Immunity and Inflammation in Health and Disease: Emerging Roles of Nutraceuticals and Functional Foods in Immune Support

Singapore

Preface

Shampa Chatterjee, Wolfgang Jungraithmayr and Debasis Bagchi

Eugène-Ernest Hillemacher’s painting on the cover of this book shows the English physician Edward Jenner injecting a small boy with cowpox to protect him against smallpox. This concept of inoculating a person with a benign or weak form of a pathogen to induce immunity from virulent forms of a similar virus is widely regarded as the foundation of immunology, a modern scientific discipline devoted toward understanding the immune system. The word immune originates from the Latin "immunis," a combination of in (not) and munis (ready for service) and thus translates in modern medical parlance into exempt from a disease. This exemption or protection arises in part due to endogenous cells, collectively called immune cells that are capable of mounting an attack on foreign agents or pathogens (bacteria, fungus, and virus) either via mechanisms of immediate recognition or priming for recognition i.e. processes that prime immune cells for recognition of pathogens. The body’s immune system is made up of innate (i.e., inborn or immediate) and adaptive (acquired or primed) immune systems. The innate immune system is the first line of defense against pathogens as it acts immediately upon pathogen attack or injury, while the adaptive immune system is triggered on later.

The immediate recognition pathway involves cells of the innate immune system that can sense microbial danger by recognizing molecular patterns in agents associated with pathogens. Recognition is in the form of binding of these molecular patterns to receptors that exist a priori on immune cells; such binding leads to the activation of complex signaling cascades that in turn mobilize a vast repertoire of immune cells. These cells either engulf pathogenic particles or initiate the generation of mediators that drive the destruction of pathogens.

Priming for recognition on the other hand is an indirect process involving cells of the adaptive immune system. These cells need to be primed to detect to distinguish foreign from self. Priming occurs when fragments from pathogenic material are processed by a subset of cells to trigger the generation of appropriate receptors for these pathogens on adaptive immune cells. Later when these adaptive immune cells differentiate and proliferate, they possess a specific receptor for that particular fragment of the pathogenic material. Upon subsequent attack by the same pathogen, this priming for recognition pathway enables the expanded population of adaptive immune cells to recognize the pathogen and initiate an attack on it or stimulate the production of antibodies against it. This pathway forms the basis of the protection conferred by immunization or vaccination. In other words, Jenner’s cowpox inoculation induced a stable memory population of adaptive immune cells that conferred protection against subsequent smallpox infection.

Both the innate and adaptive immune systems rely on recognition of the non-self from the self antigens and on the ability to activate mediators that can recruit a larger repertoire of immune cells. These mediators are inflammatory molecules that can attract immune cells to the region of injury and infection as well as to damaged, injured or infected tissue. This causes inflammation, a typical physiological response to infections and tissue injury that drives pathogen killing as well as tissue repair processes to restore homeostasis at infected or damaged sites.

However, an exaggerated or an aberrant response by the innate and adaptive immune system can lead to chronic inflammation and autoimmune disease, respectively. Indeed, chronic inflammation gives rise to a significant number of noncommunicable diseases such as cardiovascular disease, obesity and diabetes; while autoimmune malfunction (that arise from nonrecognition of self-antigenic peptides) drives rheumatoid arthritis, asthma, systemic lupus and a host of other diseases.

It is becoming increasingly clear that the inflammation-immune response, necessary as it is for protection from infection and injury, cannot be allowed to go on an overdrive. Regulation of this response is crucial to homeostasis. However, regulation or intervention at both cellular and molecular levels in the immune-inflammation processes necessitates an understanding of the signaling pathways in the form of multiple transcription factors, negative and positive feedback loops as well as synergistic mechanisms that participate in immune overdrive and in its inhibition.

Our purpose in this book has been to highlight signaling pathways associated with specific diseases that share the inflammation-immune pathway, as well as to showcase mediators that can amplify or dampen the immune and inflammation response. In doing so, we aim to reinforce the message that regulation or modulation of immune-mediated inflammation, either by molecular switches or by nutrition-lifestyle changes, forms the basis of health and well-being.

Food consumption has undergone a massive transformation in the course of the last century. Further, modern urban, technology-driven lifestyles have altered patterns of physical activity. This high calorie intake -sedentary activity paradigm impacts metabolism and has the potential to drive a low-grade chronic inflammation. At the same time, undernutrition in the form of malnourishment is well established to drive immunosuppression and impair infection resistance.

While micronutrients in our diet are important determinants of immune status, dietary components such as long- chain omega-3 fatty acids, antioxidant vitamins, flavonoids, prebiotics and probiotics can modulate chronic inflammatory conditions. These nutrients have been reported to decrease inflammatory mediator production through effects on cell signaling and gene expression (omega-3 fatty acids, vitamin E, plant flavonoids), by quenching the production of damaging oxidants (vitamin E and other antioxidants), and by promoting gut barrier function (prebiotics and probiotics). Indeed most human studies have correlated analyses of habitual dietary intake with systemic markers of inflammation. Integrated human epidemiological studies with large cohorts will likely provide further evidence of inflammation–health/disease associations, and the ability of diet to positively modulate inflammation. This would be a crucial step in developing diets with an intention to block chronic inflammatory and immune pathologies.

The book has been thematically divided into six sections. The first three sections cover the various cellular and molecular players of the immune-initiated inflammation paradigm and include the events that lead to the onset of metabolic, aging, and autoimmune related diseases. The chapters of the fourth section deal with the ramifications of a robust and excessive inflammatory response. Section five showcases the role of dietary nutrients in playing a balancing role between host defense and immune support. The sixth section comprises chapters that envision paradigm shifts in the field, whereby new futuristic directions are discussed.

Section I

Innate and Adaptive Immune Systems: Components and Regulation

Outline

Chapter 1 Innate and Adaptive Immunity: Barriers and Receptor-Based Recognition

Chapter 2 Innate Immunity at Birth: Implications for Inflammation and Infection in Newborns

Chapter 3 Redox Signaling and the Onset of the Inflammatory Cascade

Chapter 1

Innate and Adaptive Immunity

Barriers and Receptor-Based Recognition

Priyal Patel and Shampa Chatterjee,    University of Pennsylvania School of Medicine, Philadelphia, PA, United States

Abstract

The body’s defense system is made up of innate (inborn) and adaptive (acquired) immune systems. The innate immune system is the first line of defense against pathogens and consists of physical barriers (skin, epithelium, saliva, etc.) as well as immunological barriers in the form of various immune cells (monocytes, macrophages, neutrophils, etc.) that recognize molecular patterns or motifs in pathogens through pattern recognition receptors such as Toll-like receptors, NOD-like receptor proteins, C-type lectin receptors and RIG-1like receptors. The ligation of these receptors triggers a proinflammatory signaling that orchestrates the early response to infection, and also leads to subsequent activation of cells of the adaptive immune system (Tand B lymphocytes). Cells of the adaptive immune system are activated in response to antigens expressed by specific pathogens that are carried by dendritic cells and presented to T and B lymphocytes causing their transformation into cytotoxic cells that can recognize the antigens of that specific pathogen that was ingested by the dendritic cell. The activated T and B cells also give rise to clones bearing the distinct antigen receptor.

This chapter provides an overview of innate and adaptive immunity and details the crosstalk between the cells of these two systems. The defects in signaling associated with these systems and their role in inducing several inflammatory and autoimmune diseases are also discussed.

Keywords

Innate immunity; adaptive immunity; danger signals; PAMPs; DAMPs; TLR; endothelial cells; dendritic cells; antigen presenting cells; inflammasomes

1.1 Introduction

Vertebrates have evolved an immune system to stave off pathogens in the form of bacteria, fungi, viruses, parasites, or other unwanted vicious biological invasions. The vertebrate immune system comprises a complex array of receptors and signals that can sense or recognize microbial danger and respond via signaling cascades that drive inflammation and associated processes for direct killing of pathogens (Abrahimi et al., 2016). Traditionally, classified into innate (in-born or non-specific) and adaptive (acquired or specific) immunity (Figs. 1.1–1.3), it is now becoming increasingly clear that these are not discrete responses that operate in isolation (Akira and Takeda, 2004; Akira et al., 2006; Amadi et al., 2005); rather these two immune signaling pathways are synergistic with extensive crosstalk whereby mobilization of various immune cells follows a temporal sequence of immune activity (Fig. 1.2) from non-specific attack on pathogens in general to the development of immunological memory for specific pathogens. Therefore, in recent times these immune compartments are being considered as one functional unit (Bales and Kraus, 2013). While the innate immune system responds rapidly, taking minutes to hours to recognize patterns in the invading pathogen, the adaptive system takes days to weeks to respond as it uses immunological memory for recognition of antigen fragments specific to pathogens. But once the recognition of the pathogen is in place, the adaptive immune system enables the host to recognize a subsequent attack by the same pathogen (Barnes and Karin, 1997).

Figure 1.1 The immune system has numerous components both physical and immunological. The innate immune system is a nonspecific defense mechanism that comes into play almost immediately or within minutes to hours of a pathogen’s (antigen) appearance in the body. Phagocytic cells are activated by the antigens released by the pathogens. The adaptive immune system processes the antigens first. Adaptive signaling facilitates recognition and once the antigens are recognized, the adaptive immune system creates an army of immune cells specifically designed to attack that antigen.

Figure 1.2 Cells of the innate and adaptive immune system. Monocytes, macrophages, neutrophils and dendritic cells (DC) are key effector cells of the innate immune system. The response of these cells is rapid but short-lived. Natural Killer (NK) T cells (NKT) traditionally considered part of the innate immune response are now being accepted to possess the characteristics of cells of the adaptive immune system. T and B lymphocytes and plasma cells constitute the main cellular components of adaptive immunity. T lymphocytes upon contact with antigen presenting cells (APC) differentiate into cytotoxic T cells (CTL) which then express cytokines and cytolytic enzymes that drive cell death.

Figure 1.3 The interaction between an APC and T lymphocyte is crucial to activation of T-cells. The first step is the antigen presentation on APCs. Certain unstimulated cells such as the naive endothelium does not express antigens; upon activation antigens are produced. Next, co-stimulatory molecules are produced on the APCs. These bind to their corresponding counterparts on the T cell. The antigens presented to the T cell bind to the T cell receptor. Source: Adapted From Gujar SA et al. Oncolytic virus-mediated reversal of impaired tumor antigen presentation. Front Oncol. 4, 2014, 77.

In this first chapter, we provide a snapshot of the contents of the book. We introduce the vertebrate immune system, the components of this system, the signals that activate various immune cells to trigger the killing of bacterial, fungal and viral pathogens and the networks of communication that facilitate a bridging of innate and adaptive immunity. Next, we focus on the importance of the vascular wall, specifically the endothelium that lines the vessel wall in driving immune and inflammation responses via recruitment of immune cells as well as via antigen presentation. We also discuss the pathology of inflammatory diseases that are associated with unrestrained immune response and highlight the immune-inflammation-health-disease paradigm, which requires a better understanding in order to improve immunotherapy for various diseases. Finally, we highlight the necessity to integrate the inflammation and immune response so as to reconcile the need for defense against pathogens with protection against inflammation-driven chronic diseases. The roles of regulators and modulators (ligands that block inflammation signals and/or nutrients that activate immune responses) that can optimize the inflammation-immune processes are also reviewed.

1.2 The Components of the Immune Response (External and Internal Elements)

The immune system in vertebrates is a collection of host defenses ranging from the external barrier provided by mucosal and epithelial cells to recognition of pathogens by highly specific receptors that are genetically encoded on immune cells. External barriers are characterized by defenses such as the cough reflex, enzymes in tears and skin oils, the mucosal layer that traps bacteria and small particles, the skin, and stomach acids. Pathogen recognition, on the other hand, is more complex and driven by cellular machinery that responds to pathogens by recognizing conserved motifs in pathogens, as well as a number of other indicators of cell stress or death.

The cells of the innate immune system (Fig. 1.1) are dendritic cells, monocytes, macrophages, polymorphonuclear neutrophils (PMN), granulocytes, and natural killer T cells (NKT). In addition to these sentinels, the skin, the pulmonary epithelial and gut epithelial cells also act as an interface between the tissues and the environment.

During a pathogenic attack, the innate immune system is our first line of defense. It responds rapidly via receptors that recognize patterns either on pathogens or on molecules released by them. Recognition is in the form of binding of these molecules released by pathogens (also called alarmins) to receptors on immune cells leading to the activation of signaling pathways that attack the invading microbes (Bianchi, 2007; Bishayee, 2009). The receptors of the innate immune system are germ line encoded by specific genes within the DNA while the receptors of the adaptive immune system generated in developing lymphocytes (that occurs in the bone marrow and thymus) involve different variants of the genes encoding the receptor molecules. Each lymphocyte expresses receptors for only one specific antigen; the millions of lymphocytes in the body carry receptors for millions of antigens or antigenic peptides specific for pathogens. Thus lymphocytes that encounter an antigen to which their receptor binds will be activated to proliferate and differentiate (Fig. 1.3). This population of lymphocytes will be available to recognize the pathogen upon subsequent attack (Bonifaz et al., 2002).

The components of the immune response can be divided into four major categories: inducers (bacterial products such as LPS, oxidative cell damage), receptors (TLRs, NLRs), mediators (cytokines, chemokines, eicosinoids) and effectors (immune cells that respond to inflammatory mediators). These are discussed in the next section.

1.3 The Innate Immune System (Cell Types, Signaling)

The innate immune cells recognize inducers or exogenous pathogenic agents; this recognition is facilitated by receptors that can detect molecular patterns that are expressed by pathogens but are foreign to mammalian cells. Several pattern-recognition receptors (PRR) have been identified and studied over the past few years (Bishayee, 2009). These are (1) the Toll-like receptors (TLRs), (2) NOD-like receptor proteins (NLRPs), (3) C-type lectin receptors (CLRPs), and (4) RIG-1- like receptors (RLRs).

These receptors can detect conserved ligand motifs called alarmins on pathogens or on moieties released by pathogens. Alarmins are of two types: those that are associated with pathogens are termed as pathogen-associated molecular patterns (PAMPs), and those that are released from non-pathogenic damage (sterile injury) are called damage associated molecular patterns (DAMPs). Both PAMPs and DAMPs bind to PRRs (i.e., receptors) on immune cells that lead to the capturing (endocytosis) of the recognized viral or bacterial particles into the cell followed by their lysosomal degradation. The PAMP/DAMP-PRR binding results in the recruitment of adaptor proteins and the subsequent activation of a signal transduction cascade that activates mediators or proinflammatory transcription factors including NFκB (Bradley et al., 1993). The subsequent inflammatory cascade facilitates recruitment of effectors or immune cells and pathogen removal or killing (Abrahimi et al., 2016; Bishayee, 2009). The innate immune system consists of multiple cell types (Fig. 1.2) that participate in engulfing pathogens as well as in triggering inflammatory signals that drive pathogen removal. These cells are:

1. Polymorphonuclear Neutrophils (PMN) are a category of leukocytes (white blood cells) that have granules (hence PMN are considered granulocytes) within them containing proteolytic enzymes to break down bacterial protein. PMN participate within a few minutes of microbial attack or injury and later in the debridement of the injured tissue. These cells are termed as initial responders and are followed by monocyte and macrophage recruitment into the area of infection or injury (Calder et al., 2009).

2. Monocytes are leukocytes and phagocytes (a large cell that can engulf bacteria) that are in the circulation and that upon chemokine/cytokine cues reach the area of pathogen attack or injury. Monocytes have plasticity and are environmentally regulated to differentiate into macrophages or dendritic cells (Calder, 2006; Carmeliet, 2003).

3. Macrophages are large phagocytes that are considered as prolific eaters as they engulf pathogenic particles, apoptotic (dead) cells and fragments of injured tissue. Macrophages produce large amounts of some cytokines (e.g., prostaglandin E2, interleukins or IL-6, IL-10), creating a cytokine milieu consistent with chronic low-grade inflammation (Carmeliet, 2003, 2005).

4. Natural killer cells (NK) are lymphocytes (a subclass of leukocytes) in the circulation that recognize virally-infected and neoplastic cells as non-self (via their detection of major histocompatibility complex or MHC class I self antigens) (Chassaing and Gewirtz, 2014).

5. Dendritic cells: These cells lie at the interface of innate and adaptive immunity (Fig. 1.2) and sense antigens either from self or from external pathogenic materials and ingest these. Once ingested, these antigens are degraded and processed as peptide fragments which are then displayed on the dendritic cell surface within MHC class I and II molecules. The dendritic cells are considered as professional antigen presenting cells (APC). These cells migrate to lymph nodes (and spleen) where they can be in contact with cells of the adaptive immune system specifically T-lymphocytes. When T-lymphocytes come in contact with APCs such as DCs, they bind to these cells via T-cell receptors (TCRs). The antigens presented by DCs drive an APC-T cell binding via the TCRs. Overall, DCs participate in defense by their production of immune-enhancing cytokines and the mobilization of innate lymphocytes (NK, NKT, γδT) (Chen et al., 2008; Chinen and Buckley, 2010; Cines et al., 1998; Cooke et al., 2016).

Collectively the innate immune cells play a major role in anti-pathogen defenses either by direct engulfment of bacterial or viral pathogens or by upregulation of an inflammation cascade that leads to production of oxidants that cause pathogen destruction.

1.4 Inflammation and Innate Immunity

The first phase of the immune response comprises recognition of bacterial or viral pathogens by immune cells followed by either ingesting (phagocytosis or endocytosis) them or by activating a signaling cascade that causes production of oxidants to obliterate pathogens. In the second phase, the damaged tissue in the vicinity of the infection as well as damaged extracellular matrix material, and cellular detritus are ingested and removed by the immune cells. The third phase consists of repair of the damaged cells and tissue around the infection (Davidson, 2010).

The first phase or when innate immune cells recognize infection occurs via a complex cascade where PRRs on immune cells ligate alarmins, i.e., pathogen-associated molecular patterns (PAMPs), such as microbial nucleic acids, lipoproteins, and carbohydrates, or damage-associated molecular patterns (DAMPs) released from cells injured by sterile insults (heat, oxidant-induced damage, etc.). Activated PRRs then oligomerize and assemble large multi-subunit complexes that initiate signaling cascades that trigger the release of factors, promoting further recruitment and adherence of PMN and other immune cells.

The various players in the innate immune response are:

1. Alarmins (inducers)

a. PAMPs: include microbial surface components like endotoxins (LPS), proteins, lipoproteins, surface polysaccharides, glycoproteins, bacterial cell wall components such as β-glucan and α-mannan, components of the peptidoglycan bacterial cell wall and the bacterial protein flagellin, bacterial and viral nucleic acids, motifs within bacterial DNA, dsRNA, etc. (Abrahimi et al., 2016; Bishayee 2009).

b. DAMPs are endogenous molecules induced in cells that face a sterile (by oxidants such as reactive oxygen species or ROS) attack. DAMPs may also be released during cell death. DAMPs include high mobility group box-1 (HMGB-1), a DNA binding nuclear protein that binds to a cell surface receptor called receptor for advanced glycation end products (RAGE). RAGE ligation by HMGB1 leads to activation of a downstream signaling cascade that appears to be involved in activation of the cells of the adaptive immune system (Dempsey et al., 2003). RAGE is often considered a potential link between adaptive and innate responses, as RAGE ligation may affect adaptive immune responses (DiPietro, 1995).

2. Pattern recognition receptors

a. Toll Like Receptors (TLR): the major PRRs in cells are transmembrane proteins containing leucine-rich repeats that recognize bacterial and viral PAMPs. These receptors are members of the TLR family that recognize PAMPs/DAMPs in the extracellular such as TLR1, TLR2, TLR4, TLR5, TLR6, and TLR11 or in the intracellular milieu such as in endo-lysosomes (TLR3, TLR7, TLR8, TLR9, and TLR 10). TLR induced signaling occurs via adapter proteins that possess the Toll/interleukin-1 receptor (TIR) domain. These adaptor proteins, such as myeloid differentiation primary response protein (Myd88), TIR receptor domain-containing adapter protein (TIRAP), TIR domain containing adapter-inducing interferon beta (TIRF) and TIR containing adapter-inducing interferon beta-related adapter molecule (TRAM) activate a signaling pathway that drives the production of cytokines and chemokines that have anti-viral and anti-bacterial action (Dunzendorfer et al., 2004; Edele et al., 2007) (Fig. 1.4)

b. NLRP (NOD-like Receptor Protein): Certain members (NOD1,2 and 3) of the NLPR family of cytosolic PRRs can bind to PAMPs/DAMPs and activate NF-κB activation or secretion of the pro-inflammatory cytokines IL-1β and IL-18 (NLRP1, NLRP3 and NLRC4) via a process termed as inflammasome activation. This occurs when the PAMP-NLRP binding leads to the assembly of a multimeric complex comprising NLRP, adaptor protein ASC and inactive zymogen pro-caspase-1. The inflammasome acts as a scaffold where pro-caspase-1 is cleaved to active caspase-1, protease that cleaves (activates) the precursor interleukins to cytokines (pro-IL-1β and pro-IL-18). This leads to expression of IL-1β and IL-18 which are able to induce an inflammatory form of cell death known as pyroptosis.

c. The RIG-like receptor (RLR) family of PRRs are intracellular receptors for RNA viruses and comprise three members namely RIG-I, melanoma differentiation factor-5 (MDA5), and laboratory of genetics and physiology-2 (LGP-2). Activation of the RLR pathway triggers innate antiviral responses, mainly through expression of inflammatory cytokines such as interferons (Type I IFN-α, β) limit viral replication (Edfeldt et al., 2002; Fan et al., 2003; Fearon and Locksley, 1996).

Figure 1.4 (A) Innate immune signaling via Toll-like Receptor 4. Toll-like receptors (TLRs) link the pathogen with the host cell. LPS, one of the main pathogen-associated molecular patterns (PAMPs) of pathogenic bacteria, is recognized by the host through TLRs, resulting in activation of multiple downstream cell signaling cascades. The TLR-PAMP interaction recruits specific adaptor molecules which then bind the interleukin (IL)-1 receptor associated kinase (IRAK), initiating a signaling cascade. The four adaptor proteins, including myleloid differentiation primary-response protein 88 (MyD88), TIR domain-containing adaptor-inducing interferon β (TRIF), MyD88 adapter-like/TIR domain-containing adaptor protein (Mal/TIRAP), and TRIF-related adaptor molecule (TRAM), contain TIR domains that can be recruited. (B) Adaptive immune signaling. The surface of the APC expresses antigens. A T-cell binds to MHC-antigen complexes. This causes activation of the T-cell and it releases cytokines. The CD8+ cells are activated to cytotoxic lymphocytes (CTL) that cause dissolution (cytolysis) of the bacterial/viral particles. The CD4+ cells activate T-helper (TH) cells, some of which differentiate into memory cells that respond upon subsequent attack by the same pathogen. TH cells also cause differentiation of B cells into cells that produce antibodies against the antigen of the pathogen.

1.5 Induction of Adaptive Immunity (Antigen Presentation as a Key Event)

A key event of the adaptive immune response is antigen presentation (Fig. 1.3). This occurs when a subset of immune cells called dendritic cells (DC) capture and process antigens emanating from foreign agents and particles. The processed antigens are then presented within major and minor histocompatibility complexes (MHC-class I and II) on the cell surface. DCs migrate to the lymphoid organs where they arrive in the regions populated by T-lymphocytes (often referred to as T-cells). Besides DCs, other antigen presenting cells (APC) are macrophages, thymic epithelial cells, etc. However ROS and/or chemokines such as interferon gamma produced during inflammation are reported to confer several cell types such as endothelial, epithelial, etc., the ability to express MHC-II and process and present antigens (Frantz et al., 2005).

These antigenic peptides on MHC-I and/or II are recognized by T cell receptors (TCR). The peptide-MHCII-TCR binding forms the basis of adaptive immune response. Thus, antigen presentation is a pivotal event. T cells activated by DCs differentiate and/or proliferate in the thymus and possess a specific receptor for a fragment of the antigen. Fig 1.3 summarizes the APC-T cell interaction that occurs via the antigen-receptor binding.

Antigen-bearing DCs need to be in contact with T cells for periods ranging from one to more days. Sustained triggering by the antigen causes a proliferative phenotype in T cells (Girodon et al., 1997; Greenfeder et al., 2001; Grimm et al., 2002).

1.6 Cell Types in the Adaptive Immune Response

All cells of the immune system originate in the bone marrow. These are myeloid (neutrophils, basophils, eosinpophils, macrophages, and dendritic cells) and lymphoid (B lymphocyte, T lymphocyte and Natural Killer) cells as shown in Fig. 1.2. The bone marrow myeloid progenitor (stem) cells give rise to erythrocytes, platelets, neutrophils, monocytes/macrophages and dendritic cells whereas the lymphoid progenitor (stem) cell gives rise to the NK, T and B lymphocytes. For the development of T cells, the precursor T cells must migrate to the thymus where they undergo differentiation into two distinct types of T cells, the CD4+ T helper cell and the CD8+ pre-cytotoxic T cell. Within the T-helper cells, there are two types, i.e., the TH1 cells, which help the CD8+ pre-cytotoxic cells to differentiate into cytotoxic T lymphocytes (CTL), and TH2 cells, which help B cells differentiate into plasma cells, which secrete antibodies.

B and T-lymphocytes form the basis of the adaptive response. In their inactive and immature form, these cells circulate in the peripheral blood. However, once they recognize pathogens presented by an APC, the selection, growth, and differentiation of the lymphocyte begins. TH1 cells produce IFN-γ while TH2 cells produce IL-4, IL-5, and IL-13. B and T cells are small but can be distinguished by their cell surface receptors which are known as immunoglobulins (B-cell receptors) and T-cell receptors (TCR). Lymphocyte differentiation and proliferation results in the development millions of different kinds of receptors for both B-cells and T-cells(Barnes and Karin, 1997; Gupta et al., 1998).

When T cells recognize antigens or fragments of antigens from pathogens that are presented to them, they initiate direct attack of antigen-bearing cells by a cytotoxic T lymphocyte population or by stimulation of B cells to produce antibodies against the antigens. Additionally, TH cells also produce interferon gamma and other cytokines leading to inflammation and bacterial cell kill.

1.7 Crosstalk Between the Innate and Adaptive Immune Responses

As we have mentioned earlier, the innate immune cells possess receptors that recognize patterns associated with pathogens (Fig. 1.4A). These receptors are germ line encoded and therefore exist prior to pathogen attack. This enables an immediate response to bacteria, virus or other infection and injury. However this also implies that these cells have a non-specific repertoire of sensing which allows the detection of a broad range of pathogens but does not facilitate the detection of a particular bacteria or virus. Specific pathogen detection is a function carried out by the cells of adaptive immune system namely the T- and B- lymphocytes that possess receptors that can bind to specific peptide fragments associated with a particular pathogen. These receptors are expressed because of earlier priming and immunological memory whereby APC-T cell interaction and subsequent differentiation and proliferation of a subset of T cells drives pathogen killing (Fig. 1.4B).

Straddling these two branches of the immune response are the natural killer T (NKT) cells (Fig 1.2). These cells have characteristics of both innate and adaptive systems in that they possess pattern recognition receptors as well as immunoglobulins or T-cell receptors.

The crosstalk between these two systems is bidirectional, i.e., innate immune system activates the adaptive system and vice versa. While innate immune cells such as dendritic cells activate T cells via antigen presentation, adaptive immune cells activate cells of the innate immune system via IFN-γ produced by T-helper cells that in turn activate macrophages, dendritic cell subsets.

While innate components are the first line of defense, components of adaptive immunity mobilize more slowly. This is because adaptive immunity is the form of antigen presentation, APC-T cell binding, clonal expansion; and proliferation as well as formation of cytotoxic T cells is a part of the sequence of a prolonged pathway (Fig. 1.4B). This pathway also utilizes the complement cascade and adhesion molecules, which evolved as part of innate immunity. Consequently, shared mediators unite innate and adaptive immunity function as parts of an integrated immune system.

1.8 The Vascular Endothelium as a Converging Site for Both Innate and Adaptive Immunity

As integral components of the immune system, blood vessels are important in the surveillance of self. They play key roles in lymphocyte circulation and act as portals between tissue and blood compartments.

Endothelial cells (ECs) form a single cell layer called the endothelium, which lines the vasculature and lymphatic systems forming a semi-permeable barrier between blood or lymph within vessels and the surrounding tissues. The endothelium is a highly specialized, dynamic, disseminated organ with many essential functions in physiological processes. Besides serving as a physical barrier, ECs have a wide array of functions which are characterized into three major categories: trophic, tonic, and trafficking. ECs also have important immunological functions (Hahn and Schwartz, 2009). Cells of the immune system function to defend against invasive foreign pathogens and detrimental endogenous materials.

Inflammation can be seen as a vascular response (Hansson et al., 2002), where ECs become activated, display increased leakiness, enhanced leukocyte adhesiveness, and procoagulant activity, and form new vessels (Harvey et al., 2008). Thus, an immune response resulting in inflammation depends upon the ability of the microvasculature to either recruit or prevent the indiscriminate influx of immune cells into a tissue. Compared with large blood vessels, the microvascular bed constitutes the bulk of the overall endothelial surface, covering an area ~50 times greater than that of all large vessels combined (Hawiger et al., 2001).

Due to their location, ECs are one of the first cells to interact with microbial components in the circulation. EC recognition and response may be integral to early innate immune system activation. In fact, like DCs, ECs are reported to express both TLRs and NLRs as well as express chemokine receptors (Huffman et al., 2007; Ingulli, 2010). Specifically, ECs have been shown to secrete the proinflammatory cytokine interleukin-8 (IL-8) in an NLPR1-dependent manner in response to microbial stimulation.

Innate immune signaling occurs via PAMPs and DAMPs binding to receptors (TLRs) on the host cells. In this context, it needs to be mentioned that TLR1 immunoreactivity was observed in atherosclerotic endothelium (Italiani and Boraschi, 2017). TLR2 has also been detected on atherosclerotic endothelium, expressed by ECs and markedly up-regulated in vascular inflammation (Italiani and Boraschi, 2017). ECs upon stimulation with LPS, TNF-α and IFN-γ express TLR2 mRNA and protein in a NF-κB- and MyD88-dependent manner (Jakubzick et al., 2017; Kawai and Akira, 2009). The upregulation of TLR2 in EC is via neutrophil NADPH oxidase and neutropenic mice (mice without neutrophils) have been found to show decreased endothelial TLR2 expression (Jakubzick et al., 2017). This indicates a crosstalk between polymorphonuclear neutrophils and ECs that would enhance vascular defenses by up-regulating TLR2.

The impact of ECs on adaptive immunity may be exerted through their interaction with T cells. Although ECs cannot replace T and B cells, it is becoming clear that ECs can express MHC I and II class molecules and process antigens or fragments of antigens from pathogens, and can thus under some conditions act as APC. This has been reported and ECs from different species express accessory molecules required for antigen presentation, including CD80, CD86, ICOS-L, programmed death ligand 1, programmed death ligand 2, LFA-3, CD40, and CD134L (Lloyd and Hessel, 2010). As shown in Fig. 1.3, the accepted paradigm is that professional APCs such as DC present antigens to activate the recipient’s naïve T lymphocytes. The T lymphocytes thus activated cause production of inflammatory cytokines and other enzymes that cause cytolysis, injury and onset of graft rejection (Loo and Gale, 2011; Mahabeleshwar et al., 2006). The APC-T lymphocyte interaction is thus pivotal to the onset of adaptive immune response (Majno, 1998). Apart from professional APCs, other cell types can also be stimulated to be transformed into APCs. For instance, endothelial cells activated by IFN-γ or by hydrogen peroxide show expression of antigens MHC I and II and co-stimulatory molecules (Manach et al., 2004; Marelli-Berg and Jarmin, 2004; Matzinger, 2007). These moieties are the hallmarks of an APC as they facilitate APC-T cell interaction (Fig. 1.2). It is now acknowledged that the endothelium is not just a passive target of immune response but an active player in T-lymphocyte activation (Medzhitov and Janeway, 1997). However, the mechanism by which the endothelium is transformed into an APC during organ storage has not been elucidated.

EC-immune signaling also drives the process of angiogenesis (the formation of new blood vessels from pre-existing ones) which is integral to most immune-mediated conditions. As inflammation evolves, vessels expand to supply nutrients to sustain the accumulation of activated immune cells in the affected tissues (Medzhitov, 2008; Morgan et al., 1999), thus both innate and adaptive immune responses promote angiogenesis. Additionally, immune cells such as macrophages with their tissue macrophages foster vessel growth, remodeling, or regression (Hansson et al., 2002; Murdoch et al., 1999). Their proangiogenic activity is mediated by various TLRs acting in synergy with adenosine A2A receptors that up-regulate VEGF production (O’Neill et al., 2013).

1.9 Chronic Diseases of the Innate and Adaptive Immune Systems

The major function of the immune system is to recognize the non-self antigens or antigenic peptides. However an exaggerated or an aberrant response can lead to inflammation as well as to autoimmune disease (that arises from non-recognition of self antigenic peptides). Chronic inflammatory pathologies such as atherogenesis and autoimmune disorders such as rheumatic fever, rheumatoid arthritis, ulcerative colitis, myasthenia gravis, etc., are all manifestations of an exaggerated inflammation-immune response whereby cytokines and chemokines released by stimuli such as injury, denudation of vascular layer, or hormonal imbalance, accumulation of fat cells, i.e., adipocytes and/or pathogenic attack lead to recruitment of activated immune cells to the site of lesions, thus amplifying and perpetuating the inflammatory state. Chronic or long-term inflammation drives repair and remodeling process that alter cellular and tissue function.

The remodeling of the vessel that occurs with atherosclerosis involves large numbers of macrophages and T cells at the site of the atherosclerotic plaque. Thus recruitment is driven by local topology (Parisi et al., 2017) as well as by modified lipoproteins and cholesterol, while in diseases such as dermatitis or psoriasis where a bacterium like streptococcus is involved, TLR 2 activation in response to this infection leads to a high expression of TNF-α, IL-12, IL-23, and IL 17, which in turn produces psoriatic lesions (Gupta et al., 1998). In the case of asthma, an allergen challenge provokes the influx of T helper cells (specifically activated TH2) into the airways causing an increase in the levels of TH2-type cytokines and recruitment of immune cells such as eosinophils (Piotti et al., 2014; Pober et al., 1983). Airway inflammation leads to airway hyperresponsiveness and epithelial cell proliferation linked tissue remodeling. There is also clinical evidence to suggest that respiratory viral infection is also linked to the initial development of asthma as well as exacerbations that might perpetuate the disease (Pober et al., 1983).

Low-grade inflammation also characterizes metabolic syndromes and associated disorders (obesity, diabetes, etc.). Normally PMN and macrophages that show uncontrolled activity during chronic inflammation are strongly implicated as having a causal role in cancer development (Polonsky et al., 2016; Pulendran et al., 2001).

NLRP inflammasomes also participate in driving several neurologic and metabolic diseases. Misfolded protein aggregates and aberrant accumulation of certain metabolites associated with other diseased states function as DAMPs and activate the NLRP3 inflammasome, which plays a critical role in the initiation and progress of inflammation (Pulendran, 2005; Reith et al., 2005).

1.10 Improving Immunity for Prevention and Care

Studies have shown that lack of adequate nutrition is a major cause of immune dysfunction. A study from Spain (Riquelme et al., 1997) suggests that up to 85% of elderly patients with community-acquired pneumonia are malnourished. Similarly studies have shown that complete recovery form pathogen-induced infections (even though the bacteria or protozoa were present) is possible via nutritional intervention in the form of micronutrients, aminoacids, simple lipids, etc. (Sager et al., 2017). On the other hand, excessive calorie intake stimulates adipose tissue growth and promotes abdominal obesity, which along with other factors (smoking, exposure to environmental pollutants etc.) can activate the immune pathway leading to onset of inflammation. A hallmark of most chronic immune diseases is the elevated levels of inflammatory cytokines, chemokines and inflammatory growth factors. Studies carried out in individuals following calorie restrictions (enrolled in the Calorie Restriction Society, whose members voluntarily observe calorie-controlled diets) showed that consuming lower than baseline diet calories (~1800 calories/day) over several years decreased serum levels of TNF-α, proinflammatory cytokines and lipoprotein profiles (Santangelo et al., 2007).

Studies conducted in Australia among postmenopausal women demonstrated that the risk of death from inflammatory disease (digestive, respiratory, nervous system, and endocrine disorders) was nearly three times greater among women consuming a high-GI diet (a high glycemic index diet consists of high carbohydrate and sugar intake) compared with women eating a low-GI diet (Saresella et al., 2016). Intake of trans fatty acids was also found to drive inflammation. Trans 18:2 fatty acids were observed to be integrated into human aortic endothelial at twice the rate as cis 18:2 fatty acids, causing the cells to clump together and bind to arterial walls, stimulating the release of proinflammatory cytokines (Schmidt et al., 2001).

Interventions in the form of vitamin and mineral supplements cause abrogation of certain agents that are high in individuals suffering from inflammatory diseases. A supplement of retinol, β-carotene, thiamine, riboflavin, niacin, pyridoxine, folate, iron, zinc, copper, selenium, iodine, calcium, magnesium, and vitamins B12, C, D, and E when provided to healthy adults over a 12-month-long, double-blind, randomized, placebo-controlled trial, showed an elevated immune function in the form of an increase in CD4+ T numbers and NK cell activity. Importantly, infectious illness days were reduced from a mean of 48 in the placebo group to 23 in the group that received the supplement (P=.002), and antibiotic use was lowered from an average of 32 to 18 days (P=.004) (Shanker et al., 2015). Fish oil supplements (4 g/day for a minimum of 6 weeks) in the diet resulted in significantly decreased plasma levels of TNF-α with subjects with type 2 diabetes and in reduced cellular content of proinflammatory cytokines IL-1 beta, IL-6, and IL-8 in healthy subjects (Steinman and Hemmi, 2006). In subjects with rheumatoid arthritis, fish oil intake caused symptom relief (Steinman and Hemmi, 2006).

Vitamin C intake in plasma levels correlated with reduced proinflammatory prostaglandins and C-reactive protein (CRP) in a small clinical study of healthy subjects. Plasma vitamin C levels inversely correlated with symptoms of rheumatoid arthritis. Supplementation with 1 g/day of vitamin C decreased measures of oxidative stress and improved endothelial function in a clinical study of individuals with hypertension (Tam and Jacques, 2014).

Polyphenols (found in fruits, vegetables, grains, chocolate, coffee, olive oil, and tea) in the diet show powerful anti-inflammatory effects. Laboratory investigations, clinical trials, and prospective studies suggest that polyphenols inhibit enzymes decrease proinflammatory cytokine production, and block the activity of proinflammatory signaling systems (Wolinsky, 1980; Yamazaki et al., 2006; Zhou et al., 2016).

1.11 Summary

The highly specialized mammalian immune system consists of two distinct arms, innate and adaptive immunity. The cells of the innate immune system that function via recognition of molecular patterns that are expressed by pathogens also prime the cells of the adaptive immune system. The innate immune cells respond within minutes to hours of a pathogen attack. Adaptive immune responses are slow to develop upon first exposure to a new pathogen, as specific clones of B and T cells have to become activated and have to expand. However the cells of the adaptive immune system remember previous encounters with specific pathogens so that these pathogens are easily combated in subsequent infection episodes.

In subsequent chapters, innate and adaptive immune signals and their role on regulating inflammation and the how these form the common basis of multifactorial diseases will be discussed. Our understanding of these immune mechanisms and preventative measures will allow for options beyond vaccinations and drug development.

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Chapter 2

Innate Immunity at Birth

Implications for Inflammation and Infection in Newborns

Ballambattu Vishnu Bhat and Selvaraj Manoj Kumar Kingsley,    Jawaharlal Institute of Postgraduate Medical Education and Research (JIPMER), Puducherry, India

Abstract

The development of immunity at birth is vital and it involves several complex mechanisms. Since prior antigenic experience is limited, the adaptive immunity requires time for development. The newborns depend heavily on their innate immunity for survival during early life. The microbiome with the commensal flora contributes to the development of immunity in newborns. Maternal antibodies at the various mucosal surfaces are the primary checkpoints which provide immunity in the neonatal period. The reduced chemotaxis, phagocytosis and sustained activation of neonatal neutrophils and monocytes increase the susceptibility of newborns to infection. The quantitative and qualitative characteristics of dendritic cells are subset specific and their varied ontogeny and functions impair antigen presentation and reduce the vaccine responsiveness in early life. The natural killer cell functions like degranulation and release of lytic factors are reduced in early life which makes the newborns more prone to viral infections. Dietary limitation during lactation has shown to modify the functions of immune cells and increase the susceptibility to pathogenic invasion. Dietary supplements like lactoferrrin have shown to increase the levels of immunoglobulins and improve the mucosal and systemic immune response in early life.

Keywords

Newborn; Innate immunity; Mucosal immunity; Microbiome; Dietary supplements

2.1 Introduction

The neonatal period is the most crucial phase of life and survival during this period is of paramount importance. Infections account for 36% of the four million neonatal deaths every year (Lawn et al., 2005). Immunologic immaturity is one of the major reasons for the increased susceptibility of neonates to early onset sepsis (Shane and Stoll, 2014). The diminished fetal immune system may be an adaptive mechanism to prevent premature rejection by the mother (Clapp, 2006). The key players in adaptive immunity are T-lymphocytes that express markers CD4 or CD8. T-lymphocytes expressing CD4 are also known as helper T cells, and these are the most prolific cytokine producers. This subset can be further subdivided into Th1 and Th2. The Th1-type cytokines produce proinflammatory responses while Th2-type cytokines are primarily anti-inflammatory in their effects. The newborn immune response is skewed towards anti-inflammatory Th2 response, which increases their susceptibility to a diverse range of intracellular pathogens. Toll-like receptors (TLRs) are a major family of pattern recognition receptors (PRRs) that has a vital role in the innate host defense and initiation of the adaptive immune responses. The reduced infection control in newborns is associated with the impaired TLR signaling and diminished response to bacterial cell wall components (Sadeghi et al., 2006). The newborn innate immune cells have shown several unique characteristics that sometimes exceed but most often fall short of the required optimal response. The microbiome and epigenetic influences regulate the development of immunity in early life. The ontogeny of immunity in early life involves several complex mechanisms and the naïve nature of the newborn immunity increases its susceptibility to inflammation and infections.

2.2 Microbiome in Shaping the Newborn Immune System

Microbial colonization of infant microbiota begins at birth and it depends largely on the transfer of maternal microbiota during delivery. Due to several factors like maternal antibiotic usage, invasive procedures at birth, hospital environment stress, neonates, especially very low birth weight infants, are characterized by dysbiosis (Groer et al., 2015). The maternal microbiomes including the placental microbiome influence the increased incidence of preterm births (Vinturache et al., 2016). There were significant variations in the composition of placental microbiota in low birth weight and normal weight neonates (Zheng et al., 2015). Moreover when compared to babies born though vaginal route to normal weight mothers, those born to obese mothers had their microbiota enhanced in bacteroides and reduced Acinetobacter, Enterococcus etc (Mueller et al., 2016). This implies that maternal body mass index affects the development of infant microbiome during vaginal delivery. Epigenetic changes induced by in-utero variations modulate the early microbial colonization and the gastrointestinal (GI) tract development which increases the susceptibility of newborns to infection (Cortese et al., 2016). CD71+ erythroid cells are immunosuppressive, but they also prevent exaggerated activation of immune cells in intestines where frequent colonization with commensal microbes occurs after birth (Elahi et al., 2013). The disruption of the host intestinal microbiota may increase the susceptibility of neonates to necrotizing enterocolitis (Elgin et al., 2016).

The breast milk given to newborns comprises of carbohydrates, immunoglobulins, cytokines and other immunomodulatory factors (Bertino et al., 2012). It is also a source of several microbes such as Lactobacillus sp. and Bifidobacterium sp., which promote the development of newborn intestinal microbiota (Jeurink et al., 2013). Human recombinant lactoferrrin has shown to alter the fecal microbiome in very low birth weight infants (Sherman et al., 2016). Newborn mice that are undernourished had depleted microbiota with minimal diversity and were lacking in numerous microbial genetic pathways, which resulted in repressed energy exudation from dietary components (Preidis et al., 2015). Neonatal malnutrition inhibits body weight gain and reduces NO production which elevates the viability of macrophages infected with methicillin resistant Staphylococcus aureus, and also promotes reactive oxygen species (ROS) production which may enhance systemic infection with S. aureus (de Morais et al., 2016).

Early life exposure of newborn mice to a mixture of microbial extracts improved survival significantly after infection with Streptococcus pneumoniae. Preexposure to microbial extracts improved their ability to suppress bacterial growth, inhibited pneumococcal blood invasion, and enhanced the colonization of bacteria in the airway, improving airway resistance (Yasuda et al., 2010). It was later found that intratracheal exposure to microbes improved the opsonic activity of bronchoalveolar lavage fluid, which improved phagocytosis of alveolar macrophages (AMs). The maturation of dendritic cells (DCs) (CD11c+ expressing cells) in the airway was also enhanced on microbial exposure (Kasahara et al., 2012). Probiotic bacteria could modify the intestinal microbiome in newborns to tackle allergic disease. Probiotic strains such as Bifidobacterium bifidum induced partial maturation of cord blood DCs and those matured DCs stimulated T cells to secrete IL-10 and IL-4 (Niers et al., 2007).

Pregnant mouse dams exposed to antibiotics had reduced level of microbes in the intestines. The newborn pups had reduced levels of neutrophils and granulocyte macrophage progenitor cells in bone marrow and in the circulation (Deshmukh et al., 2014). The antibiotic exposure also led to a deficit in IL-17 producing intestinal cells and secretion of granulocyte colony stimulating factor (G-CSF), which led to granulocytopenia impairing the neonatal defense to Escherichia coli and Klebsiella infections. The recruitment of normal microbiota to antibiotic treated neonatal mice restored the IL-17 generation from innate lymphoid cells (ILCs) in the intestine and elevated the number of neutrophils and granulocyte macrophage colony stimulating factor (GM-CSF) levels, through the TLR4 and MyD88 pathway (Deshmukh et al., 2014). Newborn mice born to antibiotic treated mice during pregnancy showed reduced IFN-γ producing CD8+ T cells. This showed that maternal antibiotic treatment alters the development of infant GI microbiota which weakens the antiviral immune response (Gonzalez-Perez et al., 2016).

2.3 Mucosal Immunity at Birth

There is increasing evidence that the diverse and complex microbiota in early life influences the development of mucosal immunity in newborns (Malmuthuge et al., 2015). From a relatively sterile in-utero environment, the newborns are exposed to the challenges of pathogens and microbes at birth. They are mainly protected by maternal antibodies at the mucosal surfaces (Harris et al., 2006). The recognition of pathogens is facilitated via evolutionarily conserved structures on pathogens called pathogen associated molecular patterns (PAMPs) that bind to their respective PRRs. This binding of PAMPs to their respective receptors forms a major mechanism of innate mucosal immunity.

Newborns depend on the transplacental transfer of IgG in-utero and the immunoglobulins IgA, IgG, IgM in mother’s milk. Secretory IgA at the mucosal surfaces mediates the transport of commensal bacteria across the intestinal epithelia, limits the intestinal penetration of commensal bacteria, and helps maintain the host-microbiota symbiosis (Harris et al., 2006; Rol et al., 2012). The mucosal secretions from intestinal, genital or respiratory tracts contain significant amounts of immunoglobulin G (IgG). Recently a contrasting role for IgG in the neonatal period has been found which may limit the mucosal adaptive immunity. The maternally acquired IgG and IgA act against the gut microbiota and repress the mucosal T helper cell responses in early life (Koch et al., 2016). The neonatal Fc receptor (FcRn) within the mucosal tissues has significant role in triggering local immune responses needed for antitumor immunosurveillance (Baker et al., 2013). Transgenic mice that overexpress FcRn receptor show improved production of anti-thymocyte globulin, which enhanced the humoral response in rabbits (Baranyi et al., 2013). The FcRn receptor transports the IgG from the intestinal barrier to the lumen and then redirects the IgG-antigen complex to the lamina propria across the intestinal barrier. This is followed by the antigen presentation by DCs to CD4+ T cells in lymphoid structures (Yoshida et al., 2004).

Interleukin 23 (IL-23) responsive group III ILCs are well known to play a role in adult immune homeostasis; in neonates ILCs upon activation by IL-23 in neonatal intestines reportedly produced more proinflammatory mediators like IFN-γ and GM-CSF (Chen et al., 2015). The ILCs accumulate under the intestinal epithelium and aggregate along with neutrophils and cause intestinal bleeding and death in newborns. Depletion of ILCs using antibodies reduced mortality in neonatal mice (Chen et al., 2015). Gut mucosal injury in neonatal mice is characterized by macrophage rich leukocyte infiltration and is recruited to the inflamed neonatal gut mucosa by the chemokine CXCL5 (MohanKumar et al., 2012).

A combination of multiple cell types regulates immunity and inflammation. Other prototypical members of the ILC family such as the natural killer (NK) cells, DCs as well as CD4+ and CD8+ T cells participate in neonatal immunity. The profound NK cell percentage in intestinal epithelial compartment (IEC) during early life reduced other intestinal epithelial cell subpopulations such as CD3+ cells. After weaning, the NK cell percentage declined and the CD4+CD8+ regulatory T cells were the predominant cells in IEC (Pérez-Cano et al., 2005). CD103+ DCs influence the oral tolerance in neonates and the superantigen Staphylococcal enterotoxin given to neonatal mice increased the FoxP3 expression in stimulated T cells of small intestines, improving oral tolerance by CD103+DCs. This is mediated by the increased expression of retinal aldehyde dehydrogenase enzyme (RALDH) in DCs, which metabolizes Vitamin A (Stern et al., 2013). Newborn piglets respond to oral antigens with immunity, when administered along with adjuvants like CpG-oligodeoxynucleotides (CpG-ODN) (Pasternak et al., 2014). Dietary bovine lactoferrrin alters the mucosal and systemic immune cell response, improving