Dietary Omega-3 Polyunsaturated Fatty Acid Supplementation and ...

Journal of Asthma, 42:305–314, 2005 Copyright D 2005 Taylor & Francis Inc. ISSN: 0277-0903 print / 1532-4303 online DOI: 10.1081/JAS-200062950

REVIEW ARTICLE

Dietary Omega-3 Polyunsaturated Fatty Acid Supplementation and Airway Hyperresponsiveness in Asthma TIMOTHY D. MICKLEBOROUGH, PH.D.* Department of Kinesiology, Indiana University, Bloomington, Indiana, USA Asthma prevalence continues to increase despite the progress that has been made in the treatment options for asthma. Alternative treatment therapies that reduce the dose requirements of pharmacological interventions would be beneficial, and could potentially reduce the public health burden of this disease. There is accumulating evidence that dietary modification has potential to influence the severity of asthma and reduce the prevalence and incidence of this condition. A possible contributing factor to the increased incidence of asthma in Western societies may the consumption of a pro-inflammatory diet. In the typical Western diet, 20 – 25-fold more omega (n)-6 polyunsaturated fatty acids (PUFA) than n-3 PUFA are consumed, which results in the release of pro-inflammatory arachidonic acid metabolites. Eicosapentaenoic acid and docosahexaenoic acid are n-3 PUFA derived from fish oil that competitively inhibit n-6 PUFA arachidonic acid (AA) metabolism and this reduce the generation of pro-inflammatory 4-series leukotrienes (LTs) and 2-series prostaglandins (PGs) and production of cytokines from inflammatory cells. These data are consistent with the proposed pathway by which dietary intake of n-3 PUFA modulates lung disease. This article will review the existing information concerning the relationship between n-3 PUFA supplementation and airway hyperresponsiveness in asthma. It includes studies assessing the efficacy of n-3 PUFA supplementation in exercise-induced bronchoconstriction. This review will also address the question as to whether supplementing the diet with n-3 PUFA represents a viable alternative treatment regimen for asthma. Keywords bronchial responsiveness, exercise-induced asthma, exercise-induced narrowing, exercise-induced bronchoconstriction, fish oil, polyunsaturated fatty acids

I NTRODUCTION Asthma is one of the most common chronic diseases in the world and it is estimated that approximately 300 million people of all ages and ethnic backgrounds suffer from asthma (1). Approximately 20.3 million Americans (6.3 million children) had asthma in 2001; 73.4 per 1000 population (2). The rate of asthma tends to increase as communities become urbanized and adopt western lifestyles (1). Research into the causation of asthma, and the efficacy of primary and secondary intervention strategies, represent priority areas in asthma research. However, despite the progress that has been made in the treatment of asthma, the prevalence and burden of this disease has continued to increase (1, 3). Asthma is a chronic inflammatory disease of the airways and causes symptoms such as wheezing, breathlessness, chest tightness, excessive mucus production, and cough (4). Long-term airway remodeling is characteristic of asthma and may be associated with an increase in airway hyperresponsiveness to a variety of stimuli (4). Treatment of asthma almost exclusively involves the use of pharmacological medications. Indeed, inhaled corticosteroids, longacting b2-agonists, and short-acting b2-agonists have proven highly effective as medications in relief of symptoms, while daily medications such as leukotriene receptor antagonists and leukotriene enzyme inhibitors

have recently proven highly effective in asthma therapy (5). However, these medications are not without real and potential side effects. Prolonged use of some medications may result in reduced efficacy, or tachphylaxis. For example, reversal of an asthma attack, such as exerciseinduced asthma, is ineffective when short-acting b2agonists are used daily (6), and daily use of long-acting b2-agonists in the management of exercise-induced asthma in children has recently been questioned (7). Therefore, alternative therapies for treatment, such as dietary manipulation, that reduces the dose requirements of pharmacological medication would be of benefit to the asthmatic, and potentially reduce the public health burden of this disease (8–17). Dietary factors and their relationship to the severity of asthma have been repeatedly examined (10, 11, 15–24), while there have been few attempts to evaluate dietary change as a modifier of airway hyperresponsiveness following exercise in asthmatics (25–30) and nonasthmatics with exercise-induced bronchoconstriction (EIB) (13, 31–37). This article will review the existing information concerning the relationship between omega (n)-3 polyunsaturated fatty acids (PUFA) and airway hyperresponsiveness in asthma and EIB, and, in particular, address the question of whether supplementing the diet with n-3 PUFA represents a viable alternative treatment therapy for asthma and EIB. O MEGA-3

*Corresponding author: Dr. Timothy D. Mickleborough, Ph.D., Department of Kinesiology, Indiana University, 1025. E 7th St., HPER 112, Bloomington, IN 47401, USA; Fax: (812) 855-3193; E-mail: tmickleb @indiana.edu

POLYUNSATURATED FATTY ACIDS AND ASTHMA

Changes in patterns of dietary consumption, associated with the development of a more affluent lifestyle, may have 305

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F IGURE 1.—Metabolism of dietary omega-3 and -6 polyunsaturated fatty acids (PUFA) after consumption through the 5-lipoxygenase enzymatic and cyclooxygenase pathways and consequent effects on airway inflammation and airway narrowing. LT, leukotriene; PG, prostaglandin; TX, thromboxane).

contributed to the rise in asthma over the past few decades (38, 39). The idea that diet may be important in asthma is long established, but both the precise formulation of the concept and its popularity has varied widely over time. Diet has been thought to influence asthma through the ingestion of allergens, the ingestion of mediators, the ingestion of mediator precursors (in particular some fatty acids), the ingestion of electrolytes, and the ingestion of antioxidants. Since the epidemiological studies of Dyerberg et al. (40) in the early 1970s, which demonstrated that Greenland Inuit eating diets high in fish oil have a lower incidence of

thrombosis, coronary heart disease, and myocardial infarction, interest has been focused on n-3 PUFA. Compared with a Caucasian control group, the content of n-3 PUFA (contained in fish oil), especially eicosapentaenoic acid (EPA), was increased in thrombocytes of Greenland Inuit. Since then, numerous studies have been performed in vitro and in vivo that showed fewer inflammatory properties of n-3 PUFA compared with n-6 PUFA (41) in coronary heart disease, lipid disorders, and diabetes mellitus. In the typical Western diet, 20–25-fold more n-6 PUFA than n-3 PUFA is consumed (42). This predominance of n-6 PUFA is due to the abundance of dietary linoleic acid (18:2n-6), which is

OMEGA-3 FATTY ACIDS AND ASTHMA present in high concentrations in soy, corn, safflower, and sunflower oils. By contrast, there is a low intake of the n-3 homologue of linoleic acid, a-linolenic acid (18:3n-3), which is present in leafy green vegetables and in flaxseed and canola oils. Once ingested, the 18-carbon fatty acids are desaturated and elongated to 20-carbon n-6 PUFA. Linoleic acid is converted to arachidonic acid (AA) and alinolenic acid is converted to EPA (20:5n-3) (Figure 1). Compared with linoleic acid there is little dietary intake of AA and EPA, which are present in meat and fish, respectively. Linoleic acid and a-linolenic are necessary for a complete diet and cannot be synthesized in vertebrates; therefore, they are essential fatty acids. As a consequence, the relative dietary amounts of n-6 and n-3 PUFA are determinants of the relative cellular amounts of linoleic acid and a-linolenic acid. There has been increased emphasis on the beneficial effects for cardiovascular health of replacing lard and dairy fats rich in saturated fatty acids. This has led to increased consumption of vegetable oils rich in omega-6 PUFA and a simultaneous decrease in consumption of oily fish and leafy vegetables, the major sources of n-3 PUFA. This dietary shift is characterized by a fall in consumption of saturated fats and an increase in n-6 PUFA (43). The antiinflammatory properties of n-3 PUFA such as EPA and docosahexaenoic acid (DHA) and generally pro-inflammatory properties of dietary n-6 PUFA (44, 45), such as linoleic acid, suggest that these dietary trends may have predisposed some individuals to inflammatory disorders, such as rheumatoid arthritis, inflammatory bowel diseases, and asthma. Omega-3 PUFA, such as EPA and DHA in fish oils, compete with AA as substrates for the formation of pro-inflammatory mediators, such as leukotrienes (LTs) and prostaglandins (PGs) (46), and can mediate effects via direct action on neutrophil and monocyte production of mediators, chemotactic responses, and production of cytokines (46, 47). Observational Studies Epidemiological studies suggest that a diet high in marine n-3 PUFA (fish oil) may have beneficial effects on airway hyperresponsiveness in asthma. Black et al. (48) suggested that the reason asthma prevalence has increased over the last two decades is due to an alteration in dietary consumption of fatty acids, with a marked increase in the intake of n-6 PUFA and a decrease in saturated fatty acids. Support for the hypothesis is available from a case-control study showing an association of higher fat intake with adult onset wheeze in Scotland (49), and a cohort study from Malmo in which men with asthma had a higher intake of dietary fat (50). Dunder et al. (51) found that atopic children consumed more margarine (rich in n-6 PUFA) and less butter than did nonatopic children, which supports the importance of diet in the development of atopic disease in children. Haby and colleagues (52) assessed the prevalence of, and risk factors for, asthma in preschool children and found that high n-6 PUFA and low n-3 PUFA consumption was associated with increased risk of asthma. In a crosssectional study, Hodge et al. (53) found an inverse relationship between weekly oily fish intake over the

307 course of 12 months and the prevalence of asthma in schoolchildren. Antova and colleagues (54) examined the role of nutrition in children’s respiratory health within the cross-sectional Central European Study on Air Pollution and Respiratory Health (CESAR), and found that low fish intake was the most consistent predictor of poor respiratory health. Taken together, these studies suggest that consumption of oily fish is associated with a reduced risk of asthma in childhood. In addition, Patal and coworkers (55) demonstrated an association between consumption of oily fish and symptomatic wheeze in individuals with and without physician-diagnosed asthma. Their data suggest that regular consumption of oily fish may be protective of symptomatic asthma. Woods and colleagues (56), in a community-based cross sectional study, sought to determine whether plasma long-chain n-3 PUFA levels, as a measure of dietary intake, was protective against asthma in young adults. These authors found that plasma n-3 PUFA are not associated with a reduced risk of asthma or atopy among young adults. However, their results did suggest that gamma (g)-linolenic acid (n-6 PUFA) has the strongest association with asthma. Due to the fact that this was a cross-sectional study, the authors were unable to establish a cause and effect relationship for the PUFA – asthma associations found and suggested that the role of dietary fats in asthma warrants further research. Oddy and colleagues (57) recently measured patterns of fish intake and investigated the extent to which the frequency and type of fish would predict the serum phospholipid levels of very-long-chain n-3 PUFA in children with and without asthma. Although these authors observed no differences in frequency or category of fish consumed between the asthmatic and nonasthmatic children, they did find that frequency of fish consumption was related to serum phospholipid levels of EPA and DHA. However, these data provide no insight into whether or not fish consumption influences asthma symptoms, airway hyperresponsiveness, and medication use. Interventional Studies The potential therapeutic effect of a diet rich in fish oil on asthmatic symptoms has been examined repeatedly, with clinical data on the effect of fish oil supplementation in asthma being equivocal. Some interventional studies have shown no clinical improvement in asthmatic symptoms (25, 58–61); however, other studies show an improvement in asthmatic status (44, 45, 62–65) following n-3 PUFA supplementation/modification. An early short-term trial (8 weeks) of up to 4 g/day of EPA in 12 patients with severe asthma showed no clinical benefit, despite demonstrating profound suppression of neutrophil chemotaxis and LT mediator production (59); however, 10 subjects were allowed to continue taking oral corticosteroids during the course of the study, which may have masked any treatment effect. Six weeks of 3 g/day of EPA had a deleterious effect on patients with aspirin-intolerant asthma (66), consistent with the known aspirin-like effect of cyclooxygenase inhibition by EPA. Further studies in milder asthmatics with 3.2 g/day for 10 weeks showed no benefit in either clinical symptoms or bronchial hyperresponsiveness (25),

308 despite demonstrating attenuation of allergen-induced latephase bronchoconstriction induced in the laboratory (62). A more prolonged trial for six months with 3.2 g/day of EPA also showed no clinical benefit in patients with polleninduced asthma and seasonal hay fever (61). In addition, Stenius-Aarniala and coworkers (60) demonstrated no clinical benefit of 10 weeks of fish oil supplementation in relatively stable asthmatics. However, their method of assessing lung function is open to question since each subject used a Peak Flow Meter at home under no supervision. Recently Surette et al. (67) showed that daily consumption of dietary g-linolenic acid (GLA) and EPA inhibited LT biosynthesis, with no change in baseline pulmonary function being observed in a population of atopic asthmatics; however, asthma severity and reliance of medication were not assessed. McDonald et al. (68) provided 2.7 g EPA and 1.8 g DHA for 10 weeks to 15 nonsmoking asthmatics and found no change in peak expiratory flow rate, medication usage, and asthma symptom scores following fish oil supplementation. Alternatively, Dry and colleagues (63) have shown positive results using a small placebo-controlled trial of low-dose EPA (1 g/day) for 12 months in 12 adult asthmatic patients and found after 9 months a small but significant improvement of 23% in FEV1. However, no details were given of concurrent medication use or confirmation of compliance by leukocyte membrane phospholipid analysis. Hodge et al. (58) demonstrated that 6-month dietary supplementation with n-3 PUFA in asthmatic children increased plasma levels of EPA and reduced stimulated tumor necrosis factor (TNF)-a and circulating eosinophils, with a concomitant improvement in peak expiratory flow and reduced medication use. Nagakura and colleagues (64) showed that dietary supplementation with fish oil (84 mg EPA and 36 mg DHA per day) over 10 months decreased asthma scores and reduced acetylcholine thresholds during an acetylcholine inhalation test in 29 children with bronchial asthma. Okamoto et al. (45) observed suppression of LTB4 and LTC4 generation by leukocytes and improvement in respiratory function following 4 weeks of perilla seed oil (n-3 PUFA) in asthmatic subjects. Payan and coworkers (69) found that high doses (4 g/day), compared to low doses (0.1 g/day), of EPA ethyl ester taken daily for 8 weeks increased LTB5 generation, and reduced AA, LTB4 and PGE2 generation by polymorphonuclear (PMN) and mononuclear leukocytes in asthmatic patients. These authors did not report pulmonary function scores, medication use, or asthma symptom scores. Villani et al. (65) supplemented seven atopic patients with 3 g/day of n-3 PUFA for only 30 days and observed a significant improvement in FEV1 with a concomitant reduction in airway resistance. Massuev (70) observed a significant attenuation of the late allergic response in 13 asthmatic patients supplemented for 2 weeks with n-3 PUFA, while in another study (71) showed that n-3 PUFA supplementation resulted in attenuation of severe attacks of asphyxia and reduced drug doses in 27 asthmatic patients. Arm et al. (62) observed that after 10 weeks of n-3 PUFA supplementation 17 atopic asthmatics showed a significant decline in the late allergic response and a suppression of inflammatory mediators (50% reduction in the capacity of

T.D. MICKLEBOROUGH PMN leukocytes to produce LTB4). Broughton et al. (44) demonstrated that supplementing the diet with 3.3 g/day of EPA and DHA daily in 27 asthmatic subjects increased EPA-derived 5-series and decreased AA-derived 4-series LTs and ameliorated methacholine-induced respiratory distress, which may be predicted by LT metabolism. A number of important points may be made regarding the conflicting results between studies assessing the efficacy of fish oil supplementation on airway hyperresponsiveness in asthma. Firstly, the dosage (0.1 to 4 g/day) and duration (3 weeks to 12 months) of fish oil supplementation varied considerably between studies. Secondly, the heterogeneity of asthmatic patients between studies was not accounted for. Thirdly, asthmatic patients do not always have highly reproducible responses to bronchial challenge testing (methacholine/histamine), particularly when assessment of peak expiratory flow rate is the solitary measurement (72, 73). Fourthly, the grade of fish oil supplementation varied among studies. Pharmaceutical-grade fish oil has only recently become available and enables the experimental evaluation of the specific mechanism of n-3 PUFA action without the confounding variables of impurity. In addition, pharmaceutical-grade fish oil has a higher percentage of total long-chain n-3 PUFA than does a lower-grade fish oil. Fifth, in only one study (58), was dietary manipulation (using plant-derived oils) performed as part of the treatment phase. This study demonstrated a significant improvement in peak expiratory flow and a reduction in asthma medication use on the n-3 PUFA diet (canola oil and canola-based margarines and salad dressings), while a decrement in resting peak expiratory flow and increased medication use was observed on the n-6 PUFA diet (sunflower oil and sunflower oil – based margarines and salad dressings). Interestingly, Woods et. al. (74) in The Cochrane Database of Systematic Reviews, assessing the efficacy of fish oil for asthma in adults and children were unable to determine the effect of fish oil supplementation in asthma or answer the question whether increasing dietary marine n-3 PUFA by increased fish intake results in improved asthma control. These authors identified 22 studies for possible inclusion; however, the authors only included nine studies. Reasons for noninclusion were 1) not a randomized controlled trial (4 studies), 2) not using marine fatty acids in asthma (3 studies), 3) no outcome measures reported (3 studies), and 4) an inadequate intervention period (1 study). None of the studies reported asthma exacerbations, health status (quality of life), or hospital admissions. These authors stressed that further studies should address these issues. O MEGA-3

POLYUNSATURATED FATTY ACIDS AND EXERCISE-INDUCED BRONCHOCONSTRICTION

Exercise is a powerful trigger of asthma symptoms and may result in asthmatic patients avoiding physical activity resulting in detrimental consequences to their physical and social well-being. Approximately 90% of asthmatics and a high prevalence of nonatopic elite athletes are hyperresponsive to exercise and experience exercise-induced bronchoconstriction (EIB) (75, 76). Exercise-induced bronchoconstriction (EIB), exercise-induced asthma or, exercise-induced airway narrowing are synonymous terms that

OMEGA-3 FATTY ACIDS AND ASTHMA

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F IGURE 2.—The percent change in FEV 1 from pre- to postexercise in subjects with exercise-induced bronchoconstriction (EIB) and control subjects across the three diets. Reductions in FEV 1 in excess of 10% represents abnormal pulmonary function. Letters ( a,b) refer to comparisons by diet within respective time period. Different letters designate significant difference ( p < 0.017). Closed circles, subjects with EIB with normal diet; open circles, subjects with EIB with placebo diet, closed inverted triangles, subjects with EIB with omega-3 (n-3) polyunsaturated fatty acids (PUFA) diet; open inverted triangles, control subjects with normal diet; closed squares, control subjects with placebo diet; open squares, control subjects with n-3 PUFA diet. (Mickleborough TD, Murray RL, Ionescu AA, Lindley MR. Fish oil supplementation reduces the severity of exerciseinduced bronchoconstriction in elite athletes. American Journal of Respiratory and Critical Care Medicine, 2003; 168:1181 – 1189. Official Journal of the American Thoracic Society. D American Thoracic Society.)

describe a condition in which vigorous physical activity triggers acute airway obstruction in individuals with heightened airway reactivity (77). Because exercise does not cause asthma, the most accurate description of this condition is EIB (78). Exercise-induced bronchoconstriction is not an isolated disorder or specific disease, but rather often part of the asthmatic diathesis where exercise is one of many stimuli that may induce airflow limitation, and it is a prominent and troublesome feature of asthma. A characteristic evident in individuals with EIB is a marked decrease in exercise capacity and breathlessness upon exertion. Individuals who demonstrate EIB are often asthmatics; however, significant numbers of healthy, nonasthmatic individuals also demonstrate EIB. These individuals are often referred to as having solitary EIB. For the alert practitioner, EIB provides the ideal opportunity to detect and better manage asthma, particularly since 29%– 51% of asthma is silent or undetected unless the subjects are exercised (79). Eighty to ninety percent of asthmatics have EIB, depending on the study (80), while the percent of nonasthmatics who have EIB in the general population is unknown. It does not typically present a medical emergency or is not cause for a hospital emergency/admittance, so data on prevalence within the general population is sparse. The best estimate of the prevalence of EIB for the general population is 3%–10% (81). Estimates range as high as 19.3% in a sample of Australian school children (82). Estimates of EIB for various athletic populations are generally higher. For example, a survey of the 1984 U.S. Olympic team indicated that approximately 11% had EIB

(83). More recently, Wilber reported that this prevalence was 23% for the 1998 U.S. Winter Olympic team (84). Thus, the prevalence of EIB is high in the asthmatic population, and likely reaches significant numbers in the nonasthmatic population. Only one study to date has evaluated the effect of fish oil supplementation on the airway hyperresponsiveness to exercise in patients with asthma (25). After 10 weeks of daily supplementation with 3.2 g EPA and 2.2 g DHA, subjects underwent a histamine challenge, exercise challenge, and blood neutrophil studies. Although there was a significant increase in EPA and DHA neutrophil content and a 50% inhibition of total LTB synthesis (LTB4 and LTB5), there was no detectable change in the clinical outcome (e.g., histamine response, exercise response, specific conductance of the airway, or symptom scores). Recently, Mickleborough and colleagues (37) conducted a study assessing the effect of 3 weeks of fish oil (3.2 g EPA and 2.2 g DHA) supplementation on the severity of EIB in elite athletes. Ten nonasthmatic elite athletes with documented EIB and 10 elite athletes without EIB (control subjects) participated in a randomized cross-over doubleblind trial. This study demonstrated for the first time that 3 weeks of fish oil supplementation reduces the severity of EIB and resulted in a significant suppression of several proinflammatory mediators in nonatopic elite athletes who exhibit ‘‘asthma-like symptoms’’ following exercise (37). The airway response to exercise was used to assess changes in nonspecific bronchial responsiveness during dietary supplementation with n-3 PUFA. The fish oil diet had no effect on baseline pulmonary function in EIB and control

310 subjects or following exercise in control subjects. However, in the group of athletes who had a history of exerciseinduced narrowing, the fish oil diet reduced the fall in FEV1 at 15 minutes postexercise by almost 80% (Figure 2) in conjunction with a greater than 20% reduction in bronchodilator use. In addition, the increase in tissue phospholipid n-3 PUFA concentration in EIB subjects was coincident with a significant suppression of the proinflammatory eicosanoids LTE4, PGD2 metabolite 9a, 11b-PGF2, and LTB4 and pro-inflammatory cytokines IL1b and TNF-a. The divergent findings between the Mickleborough et al. study (37) and that of Arm and colleagues (25) are difficult to resolve, especially since the Arm et al. study had a longer supplementation period with an identical fish oil dosage. The negative findings observed by Arm et al. (25) may be due to the methodological and statistical limitations of their study. These authors exercised a cohort of mild asthmatics at very low exercise intensity (80% predicted maximal oxygen consumption for 8 min at ambient temperature and humidity). It is generally accepted that inhaling cold-dry air at high ventilation rates initiates EIB. Rundell and coworkers (114) have shown that out of 23 subjects who tested positive for EIB in cold-dry air, 18 (78%) subjects tested negative in ambient conditions (21°C and 50% relative humidity). This suggests that the exercise protocol performed in ambient conditions in the Arm et al. study (25) may have been less sensitive to identifying changes in airway hyperresponsiveness following exercise due to inadequate environmental stress. In addition, an assessment of the numbers used in the airway response to exercise of Arm and coworkers’ study (25) (5 subjects receiving placebo and 6 subjects receiving fish oil supplementation) suggests insufficient patients to detect a statistical difference and avoid a type I error. The mechanism of exerciseinduced airway narrowing in elite athletes may be different from the mechanism of exercise-induced airway narrowing in patients with common asthma (85), and it has been suggested that this may explain the disparity between the two studies (25, 37). Godfrey (86) has suggested that all individuals who exhibit EIB by demonstrating reductions in postexercise pulmonary function are asthmatic to some degree However, recent evidence of airway remodeling in cross-country skiers with EIB (87–89), and the fact it has been shown that inhaled corticosteroids appear to have no effect on airway inflammatory markers or obstructive symptoms in athletes with EIB (90), indicates a different pathphysiology in EIB compared to common asthma. Evidence for this concept comes from Sue-Chu et al. (88) who reported a higher frequency of lymphoid aggregates in endobronchial biopsies from a population of young elite cross-country ski athletes, who exhibit asthma-like symptoms, compared to healthy control subjects. In addition, an increase in the number of neutrophils has been observed in the sputum of elite swimmers after training (91) and an increased neutrophil concentration in BALF has been observed in a canine model of hyperpnea with cold dry air (92, 93), providing further evidence that the inflammatory processes in athletes with EIB may be different to that of individuals with common asthma. However, further

T.D. MICKLEBOROUGH studies are needed to asses the efficacy of fish oil on airway hyperresponsiveness following exercise in asthma. C ELLULAR

AND MOLECULAR MECHANISM OF ACTION OF OMEGA-3 POLYUNSATURATED FATTY ACIDS

Many of the anti-inflammatory effects of n-3 PUFA appear to be exerted at the level of altered gene expression and have been demonstrated only a limited number of times in vitro. Thus, the extent of these effects in vivo is not yet clear. Increasing evidence suggests that PUFA are not only the precursors of eicosanoids and other lipid mediators, but can also modulate signal transduction pathways and transcription factors such as nuclear factor-kappaB (NFkB) (94–96). It has been suggested that that NF-kB plays a pivotal role in the pathogenesis of asthma (97–101), since macrophages of induced sputum and bronchial epithelial cells from stable asthmatics exhibit increased NF-kB activity compared with cells from healthy individuals (100). Lee and coworkers (102, 103) recently demonstrated that activation of the NF-kB pro-inflammatory pathway, and cyclooxygenase-2 (COX-2) expression by saturated fatty acids and inhibition of this induction by n-3 PUFA, are mediated through a common signaling pathway derived from toll-like receptor 4 (Tlr-4). This suggests that if activation of Tlr-4 is modulated by n-3 PUFA, then signaling pathways downstream, such as NF-kB, and ensuing cellular responses [e.g., inducible nitric oxide, pro-inflammatory cytokines, TNF-a, IL-1b and eicosanoids (prostanoids and LTs)] should also be modified (102, 103). Indeed, it has been demonstrated that pro-inflammatory cytokine inhibition in murine M1 by n-3 PUFA is mediated, in part, through inactivation of NF-kB (104, 105) and inhibition of COX-2 and PGE2 expression in blood monocytes with a Tlr-4 agonist (102). Therefore, since Tlr-4 conveys signals as a part of innate immunity from the endotoxin receptor (CD14) on the surface of macrophages to the inner cell, a downregulation of nuclear transcription factors, such as NF-kB formation of cytokines might be reduced after fish oil ingestion (106). It has been suggested that EPA may alter LT and PG metabolism and DHA may act directly at the membrane level in a mechanism related to phospholipid modification (107). DHA has little effect on AA metabolism by the 5-lipoxygenase (LO) pathway, whereas EPA is an inhibitor of both the cyclooxygenase (COX)-2 and 5-LO pathway (108, 109). Eicosapentaenoic acid and DHA, derived from fish oil, competitively inhibit n-6 PUFA AA metabolism, thus reducing the generation of inflammatory 4-series LT and 2-series PG prostanoids (PGs and thromboxanes) (46), and the production of cytokines from inflammatory cells (47). The EPA-derived metabolites (5-series LTs and 3series prostanoids) have lower biological activity compared with the analogous AA derivatives. After the enzymatic conversion of EPA, the 5-series cysteinyl (cyst) LTs (LTB5, LTC5, LTD5, LTE5) are generated; these LTs have partially antagonistic biological effects compared with AA derivatives (110, 111) (Figure 1). The 4-series cyst LTs increase vascular permeability and contract smooth muscle cells, causing bronchoconstriction and vasoconstriction (110). The bronchoconstrictive and chemotactic potency

OMEGA-3 FATTY ACIDS AND ASTHMA of LTB5 is two orders of magnitude lower than is the activity of LTB4 (41) (Figure 1). Consuming fish oil results in partial replacement of AA in inflammatory cell membranes by EPA (46, 47), and, thus, demonstrates a potentially beneficial anti-inflammatory effect of n-3 PUFA. Supplementing the diet with n-3 PUFA has been shown to reduce AA concentrations in neutrophils and neutrophil chemotaxis, reduce LT generation (46, 69) and reduce airway late response to allergen exposure (62). These data are consistent with the proposed pathway by which dietary intake of n-3 PUFA modulates lung disease. The recent availability of pharmaceutical-grade n-3 PUFA fish oil has greatly facilitated the study of potential mechanisms of n-3 PUFA-mediated signal pathways in vitro. Novak et al. (105) recently demonstrated that a 4-hr pretreatment with a high-purity n-3 PUFA emulsion reduced LPS-stimulated M1 TNF-a production by 46%, while n-6 PUFA pretreatment did not alter TNF-a production compared to medium alone. Omega-3-PUFA, therefore, seems to interfere with early inflammatory signal transduction processes and is, thus, capable of blunting hyperinflammation. Elucidating the mechanism of this modulation could help us to understand how dietary n-3 PUFA achieve their specific effects on airway hyperresponsiveness in asthma. S UMMARY The role of dietary n-3 PUFA supplementation on airway hyperresponsiveness remains largely undefined, however the present evidence suggests that n-3 PUFA may have a protective effect on airway function in asthmatics (44, 45, 62–65) and nonasthmatics with EIB (37). Since asthmatic individuals with hyperresponsive airways produce increased quantities of LTs compared with healthy individuals (78, 112), dietary interventions that decrease the capacity to synthesize these and other pro-inflammatory mediators are warranted. It can be hypothesized that pharmacological medication use could be decreased in some patients with asthma in concert with increased n-3 PUFA supplementation if both the drug and n-3 PUFA are exerting their therapeutic effects through the same molecular actions. This might also apply to new drugs or new treatment modalities that aim to suppress cytokine concentrations. There may be an opportunity for beneficial additive effects with n-3 PUFA supplementation to increasing the intake of n-3 PUFA. Thus, the possibility exists for drug-diet interactions that confer greater antiinflammatory benefits than does either agent alone or similar anti-inflammatory effects with less toxicity. It is also possible the absolute and relative quantities of other PUFA and saturated fatty acids are equally significant (113), and that an excess of n-6 PUFA may be important. Given the small number of studies that have been conducted to date and the limited range of clinically important outcomes that have been reported, there is a need for further research in this area. To date, in the studies involving the efficacy of n-3 PUFA supplementation in AHR, the total number of asthmatic subjects studied has only been 187, methodologies have been variable, and the outcome measures of asthma exacerbations, hospital

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