Macrophages in Vertebrate Immunity

#### **Chapter 2**

## Macrophage: A Key Player of Teleost Immune System

*Ragini Sinha*

#### **Abstract**

Fish, the free-living organisms, residing in aquatic environment, are earliest vertebrates with fully developed innate and adaptive immunity. Immune organs homologous to those of mammalian immune system are found in fish. Macrophages are best known for their role in immunity, basic function of which being cytokine production and phagocytosis. Due to environmental adaptation and whole genome duplication, macrophages in teleost are differently modulated (pro-inflammatory, M1-type, and anti-inflammatory/regulatory, M2-type) and perform a variety of different functions as compared with those of mammals. Phagocytosis is a major mechanism for removing pathogens and/or foreign particles in immune system and therefore is a critical component of the innate and adaptive immune system. One of the most competent phagocytes in teleost is found to be macrophages/monocytes. Increasing experimental evidence demonstrates that teleost phagocytic cells can recognize and destroy antigens to elicit adaptive immune responses that involve multiple cytokines. A detail understanding of teleost macrophages and phagocytosis would not only help in understanding the immune mechanism but will also help in disease prevention in teleost.

**Keywords:** inflammatory response, cytokine production, macrophages, phagocytosis, teleost

#### **1. Introduction**

Fish, the first vertebrate group, appeared in evolution after adaptive radiation during the Devonian period, presenting the most successful and diverse group of vertebrates. Importantly, immune organs homologous to those of the mammalian system are found in fish. This population possesses complicated innate immune networks and are the earliest vertebrates that have fully developed both arms of the immune system, i.e., innate and adaptive immunity [1]. Macrophage lineage cells are integral to fish immune responses like any other vertebrate, and hence, recent fish immunology research focuses on fish macrophage biology. Macrophages are one of the most important immune cells that bridge the innate and adaptive immunity. It plays a crucial role in tight regulation of immune response by secreting different immune mediators [2, 3]. Macrophages are present in most animal tissues and play crucial roles in host protection and homeostasis. They are known by different names such as amebocytes, hemocytes, coelomocytes, granulocytes, monocytes, and macrophages,

but have similar morphology and comparable functions [4–6]. Due to whole genome duplication and environmental adaptation, teleost monocyte/macrophages possess a variety of different functions and modulation compared with those of mammals. The basic functions of macrophages are production of cytokines and phagocytosis in vertebrates. Monocytes give rise to macrophages during inflammatory conditions in both mammals and fish [7]. Macrophages play multiple roles in immune system. Macrophages are potent innate immune cells, which exert a crucial antimicrobial defense through phagocytosis and release of different antimicrobial mediators, including reactive oxygen and nitrogen species (ROS and RNS). Additionally, they also serve as professional antigen presenting cells (APCs) to activate the adaptive immune system (T and B cells) [8]. Macrophages pose the phagocytic activity, which is the initial step in the immune response in fish and is the major line of defense for all foreign material, including pathogenic agents [9]. Measurement of macrophage activation serves as a bio-indicator and reveals the impact of environmental stress as well as chemical contamination of the aquatic bodies.

#### **2. Development of macrophages**

Teleost blood cell development occurs within primitive waves of hematopoiesis [7]. In mammals, macrophages are predominantly derived from the hematopoietic precursors born in the yolk sac (YS) and the aorta-gonad-mesonephros (AGM) where embryonic and adult hematopoiesis occurs [10–14]. Likewise, macrophages originate from the rostral blood island (RBI) and ventral wall of dorsal aorta (VDA), the fish hematopoietic tissue equivalent to the mammalian YS and AGM for myelopoiesis, respectively [15–19]. During primitive hematopoiesis, embryonic mesoderm becomes committed to produce monopotent hematopoietic precursors in the rostral blood island that give rise to macrophages [20–22]. Following monopoiesis, first multilineage progenitor cells arise, known as erythromyloid progenitors (EMPs), which can develop into both erythroid and myeloid cells. Later, a population of hematopoietic stem cells (HSCs) arises in the AGM. The existence of renal marrow-derived HSCs has been documented in both zebrafish and ginbuna carp [23, 24]. The progenitor cells that are found in the kidney have been shown to be able to differentiate into erythrocytes, lymphocytes, thrombocytes, granulocytes, and monocytes. Monocytes mainly exist in the bone marrow, blood, and spleen. They can differentiate into inflammatory macrophages and dendritic cells during inflammation [25, 26]. Macrophages reside in a variety of tissues including lymphoid and non-lymphoid ones. Until recently, tissue macrophages were believed to arise from circulation monocyte precursors in response to different stimuli [27]. Recent evidence by fate-mapping blood cell lineages suggests that contribution of monocytes is limited in maintaining the population of tissue macrophages. Instead, tissue macrophages are "seeded" during primary haematopoiesis and self-maintain the resident population like that of the mammals [28–30]. There is a specific group of cytokines that act as hematopoietic group of cytokine, which can regulate the development of multiple cell lineages and can act individually or concurrently to stimulate a specific response. Hematopoietic cytokines are produced by a variety of cell types, which can act in paracrine, endocrine, juxtracrine, or autocrine manner on the target cells for their renewal and development [31, 32]. Cytokine sensitivity is determined by a complex regulatory network, a hematopoietic cytokine may induce different developmental changes in different circumstances. Specific cell lineage can be responsive to certain cytokines.

#### **3. Role of transcription factor in macrophage development**

The regulation of hematopoiesis is carried out in and orchestrated manner involving cell-cell and cell-extracellular matrix. Transcription factors play a critical role in determining the fate of development of macrophages. Transcription factors are DNA-binding proteins that recognize specific domains. Improper expression of transcription factors and activity results in serious consequences within the hematopoietic system including inhibition of proliferation [33, 34]. Synergistic interactions between transcription factors are generally required for the activation of specific genes. Apart from that, negative interaction between transcription factors is also necessary for the control of hematopoiesis [31].

#### **3.1 Role of colony-stimulating factor-1**

Macrophage colony-stimulating factor-1 (CSF-1) is an important growth and differentiation factor of both fish and mammalian macrophages [35]. The survival, proliferation, differentiation, and functionality of most of the macrophage lineage cells are governed by CSF-1 through binding to its cognate receptor (CSF-1R). CSF-1R is expressed exclusively on committed myeloid precursors and derivative macrophage populations [36–42]. CSF-1 has recently been identified in several fish species including trout [43], zebrafish [43], and goldfish [43, 44]. Recombinant trout CSF-1 was found to promote the proliferation of trout head kidney leucocytes [43]. Reports suggest that the recombinant goldfish CSF-1 (rg-CSF-1) induced chemotactic response and enhanced antimicrobial functions of macrophages. It plays a central role in regulation of goldfish pro-inflammatory macrophage responses [35]. Many teleost fish species have two distinct CSF-1 genes (CSF-1.1 and CSF-1.2) [43], which happen to work by upregulating pro-inflammatory components [45, 46]. A variety of cytokines can induce the production of CSF-1 by monocytes and macrophages, such as GM-CSF [47], TNF-α [48], IL-1 [49, 50], and INF-γ [51, 52]. The capacity of monocyte/macrophages to produce CSF-1 suggests that these cells can auto-regulate their own proliferation and functions [31]. CSF-1 also stimulates the production of several cytokines including G-CSF, GM-CSF, IL-1, IL-6, IL-8, and TNF-α and interferons [38, 53]. Cyprinid fish produce a soluble CSF-1 receptor (sCSF-1R) that downregulates their pro-inflammatory responses by reducing available soluble CSF-1. The sCSF-1R is produced by mature macrophages and not by monocytes and efficiently removes a variety of inflammatory events including macrophage chemotaxis, phagocytosis, and production of ROS intermediates and recruitment of leukocytes [54]. Circulating CSF-1 can effectively be cleared by the process of CSF-1 receptor-mediated internalization followed by intracellular destruction of the growth factor. Liver and splenic macrophages have been demonstrated to be capable of absorbing approximately 94% of the circulating CSF-1 [55, 56]. Adding CSF-1 to primary cultures has proven to increase the longevity of the cultures and can drive the culture from a heterogeneous population of progenitor monocyte and macrophage cells, toward a homogeneous population of macrophages [57].

#### **4. Activation of macrophages**

Macrophage activation occurs under various intracellular as well as environmental influences. Based on the activation cue and the following effector functions, macrophages have been broadly classified in two types: classically activated macrophages (M1) induced in a T helper 1 (TH1) cytokine environment and alternatively activated macrophages (M2) induced in a T helper 2 (TH2) cytokine environments [58]. In a different terminology, M1 macrophages have been termed to be "inflammatory," whereas M2 macrophages have been termed to be "healing" in nature. There have been studies indicating four different phenotypes of macrophages, which are innate activated, classically activated, and alternatively activated and regulatory macrophages. Classically activated macrophages present higher respiratory burst activity and iNOS expression as compared with innate activated macrophages [59]. Macrophages that are activated by microbial stimulus and innate danger signals without any influence of adaptive immune cells lead to the formation of the M1 population [5, 59]. M2 macrophages that form in the presence of TH2 cytokines can again be classified into three groups: activated by IL-4/IL-13 or M2a macrophages [60], stimulated by Toll-like receptor (TLR) ligands in combination with second signal or M2b, developed in response to IL-10 or M2c [60].

#### **4.1 M1 macrophage activation**

Innate activation of M1 macrophages is induced by microbial stimulus, which can be detected by various receptors on the macrophage surface [61]. These microbial stimuli can activate macrophages through a large array of pattern recognition receptors (PRRs) [62]. Fish species poses a wide variety of PRRs both putative mammalian orthologues and fish-specific family members [63] and can be activated in the absence of exogenous cytokines. M1 macrophages are induced by pathogen associated molecular patterns (PAMPs) such as lipopolysaccharides (LPSs), a major component of outer membrane of Gram-negative bacteria [5, 6]. A number of publications show that in vitro stimulation of fish macrophages with LPS leads to increased respiratory burst activity and increased secretion of pro-inflammatory cytokines [64]. Classically activated macrophages require a microbial stimulus plus the presence of the cytokine INFγ (**Figure 1**) [65]. INFγ has been sequenced in fugu [66], rainbow trout [67], zebrafish [68], Atlantic salmon [69], catfish [70], common carp [71], goldfish [72], Atlantic cod [73], and flounder [74]. Certain fish species possess two distinct types of INFs. Both the isoforms, initially named INFγ1 and INFγ2, contain typical INFγ motifs and are now referred to as INFγ- related (INFγrel) and INFγ, respectively [75]. In carp and in grass carp, both isoforms are regulated by different stimuli [71, 76], *in vivo* bacterial infection in zebrafish embryo indicated that INFγ and INFγrel act partly redundantly, they have largely overlapping functions [77]. Goldfish INFγrel induced significantly higher phagocytosis and nitrite production in monocytes and macrophages, respectively, when compared with INFγ [72]. Research studies suggest that most probably INFγrel proteins are antiviral proteins without direct effects on M1/M2 polarization in fish [78]. It is particularly notable that certain teleosts possess two INFγ-receptor-binding chains (IFNGR1-1 and IFNGR1-2) in comparison to other vertebrates that have a single INFγ receptor 1 (INFGR1) [72, 79, 80]. These suggest that fish have adopted very unique strategies surrounding their M1 activation cytokine system. INFγ as a combination stimulus with LPS induces inflammatory M1 population. These macrophages show higher respiratory burst activity and nitric oxide synthase expression [62].

Classically activated macrophages are induced by a combination of INFγ and TNFα [81, 82]. Like its mammalian counterpart, teleost TNFα is one of the markers of M1 macrophages [83, 84]. Multiple isoforms of TNFα have been found in a

*Macrophage: A Key Player of Teleost Immune System DOI: http://dx.doi.org/10.5772/intechopen.103804*

#### **Figure 1.**

*M1 and M2 macrophage activation.*

variety of fishes. These isoforms have been shown to enhance inflammatory gene expressions, macrophage chemotaxis, and phagocytosis [85–96]. Functional evaluation of fish TNFα has discovered some contradictory results. In some fish species, recombinant TNFα (rTNFα) was found to hardly activate macrophages [97–99], whereas the trout and goldfish TNFα1 and 2 are shown to be active in macrophages [87, 100]. Two different TNF receptors have been found in goldfish, namely TNF-R1 and TNF-R2, which bind the goldfish TNFα1 and TNFα2 in a homodimeric conformation unlike the trimeric conformations of mammalian TNF ligands and receptors [101]. The bacterial LPS readily induces the TNFα gene expression, which in turn plays a major role in polarizing the macrophages [99, 102]. From different studies it is clearly understood that fish possess a well-defined M1 polarization upon microbial stimuli.

#### **4.2 M2 macrophage activation**

M2 macrophages also known as alternatively activated macrophages can be generally characterized as having "anti-inflammatory" or "pro-healing" phenotypes (**Figure 1**) when developed in the presence of TH2 cytokines IL-4 and/or IL-13 [103]. To date, at least two genes have been identified in fish that share homology with both the mammalian IL-4 and IL-13 cytokines (IL-4/13A and IL-4/13B) [104] even though variable number of copies of these genes are present in different fish due to genome duplication events [105]. Of the two may be IL-4/13A shows complete synteny with other genes in TH2 cytokine complex [106]. There is a common homodimeric receptor subunit called IL-4Rα for both cytokines (IL-4 and IL-13) found in mammalian vertebrates [107], paralogues of which, IL-13Rα1 and IL-13Rα2, have also been identified in teleosts [108, 109]. Teleost recombinant IL-4/13A and IL-4/13B have anti-inflammatory roles including upregulation of immunosuppressive genes (TGF-β, IL-10, SAP1, and SOC3) and downregulation of pro-inflammatory cytokine gene expressions (TNFα, IL-1β, and INFγ) [110–112]. These M2 macrophages show increased arginase activity. In M1 macrophage, the iNOS enzyme converts L-arginine to L-cutrulin and NO. By contrast, in M2 macrophages, the enzyme arginase, a manganese metallo-enzyme, converts L-arginine to L-ornithine and urea [113, 114]. Mammals possess two arginase isoforms including arginase-1 located in cytosol and arginase-2 located in mitochondria [115]. Teleosts possess both the forms arginase-1 and arginase-2, which are found to be mitochondrial forms unlike that of their vertebrate counterparts. In carp, arginase-1 gene expression was found mainly in the mid kidney, whereas arginase-2 expression was found in all organs with the liver having the maximum expression [116]. Under stimulation of exogenous cAMP, carp head kidney-derived macrophages show upregulation of arginase-2 but not arginase-1 expression, suggesting that arginase-2 might be an excellent marker of M2 macrophages in fish.

M2 macrophages that are deactivated by glucocorticoids or by cytokines such as TGF-β or IL-10 are also referred to as regulatory macrophages. Glucocorticoids diffuse across plasma membrane and alter the expression of immune-related genes [60]. It has been shown to be a strong inhibitor of NO production in goldfish macrophages [117] and increases fish susceptibility to diseases due to its immunosuppressive nature [118–120]. Grass carp recombinant IL-10 and recombinant TGF-β1 have found to attenuate LPS-stimulated inflammatory gene expressions in monocyte/macrophages [121]. The goldfish TGFβ downregulates the nitric oxide response of TNFα-activated macrophages [122]. Mammalian IL-10 functions through IL-10R1 and IL-10R2 leading to activation of STAT3 [123]. Similar to mammalian IL-10, carp IL-10 acts through a signaling pathway involving phosphorylation of STAT3 and leading to upregulation of SOCS-3 expression [124]. An IL-10R1 has been found in zebrafish, goldfish, and grass carp [125, 126], whereas IL-10R2 has been found in rainbow trout [127]. These cytokines demonstrate an evolutionary conserved role in fish immunology.

#### **5. Function of macrophages**

Macrophages and monocytes serve as professional phagocytes in fish [128]. Phagocytosis is a specific type of endocytic process by which cell engulfs solid particulate targets. These solid particles (including microbial pathogens) are internalized to form phagolysosome followed by antigen degradation [129–132]. Phagocytosis plays

#### *Macrophage: A Key Player of Teleost Immune System DOI: http://dx.doi.org/10.5772/intechopen.103804*

an essential role of linking the innate and adaptive immune response in vertebrates. It is well established that fish have both the innate and adaptive immune system in which macrophages happen to play a crucial role. The phagocytic mechanism depends on recognition of the foreign particle by cell surface receptors and killing by oxygen radicals [133, 134]. Phagocytosis plays a crucial role in the macrophage inflammatory immune response through hydrophobic interaction between the phagocytic membrane and the target particles. The multiple receptors present on the phagocyte can recognize their targets coated with opsonin molecules and form the phagosome by engulfing them [135]. Lysosome then fuses with the phagosome to form the phagolysosome, the vesicles in which the internalized microbes would be killed and degraded. Potent antimicrobial compounds including degradative enzymes (proteases, nucleases, phosphatases, lipases) and antimicrobial peptides (basic proteins and neutrophilic peptides) are generated by active phagocytes, which help in destruction of the phagocytosed pathogens [136–141]. Both M1 and M2-type macrophages form phagolysosomes. Reports suggest that M1 macrophages form a phagosome with relatively neutral pH as compared with M2 macrophages that form phagosomes with acidic pH [142]. Macrophages are known to be "professional" phagocytes along with polymorphonuclear cells (PMNs), monocytes, and dendritic cells in vertebrates. Apart from this, some "amateur" phagocytic cells (epithelial cells, fibroblasts, and B lymphocytes) show a lower degree phagocytic activity [129, 143]. Research suggests that succinate is critical in controlling phagocytosis in macrophages. Exogenous methyl-succinate was found to enhance phagocytosis, pro-inflammatory cytokine production, and expression of phagocytic genes [46].

The destruction of the internalized microorganism occurs by robust production of ROS (reactive oxygen species) by active macrophages. The multi-component enzyme NADPH assembles on the phagosome membrane during macrophage respiratory burst, which transfers electrons from NADPH to molecular oxygen-producing superoxide anion [144]. The functional sites of fish and mammalian NADPH oxidase are highly conserved. All of the components of NADPH oxidase have been found in teleosts, and fish ROS generation has been well documented following PAMP stimulation [145–148] and antimicrobial responses [149, 150].

Classically activated M1 macrophages abundantly express high levels of inducible nitric oxide synthase enzyme iNOS, which catalyze the conversion of L-arginine to L-citruline, resulting in the production of nitric oxide (NO) [151]. iNOS serves as a marker of M1 macrophage and is upregulated in response to INFγ, TNFα, and microbial compounds [82]. The fish iNOS has been characterized with marked similarity to the mammalian enzyme counterpart. The fish iNOS gene expression is induced by antimicrobial and inflammatory stimuli including cleaved transferring products [152, 153]. iNOS plays an important role in protection of fish from a variety of pathogens.

Another hallmark of M1 macrophages is upregulation of the expression of indoleamine2,3-dioxygenase (IDO) enzyme that depletes local tryptophan levels [154]. Tryptophan degradation produces certain metabolites that may inhibit T cell proliferation. Teleost IDO is less effective in tryptophan degradation as compared with their mammalian counterparts [155].

#### **6. Conclusion**

Teleosts are found throughout the world and are highly susceptible to variations caused by natural as well as man-made external changes, which affect their immune system. Macrophages are one of the basic immune cells found in teleosts like their mammalian counterparts, which play a crucial role in bridging the innate and adaptive immunity in fish. Macrophages of teleost fish exhibit many functions from that of homeostasis to host immune defense. They possess the phagocytic activity, which is initial step of defense in fish immunity. Measurement of macrophage activation serves as a bioindicator of fish health. Teleosts have shown to have different macrophage polarizations (M1 and M2) pathways under different stimuli, which provides a great support in understanding the evolutionary development of fish immune system. Despite having multiple isoforms of key macrophage cytokines in fish, functional studies of these have been limited. Whole-genome duplication events are responsible for the availability of multiple isoforms of immune mediators in different fish [156]. A greater understanding of teleost macrophages and their function with growing genetic resources would help widely in deciphering the minutes of fish immune system and its evolutionary linkage with that of their mammalian counterparts.

#### **Acknowledgements**

RS is thankful to the Council of Scientific and Industrial Research (CSIR), Human Resource Development Group, Govt. of India for her NET-SRF fellowship (Grant No. 09/202(0078)/2018-EMR-I). The author is thankful to Prof. Dipak Kumar Mandal for his guidance and support and to Monisha Das and Nibedita Sharma for their constant encouragement.

### **Conflict of interest**

The author declares no conflict of interest.

#### **Note/thanks/other declarations**

The author thanks the Head of the Department of Zoology, for providing the assistance in the research work.

## **Author details**

Ragini Sinha Department of Zoology, Visva-Bharati University, Santiniketan, West Bengal, India

\*Address all correspondence to: raginniissinha5@gmail.com

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Macrophage: A Key Player of Teleost Immune System DOI: http://dx.doi.org/10.5772/intechopen.103804*

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#### **Chapter 3**
