2.1 Immune system
As explained in the introduction before, the immune system can be divided into two parts; the innate and adaptive system. The innate immune response reacts fast and nonspecifically when a pathogen is encountered, whereas the adaptive immune response is slower to develop but specific. An important function of the innate immune system is the acute inflammation when the body is being attacked. This process develops within minutes after tissues are damaged and promote three types of signals. The first signal is given when damaged cells release molecules (damage-associated molecular patterns, DAMPs). DAMPs trigger the release of cytokines, chemokines and enzymes. The second signal is given by pathogen-associated molecular patterns (PAMPs) provided by invading microbes. These also trigger the release of cytokines, chemokines and enzymes. Lastly, tissue damages causes pain and sensory nerves to release bioactive peptide which attract leukocytes and increase local blood flow.
DAMPs and PAMPs can bind to pattern-recognition receptors (PRRs) on macrophages, dendritic cells and mast cell which causes them to be activated. This causes the production of a mixture of molecules that trigger inflammation, inhibit microbial growth and initiate the first steps in adaptive immunity [Tizard, 2013].
One of the most well-known cells in this process is the macrophage. In this chapter the macrophage, and the monocyte, will be thoroughly described. In addition, the cytokines involved in the inflammation process and secreted by or involved with macrophages will be explained. There are differences between the known immune system in mammals and birds. Therefore, the avian immune system will be evaluated in this report by researching literature.
2.1.1 Monocytes and macrophages
The macrophage is a large tissue cell and widely distributed in all organs and connective tissue. It is responsible for removing damaged tissue, cells and bacteria by performing phagocytosis. In addition, it is an antigen-presenting cell and can secrete a large variety of cytokines. The central and many functions of the macrophage on both the innate and adaptive system can clearly be seen in figure 1. It especially acts as an important source of inflammatory cytokines like tumour necrosis factor α (TNF-α), IL-1 and IL-6. These and other cytokines involved with macrophages will be further explained in paragraph 2.1.2 Cytokines.
The precursor of the macrophage is the monocyte, which is also responsible for removing damaged tissue and microorganisms. They circulate in the bloodstream and can also be found in the spleen. After entering into the tissue, especially during inflammatory reactions, they will differentiate into macrophages [Abbas et al., 1994 ; Playfair & Chain, 2012 ; Tizard, 2013].
iNOS and NO
Phagocytosis can be described as intracellular killing of bacteria. It requires the uptake of oxygen by the phagocytic cell and a chain of enzyme reactions to produce ROS, which is called the respiratory burst. This process can be seen in figure 2. During this event, NADPH oxidase is activated and superoxide dismutase (SOD) is produced. This causes oxygen to be reduced to superoxide (O2−), hydrogen peroxide (H2O2), hydroxyl ions (OH−) and singlet oxygen (O2). These reactive oxygen species (ROS) are lethal to bacteria. Excessive ROS production can even cause damage to host tissues [Abbas et al., 1994 ; Playfair & Chain, 2012].
Another reactive oxygen compound that is highly toxic to microorganisms in large amounts is nitric oxide (NO). It is produced from arginine by the action of an enzyme called inducible nitric oxide synthase (iNOS) as can be seen in figure 3 [Abbas et al., 1994 ; Playfair & Chain, 2012]. iNOS activity or NO production can be used as an indicator for activated macrophages.
2.1.2 Cytokines
Cytokines are mediator molecules that are secreted by signalling cells and spread to nearby receiving cells, where they bind to their receptors and can trigger their responses [Tizard, 2013]. The inflammatory response is organised by several cytokines, which are produced by macrophages and other cell types. The most important ones are TNF-α, IL-6 and IL-1. These cytokines are pleiotropic, which means they have multiple functions. They initiate changes in the vascular endothelium that promote leukocyte entry into the inflammatory site, induce the acute phase response and the process of tissue repair Abbas et al., 1994 ; Playfair & Chain, 2012]. These and other cytokines involved with macrophages will be individually explained in the text below and an overview can be seen in Table 1.
TNFα
TNFα can be produced in two forms: soluble and membrane-bound. The membrane bound form is cleaved from the cell surface by a protease called TNFα convertase and becomes soluble. TNFα production is stimulated through toll-like receptors (TLRs) and also by the neurotransmitter neurokinin-1, a molecule secreted by nerves. There are two TNF receptors, TNFR1 is found on most cells and TNFR2 is restricted to the cells of the immune system and responds only to cell-associated TNF [Tizard, 2013].
TNFα triggers the release of chemokines and cytokines from nearby cells and promotes the adherence, migration, attraction and activation of leukocytes.
TNFα plays a leading role in changes to vascular endothelium that are critical in the inflammatory response by stimulating, in combination with IL-1, the production of adhesion molecules on the inner surface of blood vessels. TNFα attracts neutrophils to the tissue damage, increases their adherence to vascular endothelium and enhance their ability to kill microbes. Together with IL-1, TNFα induces macrophages to increase their own synthesis whereby it amplifies and prolongs inflammation.
By enhancing antigen presentation and T cell activation, it facilitates the transition from innate to adaptive immunity. When a body is severely infected or injured, excessive TNF can get into the circulation. This will lead to shock and multiple organ damage [Abbas et al., 1994 ; Playfair & Chain, 2012 ; Tizard, 2013].
IL-1
Interleukin-1 is an important pro-inflammatory cytokine. CD14 and TLR4 stimulate macrophages to produce IL-1a and IL-1b. IL-1b is ten to fifty times more produced than IL-1a and cleaved by caspase-1, whereas IL-1a remains attached to the cell. Therefore, IL-1a only acts on cells in direct contact with the macrophage. IL-1b acts, like TNFα, on nearby cells to initiate and amplify inflammation. It makes vascular endothelial cells adhesive for neutrophils and stimulates other macrophages to synthesise NOS2 and COX-2. During infection, IL-1b circulates in the bloodstream. IL-1 acts on the temperature control centre in the hypothalamus and thereby causes fever and other sickness behaviour together with TNFα. It acts on muscle cells to mobilise amino acids, causing pain and fatigue. It acts on liver cells to induce the production of new proteins, called acute-phase proteins, that assist in the defence of the body [Abbas et al., 1994 ; Playfair & Chain, 2012 ; Tizard, 2013].
Important IL-1 receptors are CD121a and CD121b. CD121a is a signalling receptor, and CD121b inhibits IL-1 functions by acting as an IL-1 antagonist. Together with he multi-molecular complex inflammasome it regulates IL-1 activity [Abbas et al., 1994 ; Playfair & Chain, 2012 ; Tizard, 2013].
IL-6
Interleukin 6 (IL-6) is produced by macrophages, T cells and mast cells. Il-1 and TNFα can trigger its production. IL-6 has an influence on both inflammation and adaptive immunity. It promotes some aspects of inflammation and is a major mediator of the acute phase reaction and, septic shock and antibacterial resistance. In addition, IL-6 has an anti-inflammatory role as it inhibits activities of TNFα and IL-1 and induces IL-10 production which is a suppressive cytokine [Abbas et al., 1994 ; Playfair & Chain, 2012 ; Tizard, 2013].
IL-12
Interleukin 12 (IL-12) is a cytokine produced by macrophages, dendritic cells, B cells and neutrophils. IL-12 determines the Th1/Th2 polarisation. In presence of IL-12, Th1 cell will develop and in absence Th2 cells will develop.
IL-23. When neutrophils die, they are removed by macrophages. During this process, macrophages will produce IL-23 which promotes IL-17 production by lymphocytes. IL-17 will stimulate G-CSF production and stem cell activity to increase the rate of neutrophil production [Abbas et al., 1994 ; Playfair & Chain, 2012 ; Tizard, 2013].
IFN
Interferons IFNα and IFNβ are signals of innate immunity, that activate a broad range of antiviral mechanisms in many types of cell. They are produced by almost all cells, but plasmacytoid dendritic cells produce 1000 times more than any other cell type. In contrast, IFNγ is only weakly antiviral, but is a major regulator of macrophage activation. All three cytokines bind to type II receptors, and activate signals broadly similar to type I. The inhibitory cytokine IL-10 also binds to a type II receptor. [Abbas et al., 1994 ; Playfair & Chain, 2012].
2.1.3 Avian immune system
The avian immune system has showed to differ on many fronts from the mammalian immune system. For instance, chickens do not have lymph nodes, have no functional eosinophils and interleukin 5 (IL-5) is a pseudogene [Kaiser et al., 2005]. However, when chicken monocytes are stimulated they produce nitric oxide (NO) and express inducible NO synthase (iNOS). In addition, they secrete pro-inflammatory cytokines IL-1b and IL-6 [Haiqi et al., 2011]. These are the same as an inflammatory response in mammals would be.
Nonetheless, another important factor in the inflammatory response is TNFα. This cytokine makes a large difference between mammals and chicken as that TNFα is not yet identified in avian species [Hong et al., 2006]. Even in fish and amphibians TNFα has been described [Rohde et al., 2018]. However, 10 members of the tumour necrosis factor superfamily (TNFSF) are identified in the chicken genome by Kaiser et al together with 23 ILs, 8 type I interferons (IFNs), IFN- , 1 colony-stimulating factor (GM-CSF), 2 of the 3 known transforming growth factors (TGFs) and 24 chemokines [Kaiser et al., 2005]. Hong et al isolated a full- length cDNA encoding the chicken homologue of LPS-induced TNF-a factor (LITAF), transcription factor, with an open reading frame of 148 amino acids and a predicted molecular mass of 16.0 kDa. With a RT-PCR analysis they found that LITAF mRNA was expressed in the spleen and were up-regulated when macrophages were stimulated [Hong et al., 2006]. An avian ortholog of TNFα has been identified as chicken TNFα (chTNFα) [Rohde et al., 2018]. chTNFα is encoded by a highly GC-rich gene and shows a 45% homology with the mammalian equivalent. Even full sequences for homologs of TNFα receptors 1 and 2 (TNFR1, TNFR2) are obtained in this recent study. It seems that chTNF-α mRNA is strongly induced by lipopolysaccharide (LPS) stimulation of monocyte derived, splenic and bone marrow macrophages. The cytokine induces an NFkB-luciferase reporter. Rohde et al conclude with that there is a functional TNF-a/TNF-a receptor system in birds [Rohde et al., 2018].
2.2 Trained innate immunity
Although the immune system has been divided into innate (fast and nonspecifically) and adaptive (slow and specific) for a long time, this distinction has been questioned recently. Plants and invertebrates that do not have an adaptive immune seem to have protection against reinfection [Kurtz, 2005]. Other studies show that also mammals demonstrate cross protection between infections with different pathogens. These studies have led to the hypothesis that innate immunity can be influenced by previous infections, which has led to the term trained immunity [Medzhitov & Janeway, 2000 ; Kurtz, 2005 ; Bowdish et al., 2007 ; Netea et al., 2011 ; Quintin et al., 2014 ; Netea et al., 2016]
Most of previous studies are done with human and rats. These studies form an important base and thus need to be researched. The effect of β-glucan on monocytes found in the literature will be discussed. In addition, the mechanism of trained innate immunity will be explained in this chapter.