An Immunologist's Perspective on Nutrition, Immunity, and Infectious

M. H. Kogut *2 and K. Klasing †

* Southern Plains Agricultural Research Center, USDA-ARS, College Station, TX 77845; and † Department of Avian Sciences, University of California, Davis 95616

Primary Audience: Poultry Nutritionists, Immunologists, Veterinarians, Researchers

SUMMARY The immune system is a multifaceted arrangement of membranes (skin, epithelial, and mucus), cells, and molecules whose function is to eradicate invading pathogens or cancer cells from a host. Working together, the various components of the immune system perform a balancing act of being lethal enough to kill pathogens or cancer cells yet specific so as not to cause extensive damage to “self” tissues of the host. A functional immune system is a requirement of a healthy life in modern animal production. yet infectious diseases still represent a serious drain on the economics (reduced production, cost of therapeutics, and vaccines) and welfare of animal agriculture. The interaction involving nutrition and immunity and how the host deals with infectious agents is a strategic deter-

minant in animal health. Almost all nutrients in the diet play a fundamental role in sustaining an optimal immune response, such that deficient and excessive intakes can have negative consequences on immune status and susceptibility to a variety of pathogens. Dietary components can regulate physiological functions of the body; interacting with the immune response is one of the most important functions of nutrients. The pertinent question to be asked and answered in the current era of poultry production is whether the level of nutrients that maximizes production in commercial diets is sufficient to maintain competence of immune status and disease resistance. This question, and how to answer it, is the basis of this overview. Clearly, a better understanding of the interactions between the immune signaling pathways and productivity signaling could provide the basis for the formulation of diets that optimize disease resistance. By understanding the mechanisms of nutritional effects on the immune system, we can study the specific interactions that occur between diet and infections. This mechanism-based framework allows for experiments to be interpreted based on immune function during an infection. Thus, these experiments would provide a “real world” assessment of nutritional modulation of immune protection separating immune changes that have little impact on resistance from those that are truly important. Therefore, a coordinated account of the temporal changes in metabolism and associated gene expression and production of downstream immune molecules during an immune response and how nutrition changes these responses should be the focus of future studies. These studies could be answered using new “-eomics” technologies to describe both the local immune environments and the host-pathogen interface. Key words: nutrition, avian immunity , infectious disease, nutrigenomics 2009 J. Appl. Poult. Res. 18 :103–110 doi: 10.3382/japr.2008-00080

1 Papers from the Informal Nutrition Symposium, “Modulating Immunity: The Role of Nutrition, Disease, Genetics, and Epi- genetics,” were presented at the Poultry Science Association 97th Annual Meeting in Niagara Falls, Ontario, Canada, on July 20, 2008. 2 Corresponding author: [email protected] 104 JAPR: Symposium

DESCRIPTION OF PROBLEM maintain competence of immune status and dis- This paper is not a review of nutritional im- ease resistance. This question and how to answer pact on the avian immune system. There are it will be the basis of this overview. We make several recent reviews that do a much more no attempt to provide an ultimate answer to the thorough job than can be approached here [1–5]. question, but instead provide an outline for fu- However, we will use Klasing’s unique perspec- ture studies that should provide the foundation tive on this topic as presented at the Gordon Me- for the answer to the question. Furthermore, we morial Lecture on “Nutrition and the Immune should all keep in mind that in discussing the System” to the World Poultry Science Associa- interaction of nutrition (diet) on infection and tion (United Kingdom branch) as the basis for immunity, there is a need to consider the agenda this overview of the topic [3]. of the bird, determined by its genotype through The US poultry industry is the most produc- metabolic and physiologic mechanisms, in de- tive in the world, with 15 million metric tons veloping and maintaining an optimal immune of broiler meat produced in 2004. The industry system during an infection. is fully integrated and supports the intensive production of 8 billion birds annually. The pro- OVERVIEW OF AVIAN duction of a large volume of meat that meets IMMUNE RESPONSE consumer demands requires birds to be grown intensively at high population densities. In part, The immune system is a multifaceted ar- because of these intensive rearing conditions, rangement of membranes (skin, epithelial, mu- infectious diseases remain a threat to the poultry cus), cells, and molecules whose function is to industry. Infectious diseases represent a serious purge a host of invading pathogens or cancer drain on the economics (reduced production, cells. Working together, the various components cost of therapeutics and vaccines) and welfare of the immune system perform a careful balance of animal agriculture. The outcome of a host- of being lethal enough to kill pathogens or can- pathogen encounter is determined by virulence cer cells yet specific so as not cause extensive factors of the pathogen and defense factors of damage to healthy “self” tissues of the host. A the host. functional immune system is a requirement of a From an immunologist’s point of view, the “healthy” life in modern animal production. amount of information on poultry nutrition is The foremost function of an immune re- vast when compared with the field of avian im- sponse is to identify and eliminate infection. The munology, which is essentially in its infancy. immune system of vertebrates is made up of 2 For example, many required nutrients have been functional elements, the innate and the acquired, identified, the concentrations of these essential which contrast in their mechanisms of pathogen nutrients in feedstuffs and their availability to recognition [6]. The innate system uses germ- the bird are known, and minimal concentrations line encoded receptors, known as pattern rec- of many nutrients required for maximum pro- ognition receptors (PRR), that recognize the duction have all been defined. However, when evolutionarily conserved molecular components this information is applied to the interactions of {i.e., pathogen-associated molecular patterns nutrition (diet) and immune status, the outcome (PAMP) of infectious microbes [7–10]}. The is less definitive. To be sure, it has been shown acquired response uses highly specific antigen that almost all nutrients in the diet play a funda- receptors on T and B lymphocytes that are gen- mental role in sustaining an optimal immune re- erated by random processes by gene rearrange- sponse, such that deficient and excessive intakes ment [7, 11]. Therefore, the antigen receptors of can have negative consequences on immune sta- the acquired immune system can be produced tus and susceptibility to a variety of pathogens. for any given antigen. However, based on the current literature, the pertinent question to be asked and answered in Innate Immune Response the current era of poultry production is whether the concentration of nutrients that maximizes The host immune response to pathogens in production in commercial diets is sufficient to the earliest stages of infection is a critical deter- Kogut and Klasing: informal nutrition symposium 105 minant of disease resistance and susceptibility. cytokines, antiapoptotic factors, and antimicro- These early responses are dedicated to the con- bial peptides [12–14]. Although a certain degree tainment of the pathogens, holding infections of redundancy exists between signals induced to a level that can be resolved by the ensuing by various PRR, in general, no single PRR is development of acquired immune mechanisms. likely to be the sole mediator of activation of the The innate immune system is a rapidly induced, innate immune response. Therefore, a variety of phylogenetically conserved response of all mul- pathogens, each containing different PAMP, can ticellular organisms [6] that depends on PRR interact with a certain combination of PRR on recognition of PAMP on or in major groups of or in a host cell. The variety of PRR complexes microbes. The PAMP are critical for pathogen triggers specific intracellular signal transduction replication, survival, or both and are unique to pathways that will induce specific gene expres- large groups of microorganisms and not host sion profiles, particularly cytokine/chemokine cells, thus providing the host with an efficient, expression, best suited for the invasive pathogen nonself means of detecting invading pathogens. [15–20]. Furthermore, the induction of cytokine Several well-known examples described are li- mRNA transcripts is regulated by molecular popolysaccharides of all gram-negative bacte- bridges known as transcription factors. The ac- ria, lipoteichoic acids of gram-positive bacteria, tivation of transcription factors, such as NF-κB, double-stranded RNA of RNA viruses, mannans activation protein-1, and interferon regulatory of yeast cell walls, unmethylated CpG motifs of factors 3, 5, and 7, are required step(s) in intra- bacterial DNA and not eukaryotic DNA, lipo- cellular signaling that result in changes in gene proteins and peptidoglycans of all bacteria, and expression. the glycolipids of mycobacteria [6–8]. Over the last few years, it has become read- Recognition of PAMP induces various ex- ily apparent that an essential function of the intracellular activation cascades and intracellular nate immune system is to instruct the lympho- signaling pathways, leading to the inflammatory cytes to develop a particular effector response response, recruitment of phagocytic cells for to a specific pathogen [6, 11]. Because PAMP clearance of the pathogens, and mobilization of are produced exclusively by microbes and not professional antigen-presenting cells. The PRR by the host, recognition by host PRR signals the are present in 2 separate compartments of the presence of pathogens. The PAMP recognition host: cell membranes and cell cytoplasm. The activates effector mechanisms of innate host PRR on cell membranes have assorted functional defenses, including phagocytosis, the synthe- activities, including promotion of phagocytosis, sis of antimicrobial peptides, and the induction presentation of PAMP to other PRR, and the ini- of respiratory burst and nitric oxide synthase. tiation of major intracellular signaling pathways. Furthermore, PAMP induce the expression and Cytoplasmic PRR detect intracellular viruses production of proinflammatory cytokines and and bacteria, resulting in the production of type I chemokines. These endogenous signals orches- interferons and activating inflammasomes. Both trate the recruitment of leukocytes to the site families of PRR are essential to impart global of infection and regulate the activation of the antimicrobial responses to the host. Each family suitable effector mechanisms by controlling dif- of PRR is differentially expressed to optimize ferentiation of T lymphocytes into effector cells their ability to respond to a variety of pathogens of a particular type. Last, and most important, activating specific intracellular signaling path- recognition of PAMP by PRR induces the ex- ways that lead to the release of cytokine profiles pression of costimulatory molecules by antigen- specific for particular PAMP. presenting cells (i.e., natural adjuvant activity) Recognition of PAMP by PRR, either alone and leads to the development of highly antigen- or in heterodimerization with other PRR [Toll- specific memory cells [6, 8, 21]. The ability of like receptors (TLR), nucleotide-binding oli- the lymphocytes to differentiate into effector gomerization domain proteins, retinoic acid- cells is dependent on, and controlled by, sig- inducible gene-I, and C-type lectins], induces nals produced by the cells of the innate system intracellular signals responsible for the activa- [6, 22]. Thus, it can be seen that adjuvants are tion of genes that encode for proinflammatory primarily PAMP that induce the innate immune 106 JAPR: Symposium response to produce the required costimulatory because of the somatic rearrangement of im- molecules that result in protection. munoglobulin and T-cell receptor genes to cre- In chickens, heterophils, the avian polymor- ate clones of lymphocytes that express distinct phonuclear leukocyte equivalent to the mam- antigen receptors. The receptors on lymphocytes malian neutrophil, and macrophages ingest and are generated by somatic mechanisms during the kill a variety of microbial pathogens. Function- ontogeny of each individual and thus generate a ally, both cell types exhibit an assortment of cy- diverse repertoire of antigen receptors with ran- toskeletal and biochemical activities that include dom specificities on the lymphocytes. After the adhesion, chemotaxis, phagocytosis, and the elimination of an infection, the antigen-specific microbicidal activities of degranulation, respi- clones remain expanded as memory lympho- ratory burst, or both that can be easily assayed cytes that provide a more rapid response to sec- in vitro [23–25]. We have been investigating ond exposure to the same antigen. Recognition whether the avian heterophils and macrophages of an antigen by T or B lymphocytes requires have a more direct contribution to host defenses the antigen to be presented bound to cell surface than their traditional role as professional phago- proteins called major histocompatibility com- cytes. We have shown that types of phagocytes plex class I or II and costimulatory molecules can be induced to produce proteins that function [38, 39]. It is important to note that the ability of to regulate inflammatory and immune responses the acquired response to differentiate self from including TLR, nitric oxide, and cationic antimi- nonself antigens is not absolute. crobial peptides [26, 27]. Both types of phago- Acquired immunity differs from the innate cytic cells are also capable of rapid changes in responses by possessing specificity in recogni- proinflammatory cytokine and inflammatory tion of foreign invaders (antigens) and the de- chemokine gene expression following receptor- velopment of memory (i.e., re-exposure to an mediated phagocytosis [24–26]. Because het- antigen results in a more rapid response than was erophils and macrophages represent the first elicited during the primary exposure). Acquired cell types encountering and interacting with an immunity is mediated by 2 different types of re- inflammatory or etiologic agent, the ability to sponses depending on the type of lymphocyte synthesize and release an array of cytokines pro- that primarily responds to the antigen: humoral vides evidence for their role in both the immu- (B lymphocyte) or cell-mediated (T lympho- noregulation and pathophysiology of disease. cyte) [39]. Humoral immunity is characterized There are 8 known TLR in chickens (TLR 2, by the production of antibodies by the B lym- type 1 and type 2, TLR3, 4, 5, 7, 8, 15, and 16, phocytes in response to extracellular antigen formally 1/6/10) [26–33]. We have found that recognition, whereas cell-mediated immunity is heterophils constitutively express all 8 known characterized by the recognition of infected or chicken TLR [26]. However, chicken mono- transformed host cells by T lymphocytes. Two cytes constitutively express all but TLR5 and types of phenotypically different T lymphocytes TLR8 [34], whereas macrophages constitutively perform different effector functions to control express all but TLR3, 7, and 8 [27–33]. Stimu- the host response: T cytotoxic lymphocytes lyse lating heterophils and monocytes with specific viral-infected or neoplastically transformed host TLR agonists stimulates oxidative burst, nitric cells, whereas T-helper cells activate B lympho- oxide production, and degranulation [35–38] cytes to produce antibodies, inhibit pathogen and induces upregulation of proinflammatory proliferation in host cells through the production cytokines and inflammatory chemokines [26, of effector cytokines such as interferon-γ, and 34]. direct further cell-mediated immunity through the production of interleukin-2 [38, 39]. Acquired Immunity NUTRITION-INFECTION- The acquired immune response is an induc- IMMUNITY INTERACTIONS ible response found only in vertebrates. The acquired host defenses, mediated by T (thymus- Nutrition derived) and B (bursa-derived) lymphocytes, The interaction involving nutrition and im- are infinitely adaptable to antigenic response munity and how the host deals with infectious Kogut and Klasing: informal nutrition symposium 107 agents is a strategic determinant in animal • Modulating signaling in leukocytes = health. Traditionally, the nutrition-immune- what nutrients up- or downregulate im- infection interaction has concentrated on the mune cell function? Examples: vitamin D, role of nutrients in upregulating host defenses, polyunsaturated fatty acids for eicosonoid whereas the effect of infectious agents has been production. on host nutritional requirements. Klasing [3] • Protecting against immunopathology = has suggested that to interpret the available and what nutrients can be used to prevent or future literature as well as to establish whether manage inflammation, or both? Examples: changes in diet are beneficial or detrimental, a antioxidants such as vitamins E and C for first-principles approach to experimental design repair of local oxygen radical damage by should be adopted. These principles can be cate- phagocytes. gorized as mechanisms by which diet can affect • Influencing intestinal dynamics = what nu- immunity and the response to infections. Simply trients can stabilize microflora that equals put, experiments would be designed by evaluat- optimal growth and health? Example: di- ing the effect of a diet on 1 of 5 specific immune etary fiber provides growth substrates for mechanisms. By understanding the mechanisms microflora. of nutritional effects on the immune system, we can study the specific interactions that occur between diet and infections. This mechanism- Immunity based framework allows for experiments to be Until recently, one of the main problems interpreted based on immune function during in avian immunology with application of the an infection. Thus, these experiments would first-principles approach to nutrition-immunity provide a real-world assessment of nutritional interactions is the lack of understanding and modulation of immune protection, separating characterization of the specific cellular and mo- immune changes that have little impact on resis- lecular mechanisms of the immune response, tance from those that are truly important. Thus, in particular innate immunity. However, with detailing specific mechanisms of how a diet the clarification of more cellular and molecular can affect immunity, one can then evaluate how avian innate immune pathways, from pathogen these mechanisms relate to various nutrients and recognition by specific cells to the generation pathogens. For example, knowing how a diet or of effector peptides and production of inflam- nutritional supplements induce temporal chang- matory and regulatory cytokines, the connection es in host metabolism and the associated func- between nutrition and immunity can be further tion of immune cells and expression of immune delineated. Some recent fundamental discover- response genes during different stages of an im- ies in avian immunology are outlined in Table 1. mune response could result in the formulation Thus, the ability to evaluate the effect of diet on of affordable diets that optimize disease resis- specific immune mechanisms can now be evalu- tance to a variety of pathogenic organisms. Each ated at the cellular and molecular level. We can mechanism provides an applicable hypothesis study the effect of nutritional status on the func- that can be tested, answered, and these science- tioning immune system in practical applications. based results provided to commercial interests These include the use of immune parameters to for the development of least-cost diets: assess the nutritional status of flocks, to evalu- • Substrates for cells of the immune system ate the efficacy of nutritional therapy, and to for- = what is the priority of immune cells for mulate dietary recommendations to achieve the critical nutrients vs. nonimmune cells? immune responses needed to prevent specific in- Examples: amino acids as substrates for fectious diseases at the national, regional, or lo- cytokine production by leukocytes, pro- cal level. The availability of the chicken genome tein production by liver cells. sequence provides the opportunity to resolve • Supplying critical nutrients to pathogens = questions concerning the molecular components what nutrients are important for pathogens of the avian immune system. Key developments as well as immune cell function? Exam- in molecular, genetic, and cellular biological ples: iron, biotin. techniques provide us with new approaches to 108 JAPR: Symposium

Table 1. Recent fundamental findings in avian 5. The immune system is regulated by fac- immunology that will aid in the first-principles approach tors other than simply the degree of anti- to studying the nutrition-immunity-infection interaction genic stimulation and immediate nutrient Immune response finding1 References supply, as evidenced by the fact that if a strong immune response were resource- Acquired Th1/Th2 cytokines [40, 41] cheap, then all hosts would be expected cDNA microarray [42] to respond vigorously to an infection. Innate 6. Clearly, a better understanding of the in- TLR expression [26, 29] teractions between the immune signaling Chemokine expression/production [43] pathways and productivity signaling, for Dendritic cells [44, 45] Defensins/antimicrobial peptides [46–48] example, food intake, could provide the

1 basis for the formulation of diets that op- Th1/Th2 = T helper cells; TLR = Toll-like receptors. timize disease resistance. 7. Although nutrients can modulate immune use the genome to investigate the nutritional ef- function, nutrient status and intake must fects on functional genomics (the study of how also be considered in immune response and why a given gene behaves in a certain way studies during infection. Variations in under specific conditions), pathogenomics (the nutrient uptake undoubtedly contribute study of how genes behave when pathogens to variations in immune responsiveness interact with their host) of the avian innate re- to pathogens between individual birds sponse to pathogens, and also to develop and and possibly between groups of birds. apply the specialized field of nutrigenomics (the study of the influence of nutrition on gene and protein expression) to poultry production. REFERENCES AND NOTES

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