 16 h after infection in the EPA + DHA group (from 52 x 1010 ± 29 x 1010 to 10 x 1010 ± 5 x 1010 CFU/L, respectively) but did not change in the control group (from 22 x 1010 ± 10 x1010 to 23 x1010 ± 11 x1010 CFU/L, respectively). Therefore, the EPA + DHA diet improved bacterial clearance in the late stage of infection.


Figure 5
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  		FIGURE 5  Numbers of bacteria in the lung of mice fed control or EPA + DHA diets 2, 4, 8, and 16 h after P. aeruginosa infection. Data are expressed as mean ± SEM for n x1010 CFU of P. aeruginosa/L, n = 10–15. Asterisks indicate differences between the control diet and the EPA + DHA diet for each day of the experiment, P < 0.05; **P < 0.01; ***P < 0.001 (2-way ANOVA test followed by Bonferroni correction).

 

   Discussion
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
LITERATURE CITED
 
This study demonstrates that EPA and DHA modify the responses of pro- and antiinflammatory molecules to acute pulmonary infection with P. aeruginosa. The production of proinflammatory cytokines is delayed during the first 2 h of infection and is upregulated by 8 h after infection. The overexpression of proinflammatory cytokines coincides with exacerbation of the antiinflammatory response. In contrast, overexpression of proinflammatory cytokines in the control group occurred earlier and the antiinflammatory response was reduced 90–95% compared with the antiinflammatory response observed in the EPA + DHA group. This kinetic modulation and exacerbation of antiinflammatory parameters by EPA and DHA was associated with improved bacterial clearance, reduced lung injury, and increased survival during the first 24 h of infection. These results complement published data on the antiinflammatory properties of EPA and DHA diets in other models of lung infection/inflammation. In 1990, Peck et al. (30) reported better survival in mice fed fish oil compared with mice fed safflower oil using a burn-injury model of mice infected with P. aeruginosa. The authors attributed the improvement to low splenic macrophage production and a reduction in prostaglandin E2 production. In the same way, Freedman et al. (31) showed that DHA decreased eicosanoid production in cftr–/– mice but not in wild-type mice with lipopolysaccharide (LPS)-induced lung inflammation. Our analysis of the early response to lung aggression offers a new insight into how the antiinflammatory properties of an EPA + DHA diet may minimize the consequences of P. aeruginosa infection.

The delay in the proinflammatory response suggests that activation of signaling pathways such as the NF-{kappa}B pathway are postponed. Toll-like receptors (TLR) and the myeloid differentiation primary response gene 88 (MyD88) are involved in the development of early, nonspecific host responses to P. aeruginosa (32). TLR are recruited within cell membrane microdomains (rafts) upon stimulation by bacterial products (26,33). As we reported previously, our diet changes lung membrane composition (16). These changes in the composition of phospholipids, which presumably extend to cell membrane microdomains (34,35), may modify the fluidity of cell membranes and alter their properties (36–38). Lee et al. (39,40) used an LPS-stimulated macrophage culture model to show that TLR-4 and its inflammatory downstream cascade are affected by DHA. Our in vivo approach confirms the previous results obtained using monocytes, neutrophils, and epithelial cell cultures challenged with LPS or bacteria (41,42). Our study demonstrated a temporal effect of EPA and DHA on the expression and level of cytokines, which may explain discrepancies between the results of in vitro studies in which cytokines were studied at various times after infection. The EPA- and DHA-induced reduction in proinflammatory cytokine expression was transient, because it was followed by increased expression. This increase is consistent with our previous results, which showed that levels of TNF{alpha} increased 24 h after acute lung infection in mice (43). The overexpression of proinflammatory cytokines 8 h after infection was not caused by an elevated bacterial load or an elevated number of neutrophils, but may represent activation of proinflammatory signaling pathways. Pro- and antiinflammatory responses constitute host-pathogen interactions (33). We showed that pro- and antiinflammatory responses were 5- to 20-fold and 3- to 10-fold greater, respectively, and concomitant in mice fed EPA + DHA compared with mice fed the control diet (Fig. 6). However, we could not determine whether overexpression of the proinflammatory response induces overexpression of the antiinflammatory response directly or whether these 2 events originate simultaneously followed by a delay in the antiinflammatory response.


Figure 6
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  		FIGURE 6  General representation of the effect of EPA and DHA on the kinetics of proinflammatory, antiinflammatory, PPAR{alpha}, and PPAR{gamma} gene expression. The graphs depict mean mRNA levels for proinflammatory genes (CXCL1 and IL-6), antiinflammatory genes (A20, IL-10, I{kappa}B{alpha}), and PPAR{alpha} and PPAR{gamma} genes for each interval (expressed in minutes).

 
(n-3) LCPUFA-induced overexpression of PPAR{alpha} and PPAR{gamma} mRNA may represent a key point in the control of inflammation. PPAR are expressed in various types of cells that play important roles in inflammation and immunity. PPAR{alpha} is abundant in macrophages, monocytes, and endothelial cells (44,45) and PPAR{gamma} is abundant in alveolar epithelial cells and the bronchial submucosa (46,47). A recent study indicated that PPAR{alpha}-deficient mice have abnormally prolonged responses to various inflammatory stimuli (48). Ligands of PPAR{alpha} and PPAR{gamma} inhibit the expression of proinflammatory genes in response to cytokine activation (13). This antiinflammatory effect of PPAR ligands is dependent upon inhibition of NF-{kappa}B activation, which increases the expression of PPAR{alpha} (11). EPA and DHA and their metabolites are natural PPAR ligands and induce PPAR{gamma} mRNA expression (49). Overexpression of PPAR{alpha} and PPAR{gamma} mRNA was only observed in mice fed EPA/DHA (Fig. 6). Despite these observations, we could not determine the specific roles of PPAR{alpha} and PPAR{gamma} in the antiinflammatory response, because did not have access to antagonists or knockout mice for both markers. The preferential association of PPAR{alpha} with macrophages, which were most prevalent 4 h after infection, could explain the temporal difference in maximum expression of PPAR{alpha} (4 h after infection) and PPAR{gamma} (8 h after infection).

Macrophage and neutrophil influx to the site of infection is an important component of the host response. Both cell types enhance the production of proinflammatory cytokines and take part in the clearance of bacteria. According to the overexpression of CXCL1 mRNA in EPA + DHA mice 8 h after infection, the number of neutrophils increased with time and was higher than in control mice 16 h after infection. These results confirm those of our previous study with a model of chronic P. aeruginosa chronic infection, which showed that the influx of neutrophils in BALF is high 24 h after infection (16).

The EPA + DHA group had reduced lung injury 8 h after infection and promoted efficient clearance of bacteria 16 h after infection. Although mortality was similar in the 2 groups after 48 h of infection, the EPA + DHA group had postponed death during the first 24 h. This delay indicated that the (n-3) LCPUFA diet conferred resistance against P. aeruginosa lung infection. Long-term administration of (n-3) LCPUFA may strengthen host resistance, boost the effectiveness of specific therapeutic intervention, and prevent P. aeruginosa infection. The amount of PUFA provided to the mice was not excessive in human terms. EPA + DHA constituted 15% of total fat intake and 2.4% of total energy intake. This corresponds to an intake of 5–6 g/d of EPA + DHA in humans (most interventional studies in humans have used doses ranging from 0.6 to 2.7 g/d). Further investigations are needed to confirm the beneficial effect of our (n-3) LCPUFA diet in preventing P. aeruginosa infection in mice with cystic fibrosis.

We demonstrated that the balance between pro- and antiinflammatory activities during infection is altered by an EPA + DHA diet and results in improved host response and clinical outcomes. The balance between pro- and antiinflammatory activities is the key to reducing lung damage and improving resistance to P. aeruginosa infection. The EPA + DHA diet may be of use in the treatment of diseases in which this bacterium is frequently present, such as pulmonary disease and cystic fibrosis.

In conclusion, the EPA + DHA diet increases host resistance to P. aeruginosa infection. This suggests that an EPA + DHA diet may be used as a preventive treatment against the initial colonization of P. aeruginosa, as an adjunct to antibiotic treatment, and to reduce morbidity.


   ACKNOWLEDGMENTS
 
We thank Dr. Jean-Claude Sirard (INSERM U801, Pasteur Institute of Lille) for the primer sequences.


   FOOTNOTES
 
1 Supported by a grant from the French association Vaincre la Mucoviscidose and Danone Research, Germany. Back

2 Author disclosures: H. Tiesset, M. Pierre, J.-L. Desseyn, B. Guéry, C. Beermann, C. Galabert, F. Gottrand, and M.-O. Husson, no conflicts of interest. Back

3 Supplemental Tables 1–3 are available with the online posting of this paper at jn.nutrition.org. Back

11 Abbreviations used: BAL, bronchoalveolar lavage; BALF, bronchoalveolar lavage fluid; CFU, colony-forming unit; DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid; IL, interleukin; LCPUFA, long-chain PUFA; LPS, lipopolysaccharide; MYD88, myeloid differentiation primary response gene 88; NF-{kappa}B, nuclear factor-{kappa}B; Qb, lung blood volume; TLR, toll-like receptor; TNF{alpha}, tumor necrosis factor-{alpha}. Back

Manuscript received 9 July 2008. Initial review completed 4 August 2008. Revision accepted 17 October 2008.


   LITERATURE CITED
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
 LITERATURE CITED
 

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