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© 2008 American Society for Nutrition J. Nutr. 138:1336-1342, July 2008

Nutritional Immunology

Cis-9,trans-11-Conjugated Linoleic Acid Inhibits Allergic Sensitization and Airway Inflammation via a PPAR{gamma}-Related Mechanism in Mice1–3,

Anke Jaudszus4, Martin Krokowski5, Peter Möckel4, Yasemin Darcan5, Anzhela Avagyan5, Paolo Matricardi5, Gerhard Jahreis4,7 and Eckard Hamelmann5–7*

4 Institute of Nutrition, Friedrich Schiller University, 07743 Jena, Germany; 5 Department of Pediatric Pneumology, Charité University Medicine, 13353 Berlin, Germany; and 6 University Children Hospital, Ruhr University, 44791 Bochum, Germany

* To whom correspondence should be addressed. E-mail: eckard.hamelmann{at}charite.de.


   ABSTRACT
TOP
 ABSTRACT
Introduction
 Materials and Methods
 Results
 Discussion
 LITERATURE CITED
 
Milk consumption from early childhood on has been found to be inversely correlated with allergic sensitization and the onset of bronchial asthma. We tested whether cis-9,trans-11-conjugated linoleic acid (c9,t11-CLA), naturally occurring in milk fat, may prevent allergic sensitization and inhibit airway inflammation in a murine asthma model. BALB/c mice were fed a diet enriched in 1 wt% of c9,t11-CLA or a control diet 7 d prior to and for 32 d during sensitization [d 1 and 14, 100 mg/L ovalbumin (OVA) in adjuvant vs. PBS] and airway challenges (d 28–30, 1% OVA in PBS vs. PBS). Subgroups of mice were coadministered 20 µmol/L of the selective PPAR{gamma} antagonist GW9662 during each OVA challenge. C9,t11-CLA feeding resulted in significantly reduced IgE production and allergen-induced in vivo airway hyperresponsiveness. Further, less mucous plugging of segmental bronchi and significantly reduced interleukin-5 and eosinophils were determined in bronchoalveolar lavage fluids of c9,t11-CLA-fed mice. C9,t11-CLA feeding prevented the downregulation of PPAR{gamma} mRNA in the lung tissues observed after allergen sensitization and airway challenges in control mice. The inhibitory effects of c9,t11-CLA on airway inflammation were partially prevented by coadministration of GW9962. Further, c9,t11-CLA feeding resulted in a significantly lower concentration of the eicosanoid precursor, arachidonic acid, in tissue lipids. These findings demonstrate that dietary c9,t11-CLA can reduce allergic airway inflammation, most likely via a PPAR{gamma}-related mechanism and by reducing eicosanoid precursors. They give new insights into the fatty acid-mediated mechanism of immunomodulation and may represent a step toward an attractive novel strategy in the dietary prevention and treatment of allergic asthma.


   Introduction
TOP
 ABSTRACT
 Introduction
Materials and Methods
 Results
 Discussion
 LITERATURE CITED
 
The recent epidemic trend of childhood allergy in most developed countries has been related to lifestyle changes typical of westernization, such as hygiene, an indoor life, reduced physical activity, and a modified diet. In particular, for the diet hypothesis, epidemiological and cross-sectional evidence has been relatively consistent in suggesting that an unbalanced high intake of (n-6) PUFA, like linoleic acid (LA)8 [18:2(n-6)] (1,2), may contribute to the onset of allergic sensitization and asthma (3,4). Mechanistic explanations conferred plausibility. Once ingested, LA is enzymatically metabolized to arachidonic acid (AA) [20:4(n-6)], which acts as a substrate for the cyclooxygenase (COX) and lipoxygenase (LOX) enzyme systems for subsequent formation of prostaglandins and thromboxanes of the 2-series and leukotrienes of the 4-series, which are closely involved in the pathophysiology of allergic sensitization and asthma (5).

Recent evidence, however, suggested a more complex scenario; while excessive LA generates proinflammatory mediators, its conjugated derivatives subsumed to the term conjugated linoleic acids (CLA) were shown to possess antiinflammatory properties and may thus exert beneficial effects on different immune functions (6). This concept is supported by current epidemiological studies showing an inverse association between asthmatic disease and the dietary intake of ruminant full-cream milk and butter (7,8), a main source of CLA, which originate from natural in vivo biohydrogenation in the rumen (9). Moreover, CLA can be endogenously converted from vaccenic acid in rodents (10) and humans (11). CLA comprise a group of various positional (e.g. 8/10, 9/11, 10/12, 11/13) and geometric (c/c, c/t, t/c, t/t) isomers; among these, the cis-9,trans-11-CLA (c9,t11-CLA) isomer not only is the predominant naturally occurring one but also seems to exert an antiinflammatory action (12; for comparison to t10,c12-CLA, see also 13). Similar to antiinflammatory fatty acid-like synthetic drugs (14,15), c9,t11-CLA can bind to and activate the nuclear transcription factor PPAR{gamma} (16), whose ligand-dependent activation dramatically inhibits cellular immune responses and production of inflammatory mediators (17–22). The antiinflammatory action of c9,t11-CLA has been linked to PPAR{gamma} (23–25). Moreover, cellular uptake of c9,t11-CLA has been shown to entail a profound decline of eicosanoid release, perhaps by inhibiting the incorporation of AA into membrane phospholipids (25,26). Finally, c9,t11-CLA can inhibit COX-2 (23,25) and LOX (27) mRNA expression, thus blocking the rate-limiting step of prostaglandin, thromboxane, and leukotriene synthesis.

In light of these antiinflammatory properties of c9,t11-CLA, it is therefore very intriguing to analyze possible effects of this compound in regard to allergen-induced immune response and disease. We therefore investigated in this study whether systemically applied c9,t11-CLA may inhibit allergen-induced airway inflammation in a murine model of asthma.


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

    Animals and diets. All experimental protocols were approved by the animal care committee (Berlin Office for Occupational Safety, Protection of Health and Technical Safety-LaGetSi). Female 6- to 8-wk-old BALB/c mice purchased from Harlan/Winkelmann were accustomed to the housing conditions (pathogen-free, 22°C, 12-h-light/12-h-dark cycle) for 5 d before they were randomly assigned to 1 of 2 experimental diet groups. A commercial ovalbumin (OVA)-free basal mouse diet (V1535; ssniff) was enriched with 1.5% by weight of diet of an esterified CLA preparation free of t10,c12-CLA (66% c9,t11-CLA; Cognis Illertissen), allowing an application of 1 wt% of c9,t11-CLA. For the control diet, sunflower oil (Bombastus-Werke AG) was added in sufficient quantity to the basal diet to balance the c9,t11-CLA with LA (diet composition shown in Table 1). We intended to test whether c9,t11-CLA can inhibit the development of allergic airway inflammation primarily by affecting the systemic sensitization. Thus, for a primary prevention approach, diets were given 7 d prior to and for 32 d during the complete sensitization and airway challenge protocol (Fig. 1). Mice had free access to food and water throughout the entire study. Body weight and mean diet consumption were documented for each mouse every third day.


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  		TABLE 1 Composition of the experimental diets

 

Figure 1
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  		FIGURE 1  Asthma induction protocol and feeding systems of the primary prevention approach. Both diet groups were subdivided into OVA and PBS groups for sensitization (d 0 and 14) and airway challenge (d 28–30). A 5th experimental group corresponding to the c9,t11-CLA OVA group received 20 µmol/L GW9662, a selective PPAR{gamma} inhibitor, by nebulization 20 min immediately before and during each OVA challenge on d 28–30. Dietary intervention started 7 d prior to the first intraperitoneal sensitization. On d 31, whole-body plethysmography was conducted to assess in vivo AR. On d 32, mice were killed for further analyses.

 
    Sensitization and airway challenge. Mice of both diet groups were subdivided into the treatment groups and either sensitized by intraperitoneal injection of OVA (Grade VI, 100 g/L; Sigma) or sham-sensitized with PBS (Seromed, Biochrom), emulsified in adjuvant [10 g/L Al(OH)3; Pierce Chemical] in a total volume of 200 µL on d 0 and 14 of the protocol. After 14 d, mice were challenged via the airways with OVA (1% wt:v in PBS) or PBS daily for 20 min on 3 consecutive days (d 28–30) by ultrasonic nebulization (Fig. 1). To delineate a receptor-mediated mode of action, 1 experimental group corresponding to the c9,t11-CLA OVA group received 20 µmol/L of the selective PPAR{gamma} antagonist GW9662 (2-chloro-5-nitro-N-phenylbenzamide, Axxora) by nebulization 20 min immediately before and during each challenge (14,15) on d 28–30.

In vivo airway reactivity

On d 31, whole-body plethysmography (Buxco) was conducted to assess in vivo airway reactivity (AR) to increasing doses of inhaled metacholine (Mch) as described (28). Data are expressed as -fold increase in the dimensionless parameter enhanced pause (Penh) of baseline values after PBS.

Immunoglobulin measurement

On d 32, blood was collected from tail veins into heparinized tubes before mice were killed. Total IgE, OVA-specific IgE, and IgG1 antibody titers were measured by ELISA as previously described (29), related to pooled standards, and expressed as µg/L or ELISA units/L.

Bronchoalveolar lavage

Bronchoalveolar lavage (BAL) was performed via a tracheal tube with PBS (2 x 800 µL) and total cell count was determined using a hemocytometer. We assessed differential cellular content of lavage fluid microscopically due to morphological criteria on cytospin preparations after Diff-Quik staining (Dade Behring AG). Interleukin (IL)-5 protein content of BAL fluid was measured by ELISA according to the manufacturer's protocol (BD Pharmingen).

Histology

For histology, left lung lobes were embedded in TissueTek and 4-µm sections of the frozen samples were prepared and stained with periodic acid Schiff (PAS) for detection and semiquantitative scoring of goblet cell hyperplasia and mucopolysaccharide accumulation (score: 0, no mucus; 1, few cells secreting mucus; 2, many cells secreting mucus; 3, extensive mucus).

TaqMan-quantitative RT-PCR

Right lobes were used for PPAR{gamma} gene expression analysis by TaqMan technique for real time quantitative RT-PCR. Total RNA was extracted from the homogenized samples using a commercially available RNA isolation kit (Invitek) and subjected to a 1-step PCR multiplex amplification with β-actin (71 bp) as housekeeping gene to normalize PPAR{gamma} (141 bp). The following were added to 10 µL sample template and processed in triplicates in 96-well optical plates (Applied Biosystems, Applera): 40 µL of an optimized reaction preparation containing TaqMan Universal PCR Master mix (Applied Biosystems, Roche), target primers at a concentration of 300 nmol/L each (PPAR{gamma} forward, 5'-TAACTGCCGGATCCACAAA-3' and reverse, 5'-ATCTCCGCCAACAGCTTCT-3'; BioTez Berlin-Buch), 100 nmol/L fam-labeled probe of PPAR{gamma} (5'fam-CTGTCGGTTTCAGAAGTGCCTTGC-3'tam; MWG-Biotech), housekeeping primers at a concentration of 20 nmol/L each (β-actin forward, 5'-GTTTGAGACCTTCAACACCCCA-3' and reverse, 5'-GACCAGAGGCATACAGGGACA-3'), 100 nmol/L vic-labeled probe of β-actin (5'vic-CCATGTACGTAGCCATCCAGGCTGTG-3'tam), 10 U/µL RNasin (Promega), 10 U/µL Moloney murine leukemia virus-H RT (Promega), and diethylpyrocarbonate-water. PCR conditions were as follows: 30 min at 48°C for RT reaction, 10 min at 95°C as a hot start followed by 40 cycles of 15 s at 95°C for denaturation, and 1 min at 60°C for annealing extension. As an internal standard, serial dilutions of murine bronchial epithelial cell line template of a known amount were included in duplicate in each PCR analysis. TaqMan analysis was conducted on a 7300 Real Time PCR system and documented by the SDS software v1.3.2 (Applied Biosystems, Applera).

Fatty acid analysis

In brief, the lipids of white adipose tissue, liver, erythrocytes, and spleen were extracted from homogenized samples using a methanol/chloroform mixture according to Bligh and Dyer (30). Further, a base-catalyzed methylation was performed with sodium methylate and 1,1,3,3-tetramethylguanidine (Sigma-Aldrich) before TLC was conducted on layers of silica gel using hexane:diethyl ether:acetic acid (85:15:1, v:v:v) for purification of FAME. The resulting FAME were separated in a GC (GC 17a V3, Shimadzu) as previously described (31) using a fused-silica capillary column (DB 225 MS, 60-m x 0.25-mm i.d., 0.25-µm film thickness; J & W Scientific), suitable for achieving separation of FAME ranging from C4 to C26 and were downstream detected by flame ionization. GC conditions were as follows: injection modus, split 1:20, split flow 150 mL/min; injector temperature, 260°C; temperature program, initial isothermal period of 2 min at 70°C, temperature increase of 10°C/min to 180°C, 2°C to 220°C/min for 5 min, 2°C/min to 230°C for 7 min; detector temperature, 270°C; carrier gas, H2; and linear velocity, 42 cm/s.

Statistical analysis

A 2 x 2 factorial ANOVA was conducted to determine the effects of treatment (PBS or OVA) and diet (control or c9,t11-CLA) and the interaction between the factors, followed by Student-Newman-Keuls post hoc testing. Treatment and diet did not interact for any analyzed parameter except for PPAR{gamma} mRNA expression with hybride interaction determined (P = 0.05; 2-way interaction ANOVA). Thus, for this parameter, only the main effect of diet was interpretable and we performed comparisons between the groups with unpaired Student's t test. For Penh determination, ANOVA for repeated measures was used. Differences between the c9,t11-CLA OVA groups with or without GW9662 and the data on tissue concentration of fatty acids were evaluated by 1-way ANOVA. Data are reported as means ± SEM of at least 6 mice per group. Differences were considered significant at P ≤ 0.05. Statistical analysis was carried out with SPSS software v11.5.


   Results
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
Discussion
 LITERATURE CITED
 
    Systemic allergen sensitization. Oral administration of c9,t11-CLA 7 d prior to and for 32 d during allergen (OVA) sensitization and airway challenges depressed the production of total IgE antibodies compared with the control diet (–33.2%; P = 0.034; Fig. 2A). With regard to OVA-specific immunoglobulin production, the c9,t11-CLA-mediated reduction in plasma antibody titers was prominent for OVA-specific IgE (–42.1% vs. control diet OVA; nonsignificant, P = 0.185; Fig. 2B) and significant for OVA-specific IgG1 (–31.8% vs. control diet OVA; P = 0.048; Fig. 2C) and OVA-specific IgG2a (–76% vs. control diet OVA; P = 0.018; Fig. 2D). These data suggested that the c9,t11-CLA diet had a general effect on allergen-specific Ig production, thus not only inhibiting Th2-dependent (IgE and IgG1) but also suppressing Th1-skewed (IgG2a) immune responses.


Figure 2
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  		FIGURE 2  Plasma titers of total IgE and OVA-specific IgE, IgG1, and IgG2a antibodies were measured in OVA- or PBS-treated mice fed either a c9,t11-CLA-enriched or control diet prior to and during immunization. Data are means ± SEM, n = 6. Means without a common letter differ, P ≤ 0.05. 1 2-way ANOVA.

 
    In vivo AR. Penh values, reflecting the AR in response to aerosolized Mch and the degree of airway obstruction, increased dose dependently in OVA-sensitized and -challenged mice compared with nonsensitized controls. Mice in the c9,t11-CLA diet group displayed reduced in vivo airway responses compared with their OVA-treated and control diet-fed counterparts (P = 0.018 vs. control diet OVA, ANOVA for repeated measures; Fig. 3). To analyze if the mechanism underlying the action of c9,t11-CLA involved PPAR{gamma} ligation, we coadministered a specific receptor blocker. Application of aerosolized GW9662 to c9,t11-CLA-fed mice immediately before and during each allergen challenge failed to continuously abrogate the c9,t11-CLA-mediated suppression of airway hyperresponsiveness (AHR). However, a restoration of the OVA-mediated enhancement in Penh was observed at 25 g/L Mch (P = 0.041 vs. c9,t11-CLA OVA; 1-way ANOVA; Fig. 3). Response curves of PBS-treated mice only slightly inclined and were not affected by the diets (Fig. 3).


Figure 3
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  		FIGURE 3  AR in response to increasing concentrations of Mch was determined by whole-body plethysmography of OVA- or PBS-treated mice fed either a c9,t11-CLA-enriched or control diet prior to and during immunization. An immunized subgroup fed with c9,t11-CLA additionally received GW9662 during the allergen challenges. Data are means ± SEM, n = 6, of -fold increase of baseline Penh values after PBS. Index graphs without a common letter differ, P ≤ 0.05 (ANOVA for repeated measures). *Different from c9,t11-CLA OVA at 25 g/L Mch, P ≤ 0.05 (1-way ANOVA).

 
    Airway inflammation. IL-5 protein content of BAL fluid of OVA-sensitized and -challenged mice was reduced by ~50% after c9,t11-CLA feeding prior to and during the immunization period (P = 0.035 vs. control diet OVA; Fig. 4B). More apparent than for airway function, this inhibition in airway inflammation due to c9,t11-CLA feeding was abrogated when mice received GW9662 for PPAR{gamma} blocking immediately before and during each OVA challenge (P = 0.05 vs. c9,t11-CLA OVA), strongly suggesting a receptor-mediated mode of action.


Figure 4
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  		FIGURE 4  Differential cell count (A) and IL-5 protein content (B) in BAL fluids of OVA- or PBS-treated mice fed either a c9,t11-CLA-enriched or control diet prior to and during immunization were determined 48 h after the last airway challenge. Data are means ± SEM, n = 6. Means without a common letter differ, P ≤ 0.05. 1 2-way ANOVA. *Different from c9,t11-CLA OVA, P ≤ 0.05 (1-way ANOVA).

 
Systemic sensitization and allergen challenges elicited substantial cellular infiltration into the airways that was greater in the control diet group than in the c9t11-CLA group (214 ± 44 vs. 123 ± 17 x 106/L; P = 0.05; Fig. 4A). Eosinophilic influx was most pronounced in the immunized and control diet-fed mice (142 ± 33 x 106/L; Fig. 4A). In contrast, allergen-induced pulmonary eosinophilia occurred to a markedly lesser extent in c9,t11-CLA-fed mice (55 ± 8 x 106/L; P = 0.02 vs. control OVA). Administration of aerosolized GW9662 just failed to significantly revert the c9,t11-CLA-mediated inhibition in eosinophilic influx. However, total cell content increased in the BAL fluids after GW9662 treatment (215 ± 40 x 106/L; P = 0.05 vs. c9,t11-CLA OVA; Fig. 4A). BAL fluids of PBS controls contained few cells, mainly identified as monocytes.

Goblet cell hyperplasia and related mucopolysaccharide accumulation, another indicator of airway inflammation, were comparatively diminished after c9,t11-CLA feeding prior to and during allergen sensitization and airway challenges (score, 0.9 ± 0.2 vs. 2.3 ± 0.3 for control diet OVA; Fig. 5). The lungs of PBS-treated animals displayed no histopathological alterations regardless of which diet was fed (not shown).


Figure 5
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  		FIGURE 5  For detection of mucopolysaccharide accumulation, 4-µm sections through bronchi of distal airways were stained with PAS. Pictures are representative for the respective diet group or treatment. Original magnification x2.5 (upper photographs) and x20 (lower photographs). PAS score was semiquantitatively assessed by microscopically evaluating goblet cell hyperplasia and mucopolysaccharide accumulation of at least 6 bronchi/bronchioles per mouse, n = 6.

 
    PPAR{gamma} mRNA expression in OVA lungs. Gene expression analysis revealed that the development of allergic airway disease was inversely correlated with PPAR{gamma} mRNA production in the lungs. OVA treatment caused a 65% reduction of PPAR{gamma} mRNA expression in lungs of control diet-fed animals compared with PBS sham-sensitization (P = 0.004; Fig. 6). In contrast, c9,t11-CLA fed prior to and during allergen sensitization and airway challenges prevented the allergen-related decrease in PPAR{gamma} mRNA expression and restored the levels to that of nonsensitized controls (P ≤ 0.001 vs. control diet OVA). Only marginal amounts of PPAR{gamma} mRNA were detectable in the lungs of c9,t11-CLA-fed mice after GW9662 treatment (P ≤ 0.001 vs. c9,t11-CLA OVA; Fig. 6). PPAR{gamma} mRNA expression in PBS mice did not differ among the diet groups.


Figure 6
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  		FIGURE 6  Expression of PPAR{gamma} mRNA in the lung tissue of OVA- or PBS-treated mice fed either a c9,t11-CLA-enriched or control diet prior to and during immunization was determined by quantitative real-time PCR. Data are means ± SEM, n = 6, of relative (β-actin-normalized) mRNA levels of PPAR{gamma}. Means without a common letter differ, P ≤ 0.05 (unpaired Student's t test).

 
    C9,t11-CLA tissue accumulation and eicosanoid precursory fatty acid distribution. We further intended to delineate if feeding with c9,t11-CLA vs. control diet influenced the distribution of eicosanoid precursory fatty acids. Therefore, we assessed the fatty acid spectrum in various organs. In general, the different diets causally determined recovery of abundant amounts of the main fatty acids of the respective supplemented oil in all analyzed tissues. According to physiological fatty acid metabolism, c9,t11-CLA accumulated not only in lipogenic tissues, such as white adipose tissue and liver, but also in the erythrocytes as middle-term marker and the spleen as the central organ of the immune system after 39 d of feeding (Table 2; P ≤ 0.001 vs. control for all analyzed tissues). From preliminary studies, we know that c9,t11-CLA is also incorporated into the lung and the skeletal striate muscle in a time-dependent manner according to dietary supplementation (data not shown). The enrichment of tissue lipids in c9,t11-CLA was associated with a decrease in the concentration of eicosanoid precursory (n-6) PUFA (P ≤ 0.001 vs. control for white adipose tissue, liver, spleen and P ≤ 0.01 vs. control for erythrocytes), primarily caused by reduction of 20:4(n-6) (AA) (P ≤ 0.01 vs. control diet; except for white adipose tissue; Table 2). Compared with the control diet, the concentration of beneficial (n-3) PUFA increased in the erythrocytes of c9,t11-CLA-fed mice (P ≤ 0.05; Table 2), accounting for a (n-6):(n-3) PUFA ratio of 5:1 (as compared with 8:1 in the control).


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  		TABLE 2 Tissue concentration of 18:2c9,t11 and 20:4(n-6) and sums of (n-6)- and (n-3) PUFA of mice fed either a c9,t11-CLA enriched or a control diet for 39 d1

 

   Discussion
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
LITERATURE CITED
 
Growing evidence suggests that the resolution phase of inflammatory states underlying many acute and chronic airway diseases closely involves the action of PPAR{gamma} (32). During airway inflammation, upregulation of the expression of genes encoding inflammatory mediators occurs in response to cytokines or other exogenous stimuli and requires binding of several transcription factors, including nuclear factor {kappa}B, activator protein 1, and/or signal transducers and activators of transcription 1, to the promoter sequences of responsive elements. PPAR{gamma} activation interferes with the regulatory influences of these transcription factors by blocking their homo- or heterodimerization, crucial for DNA binding, thus inhibiting upregulation of inflammatory gene expression (33). Because c9,t11-CLA has the potential to bind to and activate PPAR{gamma} (16), circumstantial evidence linking antiinflammatory effects with this molecule suggests a receptor-mediated mode of action (23–25). To our knowledge, the present study demonstrates for the first time that oral administration of c9,t11-CLA inhibits or reduces the allergen-induced increase in plasma Ig concentrations, development of unspecific AHR, and IL-5-associated lung eosinophilia in an animal model of asthma. This c9,t11-CLA-mediated decrease in humoral, functional, and cellular responses was accompanied by diminished pulmonary alterations like mucus accumulation, characterizing the asthmatic phenotype.

It is generally accepted that the pathological symptoms and clinical hallmarks of allergic asthma are the result of a Th2-type dominated cytokine profile with increased levels of, e.g. IL-4, IL-5, and IL-13. They may induce airway inflammation and AHR directly through effects on airway smooth muscle and bronchial mucosa (34) or indirectly via effector cells such as B-cells, mast cells, and eosinophils (35,36) and further sustain the Th2 response (37). Among the Th2 cytokines, IL-5 is a major factor promoting eosinophilia by priming the cells for heightened responsiveness, enhancing their adhesion to endothelium, increasing eosinophilic cytotoxicity, and prolonging the viability of mature eosinophils (38). Because c9,t11-CLA intervention significantly reduced the Th2 cytokine IL-5 in the BAL fluid (Fig. 4) and PPAR{gamma} ligation has been shown to inhibit Th2 cytokine production (39), it is tempting to speculate that c9,t11-CLA exerts its effects primarily by reducing a Th2 response, possibly via PPAR{gamma} activation. Consistent with this, the c9,t11-CLA diet-caused reduction in allergen-specific Ig levels (Fig. 2), essentially driven by both IL-4 and IL-13 (35), and the mucous plugging (Fig. 5) that has been linked to IL-13 (40), could be due to prevention of Th2 cytokine production for subsequent normalization of lung function (Fig. 3). Coadministration of GW9662, a selective PPAR{gamma} inhibitor, prior to and during each allergen airway challenge partially or totally abrogated the inhibitory effect of c9,t11-CLA on most of the local characteristic parameters of airway inflammation, indicating that c9,t11-CLA indeed acted through PPAR{gamma}. This receptor is constitutively expressed by alveolar macrophages (21) and dendritic cells (20), airway epithelial cells (22), endothelial cells (41), leukocytes (neutrophils, eosinophils) (14,41), lymphocytes (18), and smooth muscle cells of the pulmonary vasculature (42), all exerting pivotal roles in regulating airway homeostasis. It is likely that c9,t11-CLA intervention simultaneously inhibits different effector cell responses. Development of airway inflammation in allergen-sensitized and -challenged control mice was positively associated with a dramatic decrease in PPAR{gamma} mRNA in lung tissues. In contrast, PPAR{gamma} was highly expressed, to similar levels as those in PBS animals, in lungs of OVA-treated and c9,t11-CLA-fed mice (Fig. 6), implicating an inverse and causal relationship between PPAR{gamma} and the induction of allergic airway inflammation (43). This was further sustained by the observation that GW9662 treatment partly reconstituted the airway inflammation and almost abolished PPAR{gamma} mRNA, suggesting that PPAR{gamma} expression was subject to a negative feedback mechanism by receptor blocking. Moreover, these data suggested a regulatory role for PPAR{gamma} activating ligands on PPAR{gamma} mRNA expression and supported previous related observations (44). Because systemic deficiency of PPAR{gamma} in vivo is lethal (45), we restricted the application of the specific antagonist, GW9662, for receptor blocking to the lungs (14,15). Therefore, mechanistic explanations for c9,t11-CLA-mediated effects beyond the local compartment are limited in our model and serve no direct evidence for systemic effects of the fatty acid intervention. However, according to physiological fatty acid metabolism, c9,t11-CLA accumulated in the white adipose tissue, liver, erythrocytes, spleen (Table 2), lung, and skeletal striate muscles (data not shown) after 39 d of feeding. Therefore, it could have likely affected systemic immune cell responses as well. This might be possible, because GW9962 administration could only partly prove c9,t11-CLA's underlying mechanism. Because c9,t11-CLA was systemically administered, it is conceivable that local GW9662 application for reconstitution of AHR and chemoattraction of peripheral eosinophils was not strong enough to abrogate c9,t11-CLA's inhibitory effect on cellular influx and AHR at high Mch doses. Because AA is the main precursor of inflammatory eicosanoids and AHR is closely related to inflammatory eicosanoid release (5,46), the c9,t11-CLA-mediated decrease in AR in our model might be the result of an additional effect by reducing eicosanoid precursory fatty acids. Indeed, incorporation of c9,t11-CLA into tissue lipids occurred at the expense of AA, accounting for a significant drop of (n-6) PUFA (Table 2). In addition, c9,t11-CLA has previously been shown to reduce eicosanoid formation by inhibiting COX-2 (23,25) and LOX (27) mRNA expression. Moreover, and because the diets were free of AA, c9,t11-CLA could have likely prevented the conversion of LA to AA (47,48). The overall reduction of (n-6) PUFA and partial increase in (n-3) PUFA in our experiments led to an improvement of the (n-6):(n-3) ratio of PUFA in the c9,t11-CLA-fed mice. In the liver and erythrocytes (as a middle-term marker reflecting the dietary uptake) of c9,t11-CLA-fed mice, the (n-6):(n-3)-ratio approximated the dietary recommendation of 5:1 (49). This may very well serve plausibility to a second mode of action of c9,t11-CLA-mediated immune modulation in light of the diet hypothesis.

In conclusion, this study shows a specific inhibitory function of c9,t11-CLA on allergen-induced airway inflammation by a dual mechanism through PPAR{gamma}-mediated inhibition of Th2-related immune response and reduction of eicosanoid precursory (n-6) PUFA. Moreover, these data suggest that this fatty acid, naturally occurring in dairy products, may contribute to the protective effect of milk with respect to childhood asthma (7). For the future, oral intervention with this natural fatty acid might therefore represent an attractive novel strategy in the prevention and treatment of asthma and allergic airway disease.


   ACKNOWLEDGMENTS
 
We thank Christine Seib and Viola Kohlrautz (Department of Pediatric Pneumology, Charité University Medicine, Berlin, Germany) for their excellent technical assistance.


   FOOTNOTES
 
1 Supported by a grant from Cognis GmbH & Co. KG and in part by grants from the German Research Council (DFG Ha 2162/2-1 and DFG Ja 893/5) and the Präventions- und Informationsnetzwerk Allergie und Asthma (pina). Back

2 Author disclosures: A. Jaudszus, M. Krokowski, P. Möckel, Y. Darcan, A. Avagyan, P. Matricardi, G. Jahreis, and E. Hamelmann, no conflicts of interest. Back

3 Supplemental Table 1 is available with the online posting of this paper at jn.nutrition.org. Back

7 G. Jahreis and E. Hamelmann jointly supervised this work. Back

8 Abbreviations used: AA, arachidonic acid; AR, airway reactivity; AHR, airway hyperresponsiveness; BAL, bronchoalveolar lavage; CLA, conjugated linoleic acid; c9,t11-CLA, cis-9,trans-11-conjugated linoleic acid; COX, cyclooxygenase; IL, interleukin; LA, linoleic acid; LOX, lipoxygenase; Mch, metacholine; OVA, ovalbumin; Penh, parameter enhanced pause; PAS, periodic acid Schiff. Back

Manuscript received 17 January 2008. Initial review completed 2 February 2008. Revision accepted 24 April 2008.


   LITERATURE CITED
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
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