European Journal of Pharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎

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Modulation of inflammation and immunity by dietary conjugated linoleic acid$ Monica Viladomiu a,b, Raquel Hontecillas a,b, Josep Bassaganya-Riera a,b,n a b

Nutritional Immunology and Molecular Medicine Laboratory, Virginia Bioinformatics Institute, Virginia Tech, Blacksburg, VA 24060, USA Center for Modeling Immunity to Enteric Pathogens, Virginia Bioinformatics Institute, Virginia Tech, Blacksburg, VA 24060, USA

art ic l e i nf o

a b s t r a c t

Article history: Received 15 January 2015 Received in revised form 4 February 2015 Accepted 5 March 2015

Conjugated linoleic acid (CLA) is a mixture of positional and geometric isomers of linoleic acid. This family of polyunsaturated fatty acids has drawn significant attention in the last three decades for its variety of biologically beneficial properties and health effects. CLA has been shown to exert various potent protective functions such as anti-inflammatory, anticarcinogenic, antiadipogenic, antidiabetic and antihypertensive properties in animal models of disease. Therefore, CLA represents a nutritional avenue to prevent lifestyle diseases or metabolic syndrome. Initially, the overall effects of CLA were thought to be the result of interactions between its two major isomers: cis-9, trans-11 and trans-10, cis-12. However, later evidence suggests that such physiological effects of CLA might be different between the isomers: t-10, c-12-CLA is thought to be anticarcinogenic, antiobesity and antidiabetic, whereas c-9, t-11-CLA is mainly anti-inflammatory. Although preclinical data support a benefit of CLA supplementation, human clinical findings have yet to show definitive evidence of a positive effect. The purpose of this review is to comprehensively summarize the mechanisms of action and anti-inflammatory properties of dietary CLA supplementation and evaluate the potential uses of CLA in human health and disease. & 2015 Elsevier B.V. All rights reserved.

Keywords: Conjugated linoleic acid Inflammation Obesity Diabetes Inflammatory Bowel Disease Asthma

1. Introduction Conjugated linoleic acid (CLA), first described in 1985, refers to a class of positional and geometric isomers of conjugated dienoic derivatives of linoleic acid. As many other polyunsaturated fatty acids (PUFA) and their metabolites, dietary CLA has been proposed as a promising avenue for the development of novel and safer nutritional interventions against inflammation (Xu et al., 1999). Interest on the biological function and health benefits of dietary CLA dates back to 1987, when Ha et al. (1987) observed the ability of CLA to inhibit chemically-induced skin neoplasia in mice. This initial discovery lead to a series of studies that identified a broad range of beneficial biological properties of CLA, including but not limited to effects on weight loss, food and energy intake, alteration of body composition, cancer, enhancement of immune function, and inflammation (Lee et al., 1994; O'Shea et al., 2004; Kelley et al., 2007; Whigham et al., 2007; Mitchell and McLeod 2008). The antiobesity, anticarcinogenic, anti-inflammatory and antidiabetic ☆ Grant and funding sources: Supported by funds of the Nutritional Immunology and Molecular Medicine Laboratory. n Corresponding author at: Nutritional Immunology and Molecular Medicine Laboratory, Virginia Bioinformatics Institute, Virginia Tech, Blacksburg, VA 24060, USA. Fax: þ 1 5402312606. E-mail address: [email protected] (J. Bassaganya-Riera).

effects of CLA have been widely described in animal studies (Park et al., 1997; West et al., 1998; de Lany et al., 1999; Ostrowska et al., 1999; Park et al., 1999; Tsuboyama-Kasaoka et al., 2000; Whigham et al., 2000; Bassaganya-Riera et al., 2001a; Ryder et al., 2001; Sisk et al., 2001; Bassaganya-Riera et al., 2002; Hontecillas et al., 2002; Terpstra et al., 2002; Yamasaki et al., 2003b; Bassaganya-Riera et al., 2004; O'Shea et al., 2004; Bassaganya-Riera and Hontecillas, 2006; Evans et al., 2010; Moon, 2014). However, such effects seem to be inconsistent and less significant in humans. This review will comprehensively summarize the mechanisms of action and antiinflammatory properties of CLA supplementation in animals and humans with a focus on mucosal inflammation. Inflammation is a complex physiological response to noxious stimuli and conditions such as pathogens or non-microbial endogenous molecules that result in tissue injury and cell damage (Ferrero-Miliani et al., 2007). It is induced by chemical mediators produced by damaged host cells and serves as a protective mechanism regulated by the interaction of multiple pro-inflammatory and immunomodulatory signaling pathways that aim to eliminate harmful stimuli, remove necrotic cells and tissue, and initiate the healing process (de Cassia da Silveira et al., 2014). Such inflammatory processes require the movement and interaction of the major cells of the immune system, including basophils, neutrophils, mast cells, T cells, B cells and so on. These events are controlled by a number of extracellular mediators and regulators

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Please cite this article as: Viladomiu, M., et al., Modulation of inflammation and immunity by dietary conjugated linoleic acid. Eur J Pharmacol (2015), http://dx.doi.org/10.1016/j.ejphar.2015.03.095i

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such as cytokines, growth factors, eicosanoids, complement and peptides, along with equally complex intracellular signaling control mechanisms, which regulate immune cell maturation, activation and function as well as tissue-level homeostasis (Punchard et al., 2004; Medzhitov, 2008). Inflammation underlies the pathogenesis of many widespread diseases including inflammatory bowel disease (IBD), rheumatoid arthritis, osteoarthritis, atherosclerosis, obesity, diabetes, asthma and allergy, bacterial and viral infections, and cancer (Medzhitov, 2008; Koeberle and Werz, 2014). It is exceedingly complex and plays a crucial role in mammalian physiology. Current drug development approaches to suppress inflammation mainly focus on: (1) Agonists of the glucocorticoid receptor (glucocorticoids), (2) interference with eicosanoid biosynthesis (non-steroidal antiinflammatory drugs), and 3) Pro-inflammatory cytokine signaling blockade (Biological drugs targeting tumor necrosis α (TNFα) and interleukin 1 (IL-1) signaling) (Medzhitov, 2008). Such pharmacological strategies are strongly focused on a limited number of key molecules that are thought to be essential for each particular disease. However, some of these treatments result in poor therapeutic efficacy, significant side effects or adverse compensatory mechanisms (Medzhitov, 2008). Hence, CLA may offer an opportunity as a novel alternative and complementary intervention capable of disrupting the inflammatory process without undesired adverse side effects.

2. CLA isomers CLA is a mixture of positional and geometric octadecadienoic acid isomers derived from linolenic acid, a 18-carbon polyunsaturated fatty acid that contains two double bonds in the cis configuration (cis-9, cis-12 octadecadienoic acid) (Kennedy et al., 2010). CLA present in mammalian tissues is directly derived from diet or, in smaller amount, from the gastrointestinal microflora (Gorissen et al., 2010). CLAs are natural constituents of foods derived from animal fat tissues and dairy products as a result of lipid biohydrogenation and hepatic desaturation. They are naturally produced in the rumen of cattle as intermediates of the gut bacterial fermentation of dietary linoleic acid to stearic acid and desaturation of oleic acid derivatives (Kepler et al., 1971; Griinari et al., 2000). Specifically, microbes in the gastrointestinal tract of ruminant animals such as cows and goats convert linoleic acid into different isoforms of CLA through biohydrogenation (Medina et al., 2000), a process that changes the location and configuration of one or both double bonds of linoleic acid in such a manner than the two double bonds are no longer separated by two single bonds. This results in the formation of several dozen octadecadienoic acid isomers that contain a single pair of conjugated double bonds (two double bonds separated by a single bond) (Marcy et al., 2004). Alternatively, there is evidence that

non-ruminant animals can endogenously produce isomer cis-9, trans-11 by the delta-9 desaturation of trans-11 vaccenic acid (TVA), the primary isomer for ruminant TFAs (Corl et al., 2001, 2003; Kay et al., 2004). Bioconversion of TVA to c-9,t-11 CLA has been confirmed in mice (Santora et al., 2000), rats (Corl et al., 2003) and humans (Kuhnt et al., 2006). The proportion of CLA in dairy products ranges from 0.34% to 1.07% of total fat (2.9 to 8.92 mg CLA/g of fat), whereas CLA content in raw or processed meat product ranges from 0.12% to 0.68% (Dhiman et al., 2005; Mendis et al., 2008). In 1992, soon after CLA's first isolation from extracts of grilled ground beef, Ha et al. thoroughly studied the concentrations of CLA in different commercially available foods (Chin et al. 1992). Such efforts resulted in creation of a database containing more than 90 food items including meat, poultry, dairy products, seafood, plant oils, infant foods and processed foods. Results revealed that CLA content in common foods is highly variable, probably due to several factors such as the nutritional status or age of the animal source, thus indicating the possibility of large variations in the CLA dietary intake.. Nonetheless, the average daily intake of CLA is estimated to range from 152 to 212 mg for American non-vegetarian women and men, respectively, and 97.5 mg/day for the British (Ritzenthaler et al., 2001; Kennedy et al., 2010; Mushtaq et al., 2010). There have been 28 naturally occurring CLA isomers described to date. The c-9,t-11 CLA isomer, also known as rumenic acid, is the most predominant isomer in meats and milks from ruminant species, representing approximately 90% of total dietary CLA intake. The isomer trans-10, cis-12 comprises the remaining 10%, with negligible proportions of the other isomers (Fig. 1) (Wallace et al., 2007). Such percentages contrast with chemically synthesized and commercial preparations of CLA, which usually contain equal proportions of the two abundant isomers cis-9, trans-11 and trans-10, cis-12 (Parodi, 1997; Sebedio et al., 1999; Wang and Lee, 2013). Most of CLA's beneficial properties are elicited by its two main isomers: c9, t11-CLA and t10, c12-CLA (Khan and Vanden Heuvel, 2003). In some cases an effect is produced by only one of the isomers, whereas in other situations the effect results from the synergism of both isomers (Zabala et al., 2006; Halade et al., 2010). Moreover, individual CLA isomers can result in differential effects, and the effect of variable isomer concentrations in CLA mixtures are difficult to predict (Khan and Vanden Heuvel, 2003). For instance, t-10, c-12-CLA is involved in catabolic processes of increased lipolysis and fat oxidation, whereas c-9, t-11-CLA seems to be the active anabolic agent and is predominantly anti-inflammatory (Wang and Lee, 2013). Both isomers seem to have anti-carcinogenic properties, although they are thought to be mediated by different effects on lipid metabolism, oncogene regulation and modulation of apoptosis (Kelley et al., 2007). The majority of research to date has been performed using mixtures of CLA isomers due to the initial cost and difficulty to

Fig. 1. Structure of linoleic acid, cis-9 trans-11 CLA isomer and trans-10 cis-12 CLA isomer.

Please cite this article as: Viladomiu, M., et al., Modulation of inflammation and immunity by dietary conjugated linoleic acid. Eur J Pharmacol (2015), http://dx.doi.org/10.1016/j.ejphar.2015.03.095i

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synthesize and isolate each unique CLA isomer from vegetable oils (Kritchevsky, 2000). However, the recently improved purification, chemical synthesis and commercialization of CLA from safflower and sunflower oils have accelerated detailed investigation and identification of individual isomers or optimal isomeric blends responsible for the beneficial biological activities in each particular disease state. Hence, this review aims to describe the known protective pathways and underlying mechanisms through which CLA mediates such a diverse array of protective effects as well as discuss the need for future studies to fully characterize potential novel pathways modulated by CLA in humans.

3. Anti-inflammatory mechanisms of CLA 3.1. CLA and the immune response Beneficial effects of CLA on immune and inflammatory responses have been reported in a number of animal models (Cook et al., 1993; Bassaganya-Riera et al., 2002, 2003; Yu et al., 2002; Yang and Cook, 2003; Dilzer and Park, 2012) and human clinical trials (Albers et al., 2003; Turpeinen et al., 2008; Peterson et al., 2009), including decreased colonic inflammation, reduced antigen-induced cytokine production by immune cells, decreased adverse effects of immune challenges, and modulation of inflammatory mediators such as cytokines, prostaglandins, leukotrienes and immunoglobulins. There is a powerful body of evidence demonstrating that CLA is able to modify the immune response and prevent immune-induced wasting by influencing the production of soluble factors and inflammatory molecules (Miller et al., 1994; Oleszczuk et al., 2012). Both c-9,t-11 and t-10,c-12 CLA isomers decrease innate immune responses by lowering the activity of monocytes, macrophages, dendritic cells and natural killer cells and diminishing the production of prostaglandins and leukotrienes (O'Shea et al., 2004). Moreover, dietary c-9,t-11 and t-10,c-12 CLA mixtures (50:50 and 80:20) improve antigen-specific adaptive immune responses to bacterial and viral antigens, making it highly beneficial in immunocompromised patients whose responses are insufficient. 50:50c-9, t-11 and t-10, c-12 CLA dietary supplementation has also been shown to enhance humoral responses by increasing the production of IgG, IgM and IgA in spleen and mesenteric lymph nodes and to decrease macrophage function by reducing the synthesis of inflammatory mediators and enzymes in rats (Bassaganya-Riera et al., 2003). Moreover, c-9, t-11 CLA is also able to reduce IgE, IL-12 and PGE2 expression, all of which play key roles during allergic reactions and airway inflammation (Sugano et al., 1998). Hence, CLA differentially regulates class-specific production of immunoglobulins. Such influence on immunoglobulin production and enhanced antibody synthesis has been attributed to the t-10c-12 CLA isomer, which elicits opposing effects depending on the cytokine environment (Yamasaki et al., 2003a). 3.2. CLA as a modulator of T cell responses In 2001, we assessed the effects of dietary conjugated linoleic acid on growth, body composition and immune competence in nursery pigs of dirty and clean environments (Bassaganya-Riera et al., 2001a). Such study revealed an expansion of peripheral porcine CD8 þ lymphocyte subsets and enhancement of lymphocyte proliferation in both clean and dirty environments during 50:50c-9, t-11 and t-10, c-12 dietary CLA supplementation. These interesting data suggested that CLA enhances cellular immunity and that could be used to control inflammation resulting from diseases in which CD8 þ T cells have been identified to be critical

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in preventing pathogenesis. We ran a follow-up study with the aim to further characterize distinctive traits among such expanded CD8 cell subsets (Bassaganya-Riera et al., 2001b). We found that 50:50c-9, t-11 and t-10, c-12 dietary CLA supplementation induced in vivo expansion of porcine CD8 þ cells involving T-cell receptor (TCR)γδCD8αα T lymphocytes, CD3-CD16 þCD8αα (a porcine natural killer cell subset), TCRαβCD8αβ T lymphocytes and enhanced specific CD8 þ-mediated effector functions (e.g. granzyme activity). The expansion of peripheral blood TCRαβCD8αβ cells was positively correlated with increased percentages of CD8 þ thymocytes. Functionally, CLA enhanced the cytotoxic potential of peripheral blood lymphocytes and proliferation of TCRαβCD8αα cells. Collectively, these results indicated that dietary CLA enhances cellular immunity by modulating phenotype and effector functions of CD8 þ cells which are involved in both adaptive and innate immunity. Another study which assessed the nutritional effect of 50:50c9, t-11 and t-10, c-12 CLA dietary mixtures in the regulation of bacterially induced colitis in pigs became the first report of efficacy of CLA in ameliorating disease associated with colitis (Hontecillas et al., 2002). Although both dietary CLA supplementation and systemic bacterial immunization were able to decrease colonic epithelial erosion, only CLA treatment was able to prevent the enlargement and thickening of the colonic mucosa. Consistent with previous results, dietary CLA increased the numbers of TCRγδCD8αα cells in peripheral blood and maintained numbers of CD4 þ and CD8α þ cells in the colonic mucosa. A final porcine study revealed that the ability of CLA to delay the onset of experimental IBD correlated with the upregulation of Peroxisome Proliferator-Activated Receptor γ and its responsive genes (Bassaganya-Riera and Hontecillas, 2006), a regulatory molecule that will be discussed later on. Finally, more recent reports have shown how CLA is able to alter the differentiation of monocytes to macrophages (Tontonoz et al., 1998), an antigen-presenting cell type that plays a key role in the subsequent activation and differentiation of T and B cell subsets. Both c-9,t-11 and t-10,c-12 CLA isomers is able to reduce the production of pro-inflammatory cytokines such as TNFα (Jiang et al., 1998) and modulate the environment that favors the differentiation of lymphocytes towards a more regulatory phenotype during the antigen presentation phase. 3.3. CLA as a modulator of cytokine expression Several cell culture studies have demonstrated that CLA, particularly the c-9t-11 isomer, is able to diminish pro-inflammatory cytokine production, mainly IL-6, TNFα, IFNγ, and IL-1β, which play an important role in the pathogenesis of many chronic inflammation-mediated diseases (O'Shea et al., 2004; Peterson et al., 2009; Bassaganya-Riera and Hontecillas, 2010; Oleszczuk et al., 2012). The ability of CLA to reduce inflammation has been recently investigated in a series of detailed in vitro experiments using bovine blood. Such studies have revealed that the t-10,c-12 CLA isomer is able to reduce LPS-induced TNFα production in cultured bovine blood immune cells, whereas linoleic acid and c-9t-11 CLA treatments result in no detectable changes (Perdomo et al., 2011). Another ex vivo study showed decreased NF-κB activation and TNFα mRNA levels after adding t-10,c-12 CLA to endotoxin-stimulated pig peripheral blood mononuclear cells (PBMCs) (Kim et al., 2011). However, t-10,c-12 CLA treatment in non-stimulated PBMCs resulted in differing results as the authors report an increased NF-κB activation and TNFα production, thus suggesting that CLA is acting in a pro-inflammatory manner. These findings highlight that CLA may elicit different actions depending on the environment conditions, thus requiring further mechanistic

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investigations. The protective effects of CLA have been further investigated using several intestinal epithelial and immune cell lines. c-9,t-11 CLA pre-treatment of LPS-induced bone marrow-derived dendritic cells resulted in reduced IL-12 protein and mRNA levels (Loscher et al., 2005), a cytokine that promotes differentiation of naïve T cells to T helper 1 (Th1) cells, an effector cell subset characterized by the production of IFNγ, TNFβ and IL-2 (McGuirk et al., 2002; Mills and McGuirk 2004). CLA pre-treatment also resulted in increased surface expression of receptors for IL-10 (Loscher et al., 2005), a cytokine known to inhibit MHC class II and co-stimulatory molecule expression by macrophages and dendritic cells, thereby preventing their migration to lymph nodes and reducing their production of pro-inflammatory cytokines (Couper et al., 2008). This same study revealed that c-9,t-11 CLA increases nuclear NFκB levels and reduces cytosolic NF-κB and IκBα, suggesting that CLA pre-treatment delays the LPS-induced NF-κB translocation from the cytoplasm and into the nucleus by preventing IκBα degradation, a protein that sequesters NF-κB in the cytoplasm. Interestingly, such CLA effects were reversed in the presence of an IL-10 neutralizing antibody, thus suggesting c-9,t-11 CLA as a potential therapeutic for Th1-mediated inflammatory pathologies. In RAW 264.7 mouse cells, treatment with various CLA isomers (c-9, t-11; t-9,t-11; c-9,c-11 and t-10,c-12) decreased the mRNA levels of cyclooxygenase 2 (COX-2), TNFα, nitric oxide synthase (iNOS), IL1β, IL-6 via peroxisome proliferator-activated receptor γ (PPARγ) (Yu et al., 2002). Moreover, CLA production by probiotic bacteria induced apoptosis in HT-29 and Caco-2 intestinal epithelial cells in a PPARγ-dependent fashion (Ewaschuk et al., 2006). Therefore, the anti-inflammatory effects of CLA are partly mediated through its ability to activate PPARγ. 3.4. PPARγ-mediated anti-inflammatory mechanisms of CLA Peroxisome proliferator-activated receptors are nuclear receptors and ligand-activated transcription factors that regulate the expression of genes involved in energy regulation, glucose homeostasis and immune function (O'Shea et al., 2004). These fatty acid receptors are widely expressed in the immune system (i.e. macrophages, dendritic cells, T cells, epithelial cells) and regulate the expression of several genes involved in immune cell proliferation, apoptosis and inflammation. There are 3 PPAR isoforms: (1) PPARα, (2) PPARδ/β, and (3) PPARγ. PPARs are mostly activated by polyunsaturated fatty acids such as CLA (Fig. 2), whereas their interaction with saturated fatty acids results in weak

activation. CLA isomers c-9,t-11; t-9,t-11; and t-10,c-12 are high affinity ligands and activators of PPARα and β and induce accumulation of PPAR-responsive genes (Moya-Camarena et al., 1999a; Moya-Camarena et al., 1999b). Studies from the same group confirmed that such CLA isomers are also potent activators and ligands of PPARγ (Belury, 2002a, b; Belury et al., 2002). Examples of endogenous ligands of PPARs are eicosanoids such as prostaglandins, while synthetic ligands include thiazolidinediones and fibrates (Krey et al., 1997). When activated, PPARs heterodimerize with the retinoid X receptor (RXR) and bind to PPAR response elements (PPRE) to repress or induce transcription of target genes which affect lipid metabolism, glucose metabolism, energy balance, and immune responses (Krey et al., 1997). PPAR activation, and PPARγ activation in particular, has been reported as an important negative regulator of inflammatory responses through several different mechanisms including the transcriptional regulation of cytokines, chemokines and cell survival factors as well as the antagonism of pro-inflammatory transcription factors such as NF-κB, AP-1 and STAT (Macredmond et al., 2011). Therefore, PPARγ activation represents a promising avenue for developing safer nutritional interventions against inflammation. Thiazolidinediones were developed originally to treat diabetes mellitus due to their beneficial effects on insulin sensitivity. Subsequently, such synthetic agonists of PPARγ have been investigated for other PPARγ-mediated effects. Several in vitro and animal studies have revealed that thiazolidinediones can reduce airway inflammation by regulating the expression of pro-inflammatory and anti-inflammatory cytokines, reducing the recruitment of eosinophils and modulating NF-κB activity (Woerly et al., 2003; Kim et al., 2005; Lee et al., 2005, 2006a, 2006b). Clinical trials in asthma patients have also revealed small but significant improvements in airway hyper-reactivity after treatment with thiazolidinediones (Spears et al., 2009; Richards et al., 2010). A recent study in Ulcerative Colitis (UC) patients demonstrated that rosiglitazone (Avandia) is also efficacious in the treatment of mild to moderately active UC (Lewis et al., 2008). However, rosiglitazone is unlikely to be adopted for treating inflammatory diseases due to its significant side effects (Nesto et al., 2003; Marcy et al., 2004). Hence, the discovery of novel naturallyoccurring agonists of PPARγ that exert therapeutic and prophylactic actions against IBD with no adverse side effects is timely and necessary. In this regard, in 2004 we investigated the ability of CLA to activate PPARγ and ameliorate experimental colitis (Bassaganya-Riera et al., 2004). The molecular targets for the protective actions of CLA in the context of IBD where then unknown. By using loss-of-function studies, we were able to provide for the first time in vivo molecular evidence suggesting that dietary 50:50c-9, t-11 and t-10, c-12 CLA supplementation ameliorates colitis through a PPARγ-dependent mechanism.

4. CLA and inflammation-mediated diseases 4.1. Obesity

Fig. 2. Molecular docking ofcis-9trans-11 andtrans-10cis-12 CLA isomers with Peroxisome Proliferator-Activated Receptor γ. Interactions between conjugated linoleic acid and PPARγ predicted by Autodock Vina. Conjugated linoleic acid (Green: trans-10 cis-12 CLA; Pink: cis-9 trans-11 CLA) in stick representation surrounded by molecular surface of the binding site with coloring by element. The free energy of binding is  5.2 kcal/mol.

Obesity is a disease characterized with a systemic low-grade chronic inflammation (Kennedy et al., 2010). Several animal studies, especially in rodents, report beneficial metabolic effects resulting in reduced fat deposition and increased lean body mass during 50:50 c-9, t-11 and t-10, c-12 CLA mixture supplementation (Miner et al., 2001; Hargrave et al., 2002; Takahashi et al., 2002; House et al., 2005; Bhattacharya et al., 2006; So et al., 2009). However, later studies have confirmed that isomer t-10, c-12 rather than c-9, t11 is responsible for CLA's role in body composition and adipogenesis (Park et al., 1999). CLA's anti-obesity properties are thought to be a result of (1) reduced energy intake by

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suppressing appetite, (2) inhibition of fatty acid metabolism, adipogenesis and lipogenesis, (3) increased lipolysis or delipidation, (4) decreased adipocyte size and (5) increased fat oxidation and energy expenditure in white adipose tissue, muscle and liver tissue (Wang and Jones 2004; Kennedy et al., 2010). CLA effects seem to be smaller in human subjects. Several human trials indicate that dietary CLA supplementation results in the reduction of body weight and body fat mass as well as an improvement in lean body mass, body mass index and waist measurements in obese individuals (Dilzer and Park, 2012). However, no effects have been reported in normal weight humans (Medina et al., 2000; Zambell et al., 2000; Lambert et al., 2007; Plourde et al., 2008; Tholstrup et al., 2008). Inconsistent outcomes throughout clinical studies might be due to the large variation of (1) CLA mixture isomers used, (2) dose administered, (3) duration of the treatment, (4) study populations (age, body weight, body fat or metabolic status of the subjects), (5) gut microbiome, and (6) inflammation status. Future randomized, double-blinded, placebo-controlled clinical trials are needed to further characterize the efficacy and specificity of CLA isomers as dietary strategies for weight loss. 4.2. Type II diabetes Adipocytes are known to produce a wide range of pro-inflammatory molecules, or ‘adipokines’, including leptin, resistin, adiponectin, IL-6, MCP-1, IL-1β and TNFα. Aberrant expression of such adipokines occurs during obesity, leading to the induction of insulin resistance (Hotamisligil et al., 1993; Vendrell et al., 2004; Dandona et al., 2005). Obesity-induced insulin resistance is characterized by the infiltration of immune inflammatory cells such as macrophages and T lymphocytes (Weisberg et al., 2003). These cells are mainly found around dying adipocytes indicating a possible role in adipose tissue dead cell clearance. Inflammatory mediators released by these infiltrating cells result in the disruption of important insulin signaling pathways in adipocytes and subsequent accumulation of macrophages into the stromal vascular fraction, thus further enhancing insulin resistance (Bruun et al., 2005). The observed CLA effects in body composition and inflammation modulation make it a potential candidate for the dietary prevention and treatment of insulin resistance in the context of pre-diabetes and metabolic syndrome. The anti-diabetic effects of CLA have been examined using ob/ ob mice fed a high fat diet enriched with either c-9, t-11 CLA or linoleic acid for 6 weeks. Dietary CLA administration resulted in no significant effects regarding weight loss, food intake and adipose tissue mass. However, c-9, t-11 CLA was able to reduce fasting plasma glucose, insulin and triacylglycerol concentrations, all of them indicators of insulin resistance, while it up-regulated the production of insulin signaling pathway molecules such as GLUT4 and insulin receptor IRS-1 (Moloney et al., 2007). Moreover, c-9, t-11 CLA reduced the infiltration of macrophages into the adipose tissue as well as the production of MCP-1, CD68, IL-6 and TNFα, probably as a result of reduced NF-KB signaling in adipocytes (Moloney et al., 2007). These results suggest that alerting fatty acid composition by administering CLA may reduce the inflammation in adipose tissue that predisposes to obesity-induced insulin resistance. However, such anti-diabetic effects are not translated into T2DM patients, which suffer an increased fasting glucose concentration, exaggerated oxidative stress and reduced insulin sensitivity after t-10, c-12 CLA supplementation (Riserus et al., 2001, 2004; Wilson et al., 2009). Therefore, profound preventive and therapeutic effects of CLA seen in animal models of T2DM have yet to be rigorously verified in human studies.

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4.3. Asthma and airway inflammation Asthma is a chronic airway inflammatory disorder that involves a complex interplay between resident cells, such as epithelial cells and connective tissue, and infiltrating immune cells including eosinophils and activated T lymphocytes (Macredmond and Dorscheid, 2011). Recent meta-analysis of several prospective studies has demonstrated that obesity precedes the development of asthma with a relative risk for incident asthma in obese adults of almost 2 (Beuther and Sutherland, 2007). These data were confirmed in a second large study in which the ratio for asthma incidence was 1.21 (Hjellvik et al., 2010). Moreover, diet-mediated weight reduction has been shown to improve asthma symptoms (Stenius-Aarniala et al., 2000). Therefore, there is compelling available evidence supporting an association between obesity and asthma. It is thought that changes in adipokines might affect airway inflammation and hyper-responsiveness in obese individuals (Weiss, 2005). Leptin levels increase with obesity, resulting in a subsequent upregulation of pro-inflammatory cytokine production (Sood et al., 2006). Contrarily, obesity-related decrease in adiponectin levels can negatively regulate allergic airway inflammation (Shore et al., 2006). Therefore, weight loss, and reduction of fat mass in particular, represents a potential anti-inflammatory benefit for asthma management. Dietary CLA supplementation using variable mixtures of CLA isomers has been shown to reduce circulating plasma leptin levels, an effect that has been directly linked to fat mass reduction and food consumption (Yamasaki et al., 1999; Takahashi et al., 2002). Weight loss and modulation of adipokines following CLA treatment can have a beneficial effect in respiratory inflammation and asthma. CLA treatment has already been shown to reduce airway inflammation and hyper-reactivity through a variety of PPARγdependent and independent mechanisms. Specifically, CLA isomer c-9, t-11 is able to reduce broncho-alveaolar inflammatory cell count and lung IL-5 levels in an animal model of allergic asthma through a PPARγ-dependent mechanism (Jaudszus et al., 2008). However, the t-10, c-12 isomer seems to inhibit ligand-dependent PPARγ-activation, thus resulting in pro-inflammatory activity (Kennedy et al., 2008). Both c-9, t-11 and t-10, c-12 CLA isomers can also reduce proinflammatory eicosanoid production by regulating the transcription of cyclooxygenase 2 (COX-2) (Ringseis et al., 2006) and lipoxygenase (Ochoa et al., 2004) in both ex vivo and in vivo guinea pig models (Urquhart et al., 2002; Whigham et al., 2002; Ringseis et al., 2006). Eicosanoids are autocrine and paracrine signaling molecules resulting from arachidonic acid oxidation that play an important role in the regulation of cytokine production, antibody formation, antigen presentation and differentiation, as well as proliferation and migration of immune cells (Harizi et al., 2008). Although eicosanoid production was not reported by the authors, c-9, t-11 CLA reduced airway hyper-reactivity in a mouse model of allergic sensitization. The same isomer has also been shown to reduce pro-inflammatory cytokines production by eosinophils and bronchial epithelial cells (Jaudszus et al., 2005), as well as decrease ex vivo IL-4 production in splenocytes (Kelley et al., 2002; Yang and Cook, 2003). While c-9, t-11 CLA increases the production of IgA, IgG and IgM, it is also able to reduce allergen-induced IgE levels (Sugano et al., 1998; Yamasaki et al., 2003a; Jaudszus et al., 2008). Finally, 50:50c-9, t-11 and t-10, c-12 CLA dietary mixtures seem to have beneficial effects related to viral clearance in pigs (Bassaganya-Riera et al., 2003) and reduce rhinovirus infection and duration in humans (Bassaganya-Riera et al., 2003). In vitro studies suggest that these effects might be due to the ability of c-9, t-11 and t-10, c-12 CLA isomers to inhibit viral entry and virus-induced inflammation (Macredmond and Dorscheid, 2011).

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Although a beneficial clinical effect in overweight mild asthmatics during CLA administration was demonstrated in the only clinical trial published to date (MacRedmond et al., 2010), the limitations of such single study and lack of conclusive data preclude routinely prescribing CLA to asthma and airway inflammation patients at this time. Future studies are needed to define a role for CLA in asthma. 4.4. Inflammatory bowel disease Inflammatory Bowel Disease (IBD) is a chronic, recurring, debilitating and widespread immuno-inflammatory illness of unknown etiology with two clinical manifestations: Ulcerative Colitis (UC) and Crohn's Disease (CD) (Camilleri, 2003; Lakatos, 2006). Even though IBD therapies have improved (Camilleri, 2003), there is still a need to develop novel immuno-nutritional interventions due to the significant side effects associated with current treatments (Maconi et al., 2005). A promising avenue for the development of such nutrition-based treatments for IBD is by targeting PPARs. As discussed above, several CLA isomers are able to activate PPARγ in the nucleus of a cell. Upon CLA stimulation, PPARγ induces the transcription of anti-inflammatory genes, which have been shown to reduce clinical symptoms in Crohn's disease patients (Scrascia et al., 2003). CLA has been studied for the prevention and treatment of gut inflammation since 2002 (Bassaganya-Riera et al., 2002). By using a B. hyodysenteriae-induced colitis pig model, we found that dietary 50:50c-9, t-11 and t-10, c-12 CLA supplementation upregulates colonic PPARγ, which subsequently binds to DNA and regulates transcription of anti-inflammatory genes, thus resulting in the suppression of colonic inflammation. Specifically, dietary CLA supplementation ameliorated tissue inflammation and weight loss associated with bacterial-induced colitis (Hontecillas et al., 2002). Using Dextran Sodium Sulfate (DSS)- and CD4 T cell transfer-induced colitis in mice, we observed a reduction of TNFα mRNA levelsand an upregulation of TGF-β1 production following dietary 50:50 c-9, t-11 and t-10, c-12 CLA supplementation (Bassaganya-Riera et al., 2004). DSS challenge increased NF-κB p65 activation. However, the levels of activated NF-KB p65 were significantly lower in the colonic tissue of CLA-fed mice when compared to the control diet. Moreover, CLA induced the expression of colonic PPARγ and PPARδ and transcriptionally modulated their responsive genes involved in lipid metabolism (UCP1, UCP3, PGC1α, and CD36) and epithelial cell maturation (Gob-4 and Keratin20). PPARγ expression was induced in both healthy and DSSchallenged mice following CLA administration. Therefore, CLA prevented the shut down of colonic PPARγ usually seen after DSS challenge. Finally, loss of colonic tissue-specific PPARγ abrogated the beneficial effects of CLA. This study provided in vivo molecular evidence suggesting that dietary 50:50c-9, t-11 and t-10, c-12 CLA is able to ameliorate colitis through a PPARγ-dependent mechanism. Some bacterial strains are able to produce CLA isomers in vitro from LA (Gorissen et al., 2010). Some of these strains are contained in a bacterial mixture known as VLS#3, a probiotic with demonstrated therapeutic efficacy in patients with UC (Bibiloni et al., 2005) and in animal models of colitis (Madsen et al., 2001). Following up on these studies, we recently published novel in vivo evidence showing how VSL#3 administration changes microbial diversity and local CLA production, which results in PPARγ-dependent anti-inflammatory effects during DSS- and IL-10-deficiency-induced experimental colitis in mice (Bassaganya-Riera et al., 2012b). PPARγ is expressed in a large population of colonic cell types including epithelial cells and lamina propria mononuclear cells such as macrophages and lymphocytes. CLA has been confirmed to increase PPARγ expression and activity in adipocytes

(McNeel et al., 2003), skeletal muscle (Meadus et al., 2002), colonic mucosa and macrophages (Yu et al., 2002; Yang and Cook, 2003). However, other reports show a reduction in adipocyte PPARγ expression after CLA treatment (Kennedy et al., 2008; Miller et al., 2008). Therefore, there is a need to further characterize the cellspecific PPARγ expression and determine the main cellular source responsible for the therapeutic effect of CLA during experimental IBD (Dubuquoy et al., 2006). More recently, we have investigated the immunoregulatory efficacy of CLA in CD patients (Bassaganya-Riera et al., 2012a). Thirteen patients with mild to moderately active CD were enrolled in an open-label study of CLA. Patients received a capsule containing 6 g of 50% c-9, t-11 and 50% t-10, c-12 CLA daily for 12 weeks. Treated patients expressed a decreased disease activity (calculated using the CD activity index-CDAI) and increased quality of life (assessed using the Inflammatory Bowel Disease Questionnaire-IBDQ), which correlated with a significant suppression of the ability of peripheral blood CD4 þ and CD8 þ T cell subsets to produce pro-inflammatory cytokines including IFNγ, TNFα and IL17 and lymphoproliferation at week 12 post-treatment. Overall, CLA supplementation was well tolerated and ameliorated disease in patients with CD. Thus, this human study validated previous findings obtained using experimental models of colitis in mice and pigs. 4.5. Inflammation-driven colorectal Cancer Along with hereditary syndromes of familial adenomatous polyposis and hereditary nonpolyposis, IBD is among the top three high-risk conditions for the development of colorectal cancer (Xie and Itskowitz, 2008), the third most commonly diagnosed cancer in the United States (Jemal et al., 2008). The relative risk of developing colorectal cancer in UC patients correlates with the extent and duration of the disease (Eaden et al., 2001; Xie and Itzkowitz, 2008). Specifically, it has been estimated that the risk increases by 0.5–1% yearly after 8–10 years of IBD initial diagnosis (Munkholm, 2003). In 2010, we reported that dietary 50:50c-9, t-11 and t-10, c-12 CLA ameliorates inflammation-driven colorectal cancer in mice (Evans et al., 2010). Particularly, CLA upregulated the levels of regulatory CD4 þ T cells in mesenteric lymph nodes of wild type mice. However, no differences were seen in immune cell-specific PPARγ null mice, suggesting that CLA is able to modulate the Treg compartment in a PPARγ-dependent fashion. These findings are in line with previous reports which demonstrated that PPARγ is required for appropriate Treg cell function (Hontecillas and Bassaganya-Riera 2007; Wohlfert et al., 2007). In a more recent study published in 2012 (Bassaganya-Riera et al., 2012c), we showed how dietary 50:50c-9, t-11 and t-10, c-12 CLA or VSL#3 treatments resulted in a faster recovery during acute inflammation and lowered disease severity during the chronic, tumor-bearing phase of disease during chemically-induced colorectal cancer in mice. Both treatments were able to reduce colonic adenoma and adenocarcinoma formation. Whereas VSL#3 increased the mRNA levels of TNFα, angiostatin and PPARγ, CLA treatment decreased colonic COX-2 expression. However, only the probiotic VSL#3 was able to modulate adaptive T cell responses as shown by an increased IL-17 expression in MLN CD4 þ T cells and accumulation of mucosal Treg cells and memory CD4 þ T cells. As follow up of these ongoing studies, placebo-controlled, large-scale studies should investigate the interaction between CLA supplementation, gut microbiota and mucosal immunity. 5. Conclusions With the emergence of inflammatory and immune-mediated

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diseases, there is an urgent need to search for nutrition-based interventions that address many of the risk factors. Although preclinical data support a benefit of CLA supplementation within various areas of health and well-being, human data have yet to show convincing evidence of a positive effect. To date, there is no clear consensus regarding the role of CLA in inflammation-related diseases. Moreover, data suggest there are likely to be isomerspecific effects and mechanisms which have not been completely characterized yet. Although cumulative data suggests that CLA is well-tolerated in humans, some cases of mild gastrointestinal disturbances and increased insulin resistance have been reported after long-term CLA treatment. Evidence of such adverse side effects is not conclusive. Nonetheless, additional human studies and further animal research are being conducted to unveil CLA's beneficial mechanisms, optimal dosage, long-term management and target population. Such studies, in combination with computational and animal modeling, will accelerate the development of CLA-based nutritionals and medical foods, and eventually contribute to the effort to reduce chronic disease in humans.

References Albers, R., van der Wielen, R.P., et al., 2003. Effects of cis-9, trans-11 and trans-10, cis-12 conjugated linoleic acid (CLA) isomers on immune function in healthy men. Eur. J. Clin. Nutr. 57, 595–603. Bassaganya-Riera, J., Hontecillas-Magarzo, R., et al., 2001a. Effects of dietary conjugated linoleic acid in nursery pigs of dirty and clean environments on growth, empty body composition, and immune competence. J. Anim. Sci. 79, 714–721. Bassaganya-Riera, J., Hontecillas, R., 2006. CLA and n-3 PUFA differentially modulate clinical activity and colonic PPAR-responsive gene expression in a pig model of experimental IBD. Clin. Nutr. 25, 454–465. Bassaganya-Riera, J., Hontecillas, R., 2010. Dietary conjugated linoleic acid and n-3 polyunsaturated fatty acids in inflammatory bowel disease. Curr. Opin. Clin. Nutr. Metab. Care 13, 569–573. Bassaganya-Riera, J., Hontecillas, R., et al., 2002. Colonic anti-inflammatory mechanisms of conjugated linoleic acid. Clin. Nutr. 21, 451–459. Bassaganya-Riera, J., Hontecillas, R., et al., 2012a. Conjugated linoleic acid modulates immune responses in patients with mild to moderately active Crohn’s disease. Clin. Nutr. 31, 721–727. Bassaganya-Riera, J., Hontecillas, R., et al., 2001b. Dietary conjugated linoleic acid modulates phenotype and effector functions of porcine CD8( þ ) lymphocytes. J. Nutr. 131, 2370–2377. Bassaganya-Riera, J., Pogranichniy, R.M., et al., 2003. Conjugated linoleic acid ameliorates viral infectivity in a pig model of virally induced immunosuppression. J. Nutr. 133, 3204–3214. Bassaganya-Riera, J., Reynolds, K., et al., 2004. Activation of PPAR gamma and delta by conjugated linoleic acid mediates protection from experimental inflammatory bowel disease. Gastroenterology 127, 777–791. Bassaganya-Riera, J., Viladomiu, M., et al., 2012b. Probiotic bacteria produce conjugated linoleic acid locally in the gut that targets macrophage PPAR gamma to suppress colitis. PloS One 7, e31238. Bassaganya-Riera, J., Viladomiu, M., et al., 2012c. Immunoregulatory mechanisms underlying prevention of colitis-associated colorectal cancer by probiotic bacteria. PloS one 7, e34676. Belury, M.A., 2002a. Dietary conjugated linoleic acid in health: physiological effects and mechanisms of action. Annu. Rev. Nutr. 22, 505–531. Belury, M.A., 2002b. Inhibition of carcinogenesis by conjugated linoleic acid: potential mechanisms of action. J. Nutr. 132, 2995–2998. Belury, M.A., C.S., M., Lub, M., Shib, L., Leesnitzerc, L.M., Blanchardc, S.G., 2002. Conjugated linoleic acid is an activator and ligand for peroxisome proliferatoractivated receptor-gamma (PPARγ). Nutr. Res., 817–824. Beuther, D.A., Sutherland, E.R., 2007. Overweight, obesity, and incident asthma: a meta-analysis of prospective epidemiologic studies. Am. J. Respir. Crit. Care Med. 175, 661–666. Bhattacharya, A., Banu, J., et al., 2006. Biological effects of conjugated linoleic acids in health and disease. J. Nutr. Biochem. 17, 789–810. Bibiloni, R., Fedorak, R.N., et al., 2005. VSL#3 probiotic-mixture induces remission in patients with active ulcerative colitis. Am. J. Gastroenterol. 100, 1539–1546. Bruun, J.M., Lihn, A.S., et al., 2005. Monocyte chemoattractant protein-1 release is higher in visceral than subcutaneous human adipose tissue (AT): implication of macrophages resident in the AT. J. Clin. Endocrinol. Metabol. 90, 2282–2289. Camilleri, M., 2003. GI clinical research 2002–2003: the year in review. Clin. Gastroenterol. Hepatol.: Off. Clin. Pract. J. Am. Gastroenterol. Assoc. 1, 415–420. Chin SF, L.W., Storkson, J.M., Ha, J.M., Pariza, M.W., 1992. Dietary sources of conjugated dienoic isomers of linoleic acid, a newly recognized class of anticarcinogens. J. Food Compos. Anal., 185–197. Cook, M.E., Miller, C.C., et al., 1993. Immune modulation by altered nutrient metabolism: nutritional control of immune-induced growth depression. Poult. Sci.

7

72, 1301–1305. Corl, B.A., Barbano, D.M., et al., 2003. cis-9, trans-11 CLA derived endogenously from trans-11 18:1 reduces cancer risk in rats. J. Nutr. 133, 2893–2900. Corl, B.A., Baumgard, L.H., et al., 2001. The role of Delta(9)-desaturase in the production of cis-9, trans-11 CLA. J. Nutr. Biochem. 12, 622–630. Couper, K.N., Blount, D.G., et al., 2008. IL-10: the master regulator of immunity to infection. J. Immunol. 180, 5771–5777. Dandona, P., Aljada, A., et al., 2005. Metabolic syndrome: a comprehensive perspective based on interactions between obesity, diabetes, and inflammation. Circulation 111, 1448–1454. de Cassia, da Silveira, Andrade, E.S.R.,, L.N., et al., 2014. A review on anti-inflammatory activity of phenylpropanoids found in essential oils. Molecules 19, 1459–1480. de Lany, J.P., Blohm, F., et al., 1999. Conjugated linoleic acid rapidly reduces body fat content in mice without affecting energy intake. Am. J. Physiol. 276, R1172–R1179. Dhiman, T.R., Nam, S.H., et al., 2005. Factors affecting conjugated linoleic acid content in milk and meat. Crit. Rev. Food Sci. Nutr. 45, 463–482. Dilzer, A., Park, Y., 2012. Implication of conjugated linoleic acid (CLA) in human health. Crit. Rev. Food Sci. Nutr. 52, 488–513. Dubuquoy, L., Rousseaux, C., et al., 2006. PPARgamma as a new therapeutic target in inflammatory bowel diseases. Gut 55, 1341–1349. Eaden, J.A., Abrams, K.R., et al., 2001. The risk of colorectal cancer in ulcerative colitis: a meta-analysis. Gut 48, 526–535. Evans, N.P., Misyak, S.A., et al., 2010. Conjugated linoleic acid ameliorates inflammation-induced colorectal cancer in mice through activation of PPARgamma. J. Nutr. 140, 515–521. Ewaschuk, J.B., Walker, J.W., et al., 2006. Bioproduction of conjugated linoleic acid by probiotic bacteria occurs in vitro and in vivo in mice. J. Nutr. 136, 1483–1487. Ferrero-Miliani, L., Nielsen, O.H., et al., 2007. Chronic inflammation: importance of NOD2 and NALP3 in interleukin-1beta generation. Clin. Exp. Immunol. 147, 227–235. Gorissen, L., Raes, K., et al., 2010. Production of conjugated linoleic acid and conjugated linolenic acid isomers by Bifidobacterium species. Appl. Microbiol. Biotechnol. 87, 2257–2266. Griinari, J.M., Corl, B.A., et al., 2000. Conjugated linoleic acid is synthesized endogenously in lactating dairy cows by Delta(9)-desaturase. J. Nutr. 130, 2285–2291. Ha, Y.L., Grimm, N.K., et al., 1987. Anticarcinogens from fried ground beef: heataltered derivatives of linoleic acid. Carcinogenesis 8, 1881–1887. Halade, G.V., Rahman, M.M., et al., 2010. Differential effects of conjugated linoleic acid isomers in insulin-resistant female C57Bl/6J mice. J. Nutr. Biochem. 21, 332–337. Hargrave, K.M., Li, C., et al., 2002. Adipose depletion and apoptosis induced by trans-10, cis-12 conjugated linoleic Acid in mice. Obes. Res. 10, 1284–1290. Harizi, H., Corcuff, J.B., et al., 2008. Arachidonic-acid-derived eicosanoids: roles in biology and immunopathology. Trends in molecular medicine 14, 461–469. Hjellvik, V., Tverdal, A., et al., 2010. Body mass index as predictor for asthma: a cohort study of 118,723 males and females. Eur. Respir. J. 35, 1235–1242. Hontecillas, R., Bassaganya-Riera, J., 2007. Peroxisome proliferator-activated receptor gamma is required for regulatory CD4 þ T cell-mediated protection against colitis. J. Immunol. 178, 2940–2949. Hontecillas, R., Wannemeulher, M.J., et al., 2002. Nutritional regulation of porcine bacterial-induced colitis by conjugated linoleic acid. J. Nutr. 132, 2019–2027. Hotamisligil, G.S., Shargill, N.S., et al., 1993. Adipose expression of tumor necrosis factor-alpha: direct role in obesity-linked insulin resistance. Science 259, 87–91. House, R.L., Cassady, J.P., et al., 2005. Conjugated linoleic acid evokes de-lipidation through the regulation of genes controlling lipid metabolism in adipose and liver tissue. Obes. Rev.: Off. J. Int. Assoc. Stud. Obes. 6, 247–258. Jaudszus, A., Foerster, M., et al., 2005. Cis-9,trans-11-CLA exerts anti-inflammatory effects in human bronchial epithelial cells and eosinophils: comparison to trans-10,cis-12-CLA and to linoleic acid. Biochim. Biophys. Acta 1737, 111–118. Jaudszus, A., Krokowski, M., et al., 2008. Cis-9,trans-11-conjugated linoleic acid inhibits allergic sensitization and airway inflammation via a PPARgamma-related mechanism in mice. J. Nutr. 138, 1336–1342. Jemal, A., Siegel, R., et al., 2008. Cancer statistics, 2008. CA: Cancer J. Clin. 58, 71–96. Jiang, C., Ting, A.T., et al., 1998. PPAR-gamma agonists inhibit production of monocyte inflammatory cytokines. Nature 391, 82–86. Kay, J.K., Mackle, T.R., et al., 2004. Endogenous synthesis of cis-9, trans-11 conjugated linoleic acid in dairy cows fed fresh pasture. J. Dairy Sci. 87, 369–378. Kelley, D.S., Warren, J.M., et al., 2002. Similar effects of c9,t11-CLA and t10,c12-CLA on immune cell functions in mice. Lipids 37, 725–728. Kelley, N.S., Hubbard, N.E., et al., 2007. Conjugated linoleic acid isomers and cancer. J. Nutr. 137, 2599–2607. Kennedy, A., Chung, S., et al., 2008. Trans-10, cis-12 conjugated linoleic acid antagonizes ligand-dependent PPARgamma activity in primary cultures of human adipocytes. J. Nutr. 138, 455–461. Kennedy, A., Martinez, K., et al., 2010. Antiobesity mechanisms of action of conjugated linoleic acid. J. Nutr. Biochem. 21, 171–179. Kepler, C.R., Tucker, W.P., et al., 1971. Biohydrogenation of unsaturated fatty acids. V. Stereospecificity of proton addition and mechanism of action of linoleic acid delta 12-cis, delta 11-trans-isomerase from Butyrivibrio fibrisolvens. J. Biol. Chem. 246, 2765–2771. Khan, S.A., Vanden Heuvel, J.P., 2003. Role of nuclear receptors in the regulation of gene expression by dietary fatty acids (review). J. Nutr. Biochem. 14, 554–567.

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Kim, D.I., Kim, K.H., et al., 2011. Trans-10, cis-12-conjugated linoleic acid modulates NF-kappaB activation and TNF-alpha production in porcine peripheral blood mononuclear cells via a PPARgamma-dependent pathway. Br. J. Nutr. 105, 1329–1336. Kim, S.R., Lee, K.S., et al., 2005. Involvement of IL-10 in peroxisome proliferatoractivated receptor gamma-mediated anti-inflammatory response in asthma. Mol. Pharmacol. 68, 1568–1575. Koeberle, A., Werz, O., 2014. Multi-target approach for natural products in inflammation. Drug Discov. Today. Krey, G., Braissant, O., et al., 1997. Fatty acids, eicosanoids, and hypolipidemic agents identified as ligands of peroxisome proliferator-activated receptors by coactivator-dependent receptor ligand assay. Mol. Endocrinol. 11, 779–791. Kritchevsky, D., 2000. Antimutagenic and some other effects of conjugated linoleic acid. Br. J. Nutr. 83, 459–465. Kuhnt, K., Kraft, J., et al., 2006. Trans-11-18: 1 is effectively Delta9-desaturated compared with trans-12-18: 1 in humans. Br. J. Nutr. 95, 752–761. Lakatos, P.L., 2006. Recent trends in the epidemiology of inflammatory bowel diseases: up or down? World J. Gastroenterol. 12, 6102–6108. Lambert, E.V., Goedecke, J.H., et al., 2007. Conjugated linoleic acid versus high-oleic acid sunflower oil: effects on energy metabolism, glucose tolerance, blood lipids, appetite and body composition in regularly exercising individuals. Br. J. Nutr. 97, 1001–1011. Lee, K.N., Kritchevsky, D., et al., 1994. Conjugated linoleic acid and atherosclerosis in rabbits. Atherosclerosis 108, 19–25. Lee, K.S., Park, S.J., et al., 2005. PPAR-gamma modulates allergic inflammation through up-regulation of PTEN. FASEB J.: Off. Publ. Fed. Am. Soc. Exp. Biol. 19, 1033–1035. Lee, K.S., Park, S.J., et al., 2006a. Modulation of airway remodeling and airway inflammation by peroxisome proliferator-activated receptor gamma in a murine model of toluene diisocyanate-induced asthma. J. Immunol. 177, 5248–5257. Lee, S.Y., Kang, E.J., et al., 2006b. Peroxisome proliferator-activated receptor-gamma inhibits cigarette smoke solution-induced mucin production in human airway epithelial (NCI-H292) cells. Am. J. Physiol. Lung Cell. Mol. Physiol. 291, L84–L90. Lewis, J.D., Lichtenstein, G.R., et al., 2008. Rosiglitazone for active ulcerative colitis: a randomized placebo-controlled trial. Gastroenterology 134, 688–695. Loscher, C.E., Draper, E., et al., 2005. Conjugated linoleic acid suppresses NF-kappa B activation and IL-12 production in dendritic cells through ERK-mediated IL-10 induction. J. Immunol. 175, 4990–4998. Maconi, G., Colombo, E., et al., 2005. Prevalence, detection rate and outcome of cytomegalovirus infection in ulcerative colitis patients requiring colonic resection. Dig. Liver Dis.: Off. J. Ital. Soc. Gastroenterol. Ital. Assoc. Stud. Liver 37, 418–423. Macredmond, R., Dorscheid, D.R., 2011. Conjugated linoleic acid (CLA): is it time to supplement asthma therapy? Pulm. pharmacol. Ther. 24, 540–548. MacRedmond, R., Singhera, G., et al., 2010. Conjugated linoleic acid improves airway hyper-reactivity in overweight mild asthmatics. Clin. Exp. Allergy: J. Br. Soc. Allergy Clin. Immunol. 40, 1071–1078. Madsen, K., Cornish, A., et al., 2001. Probiotic bacteria enhance murine and human intestinal epithelial barrier function. Gastroenterology 121, 580–591. Marcy, T.R., Britton, M.L., et al., 2004. Second-generation thiazolidinediones and hepatotoxicity. Ann. Pharmacother. 38, 1419–1423. McGuirk, P., McCann, C., et al., 2002. Pathogen-specific T regulatory 1 cells induced in the respiratory tract by a bacterial molecule that stimulates interleukin 10 production by dendritic cells: a novel strategy for evasion of protective T helper type 1 responses by Bordetella pertussis. J. Exp. Med. 195, 221–231. McNeel, R.L., Smith, E.O., et al., 2003. Isomers of conjugated linoleic acid modulate human preadipocyte differentiation. In vitro Cell. Dev. Biol. Anim. 39, 375–382. Meadus, W.J., MacInnis, R., et al., 2002. Prolonged dietary treatment with conjugated linoleic acid stimulates porcine muscle peroxisome proliferator activated receptor gamma and glutamine-fructose aminotransferase gene expression in vivo. J. Mol. Endocrinol. 28, 79–86. Medina, E.A., Horn, W.F., et al., 2000. Conjugated linoleic acid supplementation in humans: effects on circulating leptin concentrations and appetite. Lipids 35, 783–788. Medzhitov, R., 2008. Origin and physiological roles of inflammation. Nature 454, 428–435. Mendis, S., Cruz-Hernandez, C., et al., 2008. Fatty acid profile of Canadian dairy products with special attention to the trans-octadecenoic acid and conjugated linoleic acid isomers. J. AOAC Int. 91, 811–819. Miller, C.C., Park, Y., et al., 1994. Feeding conjugated linoleic acid to animals partially overcomes catabolic responses due to endotoxin injection. Biochem. Biophys. Res. Commun. 198, 1107–1112. Miller, J.R., Siripurkpong, P., et al., 2008. The trans-10, cis-12 isomer of conjugated linoleic acid decreases adiponectin assembly by PPARgamma-dependent and PPARgamma-independent mechanisms. J. Lipid Res. 49, 550–562. Mills, K.H., McGuirk, P., 2004. Antigen-specific regulatory T cells–their induction and role in infection. Semin. Immunol. 16, 107–117. Miner, J.L., Cederberg, C.A., et al., 2001. Conjugated linoleic acid (CLA), body fat, and apoptosis. Obes. Res. 9, 129–134. Mitchell, P.L., McLeod, R.S., 2008. Conjugated linoleic acid and atherosclerosis: studies in animal models. Biochem. Cell Biol. ¼Biochim. Biol. Cell. 86, 293–301. Moloney, F., Toomey, S., et al., 2007. Antidiabetic effects of cis-9, trans-11-conjugated linoleic acid may be mediated via anti-inflammatory effects in white adipose tissue. Diabetes 56, 574–582. Moon, H.S., 2014. Biological effects of conjugated linoleic acid on obesity-related cancers. Chem.-Biol. Interact. 224C, 189–195.

Moya-Camarena, S.Y., Van den Heuvel, J.P., et al., 1999a. Conjugated linoleic acid activates peroxisome proliferator-activated receptor alpha and beta subtypes but does not induce hepatic peroxisome proliferation in Sprague-Dawley rats. Biochim. Biophys. Acta 1436, 331–342. Moya-Camarena, S.Y., Vanden Heuvel, J.P., et al., 1999b. Conjugated linoleic acid is a potent naturally occurring ligand and activator of PPARalpha. J. Lipid Res. 40, 1426–1433. Munkholm, P., 2003. Review article: the incidence and prevalence of colorectal cancer in inflammatory bowel disease. Aliment. Pharmacol. Ther. 18 (Suppl 2), S1–S5. Mushtaq, S., Heather Mangiapane, E., et al., 2010. Estimation of cis-9, trans-11 conjugated linoleic acid content in UK foods and assessment of dietary intake in a cohort of healthy adults. Br. J. Nutr. 103, 1366–1374. Nesto, R.W., Bell, D., et al., 2003. Thiazolidinedione use, fluid retention, and congestive heart failure: a consensus statement from the American Heart Association and American Diabetes Association. Circulation 108, 2941–2948. O’Shea, M., Bassaganya-Riera, J., et al., 2004. Immunomodulatory properties of conjugated linoleic acid. Am. J. Clin. Nutr. 79, 1199S–1206S. Ochoa, J.J., Farquharson, A.J., et al., 2004. Conjugated linoleic acids (CLAs) decrease prostate cancer cell proliferation: different molecular mechanisms for cis-9, trans-11 and trans-10, cis-12 isomers. Carcinogenesis 25, 1185–1191. Oleszczuk, J., Oleszczuk, L., et al., 2012. Biological effects of conjugated linoleic acids supplementation. Pol. J. Vet. Sci. 15, 403–408. Ostrowska, E., Muralitharan, M., et al., 1999. Dietary conjugated linoleic acids increase lean tissue and decrease fat deposition in growing pigs. J. Nutr. 129, 2037–2042. Park, Y., Albright, K.J., et al., 1997. Effect of conjugated linoleic acid on body composition in mice. Lipids 32, 853–858. Park, Y., Storkson, J.M., et al., 1999. Evidence that the trans-10,cis-12 isomer of conjugated linoleic acid induces body composition changes in mice. Lipids 34, 235–241. Parodi, P.W., 1997. Cows’ milk fat components as potential anticarcinogenic agents. J. Nutr. 127, 1055–1060. Perdomo, M.C., Santos, J.E., et al., 2011. Trans-10, cis-12 conjugated linoleic acid and the PPAR-gamma agonist rosiglitazone attenuate lipopolysaccharide-induced TNF-alpha production by bovine immune cells. Domest. Anim. Endocrinol. 41, 118–125. Peterson, K.M., O’Shea, M., et al., 2009. Effects of dietary supplementation with conjugated linoleic acid on experimental human rhinovirus infection and illness. Antiviral Ther. 14, 33–43. Plourde, M., Jew, S., et al., 2008. Conjugated linoleic acids: why the discrepancy between animal and human studies? Nutr. Rev. 66, 415–421. Punchard, N.A., Whelan, C.J., et al., 2004. J. Inflamm. (Lond.) 1, 1. Richards, D.B., Bareille, P., et al., 2010. Treatment with a peroxisomal proliferator activated receptor gamma agonist has a modest effect in the allergen challenge model in asthma: a randomised controlled trial. Respir. Med. 104, 668–674. Ringseis, R., Muller, A., et al., 2006. CLA isomers inhibit TNFalpha-induced eicosanoid release from human vascular smooth muscle cells via a PPARgamma ligand-like action. Biochim. Biophys. Acta 1760, 290–300. Riserus, U., Berglund, L., et al., 2001. Conjugated linoleic acid (CLA) reduced abdominal adipose tissue in obese middle-aged men with signs of the metabolic syndrome: a randomised controlled trial. Int. J. Obes. Relat. Metab. Disord.: J. Int. Assoc. Stud. Obes. 25, 1129–1135. Riserus, U., Vessby, B., et al., 2004. Effects of cis-9,trans-11 conjugated linoleic acid supplementation on insulin sensitivity, lipid peroxidation, and proinflammatory markers in obese men. Am. J. Clin. Nutr. 80, 279–283. Ritzenthaler, K.L., McGuire, M.K., et al., 2001. Estimation of conjugated linoleic acid intake by written dietary assessment methodologies underestimates actual intake evaluated by food duplicate methodology. J. Nutr. 131, 1548–1554. Ryder, J.W., Portocarrero, C.P., et al., 2001. Isomer-specific antidiabetic properties of conjugated linoleic acid. Improved glucose tolerance, skeletal muscle insulin action, and UCP-2 gene expression. Diabetes 50, 1149–1157. Santora, J.E., Palmquist, D.L., et al., 2000. Trans-vaccenic acid is desaturated to conjugated linoleic acid in mice. J. Nutr. 130, 208–215. Scrascia, M., Forcillo, M., et al., 2003. Susceptibility to rifaximin of Vibrio cholerae strains from different geographical areas. J. Antimicrob. Chemother. 52, 303–305. Sebedio, J.L., Gnaedig, S., et al., 1999. Recent advances in conjugated linoleic acid research. Curr. Opin. Clin. Nutr. Metab. Care 2, 499–506. Shore, S.A., Terry, R.D., et al., 2006. Adiponectin attenuates allergen-induced airway inflammation and hyperresponsiveness in mice. J. Allergy Clin. Immunol. 118, 389–395. Sisk, M.B., Hausman, D.B., et al., 2001. Dietary conjugated linoleic acid reduces adiposity in lean but not obese Zucker rats. J. Nutr. 131, 1668–1674. So, M.H., Tse, I.M., et al., 2009. Dietary fat concentration influences the effects of trans-10, cis-12 conjugated linoleic acid on temporal patterns of energy intake and hypothalamic expression of appetite-controlling genes in mice. J. Nutr. 139, 145–151. Sood, A., Verhulst, S.J., et al., 2006. Association of excess weight and degree of airway responsiveness in asthmatics and non-asthmatics. J. Asthma: Off. J. Assoc. Care Asthma 43, 447–452. Spears, M., Donnelly, I., et al., 2009. Bronchodilatory effect of the PPAR-gamma agonist rosiglitazone in smokers with asthma. Clin. Pharmacol. Ther. 86, 49–53. Stenius-Aarniala, B., Poussa, T., et al., 2000. Immediate and long term effects of weight reduction in obese people with asthma: randomised controlled study. BMJ 320, 827–832.

Please cite this article as: Viladomiu, M., et al., Modulation of inflammation and immunity by dietary conjugated linoleic acid. Eur J Pharmacol (2015), http://dx.doi.org/10.1016/j.ejphar.2015.03.095i

M. Viladomiu et al. / European Journal of Pharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎ Sugano, M., Tsujita, A., et al., 1998. Conjugated linoleic acid modulates tissue levels of chemical mediators and immunoglobulins in rats. Lipids 33, 521–527. Takahashi, Y., Kushiro, M., et al., 2002. Dietary conjugated linoleic acid reduces body fat mass and affects gene expression of proteins regulating energy metabolism in mice. Comp. Biochem. Physiol. Part B Biochem. Mol. Biol. 133, 395–404. Terpstra, A.H., Beynen, A.C., et al., 2002. The decrease in body fat in mice fed conjugated linoleic acid is due to increases in energy expenditure and energy loss in the excreta. J. Nutr. 132, 940–945. Tholstrup, T., Raff, M., et al., 2008. An oil mixture with trans-10, cis-12 conjugated linoleic acid increases markers of inflammation and in vivo lipid peroxidation compared with cis-9, trans-11 conjugated linoleic acid in postmenopausal women. J. Nutr. 138, 1445–1451. Tontonoz, P., Nagy, L., et al., 1998. PPARgamma promotes monocyte/macrophage differentiation and uptake of oxidized LDL. Cell 93, 241–252. Tsuboyama-Kasaoka, N., Takahashi, M., et al., 2000. Conjugated linoleic acid supplementation reduces adipose tissue by apoptosis and develops lipodystrophy in mice. Diabetes 49, 1534–1542. Turpeinen, A.M., Ylonen, N., et al., 2008. Immunological and metabolic effects of cis-9, trans-11-conjugated linoleic acid in subjects with birch pollen allergy. Br. J. Nutr. 100, 112–119. Urquhart, P., Parkin, S.M., et al., 2002. The effect of conjugated linoleic acid on arachidonic acid metabolism and eicosanoid production in human saphenous vein endothelial cells. Biochim. Biophys. Acta 1580, 150–160. Vendrell, J., Broch, M., et al., 2004. Resistin, adiponectin, ghrelin, leptin, and proinflammatory cytokines: relationships in obesity. Obes. Res. 12, 962–971. Wallace, R.J., McKain, N., et al., 2007. Isomers of conjugated linoleic acids are synthesized via different mechanisms in ruminal digesta and bacteria. J. Lipid Res. 48, 2247–2254. Wang, T., Lee, H.G., 2013. Advances in research on cis-9, trans-11 conjugated linoleic acid: a major functional conjugated linoleic acid isomer. Crit. Rev. Food Sci. Nutr. Wang, Y.W., Jones, P.J., 2004. Conjugated linoleic acid and obesity control: efficacy and mechanisms. Int. J. Obes. Relat. Metab. Disord.: J. Int. Assoc. Stud. Obes. 28, 941–955. Weisberg, S.P., McCann, D., et al., 2003. Obesity is associated with macrophage accumulation in adipose tissue. J. Clin. Investig. 112, 1796–1808. Weiss, S.T., 2005. Obesity: insight into the origins of asthma. Nat. Immunol. 6, 537–539. West, D.B., Delany, J.P., et al., 1998. Effects of conjugated linoleic acid on body fat and energy metabolism in the mouse. Am. J. Physiol. 275, R667–R672. Whigham, L.D., Cook, M.E., et al., 2000. Conjugated linoleic acid: implications for human health. Pharmacol. Res.: Off. J. Ital. Pharmacol. Soc. 42, 503–510.

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Whigham, L.D., Higbee, A., et al., 2002. Decreased antigen-induced eicosanoid release in conjugated linoleic acid-fed guinea pigs. Am. J. Physiol. Regul. Integr. Comp. Physiol. 282, R1104–R1112. Whigham, L.D., Watras, A.C., et al., 2007. Efficacy of conjugated linoleic acid for reducing fat mass: a meta-analysis in humans. Am. J. Clin. Nutr. 85, 1203–1211. Wilson, H.M., Barker, R.N., et al., 2009. Macrophages: promising targets for the treatment of atherosclerosis. Curr. Vasc. Pharmacol. 7, 234–243. Woerly, G., Honda, K., et al., 2003. Peroxisome proliferator-activated receptors alpha and gamma down-regulate allergic inflammation and eosinophil activation. J. Exp. Med. 198, 411–421. Wohlfert, E.A., Nichols, F.C., et al., 2007. Peroxisome proliferator-activated receptor gamma (PPARgamma) and immunoregulation: enhancement of regulatory T cells through PPARgamma-dependent and -independent mechanisms. J. Immunol. 178, 4129–4135. Xie, J., Itzkowitz, S.H., 2008. Cancer in inflammatory bowel disease. World J. Gastroenterol. 14, 378–389. Xu, H.E., Lambert, M.H., et al., 1999. Molecular recognition of fatty acids by peroxisome proliferator-activated receptors. Mol. Cell 3, 397–403. Yamasaki, M., Chujo, H., et al., 2003a. Immunoglobulin and cytokine production from spleen lymphocytes is modulated in C57BL/6J mice by dietary cis-9, trans11 and trans-10, cis-12 conjugated linoleic acid. J. Nutr. 133, 784–788. Yamasaki, M., Ikeda, A., et al., 2003b. Modulation of body fat and serum leptin levels by dietary conjugated linoleic acid in Sprague-Dawley rats fed various fat-level diets. Nutrition 19, 30–35. Yamasaki, M., Mansho, K., et al., 1999. Dietary effect of conjugated linoleic acid on lipid levels in white adipose tissue of Sprague-Dawley rats. Biosci. Biotechnol. Biochem. 63, 1104–1106. Yang, M., Cook, M.E., 2003. Dietary conjugated linoleic acid decreased cachexia, macrophage tumor necrosis factor-alpha production, and modifies splenocyte cytokines production. Exp. Biol. Med. (Maywood) 228, 51–58. Yu, Y., Correll, P.H., et al., 2002. Conjugated linoleic acid decreases production of pro-inflammatory products in macrophages: evidence for a PPAR gamma-dependent mechanism. Biochim. Biophys. Acta 1581, 89–99. Zabala, A., Portillo, M.P., et al., 2006. Effects of cis-9,trans-11 and trans-10,cis-12 CLA isomers on liver and adipose tissue fatty acid profile in hamsters. Lipids 41, 993–1001. Zambell, K.L., Keim, N.L., et al., 2000. Conjugated linoleic acid supplementation in humans: effects on body composition and energy expenditure. Lipids 35, 777–782.

Please cite this article as: Viladomiu, M., et al., Modulation of inflammation and immunity by dietary conjugated linoleic acid. Eur J Pharmacol (2015), http://dx.doi.org/10.1016/j.ejphar.2015.03.095i