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Genetic variations and inflammation:
NUTRITION, DISEASE, AND THE GENOME
Genetic Variations and Inflammation: A Practical Nutrigenomics Opportunity Kenneth S. Kornman, DDS, PhD, Paul M. Martha, MD, and Gordon W. Duff, BM, BCh, PhD From Interleukin Genetics, Waltham, Massachusetts, USA; and the Division of Genomic Medicine, University of Sheffield, Sheffield, United Kingdom INTRODUCTION Some individuals who are overtly healthy as they reach young adulthood will begin to experience the complications of chronic diseases such as cardiovascular disease, arthritis, osteoporosis, and Alzheimer’s disease before age 60, whereas others will reach their 80s with minimal evidence of these debilitating conditions. This simple observation defines much of the future focus of the health care delivery system and the search by individuals for prolongation of healthy and productive lives. Chronic diseases have a set of initiating factors that activate specific patterns of gene expression that in turn act to tip biochemical equilibria to a non-homeostatic state. If prolonged, these non-homeostatic equilibria produce tissue degeneration and loss of function of one or more organs and ultimately produce signs and symptoms that lead to a clinical diagnosis of disease. Although still at an early stage of development, gene expression studies are beginning to show differential expression of sets of genes in tissues from chronic diseases as compared with healthy tissues.1,2 At present the cancer field provides the most definitive data to indicate that a disease only develops if there is an altered expression of a specific set of genes. This altered pattern of gene expression may be the result of acquired and inherited factors.3 The clinical presentation of chronic diseases therefore appears to be the net result of the initiating factors as modulated by the interindividual genetic makeup and environmental variations that influence gene expression in response to the disease initiators. The practical application of predictive and preventive medicine requires early identification of the individuals who are on a path toward earlier development of disease, followed by the introduction of a targeted intervention. Ideally, one would like to modulate the biology of individuals on the more rapid disease path to prevent overt complications of disease. Because the diseasecausing patterns of gene activation may be the result of various combinations of lifestyle and genetic factors, it is necessary to evaluate and manage both types of risk factors. If the diseaserelated gene expression involves specific genetic variations, one prevention option may be to use nutritional agents to modulate the biology that results from the genetic variation.
NUTRIGENOMICS: NUTRIENT–GENE INTERACTIONS Foods provide nutrients that are used for fuel in energy metabolism and growth and for the development of structural components of the body. Some nutrients are also essential cofactors for the proper function of life-critical enzymes that are involved in various aspects of metabolism and tissue integrity. In addition, many nutrients selectively alter gene expression through transcription factor systems that regulate the activation of specific sets of genes in different tissues and under different environmental conditions.
Correspondence to: Kenneth S. Kornman, DDS, PhD, Interleukin Genetics, Inc., Waltham, MA 02452, USA. E-mail: [email protected] Nutrition 20:44 – 49, 2004 ©Elsevier Inc., 2004. Printed in the United States. All rights reserved.
Various nutrients bind to or in some way directly activate specific transcription factors,4 and other nutrients alter the oxidationreduction status of the cell to indirectly influence transcription factor activity.5 The term nutrigenomics has been defined in many ways,4,6 – 8 but it generally refers to the use of various molecular tools to explore how dietary substances interact with the genome. The ultimate goal is to determine the dietary components that are most compatible with health for a specific individual. Given our current understanding of nutrient– gene interactions, a few of the practical applications of nutrigenomics are listed below. Some of the Practical Applications of Nutrigenomics 1. Identify the genes and proteins expressed differentially in health and disease that are modifiable by nutrients. 2. Identify which genes, proteins, and metabolites are influenced by specific nutrients that are known to be beneficial or harmful. a. Identify genes, proteins, and metabolites that are altered by dietary fats associated with cardiovascular disease. b. Identify genes, proteins, and metabolites that are altered by -3 fatty acids. 3. Identify genetic variations that alter the nutrient– gene interactions in applications 1 and 2.
GENETIC VARIATIONS AND NUTRIGENOMICS: CRITERIA FOR PRACTICAL USE The three general applications of nutrigenomics described above will require many years of effort, but certain specific questions seem to be more tractable than others and may provide a demonstration of the value to be derived from such efforts. This discussion addresses how nutrigenomics may be used to identify nutrients that provide health benefits to individuals with certain genetic variations that predispose them to earlier or more severe chronic disease. Although employing knowledge of specific genetic variations to target the use of nutritional compounds is very attractive at the conceptual level, a practical application of this technology requires addressing several immediate challenges. Included among these is the simple step of determining just where in the human genome to focus the initial efforts to maximize the chance of ultimate success. The number of potential interactions between nutritional compounds and the human genome is clearly enormous. However, it is almost certain that some genes will have more of an influence than others on the future course of a disease and on the effects that specific nutritional compounds have on overall health. Therefore, by applying certain criteria when selecting genes for initial study, the task of designing an efficient strategy for moving nutrigenomic research forward can be greatly simplified. The following section includes some of the criteria we believe are most important to consider early on, followed by a brief rationale for each. 0899-9007/04/$30.00 doi:10.1016/j.nut.2003.09.008
Nutrition Volume 20, Number 1, 2004 Focus on Genes That Are Chronically Activated During Clinical Disease and That Are Modifiable by Nutritional Compounds Complex chronic diseases such as heart disease, type 2 diabetes, and osteoporosis involve the interaction of a large number of genes. Among the putative disease-related genes whose expression levels are altered by interaction with common nutritional compounds, such as -3-fatty acids and isoflavones,9 –11 are those that code for cytokines, growth factors, cholesterol-metabolizing enzymes, and lipoproteins. The influence of nutrients on transcription factors that regulate gene expression has recently been reviewed.4 Due to the chronic nature of these diseases, a prolonged modulation of gene activity, such as could be achieved through regular dietary factors that directly influence gene expression, is most likely required to alter the trajectory of these diseases over time. The Genes Have Functionally Important Variations Knowledge of gene variations, such as single nucleotide polymorphisms (SNPs), in nutrient-modifiable genes may be useful in two general ways. First, a SNP may serve as a reliable marker for the presence of other genetic factors that influence the course of disease. In this case, directing a nutrient to modify activity of that particular gene may or may not be clinically beneficial because modulating the activity of the gene being targeted may not affect the true functionally important genetic factors. In other cases, the SNP itself has an important functional impact on the protein product of the gene, which in turn directly affects the disease course. Although there are several ways that a SNP may affect its ultimate protein product, SNPs that affect the quantity of protein produced or the structure of the protein itself have received the most attention to date. Because of its direct connection with the pathophysiology of the disease, administration of nutrients that affect genes with such “functionally important” SNPs are highly likely to have clinically important effects. The Genes Are at High Leverage Points in Biological Cascades Many of the human body’s most important biological systems use a powerful “cascade” of protein–protein interactions. These cascades serve to amplify the initial signals to increase the overall activity of the system. Some familiar examples of this phenomenon include the complement cascade, the blood clotting system, and the innate immune response. Genes that encode proteins acting far upstream at, or near, the head of these cascades therefore have great leverage in determining the net effect of the system. Likewise, modifying the activity of these genes has great potential to modify the clinical course of health and disease. The SNPs Are Highly Prevalent in the Population For highly targeted, expensive prescription drugs, a focus on uncommon gene SNPs that are associated with life-threatening disease may very well make sense. However, for the practical application of genomics to the development of valuable nutritional compounds, a focus on gene SNPs that are highly prevalent in the population has substantially more value in the near term from the perspective of individual health and as a commercial opportunity. Nutrients Modifying Gene Activity Are Readily Available As nutrigenomic products first begin to enter the marketplace, it is unlikely that large numbers of people will be willing to alter their dietary behaviors in dramatic ways or to pay high prices for rare or
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expensive nutritional supplements that are intended for long-term use for health maintenance. Fortunately, there are many widely available compounds already identified that have demonstrated activity against high leverage genes involved in human disease. Much of the currently published work involves -3 fatty acids and isoflavones.9 –12 Some of these compounds may be shown to have benefits in individuals with specific genetic variations that increase the risk for disease. It is probable that the first commercially successful nutrigenomic products will use these commonly available compounds but in a more targeted manner than at present. A Known Biomarker Exists for Testing and Monitoring Nutrient Effects Measurable biomarkers for the effects of nutritional compounds are valuable in at least two ways. First, for most nutritional agents, the “active” ingredient, or combination of ingredients, is unknown. Therefore measurement of plasma levels of the active ingredient to determine proper dose level or frequency cannot be routinely done during clinical testing. Instead, a measurable biomarker that is modulated in some way by the nutrient can be used to make these otherwise impossible observations. Second, because an individual using nutritional compounds to promote health and prevent disease does not yet have clinical signs or symptoms of disease, it is sometimes impossible to know whether the nutritional supplement is having the desired effect. To help provide the individual with the critical feedback that is often the key to long-term compliance, measurement of biomarker changes (e.g., blood pressure, cholesterol, and C-reactive protein) that reflect desirable changes in the underlying biology may be invaluable. Some Potential Negative Effects of Specific Nutrient Intake Exist in Individuals With Certain Genotypes Although many nutritional compounds may not offer particular health benefits to some subsets of people, it has generally been believed that such compounds do not do the individual any harm. However, recent research has increasingly begun to question this notion. For example, -3 fatty acid supplements may reduce resistance to infection.13 Although the evaluation of the genetic basis for such variable adverse responses to nutritional compounds is still in its infancy, it is very likely that new tools of genomics research will result in many more cases being discovered in the next few years. The differential effect of certain nutritional compounds on different individuals is one of the primary justifications for using nutritional genomics to guide the development and use of nutritional products. There undoubtedly will be numerous applications of genomics in the field of nutrition, and some of these will lead to valuable new products. The above criteria describe some of the key elements that appear to be valuable in the search for near-term products based on genomics information.
NUTRITION AND GENES MAY INTERACT IN SEVERAL WAYS Common chronic diseases are complex in their biochemical processes, and many of these diseases have strong genetic influences that explain a significant part of the variance in the clinical expression of the disease. The clinical presentation of a complex disease is the result of the tissue response to a disease initiator, as influenced by the integrated effects of genetic and environmental modifiers of gene expression (Figure 1). Thus, clinical expressions of a chronic disease among different individuals exhibit a range of
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FIG. 1. Gene ⫻ environment interaction in complex diseases. A specific tissue in a homeostatic state can be induced by a disease initiator, such as hypercholesterolemia, to an activated state (i.e., activation state 1). The set of proteins that are expressed after activation is the net result of genetic variations that influence gene expression and environmental factors that alter gene and protein expressions. The effects of the disease initiator on the tissue response are therefore influenced by the genetic and environmental modifiers that may lead to multiple activation states (e.g., states 2 and 3). The observable clinical expression is the result of the expression states that have been determined by the integration of disease initiator and various gene expression modifiers. Site A marks where the effects of a nutritional component involved in disease, such as dietary fat, may be influenced by gene variations. Site B marks where genetic variations that are involved in disease may be influenced by nutritional components.
different trajectories over time, depending on the integration of the initiating factors, genetic variations, and environmental modifiers. Besides the role of nutritional components as energy sources and enzyme cofactors, certain nutritional compounds have a substantial and direct influence on gene expression and therefore must be considered as another environmental factor that has the potential to influence disease expression when a tissue is challenged. There are several definitions of the term nutrigenomics.4,6 – 8 At the most general level, nutrigenomics refers to the use of a certain set of technologies to study which components of the genome 1) mediate the body’s response to certain nutrients (Figure 1, site A) or 2) are altered in their expression by the presence of certain nutrients. As an example of the first situation, the body’s response to dietary fat appears to be strongly determined by certain gene variations such as hepatic lipase.14 In the second situation, the biologic activity of certain gene variations that modify disease expression may be modulated by nutritional factors (Figure 1, site B). The following discussion focuses on alterations caused by nutrients, i.e., how biologic activity that is due to gene variations in certain individuals may be modified by specific nutrients to reduce chronic inflammatory responses. The use of such information to optimize the selection of nutrients for specific individuals is discussed as it relates to practical opportunities to produce a favorable impact on health.
INFLAMMATION OFFERS A PRACTICAL PLACE TO START Inflammation is the Body’s Response to an External Challenge Inflammation is the first organized reaction to any injurious challenge to the body, such as a bacterial infection. The rapid, generalized inflammatory processes that occur in response to most challenges are often referred to as innate immunity to indicate an inherent inborn biological response that requires no prior learning or experience. It is a well-coordinated process that involves the migration of blood leukocytes to tissue sites of injury or infection and the activation of leukocytes to guide an amplifying cascade of biochemical and cellular events. This response has two major dimensions. The first dimension of the host response involves a complex variety of soluble mediators that are released initially from local tissue cells at the challenge site to recruit leukocytes out of the vascular space, from the freshly recruited leukocytes, and then from distant cells, such as hepatocytes, that are activated primarily to protect the well-being of the entire system. The second dimension of the host response involves metabolic regulation to mobilize energy reserves to support the immunoinflammatory response. Thus, fever, loss of appetite, and wasting of tissues are characteristic features of severe infection and severe injury.
Nutrition Volume 20, Number 1, 2004 The initial recruitment of leukocytes to the tissue involves bacterial products that are direct chemoattractants or the release of chemical mediators from damaged cells. Once the leukocytes reach the local site, cytokines that communicate among cells orchestrate a finely tuned series of processes to eradicate pathogens, repair damaged tissues, and protect the integrity of the host. Of particular importance to common chronic diseases is the fact that the body’s response is basically the same even if the initial challenge does not involve a microbial pathogen. Some biological response mechanisms do require prior experience with the challenge and these are often called acquired immunity. The innate and acquired immune responses are directed by the cytokines, which shape the response based on the nature and magnitude of the challenge. The inflammatory response must be rapid, so it employs many cascades that amplify and broaden the response after the activation of a few immediate response genes. Because the heads of the cascades have great leverage, they must also have redundant control mechanisms with substantial feedback to fine tune the response. How Does the Body Recognize so Many Different Challenges? Macrophages and dendritic cells appear to guide much of the inflammatory response by means of highly conserved, patternrecognition receptors, such as Toll-like receptors, that recognize a limited set of ligands on pathogens, called pathogen-associated molecular patterns. Recent studies have indicated that pathogenassociated molecular patterns are found not only in microbial products, such as lipopolysaccharide, but also in non-microbial challenges, such as oxidized low-density lipoprotein cholesterol.15,16 Thus, the inflammatory mechanisms will be activated in a similar manner in response to a variety of challenges, ranging from a gram-negative bacterial infection to excess cholesterol. The system is well regulated to resolve the challenge and repair the damaged tissues, but in some situations the system may fail. These failures are not well understood but likely involve failure to eliminate the challenge, as with excess cholesterol, or subtle individual differences in the balance of regulating chemicals. Such conditions result in chronic inflammatory conditions and substantial damage to involved tissues. Some components of the innate immune response, such as interleukin (IL)–1, have been shown to be critical in protection against certain microbial infections.17 An exuberant inflammatory response may enhance the clearing of bacterial infections and thus provide an evolutionary advantage.18,19 These same innate immune mechanisms have been strongly implicated in various chronic diseases, including rheumatoid arthritis, Alzheimer’s disease, osteoporosis, periodontitis, and cardiovascular disease.19,20 Therefore, it is reasonable to speculate that an individual with an exuberant inflammatory response may have an advantage with respect to early childhood infections but may be at increased risk for a more severe course of multiple chronic diseases in mid to later life. Dysregulated inflammation has long been studied as part of the classic inflammatory diseases, such as rheumatoid arthritis, but in recent years chronic inflammation has been found to underlie several major diseases that are not routinely classified as inflammatory diseases. For example, it is now known that inflammation is a major component of the process that leads to acute heart attacks21 and Alzheimer’s disease.22 IL-1 and TNF-␣ are at Key Control Points in Inflammation The innate immuno-inflammatory mediators that are released in response to challenges to the host include many molecules, such as neuropeptides, immunoglobulins, complement proteins, plateletactivating factor, cytokines, and others. The release of these mol-
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ecules is not random and their release does not occur simultaneously. Rather, the chemical mediators of the inflammatory process are released in a sequential, ordered process that involves multiple cascades. Although all of these molecules are important in various stages of the process, some have more leverage than others on the clinical outcomes. As one might imagine, the chemicals at the head of amplifying cascades should have substantial leverage on the downstream mechanisms. Among the first genes activated with any injurious challenge are the genes for IL-1 and tumor necrosis factor-␣ (TNF-␣). The two molecules activate each other and both are critical components of the inflammatory process. Some of the most relevant properties of IL-1 biological activity are the ability to initiate cyclooxygenase type 2 and inducible nitric oxide, leading to substantial expression of prostaglandin E2 and nitric oxide by cells exposed to IL-1. Other important properties include activation of adhesion molecules and regulation of collagen and bone synthesis and breakdown. The critical role of IL-1 and TNF-␣ molecules is demonstrated by the fact that recombinant drugs that block the activity of TNF-␣ (Enbrel威 and Remicade威) or IL-1 (Kineret威) are successful in the clinical control of inflammation in many patients with rheumatoid arthritis. This following discussion focuses on genetic variations in the IL-1 genes as an example of how genetic information may be used for the development of targeted nutritional products. The discussion briefly considers the following: 1. Variations in the IL-1 gene cluster are commonly found in the population. 2. Certain patterns of IL-1 gene variations are associated with increased inflammatory mediators and increased risk for selected chronic diseases. 3. IL-1 gene expression can be modulated by nutritional compounds. 4. Nutritional compounds may have different effects on individuals with different gene variations.
Variations in the IL-1 Gene Cluster Are Commonly Found in the Population IL-1 biological activity involves two agonists, IL-1␣ and IL-1, specific receptors, and a naturally occurring antagonist, IL-1 receptor antagonist. The genes for IL-1␣, IL-1, IL-1 receptor antagonist, and six newly discovered and structurally related genes are found in a 430-kb cluster on chromosome 2.23 A high degree of sequence variation recently has been shown to exist in cytokine genes. These gene polymorphisms are relatively common in regulatory regions of the cytokine genes and therefore may be functionally significant in defining interindividual differences in transcription or post-transcriptional processes. These genetic variations therefore provide a potential mechanism by which individuals may have different degrees of response to the same stimulus. Several SNPs in the IL-1 gene cluster have been identified and evaluated for their potential biological significance.19 The distribution of some of these SNPs in white subjects is shown in Table I. In addition, there is a high degree of linkage disequilibrium across the IL-1 gene cluster, and groups of alleles are inherited together as distinct IL-1 haplotypes.24 For example, in one group of white subjects, approximately 50% of the subjects carried allele 2 at the SNP locus IL-1A (⫹4845); but if the subject carried allele 2 at IL-1B (⫹3954), the carriage rate for allele 2 at IL-1A (⫹4845) went up to approximately 90%. The functional significance observed for any specific polymorphism therefore may be the result of the net effect of variations of the other IL-1 genes in that haplotype.
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Nutrition Volume 20, Number 1, 2004 TABLE I.
DISTRIBUTION OF IL-1 GENOTYPES IN CAUCASIANS
Genotype
IL-1A (⫹4845)
IL-1B (⫹3954)
IL-1B (⫺511)
IL-1RN (⫹2018)
1.1 1.2 2.2
45.7* 45.7 8.6
55.4 39 5.6
45.9 42.9 11.2
52.2 39.0 8.8
* Percentage of 499 adults with each indicated genotype. IL, interleukin
Certain Patterns of IL-1 Gene Variations Are Associated With Increased Inflammatory Mediators and Increased Risk for Selected Chronic Diseases Some people consistently produce higher levels of IL-1 proteins than do others, and certain IL-1 genetic variations have been associated with interindividual differences in IL-1 levels and other inflammatory mediators. For example, the SNP at IL-1B (⫹3954) has been associated with different levels of IL-1 produced by peripheral blood monocytes after stimulation25 and with different serum levels of C-reactive protein (Figure 2).26 The SNP at IL-1A (⫺889) has been associated with different levels of IL-1␣ in human gingival tissue fluid27; and the SNP at IL-1A (⫹4845) codes for an altered amino acid sequence in the IL-1␣ protein. Combinations of some of these specific alleles are found together in haplotypes.24 One of the patterns of IL-1 genotypes, i.e., IL-1A (⫹4845) allele 2, IL-1A (⫺889) allele 2, and IL-1B (⫹3954) allele 2, that is associated with increased levels of inflammatory mediators is also associated with increased severity of several chronic diseases, including Alzheimer’s disease,22,28,29 periodontal disease,30 –32 and others.19 This proinflammatory IL-1 genotype pattern recently has been associated with cardiovascular acute coronary events. For example, despite having total cholesterol levels below 200 mg/dL, individuals who carried two copies of IL-1A (⫹4845) allele 2 were four times (P ⬍ 0.01) more likely to have a coronary heart disease event during an 11-y monitoring period than were individuals with the same level of cholesterol but who did not carry this IL-1 genotype (unpublished data, Interleukin Genetics, Waltham, MA, USA).
FIG. 3. -3 Fatty acid supplements reduce TNF-␣ levels more predictably in individual with specific genetic variations. Subjects (n ⫽ 111) were given -3 polyunsaturated fatty acid supplements (1.8 g/d) for 12 wk.12 Peripheral blood mononuclear cells were isolated and stimulated with lipopolysaccharide, and culture supernatants were assayed for TNF-␣ levels. The figure shows the data for the subjects who were in the top tertile of TNF-␣ production before supplementation. Subjects in the top tertile had a significant (P ⬍ 0.05) reduction in TNF-␣ levels after -3 fatty acid supplementation. Subjects in the top tertile were then genotyped for the single nucleotide polymorphism in the TNF-␣ gene (TNFA[⫺308]). Most of the supplement’s benefit in lowering TNF-␣ levels was attributable to subjects who carried allele 2 at TNFA(⫺308) (P ⬍ 0.02). TNF, tumor necrosis factor. *P ⱕ 0.05
IL-1 Gene Expression Can Be Modulated by Nutritional Compounds The activation of Toll-like receptors by cell stimulants, such as oxidized low-density lipoprotein or bacterial products, leads to the expressions of IL-1␣ and IL-1 genes by a signal transduction process that includes multiple transcription factors, including nuclear factor-. When IL-1 interacts with the IL-1 type 1 receptor on a target cell, a signal transduction process is initiated that leads to expression of genes for downstream inflammatory molecules, such as the IL-6 gene. Once again, multiple transcription factors appear to be involved. Various regulatory mechanisms that are involved in expression of the IL-1 genes or IL-1 activation of other inflammatory mediators have been shown to be modifiable by various nutrients. For example, previous studies have shown that diet supplementation with -3 fatty acids33 or tocopherols34 are capable of reducing IL-1 levels. Nutrient Effect on Inflammatory Mediators Exhibits Interindividual Variation
FIG. 2. IL-1 genotype effect on mean CRP levels as predicted by the Poisson regression model after adjustment for sex, smoking, and age (n ⫽ 504). CI, confidence interval; CRP, C-reactive protein; IL-1, interleukin-1. Reprinted with permission from Berger et al.16
Prescription drugs are optimized by means of selection and chemical modifications to have high potency in relation to a specific biological target, but we know that drugs have substantial variabilities in efficacy and toxicity when used in clinical trials. Because nutritional products are usually mixtures of compounds that are not optimized to a specific target,7 one may expect even greater variability in clinical responses than in that observed for drugs. Nutritional products are often tested clinically in a relatively small number of subjects, which greatly complicates our understanding of the variability of response and, hence, the expected effectiveness in a broader population. If we assume that nutritional compounds have even the same variabilities as those observed for many drugs, some studies will be undersized and thus will lead to a high likelihood of false-negative outcomes, i.e., the nutritional agent shows no benefit in the study, even though it may actually be effective if tested in a more appropriate population. Another complication with undersized studies is that positive results may be due to unknown stratification in the population that was inadvertently
Nutrition Volume 20, Number 1, 2004 incorporated into the study sample during selection of subjects. This often leads to great variability among studies. Some of the nature of variability in clinical responses to nutritional compounds was found with respect to the inflammatory mediator, TNF-␣.12 Peripheral blood mononuclear cells were isolated from healthy males (n ⫽ 111) and stimulated with endotoxin to evaluate the levels of TNF-␣ production. Subjects were then placed on a daily supplement of fish oil that provided 1.8 g of -3 polyunsaturated fatty acid for 12 wk. The TNF-␣ production of peripheral blood mononuclear cells was then retested. The supplement had no significant effect on TNF-␣ production when all subjects were analyzed together. However, when subjects were stratified based on initial level of TNF-␣ production, the supplement significantly increased TNF-␣ production by peripheral blood mononuclear cells from subjects who were initially in the lowest tertile of TNF-␣ production. The supplement significantly decreased TNF-␣ production in subjects who were initially in the highest tertile of TNF-␣ production. In addition, for the subjects who were high TNF-␣ producers, much of the effect of the supplement in lowering the production of TNF-␣ could be attributed to subjects who carried a specific variation in the TNF-␣ gene (Figure 3). Omega-3 polyunsaturated fatty acid supplements have been shown in animals to decrease the protective effects of the immunoinflammatory response and increase adverse effects of bacterial infection.13 This finding suggests that supplementation with relatively high levels of specific nutrients may not be uniformly desirable for all individuals.
CONCLUSION One of the goals of applying genomics and proteomics technologies to nutritional science is to match individuals and specific nutrients to achieve special health benefits. Nutritional compounds that are targeted to specific high-risk individuals may have the potential to reduce the risk for developing chronic diseases. Inflammation appears to be a practical starting point to test this nutrigenomics strategy, because it is a central mechanism for multiple chronic diseases and certain nutrients are known to alter expression of inflammatory mediators. In addition, key cytokine genes, such as the IL-1 genes, have variations that appear to influence the differential expression of inflammatory mediators and the course of certain chronic diseases. Nutritional products that specifically prevent the negative effects of proinflammatory genetic variations may represent excellent preventive agents that would benefit large segments of the population.
ACKNOWLEDGMENTS The authors gratefully acknowledge Ms. Barbara Veresh for her excellent assistance in manuscript preparation.
REFERENCES 1. Matsumoto Y, Oshida T, Obayashi I, et al. Identification of highly expressed genes in peripheral blood T cells from patients with aopic dermatitis. Int Arch Allergy Immunol 2002;129:327 2. Neumann E, Kullmann F, Judex M, et al. Identification of differentially expressed genes in rheumatoid arthritis by a combination of complementary DNA array and RNA arbitrarily primed-polymerase chain reaction. Arthritis Rheum 2002;46:52 3. Munger K. Disruption of oncogene/tumor suppressor networks during human carcinogenesis. Cancer Invest 2002;20:71 4. Mu¨ ller M, Kersten S. Nutrigenomics: goals and strategies. Nat Rev Genet 2003;4:315 5. Nakamura H. Experimental and clinical aspects of oxidative stress and redox regulation. Jpn J Clin Pathol 2003;51:109 6. Fogg-Johnson N, Kaput J. Nutrigenomics: an emerging scientific discipline. FOODTech 2003;57:60
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7. Gillies P, Schmid G, Kornman K. Nutritional genomics: a new frontier in health promotion. In: German B, Watkins S, Roberts MA, eds. Nutrigenomics: the future of integrative metabolism. London: Elsevier BV (in press) 8. van Ommen B, Stierum R. Nutrigenomics: exploiting systems biology in the nutrition and health arena. Curr Opin Biotechnol 2002;13:517 9. Baumann KH, Hessel F, Larass I, et al. Dietary omega-3, omega-6, and omega-9 unsaturated fatty acids and growth factor and cytokine gene expression in unstimulated and stimulated monocytes. A randomized volunteer study. Arterioscler Thromb Vasc Biol 1999;19:59 10. Fukushima M, Shimada K, Ohashi E, et al. Investigation of gene expressions related to cholesterol metabolism in rats fed diets enriched in n-6 or n-3 fatty acid with a cholesterol after long-term feeding using quantitative-competitive RTPCR analysis. J Nutr Sci Vitaminol 2001;47:228 11. Lamon-Fava S. Genistein activates apolipoprotein A-I gene expression in the human hepatoma cell line Hep G2. J Nutr 2000;130:2489 12. Grimble RF, Howell WM, O’Reilly G, et al. The ability of fish oil to suppress tumor necrosis factor-␣ production by peripheral blood mononuclear cells in healthy men is associated with polymorphisms in genes that influence tumor necrosis factor-␣ production. Am J Clin Nutr 2002;76:454 13. Irons R, Anderson M, Zhang M, Fritsche KL. Dietary fish oil impairs primary host resistance against listeria monocytogenes more than the immunological memory response. J Nutr 2003;133:1163 14. Ordovas JM, Corella D, Demissie S, et al. Dietary fat intake determines the effect of a common polymorphism in the hepatic lipase gene promoter on high-density lipoprotein metabolism. Circulation 2002;106:2315 15. Binder CJ, Chang MK, Shaw PX, et al. Innate and acquired immunity in atherogenesis. Nat Med 2002;8:1218 16. Binder CJ, Ho¨ rkko¨ S, Dewan A, et al. Pneumococcal vaccination decreases atherosclerotic lesion formation: molecular mimicry between Streptococcus pneumoniae and oxidized LDL. Nat Med 2003;9:736 17. O’Reilly M, Silver GM, Davis JH, Gamelli RL, Hebert JC. Interleukin 1 beta improves survival following cecal ligation and puncture. J Surg Res 1992;51:518 18. Krimbrell DA, Beutler B. The evolution and genetics of innate immunity. Nat Rev Genet 2001;2:256 19. Duff GW. Genetic variation in cytokines and relevance to inflammation and disease. In: Balkwill F, ed. The cytokine network. Frontiers in molecular biology, Vol 25. Oxford: Oxford University Press, 2000:152 20. Dinarello CA. Proinflammatory cytokines. Chest 2000;118:503 21. Libby P. Inflammation in atherosclerosis. Nature 2002;420:868 22. Mrak RE, Griffin WS. Interleukin-1, neuroinflammation, and Alzheimer’s disease. Neurobiol Aging 2001;22:903 23. Nicklin M, Barton JL, Ngyyen M, Fitzgerald M, Duff GW, Kornman KS. A sequence based map of the nine genes of the human interleukin-1 cluster. Genomics 2002;79:718 24. Cox A, Camp NJ, Nicklin MJ, de Giovine FS, Duff GW. An analysis of linkage disequilibrium in the interleukin-1 gene cluster, using a novel grouping method of multiallelic markers. Am J Hum Genet 1998;62:1180 25. Pociot F, Molvig J, Wogensen L, Worsaae H, Nerup J. A taq1 polymorphism in the human interleukin-1 beta (IL-1beta) gene correlates with IL-1 beta secretion in vitro. Eur J Clin Invest 1992;22:396 26. Berger PB, McConnell JP, Nunn M, et al. C-reactive protein levels are influenced by common IL-1 gene variations. Cytokine 2002;17:171 27. Shirodaria S, Smith J, McKay IJ, Kennett CN, Hughes FJ. Polymorphisms in the IL-1a gene are correlated with levels of interleukin-1 alpha protein in gingival crevicular fluid of teeth with severe periodontal disease. J Dent Res 2000;79:1864 28. Griffin WS, Nicoll JA, Grimaldi LM, Sheng JG, Mrak RE. The pervasiveness of interleukin-1 in Alzheimer pathogenesis: a role for specific polymorphisms in disease risk. Exp Gerontol 2000;35:481 29. Grimaldi LM, Casadei VM, Ferri C, et al. Association of early-onset Alzheimer’s disease with an interleukin-1 alpha gene polymorphism. Ann Neurol 2000;47:361 30. Kornman KS, Crane A, Wang H-Y, et al. The interleukin-1 genotype as a severity factor in adult periodontal disease. J Clin Periodontol 1997;24:72 31. McGuire MK, Nunn ME. Prognosis versus actual outcome. IV. The effectiveness of clinical parameters and IL-1 genotype in accurately predicting prognoses and tooth survival. J Periodontol 1999;70:49 32. Meisel P, Siegemund A, Grimm R, et al. The interleukin-1 polymorphism, smoking, and the risk of periodontal disease in the population-based SHIP study. J Dent Res 2003;82:189 33. Meydani SN, Endres S, Woods MM, et al. Oral (n-3) fatty acid supplementation suppresses cytokine production and lymphocyte proliferation: comparison between young and older women. J Nutr 1991;121:547 34. Devaraj S, Jialal I. Low-density lipoprotein postsecretory modification, monocytes function, and circulating adhesion molecules in type 2 diabetic patients with and without macrovascular complications: the effect of {alpha}-tocopherol supplementation. Circulation 2000;102:191
Genetic Variations and Inflammation: A Practical Nutrigenomics Opportunity Kenneth S. Kornman, DDS, PhD, Paul M. Martha, MD, and Gordon W. Duff, BM, BCh, PhD From Interleukin Genetics, Waltham, Massachusetts, USA; and the Division of Genomic Medicine, University of Sheffield, Sheffield, United Kingdom INTRODUCTION Some individuals who are overtly healthy as they reach young adulthood will begin to experience the complications of chronic diseases such as cardiovascular disease, arthritis, osteoporosis, and Alzheimer’s disease before age 60, whereas others will reach their 80s with minimal evidence of these debilitating conditions. This simple observation defines much of the future focus of the health care delivery system and the search by individuals for prolongation of healthy and productive lives. Chronic diseases have a set of initiating factors that activate specific patterns of gene expression that in turn act to tip biochemical equilibria to a non-homeostatic state. If prolonged, these non-homeostatic equilibria produce tissue degeneration and loss of function of one or more organs and ultimately produce signs and symptoms that lead to a clinical diagnosis of disease. Although still at an early stage of development, gene expression studies are beginning to show differential expression of sets of genes in tissues from chronic diseases as compared with healthy tissues.1,2 At present the cancer field provides the most definitive data to indicate that a disease only develops if there is an altered expression of a specific set of genes. This altered pattern of gene expression may be the result of acquired and inherited factors.3 The clinical presentation of chronic diseases therefore appears to be the net result of the initiating factors as modulated by the interindividual genetic makeup and environmental variations that influence gene expression in response to the disease initiators. The practical application of predictive and preventive medicine requires early identification of the individuals who are on a path toward earlier development of disease, followed by the introduction of a targeted intervention. Ideally, one would like to modulate the biology of individuals on the more rapid disease path to prevent overt complications of disease. Because the diseasecausing patterns of gene activation may be the result of various combinations of lifestyle and genetic factors, it is necessary to evaluate and manage both types of risk factors. If the diseaserelated gene expression involves specific genetic variations, one prevention option may be to use nutritional agents to modulate the biology that results from the genetic variation.
NUTRIGENOMICS: NUTRIENT–GENE INTERACTIONS Foods provide nutrients that are used for fuel in energy metabolism and growth and for the development of structural components of the body. Some nutrients are also essential cofactors for the proper function of life-critical enzymes that are involved in various aspects of metabolism and tissue integrity. In addition, many nutrients selectively alter gene expression through transcription factor systems that regulate the activation of specific sets of genes in different tissues and under different environmental conditions.
Correspondence to: Kenneth S. Kornman, DDS, PhD, Interleukin Genetics, Inc., Waltham, MA 02452, USA. E-mail: [email protected] Nutrition 20:44 – 49, 2004 ©Elsevier Inc., 2004. Printed in the United States. All rights reserved.
Various nutrients bind to or in some way directly activate specific transcription factors,4 and other nutrients alter the oxidationreduction status of the cell to indirectly influence transcription factor activity.5 The term nutrigenomics has been defined in many ways,4,6 – 8 but it generally refers to the use of various molecular tools to explore how dietary substances interact with the genome. The ultimate goal is to determine the dietary components that are most compatible with health for a specific individual. Given our current understanding of nutrient– gene interactions, a few of the practical applications of nutrigenomics are listed below. Some of the Practical Applications of Nutrigenomics 1. Identify the genes and proteins expressed differentially in health and disease that are modifiable by nutrients. 2. Identify which genes, proteins, and metabolites are influenced by specific nutrients that are known to be beneficial or harmful. a. Identify genes, proteins, and metabolites that are altered by dietary fats associated with cardiovascular disease. b. Identify genes, proteins, and metabolites that are altered by -3 fatty acids. 3. Identify genetic variations that alter the nutrient– gene interactions in applications 1 and 2.
GENETIC VARIATIONS AND NUTRIGENOMICS: CRITERIA FOR PRACTICAL USE The three general applications of nutrigenomics described above will require many years of effort, but certain specific questions seem to be more tractable than others and may provide a demonstration of the value to be derived from such efforts. This discussion addresses how nutrigenomics may be used to identify nutrients that provide health benefits to individuals with certain genetic variations that predispose them to earlier or more severe chronic disease. Although employing knowledge of specific genetic variations to target the use of nutritional compounds is very attractive at the conceptual level, a practical application of this technology requires addressing several immediate challenges. Included among these is the simple step of determining just where in the human genome to focus the initial efforts to maximize the chance of ultimate success. The number of potential interactions between nutritional compounds and the human genome is clearly enormous. However, it is almost certain that some genes will have more of an influence than others on the future course of a disease and on the effects that specific nutritional compounds have on overall health. Therefore, by applying certain criteria when selecting genes for initial study, the task of designing an efficient strategy for moving nutrigenomic research forward can be greatly simplified. The following section includes some of the criteria we believe are most important to consider early on, followed by a brief rationale for each. 0899-9007/04/$30.00 doi:10.1016/j.nut.2003.09.008
Nutrition Volume 20, Number 1, 2004 Focus on Genes That Are Chronically Activated During Clinical Disease and That Are Modifiable by Nutritional Compounds Complex chronic diseases such as heart disease, type 2 diabetes, and osteoporosis involve the interaction of a large number of genes. Among the putative disease-related genes whose expression levels are altered by interaction with common nutritional compounds, such as -3-fatty acids and isoflavones,9 –11 are those that code for cytokines, growth factors, cholesterol-metabolizing enzymes, and lipoproteins. The influence of nutrients on transcription factors that regulate gene expression has recently been reviewed.4 Due to the chronic nature of these diseases, a prolonged modulation of gene activity, such as could be achieved through regular dietary factors that directly influence gene expression, is most likely required to alter the trajectory of these diseases over time. The Genes Have Functionally Important Variations Knowledge of gene variations, such as single nucleotide polymorphisms (SNPs), in nutrient-modifiable genes may be useful in two general ways. First, a SNP may serve as a reliable marker for the presence of other genetic factors that influence the course of disease. In this case, directing a nutrient to modify activity of that particular gene may or may not be clinically beneficial because modulating the activity of the gene being targeted may not affect the true functionally important genetic factors. In other cases, the SNP itself has an important functional impact on the protein product of the gene, which in turn directly affects the disease course. Although there are several ways that a SNP may affect its ultimate protein product, SNPs that affect the quantity of protein produced or the structure of the protein itself have received the most attention to date. Because of its direct connection with the pathophysiology of the disease, administration of nutrients that affect genes with such “functionally important” SNPs are highly likely to have clinically important effects. The Genes Are at High Leverage Points in Biological Cascades Many of the human body’s most important biological systems use a powerful “cascade” of protein–protein interactions. These cascades serve to amplify the initial signals to increase the overall activity of the system. Some familiar examples of this phenomenon include the complement cascade, the blood clotting system, and the innate immune response. Genes that encode proteins acting far upstream at, or near, the head of these cascades therefore have great leverage in determining the net effect of the system. Likewise, modifying the activity of these genes has great potential to modify the clinical course of health and disease. The SNPs Are Highly Prevalent in the Population For highly targeted, expensive prescription drugs, a focus on uncommon gene SNPs that are associated with life-threatening disease may very well make sense. However, for the practical application of genomics to the development of valuable nutritional compounds, a focus on gene SNPs that are highly prevalent in the population has substantially more value in the near term from the perspective of individual health and as a commercial opportunity. Nutrients Modifying Gene Activity Are Readily Available As nutrigenomic products first begin to enter the marketplace, it is unlikely that large numbers of people will be willing to alter their dietary behaviors in dramatic ways or to pay high prices for rare or
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expensive nutritional supplements that are intended for long-term use for health maintenance. Fortunately, there are many widely available compounds already identified that have demonstrated activity against high leverage genes involved in human disease. Much of the currently published work involves -3 fatty acids and isoflavones.9 –12 Some of these compounds may be shown to have benefits in individuals with specific genetic variations that increase the risk for disease. It is probable that the first commercially successful nutrigenomic products will use these commonly available compounds but in a more targeted manner than at present. A Known Biomarker Exists for Testing and Monitoring Nutrient Effects Measurable biomarkers for the effects of nutritional compounds are valuable in at least two ways. First, for most nutritional agents, the “active” ingredient, or combination of ingredients, is unknown. Therefore measurement of plasma levels of the active ingredient to determine proper dose level or frequency cannot be routinely done during clinical testing. Instead, a measurable biomarker that is modulated in some way by the nutrient can be used to make these otherwise impossible observations. Second, because an individual using nutritional compounds to promote health and prevent disease does not yet have clinical signs or symptoms of disease, it is sometimes impossible to know whether the nutritional supplement is having the desired effect. To help provide the individual with the critical feedback that is often the key to long-term compliance, measurement of biomarker changes (e.g., blood pressure, cholesterol, and C-reactive protein) that reflect desirable changes in the underlying biology may be invaluable. Some Potential Negative Effects of Specific Nutrient Intake Exist in Individuals With Certain Genotypes Although many nutritional compounds may not offer particular health benefits to some subsets of people, it has generally been believed that such compounds do not do the individual any harm. However, recent research has increasingly begun to question this notion. For example, -3 fatty acid supplements may reduce resistance to infection.13 Although the evaluation of the genetic basis for such variable adverse responses to nutritional compounds is still in its infancy, it is very likely that new tools of genomics research will result in many more cases being discovered in the next few years. The differential effect of certain nutritional compounds on different individuals is one of the primary justifications for using nutritional genomics to guide the development and use of nutritional products. There undoubtedly will be numerous applications of genomics in the field of nutrition, and some of these will lead to valuable new products. The above criteria describe some of the key elements that appear to be valuable in the search for near-term products based on genomics information.
NUTRITION AND GENES MAY INTERACT IN SEVERAL WAYS Common chronic diseases are complex in their biochemical processes, and many of these diseases have strong genetic influences that explain a significant part of the variance in the clinical expression of the disease. The clinical presentation of a complex disease is the result of the tissue response to a disease initiator, as influenced by the integrated effects of genetic and environmental modifiers of gene expression (Figure 1). Thus, clinical expressions of a chronic disease among different individuals exhibit a range of
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FIG. 1. Gene ⫻ environment interaction in complex diseases. A specific tissue in a homeostatic state can be induced by a disease initiator, such as hypercholesterolemia, to an activated state (i.e., activation state 1). The set of proteins that are expressed after activation is the net result of genetic variations that influence gene expression and environmental factors that alter gene and protein expressions. The effects of the disease initiator on the tissue response are therefore influenced by the genetic and environmental modifiers that may lead to multiple activation states (e.g., states 2 and 3). The observable clinical expression is the result of the expression states that have been determined by the integration of disease initiator and various gene expression modifiers. Site A marks where the effects of a nutritional component involved in disease, such as dietary fat, may be influenced by gene variations. Site B marks where genetic variations that are involved in disease may be influenced by nutritional components.
different trajectories over time, depending on the integration of the initiating factors, genetic variations, and environmental modifiers. Besides the role of nutritional components as energy sources and enzyme cofactors, certain nutritional compounds have a substantial and direct influence on gene expression and therefore must be considered as another environmental factor that has the potential to influence disease expression when a tissue is challenged. There are several definitions of the term nutrigenomics.4,6 – 8 At the most general level, nutrigenomics refers to the use of a certain set of technologies to study which components of the genome 1) mediate the body’s response to certain nutrients (Figure 1, site A) or 2) are altered in their expression by the presence of certain nutrients. As an example of the first situation, the body’s response to dietary fat appears to be strongly determined by certain gene variations such as hepatic lipase.14 In the second situation, the biologic activity of certain gene variations that modify disease expression may be modulated by nutritional factors (Figure 1, site B). The following discussion focuses on alterations caused by nutrients, i.e., how biologic activity that is due to gene variations in certain individuals may be modified by specific nutrients to reduce chronic inflammatory responses. The use of such information to optimize the selection of nutrients for specific individuals is discussed as it relates to practical opportunities to produce a favorable impact on health.
INFLAMMATION OFFERS A PRACTICAL PLACE TO START Inflammation is the Body’s Response to an External Challenge Inflammation is the first organized reaction to any injurious challenge to the body, such as a bacterial infection. The rapid, generalized inflammatory processes that occur in response to most challenges are often referred to as innate immunity to indicate an inherent inborn biological response that requires no prior learning or experience. It is a well-coordinated process that involves the migration of blood leukocytes to tissue sites of injury or infection and the activation of leukocytes to guide an amplifying cascade of biochemical and cellular events. This response has two major dimensions. The first dimension of the host response involves a complex variety of soluble mediators that are released initially from local tissue cells at the challenge site to recruit leukocytes out of the vascular space, from the freshly recruited leukocytes, and then from distant cells, such as hepatocytes, that are activated primarily to protect the well-being of the entire system. The second dimension of the host response involves metabolic regulation to mobilize energy reserves to support the immunoinflammatory response. Thus, fever, loss of appetite, and wasting of tissues are characteristic features of severe infection and severe injury.
Nutrition Volume 20, Number 1, 2004 The initial recruitment of leukocytes to the tissue involves bacterial products that are direct chemoattractants or the release of chemical mediators from damaged cells. Once the leukocytes reach the local site, cytokines that communicate among cells orchestrate a finely tuned series of processes to eradicate pathogens, repair damaged tissues, and protect the integrity of the host. Of particular importance to common chronic diseases is the fact that the body’s response is basically the same even if the initial challenge does not involve a microbial pathogen. Some biological response mechanisms do require prior experience with the challenge and these are often called acquired immunity. The innate and acquired immune responses are directed by the cytokines, which shape the response based on the nature and magnitude of the challenge. The inflammatory response must be rapid, so it employs many cascades that amplify and broaden the response after the activation of a few immediate response genes. Because the heads of the cascades have great leverage, they must also have redundant control mechanisms with substantial feedback to fine tune the response. How Does the Body Recognize so Many Different Challenges? Macrophages and dendritic cells appear to guide much of the inflammatory response by means of highly conserved, patternrecognition receptors, such as Toll-like receptors, that recognize a limited set of ligands on pathogens, called pathogen-associated molecular patterns. Recent studies have indicated that pathogenassociated molecular patterns are found not only in microbial products, such as lipopolysaccharide, but also in non-microbial challenges, such as oxidized low-density lipoprotein cholesterol.15,16 Thus, the inflammatory mechanisms will be activated in a similar manner in response to a variety of challenges, ranging from a gram-negative bacterial infection to excess cholesterol. The system is well regulated to resolve the challenge and repair the damaged tissues, but in some situations the system may fail. These failures are not well understood but likely involve failure to eliminate the challenge, as with excess cholesterol, or subtle individual differences in the balance of regulating chemicals. Such conditions result in chronic inflammatory conditions and substantial damage to involved tissues. Some components of the innate immune response, such as interleukin (IL)–1, have been shown to be critical in protection against certain microbial infections.17 An exuberant inflammatory response may enhance the clearing of bacterial infections and thus provide an evolutionary advantage.18,19 These same innate immune mechanisms have been strongly implicated in various chronic diseases, including rheumatoid arthritis, Alzheimer’s disease, osteoporosis, periodontitis, and cardiovascular disease.19,20 Therefore, it is reasonable to speculate that an individual with an exuberant inflammatory response may have an advantage with respect to early childhood infections but may be at increased risk for a more severe course of multiple chronic diseases in mid to later life. Dysregulated inflammation has long been studied as part of the classic inflammatory diseases, such as rheumatoid arthritis, but in recent years chronic inflammation has been found to underlie several major diseases that are not routinely classified as inflammatory diseases. For example, it is now known that inflammation is a major component of the process that leads to acute heart attacks21 and Alzheimer’s disease.22 IL-1 and TNF-␣ are at Key Control Points in Inflammation The innate immuno-inflammatory mediators that are released in response to challenges to the host include many molecules, such as neuropeptides, immunoglobulins, complement proteins, plateletactivating factor, cytokines, and others. The release of these mol-
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ecules is not random and their release does not occur simultaneously. Rather, the chemical mediators of the inflammatory process are released in a sequential, ordered process that involves multiple cascades. Although all of these molecules are important in various stages of the process, some have more leverage than others on the clinical outcomes. As one might imagine, the chemicals at the head of amplifying cascades should have substantial leverage on the downstream mechanisms. Among the first genes activated with any injurious challenge are the genes for IL-1 and tumor necrosis factor-␣ (TNF-␣). The two molecules activate each other and both are critical components of the inflammatory process. Some of the most relevant properties of IL-1 biological activity are the ability to initiate cyclooxygenase type 2 and inducible nitric oxide, leading to substantial expression of prostaglandin E2 and nitric oxide by cells exposed to IL-1. Other important properties include activation of adhesion molecules and regulation of collagen and bone synthesis and breakdown. The critical role of IL-1 and TNF-␣ molecules is demonstrated by the fact that recombinant drugs that block the activity of TNF-␣ (Enbrel威 and Remicade威) or IL-1 (Kineret威) are successful in the clinical control of inflammation in many patients with rheumatoid arthritis. This following discussion focuses on genetic variations in the IL-1 genes as an example of how genetic information may be used for the development of targeted nutritional products. The discussion briefly considers the following: 1. Variations in the IL-1 gene cluster are commonly found in the population. 2. Certain patterns of IL-1 gene variations are associated with increased inflammatory mediators and increased risk for selected chronic diseases. 3. IL-1 gene expression can be modulated by nutritional compounds. 4. Nutritional compounds may have different effects on individuals with different gene variations.
Variations in the IL-1 Gene Cluster Are Commonly Found in the Population IL-1 biological activity involves two agonists, IL-1␣ and IL-1, specific receptors, and a naturally occurring antagonist, IL-1 receptor antagonist. The genes for IL-1␣, IL-1, IL-1 receptor antagonist, and six newly discovered and structurally related genes are found in a 430-kb cluster on chromosome 2.23 A high degree of sequence variation recently has been shown to exist in cytokine genes. These gene polymorphisms are relatively common in regulatory regions of the cytokine genes and therefore may be functionally significant in defining interindividual differences in transcription or post-transcriptional processes. These genetic variations therefore provide a potential mechanism by which individuals may have different degrees of response to the same stimulus. Several SNPs in the IL-1 gene cluster have been identified and evaluated for their potential biological significance.19 The distribution of some of these SNPs in white subjects is shown in Table I. In addition, there is a high degree of linkage disequilibrium across the IL-1 gene cluster, and groups of alleles are inherited together as distinct IL-1 haplotypes.24 For example, in one group of white subjects, approximately 50% of the subjects carried allele 2 at the SNP locus IL-1A (⫹4845); but if the subject carried allele 2 at IL-1B (⫹3954), the carriage rate for allele 2 at IL-1A (⫹4845) went up to approximately 90%. The functional significance observed for any specific polymorphism therefore may be the result of the net effect of variations of the other IL-1 genes in that haplotype.
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Nutrition Volume 20, Number 1, 2004 TABLE I.
DISTRIBUTION OF IL-1 GENOTYPES IN CAUCASIANS
Genotype
IL-1A (⫹4845)
IL-1B (⫹3954)
IL-1B (⫺511)
IL-1RN (⫹2018)
1.1 1.2 2.2
45.7* 45.7 8.6
55.4 39 5.6
45.9 42.9 11.2
52.2 39.0 8.8
* Percentage of 499 adults with each indicated genotype. IL, interleukin
Certain Patterns of IL-1 Gene Variations Are Associated With Increased Inflammatory Mediators and Increased Risk for Selected Chronic Diseases Some people consistently produce higher levels of IL-1 proteins than do others, and certain IL-1 genetic variations have been associated with interindividual differences in IL-1 levels and other inflammatory mediators. For example, the SNP at IL-1B (⫹3954) has been associated with different levels of IL-1 produced by peripheral blood monocytes after stimulation25 and with different serum levels of C-reactive protein (Figure 2).26 The SNP at IL-1A (⫺889) has been associated with different levels of IL-1␣ in human gingival tissue fluid27; and the SNP at IL-1A (⫹4845) codes for an altered amino acid sequence in the IL-1␣ protein. Combinations of some of these specific alleles are found together in haplotypes.24 One of the patterns of IL-1 genotypes, i.e., IL-1A (⫹4845) allele 2, IL-1A (⫺889) allele 2, and IL-1B (⫹3954) allele 2, that is associated with increased levels of inflammatory mediators is also associated with increased severity of several chronic diseases, including Alzheimer’s disease,22,28,29 periodontal disease,30 –32 and others.19 This proinflammatory IL-1 genotype pattern recently has been associated with cardiovascular acute coronary events. For example, despite having total cholesterol levels below 200 mg/dL, individuals who carried two copies of IL-1A (⫹4845) allele 2 were four times (P ⬍ 0.01) more likely to have a coronary heart disease event during an 11-y monitoring period than were individuals with the same level of cholesterol but who did not carry this IL-1 genotype (unpublished data, Interleukin Genetics, Waltham, MA, USA).
FIG. 3. -3 Fatty acid supplements reduce TNF-␣ levels more predictably in individual with specific genetic variations. Subjects (n ⫽ 111) were given -3 polyunsaturated fatty acid supplements (1.8 g/d) for 12 wk.12 Peripheral blood mononuclear cells were isolated and stimulated with lipopolysaccharide, and culture supernatants were assayed for TNF-␣ levels. The figure shows the data for the subjects who were in the top tertile of TNF-␣ production before supplementation. Subjects in the top tertile had a significant (P ⬍ 0.05) reduction in TNF-␣ levels after -3 fatty acid supplementation. Subjects in the top tertile were then genotyped for the single nucleotide polymorphism in the TNF-␣ gene (TNFA[⫺308]). Most of the supplement’s benefit in lowering TNF-␣ levels was attributable to subjects who carried allele 2 at TNFA(⫺308) (P ⬍ 0.02). TNF, tumor necrosis factor. *P ⱕ 0.05
IL-1 Gene Expression Can Be Modulated by Nutritional Compounds The activation of Toll-like receptors by cell stimulants, such as oxidized low-density lipoprotein or bacterial products, leads to the expressions of IL-1␣ and IL-1 genes by a signal transduction process that includes multiple transcription factors, including nuclear factor-. When IL-1 interacts with the IL-1 type 1 receptor on a target cell, a signal transduction process is initiated that leads to expression of genes for downstream inflammatory molecules, such as the IL-6 gene. Once again, multiple transcription factors appear to be involved. Various regulatory mechanisms that are involved in expression of the IL-1 genes or IL-1 activation of other inflammatory mediators have been shown to be modifiable by various nutrients. For example, previous studies have shown that diet supplementation with -3 fatty acids33 or tocopherols34 are capable of reducing IL-1 levels. Nutrient Effect on Inflammatory Mediators Exhibits Interindividual Variation
FIG. 2. IL-1 genotype effect on mean CRP levels as predicted by the Poisson regression model after adjustment for sex, smoking, and age (n ⫽ 504). CI, confidence interval; CRP, C-reactive protein; IL-1, interleukin-1. Reprinted with permission from Berger et al.16
Prescription drugs are optimized by means of selection and chemical modifications to have high potency in relation to a specific biological target, but we know that drugs have substantial variabilities in efficacy and toxicity when used in clinical trials. Because nutritional products are usually mixtures of compounds that are not optimized to a specific target,7 one may expect even greater variability in clinical responses than in that observed for drugs. Nutritional products are often tested clinically in a relatively small number of subjects, which greatly complicates our understanding of the variability of response and, hence, the expected effectiveness in a broader population. If we assume that nutritional compounds have even the same variabilities as those observed for many drugs, some studies will be undersized and thus will lead to a high likelihood of false-negative outcomes, i.e., the nutritional agent shows no benefit in the study, even though it may actually be effective if tested in a more appropriate population. Another complication with undersized studies is that positive results may be due to unknown stratification in the population that was inadvertently
Nutrition Volume 20, Number 1, 2004 incorporated into the study sample during selection of subjects. This often leads to great variability among studies. Some of the nature of variability in clinical responses to nutritional compounds was found with respect to the inflammatory mediator, TNF-␣.12 Peripheral blood mononuclear cells were isolated from healthy males (n ⫽ 111) and stimulated with endotoxin to evaluate the levels of TNF-␣ production. Subjects were then placed on a daily supplement of fish oil that provided 1.8 g of -3 polyunsaturated fatty acid for 12 wk. The TNF-␣ production of peripheral blood mononuclear cells was then retested. The supplement had no significant effect on TNF-␣ production when all subjects were analyzed together. However, when subjects were stratified based on initial level of TNF-␣ production, the supplement significantly increased TNF-␣ production by peripheral blood mononuclear cells from subjects who were initially in the lowest tertile of TNF-␣ production. The supplement significantly decreased TNF-␣ production in subjects who were initially in the highest tertile of TNF-␣ production. In addition, for the subjects who were high TNF-␣ producers, much of the effect of the supplement in lowering the production of TNF-␣ could be attributed to subjects who carried a specific variation in the TNF-␣ gene (Figure 3). Omega-3 polyunsaturated fatty acid supplements have been shown in animals to decrease the protective effects of the immunoinflammatory response and increase adverse effects of bacterial infection.13 This finding suggests that supplementation with relatively high levels of specific nutrients may not be uniformly desirable for all individuals.
CONCLUSION One of the goals of applying genomics and proteomics technologies to nutritional science is to match individuals and specific nutrients to achieve special health benefits. Nutritional compounds that are targeted to specific high-risk individuals may have the potential to reduce the risk for developing chronic diseases. Inflammation appears to be a practical starting point to test this nutrigenomics strategy, because it is a central mechanism for multiple chronic diseases and certain nutrients are known to alter expression of inflammatory mediators. In addition, key cytokine genes, such as the IL-1 genes, have variations that appear to influence the differential expression of inflammatory mediators and the course of certain chronic diseases. Nutritional products that specifically prevent the negative effects of proinflammatory genetic variations may represent excellent preventive agents that would benefit large segments of the population.
ACKNOWLEDGMENTS The authors gratefully acknowledge Ms. Barbara Veresh for her excellent assistance in manuscript preparation.
REFERENCES 1. Matsumoto Y, Oshida T, Obayashi I, et al. Identification of highly expressed genes in peripheral blood T cells from patients with aopic dermatitis. Int Arch Allergy Immunol 2002;129:327 2. Neumann E, Kullmann F, Judex M, et al. Identification of differentially expressed genes in rheumatoid arthritis by a combination of complementary DNA array and RNA arbitrarily primed-polymerase chain reaction. Arthritis Rheum 2002;46:52 3. Munger K. Disruption of oncogene/tumor suppressor networks during human carcinogenesis. Cancer Invest 2002;20:71 4. Mu¨ ller M, Kersten S. Nutrigenomics: goals and strategies. Nat Rev Genet 2003;4:315 5. Nakamura H. Experimental and clinical aspects of oxidative stress and redox regulation. Jpn J Clin Pathol 2003;51:109 6. Fogg-Johnson N, Kaput J. Nutrigenomics: an emerging scientific discipline. FOODTech 2003;57:60
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7. Gillies P, Schmid G, Kornman K. Nutritional genomics: a new frontier in health promotion. In: German B, Watkins S, Roberts MA, eds. Nutrigenomics: the future of integrative metabolism. London: Elsevier BV (in press) 8. van Ommen B, Stierum R. Nutrigenomics: exploiting systems biology in the nutrition and health arena. Curr Opin Biotechnol 2002;13:517 9. Baumann KH, Hessel F, Larass I, et al. Dietary omega-3, omega-6, and omega-9 unsaturated fatty acids and growth factor and cytokine gene expression in unstimulated and stimulated monocytes. A randomized volunteer study. Arterioscler Thromb Vasc Biol 1999;19:59 10. Fukushima M, Shimada K, Ohashi E, et al. Investigation of gene expressions related to cholesterol metabolism in rats fed diets enriched in n-6 or n-3 fatty acid with a cholesterol after long-term feeding using quantitative-competitive RTPCR analysis. J Nutr Sci Vitaminol 2001;47:228 11. Lamon-Fava S. Genistein activates apolipoprotein A-I gene expression in the human hepatoma cell line Hep G2. J Nutr 2000;130:2489 12. Grimble RF, Howell WM, O’Reilly G, et al. The ability of fish oil to suppress tumor necrosis factor-␣ production by peripheral blood mononuclear cells in healthy men is associated with polymorphisms in genes that influence tumor necrosis factor-␣ production. Am J Clin Nutr 2002;76:454 13. Irons R, Anderson M, Zhang M, Fritsche KL. Dietary fish oil impairs primary host resistance against listeria monocytogenes more than the immunological memory response. J Nutr 2003;133:1163 14. Ordovas JM, Corella D, Demissie S, et al. Dietary fat intake determines the effect of a common polymorphism in the hepatic lipase gene promoter on high-density lipoprotein metabolism. Circulation 2002;106:2315 15. Binder CJ, Chang MK, Shaw PX, et al. Innate and acquired immunity in atherogenesis. Nat Med 2002;8:1218 16. Binder CJ, Ho¨ rkko¨ S, Dewan A, et al. Pneumococcal vaccination decreases atherosclerotic lesion formation: molecular mimicry between Streptococcus pneumoniae and oxidized LDL. Nat Med 2003;9:736 17. O’Reilly M, Silver GM, Davis JH, Gamelli RL, Hebert JC. Interleukin 1 beta improves survival following cecal ligation and puncture. J Surg Res 1992;51:518 18. Krimbrell DA, Beutler B. The evolution and genetics of innate immunity. Nat Rev Genet 2001;2:256 19. Duff GW. Genetic variation in cytokines and relevance to inflammation and disease. In: Balkwill F, ed. The cytokine network. Frontiers in molecular biology, Vol 25. Oxford: Oxford University Press, 2000:152 20. Dinarello CA. Proinflammatory cytokines. Chest 2000;118:503 21. Libby P. Inflammation in atherosclerosis. Nature 2002;420:868 22. Mrak RE, Griffin WS. Interleukin-1, neuroinflammation, and Alzheimer’s disease. Neurobiol Aging 2001;22:903 23. Nicklin M, Barton JL, Ngyyen M, Fitzgerald M, Duff GW, Kornman KS. A sequence based map of the nine genes of the human interleukin-1 cluster. Genomics 2002;79:718 24. Cox A, Camp NJ, Nicklin MJ, de Giovine FS, Duff GW. An analysis of linkage disequilibrium in the interleukin-1 gene cluster, using a novel grouping method of multiallelic markers. Am J Hum Genet 1998;62:1180 25. Pociot F, Molvig J, Wogensen L, Worsaae H, Nerup J. A taq1 polymorphism in the human interleukin-1 beta (IL-1beta) gene correlates with IL-1 beta secretion in vitro. Eur J Clin Invest 1992;22:396 26. Berger PB, McConnell JP, Nunn M, et al. C-reactive protein levels are influenced by common IL-1 gene variations. Cytokine 2002;17:171 27. Shirodaria S, Smith J, McKay IJ, Kennett CN, Hughes FJ. Polymorphisms in the IL-1a gene are correlated with levels of interleukin-1 alpha protein in gingival crevicular fluid of teeth with severe periodontal disease. J Dent Res 2000;79:1864 28. Griffin WS, Nicoll JA, Grimaldi LM, Sheng JG, Mrak RE. The pervasiveness of interleukin-1 in Alzheimer pathogenesis: a role for specific polymorphisms in disease risk. Exp Gerontol 2000;35:481 29. Grimaldi LM, Casadei VM, Ferri C, et al. Association of early-onset Alzheimer’s disease with an interleukin-1 alpha gene polymorphism. Ann Neurol 2000;47:361 30. Kornman KS, Crane A, Wang H-Y, et al. The interleukin-1 genotype as a severity factor in adult periodontal disease. J Clin Periodontol 1997;24:72 31. McGuire MK, Nunn ME. Prognosis versus actual outcome. IV. The effectiveness of clinical parameters and IL-1 genotype in accurately predicting prognoses and tooth survival. J Periodontol 1999;70:49 32. Meisel P, Siegemund A, Grimm R, et al. The interleukin-1 polymorphism, smoking, and the risk of periodontal disease in the population-based SHIP study. J Dent Res 2003;82:189 33. Meydani SN, Endres S, Woods MM, et al. Oral (n-3) fatty acid supplementation suppresses cytokine production and lymphocyte proliferation: comparison between young and older women. J Nutr 1991;121:547 34. Devaraj S, Jialal I. Low-density lipoprotein postsecretory modification, monocytes function, and circulating adhesion molecules in type 2 diabetic patients with and without macrovascular complications: the effect of {alpha}-tocopherol supplementation. Circulation 2000;102:191