Innate and Adaptive Immune Cells: General Introduction Iván López-Expósito As an organ specialized in food digestion and nutrient absorption, the intestinal mucosa presents a huge surface area (almost 300 m 2 in comparison with 2 m 2 in skin) to the outside milieu and is continually exposed to foreign antigens derived from dietary constituents and the large number of microbes that reside within the intestinal lumen. In order to maintain the intestinal integrity, it is crucial to possess a fully functional associated immune system able to respond appropriately to such antigens and also to generate protective immunity to potential pathogens that employ the intestine as a primary site of entry and infection. Inappropriate responses to such antigens, apart from infections, are thought to underlie several intestinal pathologies including inflammatory bowel disease as well as food allergies (Bekiaris et al. 2014 ). Cells from both the innate and adaptive immune system can be found throughout the intestinal mucosa working together cooperatively with other cells and molecules in order to maintain intestinal functionality. Innate immunity provides effective initial defense mechanisms that take place even before infection and are poised to respond rapidly to microbes. These mechanisms react only to microbes and products of injured cells, and they are specific for structures that are common to a group of related microbes, not being able to distinguish fine differences between foreign substances. On the contrary, adaptive immune responses comprise responses that are stimulated by exposure to antigens of both microbial and non microbial origin and that increase in magnitude and defensive capabilities with each successive exposure. The main characteristics of adaptive immunity are a very high specificity for distinct molecules and the ability to remember and respond more vigorously to repeated exposures to the same antigen (Abbas et al. 2007 ). When the immune response is triggered, a wide variety of cells from both the immune system and other tissues participate. These include epithelial and Iván López-Expósito Departamento de Bioactividad y Análisis de Alimentos, Instituto de Investigación en Ciencias de la Alimentación (CIAL) (CSIC-UAM), Madrid, Spain e-mail: ivan.lopez@csic.es
142 Part III endothelial cells, neutrophils, monocytes, macrophages, dendritic cells, mast cells, natural killer cells, basophils, eosinophils, B and T cells. All of them have the ability to secrete a wide array of mediators responsible in part for their inflammatory effects (Si-Tahar et al. 2009 ). Based on the number of publications dealing with the immunomodulant/anti-inflammatory properties of food-derived compounds with bioactive properties, only monocytes, macrophages, dendritic cells, PBMCs and T cells will be covered in this part. Monocytes and Macrophages Monocytes and macrophages are essential for the development of inflammation and together with neutrophils are the phagocytic cells involved in the clearance of inert particles and microbial agents. Monocytes are bone marrow-derived cells that are continuously released into the blood. When these cells are recruited by chemotactic molecules, and leave circulation, they become activated and differentiate into macrophages under the stimulus of mediators such as TGF-β or M-CSF, between others (Gordon and Martínez 2009 ). Macrophages have a number of important functions in body defenses such as (1) capture by phagocytosis and intracellular killing of microorganisms; (2) scavenging of debris potentially harmful to tissues; (3) processing and presentation of antigens for recognition by T cells, and (4) releasing cytokines and chemokines with a major role in immune responses (Davies et al. 2013 ). The major cytokines produced by macrophages are TNF-α, IL-1β, IL-6, IL-8 and IL-33, all of which are involved in local and systemic responses. Macrophages demonstrate great developmental plasticity and may differentiate into cells with different phenotypes depending on the stimuli received as well as the tissue location. IFN-γ induces classically activated M1 macrophages (CAM), whereas IL-4, IL-13 and IL-10 induce alternatively activated M2-macrophages (AAM) (Mosser and Edwards 2008 ). In the intestinal mucosa M2-macrophages are more abundant, participating in the resolution of parasite infections, tissue remodeling, immune regulation, allergy development and tumor progression by stimulating a Th2-driven immune response (Takeuchi and Akira 2011 ). Chapter 14 describes in detail the main characteristics and differentiation protocols for THP-1 and U937, two human monocytic cells lines commonly employed in the study of the anti-inflammatory properties of food bioactives. The main food components tested, together with the methods employed to evaluate their potential anti-inflammatory activity are also explained. Dendritic Cells Dendritic cells (DC) are a widely distributed group of cells specialized in antigen sampling. In fact, they also constitute the most efficient antigen presenting cells for T cell activation, hence being the linking bridge between innate and adaptive
143 immune responses. DCs are strategically positioned at body barriers and also organ entry ports, such as the splenic marginal zone, where they remain in an immature form until they encounter an antigen (Mildner and Jung 2014 ). Once the antigen has been sampled, DCs become activated and travel towards T-cell zones, either within their respective lymphoid organ of residence or towards draining lymph nodes to ensure the activation, proliferation and differentiation of naïve T cells into their corresponding effector cells. This migration depends on the expression of the chemokine receptor CCR7 (Foster et al. 1999 ). During migration, the mature dendritic cells express high surface levels of class II major histocompatibility complex (MHC) molecules with bound antigenic peptides as well as costimulatory molecules. By the time DCs reach secondary lymphoid organs, they are able to present antigens to populations of naïve and memory T cells. Chapter 17 provides a complete overview of the different DC subsets focusing on the DCs present in the intestinal mucosa as they are among the first immune cells to come into contact with food compounds in the gastrointestinal tract and thus are instrumental in shaping the immune system s response to such exposures. In the same chapter the main DC isolation techniques as well as in vitro/ex vivo culture settings that can be applied for in vitro testing of food compounds with bioactive properties are discussed. Special attention will be paid to the potential of food-derived bioactives in inhibiting DC activation due to the relevance of DCs in initiating the inflammatory processes. Human Peripheral Blood Mononuclear Cells Human peripheral blood mononuclear cells (PBMCs) include a mixture of cells composed of lymphocytes (T cells, B cells, and NK cells), monocytes, and dendritic cells obtained from human blood or buffy coats. In humans, the frequencies of these populations vary across individuals, but lymphocytes are the most abundant, constituting in the range of 70 90 %. PBMCs are typically employed in studies where immune-regulatory effects of food bioactives are to be scrutinized. Main read-out systems include proliferation measurements, evaluation of surface activation markers by flow cytometry and quantification of the cytokine profile produced after adding the food bioactive to the cell culture. Chapter 15 fully explains the principal features and isolation procedures of PBMCs from human blood. Moreover, protocols to perform proliferation assays and evaluation of the anti-inflammatory properties with compounds from food origin are also described. T Lymphocytes or T-Cells As mentioned earlier, PBMCs are an important source of lymphocytes. Of special interest are T lymphocytes or T-cells (45 70 % of PBMCs in human peripheral blood), which are produced by stem cells in the bone marrow as progenitors and
144 Part III then migrate to the thymus where they mature into T cells. After completing their maturation, T-cells enter the bloodstream and recirculate between blood and secondary lymphoid organs until they encounter their cognate antigen. After antigen presentation by DCs, along with other appropriate stimuli, the cells may proliferate and differentiate into different subsets of effector cells (Santana and Esquivel- Guadarrama 2006 ). Originally, two main types of effector T cells, called T-helper 1 ( Th1 ) and 2 ( Th2 ) cells, were distinguished by their cytokine secretion patterns. Th1 cells secrete mainly IL-2, IFNγ and TNFα, and Th2 cells secrete IL-4, IL-13 and IL-5 (Romagnani 2000 ). Recently, a new lineage of T cells characterized by their ability to secrete a proinflammatory cytokine, IL-17, and thus designated Th17 cells has been discovered. This new T cell type has been related to autoimmune diseases (Jing and Dong 2013 ). Another subset, named regulatory T cells ( Treg ), acts by inhibiting, between others T cell responses by the production of cytokines, such as IL-10 and TGF-β and/or via cell cell interactions (Jutel and Akdis 2011 ). T cell cultures are a valuable tool in food research, especially to perform studies within the food allergy field. To study effects of food bioactives on T cells, it is necessary to activate the T cells by a polyclonal activator, either a mitogen like phytohaemagglutinin or monoclonal antibodies against CD3 and CD28. In food allergy, their main applications include analysis of immunological responses towards food protein antigens to gain further insights into the mechanisms responsible for the development of oral tolerance or for the triggering of food allergies. Chapter 16 describes the main applications in food allergy research, isolation techniques, and culture conditions for PBMC-derived T cells. Furthermore, critical parameters of the model, together with the experimental read outs are discussed. References Abbas AK, Lichtman AH, Pillai S (eds) (2007) Cellular and molecular immunology. Saunders, Philadelphia Bekiaris V, Persson EK, Agace WW (2014) Intestinal dendritic cells in the regulation of mucosal immunity. Immunol Rev 260:86 101 Davies LC, Jenkins SJ, Allen PR, Taylor PR (2013) Tissue resident macrophages. Nat Immunol 14:986 995 Foster R, Schubel A, Breitfeld D, Kremmer E, Renner-Muller I, Wolf E, Lipp M (1999) CCR7 coordinates the primary immune response by establishing functional microenvironments in secondary lymphoid organs. Cell 99:23 33 Gordon S, Martínez FO (2009) Alternative activation of macrophages: mechanism and functions. Immunity 32:593 604 Jing W, Dong C (2013) IL-17 cytokines in immunity and inflammation. Emerg Microbes Infect 2:e60 Jutel M, Akdis CA (2011) T-cell subset regulation in atopy. Curr Allergy Asthma Rep 11:139 145 Mildner A, Jung S (2014) Development and function of dendritic cells subsets. Immunity 40:642 656 Mosser DM, Edwards JP (2008) Exploring the full spectrum of macrophage activation. Nat Rev Immunol 8:958 969
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