Key contributors of the intestinal immune defence in fish

Teleost fish have three intestinal segments that can be distinguished based on morphological and functional traits. Each segment has different immune cell populations and roles (Parra et al., 2015). Macronutrient uptake occurs through the absorptive cells in the anterior intestine and uptake and transport of antigens take place in the mid intestine. The distal intestine is the immunologically-relevant segment where the antigens are sampled from lumen by the APCs (Rombout et al., 2011). In one of the early studies on rainbow trout (Oncorhynchus mykiss), immune cells such as lymphocytes, macrophages, and some plasma cells were found in the mid and distal segments of the intestine (Georgopoulou and Vernier, 1986). In Atlantic salmon, expression of several marker genes of T-lymphocytes (cd4-1, cd8a, tcra and tcrg) and B-lymphocytes (slgm, mlgm, slgt and mlgt) were relatively low in esophagus and stomach, but higher in the pyloric caeca, mid- and distal intestine. Also, the messenger

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RNA (mRNA) levels of igm and igt were found to increase from the pyloric caeca to the distal intestine (Løkka et al., 2014). In addition, in rainbow trout (Perdiguero et al., 2019), percentage of IgD⁺IgM^- cells was higher in the intestine compared to that in the spleen. Therefore, special attention should be paid to the immunological roles of mid- and distal intestine.

The first study on intestine cell isolation was performed on rainbow trout (McMillan and Secombes, 1997). Later, many researchers put in effort to isolate and characterize intestinal immune cells in other fish such as gilthead seabream (Sparus aurata) (Salinas et al., 2007) and Atlantic salmon (Attaya et al., 2018). However, lack of monoclonal antibodies and complexity of intestinal cell isolation procedures are still limiting our understanding of immune cells in fish.

1.3.1. T cells

Teleost fish have mucosal T cells, and they express genes related to T cell receptors (TCR) such as tcrab and tcrgb, cd3, cd4 and cd8, as well as mhc1 and mhc2 genes (Toda et al., 2011, Fischer et al., 2003). The presence of T cells in the intestinal epithelium and LP of carp (Cyprinus carpio L.) was demonstrated nearly two decades ago (Rombout et al., 1998). A later study on salmonids revealed the presence of more CD3ε⁺ cells in the thymus, gills, and intestine (Koppang et al., 2010). Dietary components may also influence the intestinal T cell populations - a significant increase in expression of the genes cd3pp, cd4 and cd8b was reported in the distal intestine of Atlantic salmon fed soybean meal (Bakke-McKellep et al., 2007). An induction of T cells as an intestinal inflammatory response to dietary allergens has been reported in humans (Sollid, 2002). It is worthwhile to monitor the changes in different T cell populations in fish intestine to extend our understanding on intestinal immune system of fish .

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1.3.2. B cells and immunoglobulins

Antibody or immunoglobulin (Ig) is produced by differentiated B cells called plasma cells. In teleost fish, three major Ig isotypes are expressed on the surface of B cells: IgM, IgT or IgZ and IgD. In mammals, the J chain and polymeric Ig receptor (pIgR) are essential for the transportation of IgA and IgM across IECs (Johansen et al., 2000).

However, it has been suggested that the presence of J chain may not be a requirement for the pIgR-immunoglobulin interaction in fish (Zhang et al., 2010). Rainbow trout has a pIgR and its secretory component is similar to those of mammalian IgA and IgM (Zhang et al., 2010) while fugu (Takifugu rubripes) pIgR is associated only with IgM (Hamuro et al., 2007). IgM is the main antibody in teleost fish, and probably its isotypes could have similar functions as those of mammalian IgA, which is abundant in mucous secretions (Cerutti et al., 2011). The latter neutralizes toxin and pathogenic microbes and prevents the attachment of commensal microbiota on the epithelial cells (Macpherson et al., 2008). Intestinal bacteria in rainbow trout was found to be coated with IgT, which responded to intestinal parasites in the gut, indicating their special role in mucosal immunity (Zhang et al., 2010).

The proportion of B cells within the GALT is different in teleost fishes. In carp (Rombout et al., 1998) and rainbow trout (Zhang et al., 2010), about 2-12% B cells among leukocytes isolated from LP of both anterior and posterior intestine were IgM⁺ cells. In Atlantic halibut (Hippoglossus hippoglossus), IgM⁺ cells were present within the epithelium and LP (Grove et al., 2006). In addition to their role in adaptive immunity, B-lymphocytes have been reported to perform phagocytosis. In the peritoneal cavity of mice (Parra et al., 2012) around 10-15% B cells have phagocytic ability. In teleosts, around 60% of B cells found in all systemic compartments perform phagocytosis (Li et al., 2006).

9 1.3.3. Monocytes/macrophages

In mammals, there are two macrophage subsets; classically and alternatively activated (Zhou et al., 2014). The classically activated macrophages that take part in inflammatory or microbicidal responses belong to the M1 types that are primed by interferon-gamma (IFNγ) and tumor necrosis factor-alpha (TNFα). On the other hand, the alternatively activated forms are called M2 macrophages and are involved in tissue repair and wound healing. There are three M2-macrophage subtypes; M2a subset is activated by interleukin-4 and/or interleukin-13; M2b is stimulated by immune complexes or apoptotic cells and M2c is primed by interluekin-10 (IL-10), transforming growth factor-beta (TGF-β) and/or glucocorticoid (Zhou et al., 2014). Although the polarization and functionality of mammalian macrophage subsets are clearly described, such details about fish macrophages are not yet reported. Nevertheless, Wiegertjes et al. (2016), Grayfer et al. (2018) and Hodgkinson et al. (2015) have reviewed the different types of fish macrophages.

Fish M1 macrophages that can phagocytize are classically polarized by colony stimulating factor-1 (CSF-1), IFNJ, IFNγ-related (IFNJ-rel) and TNFD1/2. The M2 types in fishes are believed to be primed by IL-4/IL-13, IL-10 and glucocorticoids (Grayfer et al., 2018, Forlenza et al., 2011). Furthermore, Wiegertjes et al. (2016) reported reliable markers for M1-macrophages (NOS-2) and M2-macrophages (arginase-2). A recent study on transcriptome of carp macrophages revealed the potential markers for M1 (il1b, nos2b and saa) and M2 (timp2b, tgm2b and arg2), which indicate that fish macrophages could have conserved functions and common transcripts to mammals (Wentzel et al., 2020b). Macrophages in fishes are stimulated by CSF-1 (Rieger et al., 2014, Rieger et al., 2013). However, the production of soluble CSF-1 receptor through CSF-1 stimulation causes the polarization to M2 form (Rieger et al., 2013). This soluble receptor evokes the expression of the anti-inflammatory cytokine IL-10 (Rieger et al., 2015). However, it should be noted that some teleosts including the Japanese pufferfish have more than one CSF1R (Williams et al., 2002), and it is not yet clear how

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these act together to promote the differentiation of macrophages. Hence, the polarization in mammals and fish cannot be presumed to be identical. Previous studies have indicated possible IFNJ-induced polarization to M1-like macrophages in goldfish (Grayfer and Belosevic, 2009) and carps (Arts et al., 2010). Although fishes are known to have two forms of this type II interferon (IFNJ and IFNJ-rel), both of them can cause M1-polarization and they have distinct and redundant functions in the differentiation (Grayfer et al., 2010, Grayfer and Belosevic, 2009). It has also been reported that IFNJ-rel strongly influences macrophage phagocytosis and nitric oxide production (Grayfer et al., 2010, Grayfer and Belosevic, 2009). In addition, carp M1-macrophages increased nitric oxide production while the M2-macrophages increased oxidative phosphorylation and glycolysis, suggesting that IFNJ may influence macrophage metabolic reprogramming (Wentzel et al., 2020a). Tumor necrosis factor alpha 1 and 2 (tnfa1 and tnfa2) in fishes are thought to have roles similar to TNFα of mammals (Nguyen-Chi et al., 2015).

As for the phagocytosis in macrophage subsets, in mammals both M1- and M2-macrophages are known to have higher phagocytic ability compared with that of naive macrophages (M0) (Lam et al., 2016). However, M2-macorphages showed higher phagocytic affinities and capacities than M1-macrophages (Schulz et al., 2019).

Furthermore, a study in mice showed that phagocytosis of Porphyromonas gingivalis by M1-macrophages produced higher expression levels of TNF-α, IL-12 and iNOS compared to those of M0- and M2-macorphages, indicating that the activation of M1-macrophages could contribute to the initiation of inflammatory responses (Lam et al., 2016). Like mammals, fish inflammatory/M1-macrophages are well known to have ability to phagocytose pathogens and produce pro-inflammatory cytokines, reactive oxygen and nitrogen intermediates as reviewed by Grayfer et al. (2018). These studies are mainly focused on head kidney-derived macrophages. As for the intestinal macrophages in teleost fishes, some studies have suggested the presence of intestinal macrophage-like cells in Atlantic cod (Gadus morhua) (Inami et al., 2009) and rainbow

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trout (Georgopoulou and Vernier, 1986). In Atlantic salmon that were anally intubated with fluorescent yeast, the microbial cells were found near the large nuclei of macrophage-like cells that were located near the epithelium, indicating that intestinal macrophage-like cells could sample antigens like mammalian macrophages (Løkka et al., 2014b). Thorough functional and morphological characterization using cell markers are warranted in fish.