A review of the functions and similarities of Melanomacrophage centres and mammalian Germinal centres and their use as a health indicator in fish Killian Murray (s3601631) Abstract The health of fish in aquaculture farms is paramount for successful yields to be achieved consistently with the need for a reliable indicator required to inform operators of any potential harm occurring to the stock. A popular candidate for such a biomarker is melanomacrophage centres (MMC), which are aggregates of pigmented cells involved in the immune response of many vertebrates which are primarily found in the liver, spleen and kidneys. Analysis indicates several similarities in structure and function between MMCs and the mammalian germinal centre (GC). Being viewed as the evolutionary precursors to the GC, there is considerable research to be conducted into how MMCs play into the adaptive immune system with no outline as to how antigen affinity maturation occurs within the hosts. The value of MMCs as a histological biomarker is high however it is necessary to investigate further into these processes so that a greater understanding of how the MMC’s role in the adaptive immune system was able to evolve into that of the GC’s role in mammals. I suggest that a comparative gene expression study be conducted to determine how MMC and GC function is similar and the exact method in which antibody affinity maturation occurs within organisms without a GC. In the following review, a detailed overview of the functions, both nonimmunological and immunological, are presented along with the environmental factors that are involved in the optimum functioning of MMC. Introduction Aquaculture and the fishing industry as a whole contributed over $5 billion in 2017/18 to the Australian economy (Fisheries Research and Development, 2019), and thus efficient methods of determining fish health are required to ensure quality and protect quantity. Infections, diet and other environmental conditions are known to cause changes in melanomacrophage centres (MMCs) and thus extensive research has gone into their use as a potential indicator of fish health. MMCs are aggregates of pigmented cells that change with conditions that the fish is subjected to in its environment. Pigmented with high concentrations of melanin, lipofuscin and haemosiderin (Agius and Roberts, 2003), these cells are distinguishable by light microscopy (Fig. 1) and are therefore, cost-effective and straightforward to survey. Whilst locations within the organism are specific to species; they are most often found in haematopoietic organs of fish including the spleen, liver and kidneys (Wolke, 1992). The structure of MMCs in the liver and kidney are less defined than those in the spleen with the latter being more organised and rigid (Falk, Press, Landsverk and Dannevig, 1995; Press, Dannevig and Landsverk, 1994). MMCs have also been found in species other than fish including amphibians and reptiles however these have not received the same attention in literature (Steinel and Bolnick, 2017). Figure 1: MMCs in spleen of stickleback (Gasterosteus aculeatus), (A) Unstained spleen slide at 50x magnification. (B) outlined in box. (Scale bar = 250 μm). (B) Unstained spleen at 200x magnification. (C) Spleen stained with H&E at 200x magnification with MMCs displayed with black arrows. (Scale bars for (B) & (C) =62.5 μm. (Steinel and Bolnick, 2017) Nonimmunological functions of MMCs The primary functions of MMCs are its involvement in both the innate and adaptive immune system however they also have roles regarding the normal function of the body. First and foremost, MMCs primarily conduct phagocytosis. Along with this, they have also been seen as a storage site for unmetabolised waste products granting them the title of “metabolic dump” (Agius, 1980). Substances that are found within the aggregates can be categorised into two groups, endogenous materials and exogenous materials (Agius, 1980). Endogenous materials include melanin, lipofuscin and haemosiderin and the exogenous are further separated via origin into experimental sources (carbon, polystyrene beads and antigens) and natural sources (metals and biological agents) (Sayed and Younis, 2017). Of the endogenous pigments found, only haemosiderin can be recycled while lipofuscin and melanin are stored, with the latter potentially being used in the organism’s immune defence (Agius and Agbede 1984). Exogenous materials, particularly the experimental products, are stored in the MMC, while natural sources are stored depending on the substance’s degradability. Another largely documented function of MMCs is the recycling of iron and whilst there are no direct experimental results, large amounts of circumstantial evidence indicate this to be the case. Degenerating erythrocytes were observed in splenic MMCs as early as 1908 (Wolke, 1992). It is also known that aggregates of the spleen contain high quantities of haemosiderin and when hyperhaemolytic crisis occurs, the pigment increased in concentration and was found in MMCs of both liver and kidneys (Wolke, 1992). Finally, a relationship between erythropoiesis and haemosiderin has been observed (Yu, Kiley, Sarot and Perlmutter, 1971). Upon being injected with radio iron labelled erythrocytes from fish into normal Trichogaster trichopterus, Yu et al. (1971) were able to measure and calculate the proportion of iron that had settled in each organ. The spleen contained the highest proportion of the metal featuring 55%. After stimulating erythropoiesis in 3 of 10 groups of fish, no immediate changes were observed to the percentage of splenic MMC area occupied by haemosiderin. Within 24 hours however it had dropped to 4.39 and 3.61 after 48 hours. Neither liver nor kidneys featured any haemosiderin in the experimental animals with some being present in the control animals. Immunological functions of MMCs Being formed by the aggregation of macrophages implies the role in phagocytosis. Whilst erythrocytes are a known target of MMCs, they have been observed to phagocytose foreign infectious materials (Brattgjerd and Evensen, 1996; Lamers. 1986). The aggregates of turtles were observed as being highly aggressive and would seek out a variety of pathogens, specifically bacteria, fungi and parasitic eggs (Johnson, Schwiesow, Ekwall and Christiansen, 1999). Being found in close proximity to the specialised capillaries called ellipsoids indicates that MMCs may filter out pathogenic substances to then consume (Ferguson, 1976). Studies conducted in vivo support this, finding that MMCs were able to quickly filter out foreign substances injected into the host organism (Brattgjerd and Evensen, 1996). The morphological similarities, along with being found in the same organs and their association with infectious disease has led to the conclusion that MMCs are the evolutionary precursor to germinal centres (GC) that are found in mammals (Fig. 2) (Agius and Roberts, 2003). GCs are sites of antibody affinity maturation that are found in secondary lymphoid organs (i.e. spleen and lymph nodes) (Allen, Okada and Cyster, 2007). GCs allow antibodies to mutate to be more specific to the antigen being presented by the Figure 2: Phylogeny showing vertebrates with and without MMC. This phylogeny is not historically to scale. (Steinel and Bolnick, 2017) pathogen during the immune response (Lamers, 1986) and are where antigens are processed for long term retention (Widdicombe et al., 2020). Unlike MMCs which are aggregates of a singular type of cell, GCs are highly structured with multiple cell types, featuring distinctive B cells, follicular dendritic cells (FDCs) and T follicular helper cell aggregates (TFC). Following the presentation of antigens to the host, antigen specific B cells accumulate and spread rapidly leading a temporary increase in GC size (Victora and Nussenzweig, 2012). During the GC response (Fig. 3), B cells undergo clonal expansion and differentiate into memory and plasma cells (Victora and Nussenzweig, 2012). FDCs are used as antigen depots for future immunity by keeping them intact on their surface as immune complexes (IC) storing them for recurrent infections. (Heesters, Myers and Carroll, 2014). Antibody affinity maturation is a microevolutionary process in which antibodies undergo somatic hypermutation (SHM), via the action of activation-induced cytidine deaminase (AID) (Pavri and Nussenzweig, 2011). The GC B cells containing the now mutated antibodies are then selected based on their affinity for the antigen (Berek, Berger and Apel, 1991). Although teleosts do not have GCs, yet there are still able to generate an affinity matured antibody response to antigen presentation (Solem and Stenvik, 2006). Studies across many species of fish describe the numerous similarities between GCs and MMCs (Steinel and Bolnik, 2017), raising the possibility that the aggregates are the site of adaptive immune response in teleosts. Melanomacrophages (MM) stained positively for CNA-42 monoclonal antibody (Vigliano, Bermúdez, Quiroga and Nieto, 2006) and express CSF1-R (Diaz-Satizabal and Magor, 2015), both of which are found within in mammalian FDCs. When injected with infectious and non-infectious materials, teleost MMCs were observed to be sites of antigen retention (Lamers and De Haas, 1985; Herraez and Zapata, 1987). Antigens that are retained are found near or within MMCs is extracellular or are trapped within ICs (Secombes and Manning, 1980). Whilst this evidence linked MMs to FDCs found in the mammalian GC, Figure 3: A diagram detailing the GC response. A specialised microenvironment which forms within B-cells after either infection or immunisation. There are two sections, the dark zone (DZ) that contains a network of CXCL12-produing reticular cells (CRCs) and is where GC B cell multiplication and SHM. Centroblasts then move into the light zone (LZ) as centrocytes through the action of CXCR5. Centrocytes capture antigen from the FDC which they then present to the TFCs in order to be selected. Centrocytes cycle back through the DZ for further rounds of proliferation and SHM (Stebegg et al., 2018) other functions including phagocytosis and blood scavenging indicated a wider gamut of cell types that the MMs act as in teleosts. Infection and immunisation of teleosts have been shown to increase MMCs in size and/or number in a similar manner to GCs (Agius, 1979; Herraez and Zapata, 1986; Widdicombe et al., 2020). Lymphoid cells were shown to be in closely located to MMCs and upon immunisation would increase (Herraez and Zapata 1986). No cell identity was discovered due to a lack of species-specific reagents used. Recent studies were able to determine that MMC adjacent immunoglobulin+ (Ig+) cells increase in size when experimentally infected (Falk, Press, Landsverk and Dannevig, 1995; Press, Dannevig and Landsverk, 1994). While assumed to be B cells, the Ig+ cells cannot be distinguished from cells expressing Ig and those binding soluble Ig or IC to their surface (Falk, Press, Landsverk and Dannevig, 1995; Press, Dannevig and Landsverk, 1994). Whilst some aggregation was observed amongst Ig+ cells, no highly compartmentalised structured was exhibited which are characteristic of mammalian GC (Falk, Press, Landsverk and Dannevig, 1995; Press, Dannevig and Landsverk, 1994). As stated prior, GCs response leads to an increase in antibody production, something not true in the MMC response (Herreaz and Zapato, 1986). AID expressing cells have been more recently observed as being in or proximal to MMCs (Saunders et al., 2010). Due to the requirement of AID expression for SHM to occur in GC, this would strongly support hypothesis of a GC-like MMC. However, no SHM has been documented in MMC (Saunders et al., 2010). These studies display a strong connection between GCs and MMCs with them likely performing similar functions but do display functional differences as well. Environmental factors and MMCs Environmental factors can cause stress in organisms that leads to a susceptibility to infection. Along with changes to the numbers of lymphocyte and granulocytes circulating, the greatest changes are the abundance of macrophage cells, a reduction in the numbers of platelets and lymphocytes and an increase in red blood cell degradation (Peters and Schwarzen, 1985). When living in polluted environments, teleosts come to reflect the lesser conditions through altered immune system activity (Kelly-Reay and Weeks-Perkins, 1994). The size of MMCs found in the kidney and spleen of Southern Bluefin Tuna have been observed to increase when the tuna is infected with Cardicola spp (Widdicombe et al., 2020). Whilst MMCs have been known to change in size with age, their changes with respect to their environment have led to the suggestion of them for use as biomarkers as a measure of pollutants both biotic and abiotic. Several studies have been conducted for histological indicators of environmental quality. Pollutants, while potentially being fatal, sublethal effects are more common (Blazer, Wolke, Brown and Powell 1987). Contaminated waters have been proven to increase the prevalence of parasites and various other diseases in multiple marine fish populations (Haaparanta, Valtonen, Hoffmann and Holmes 1996). A range of physiological and pathological responses have been posited for use as markers including incidences of tumour formation in epidermis and liver (Fabacher and Baumann 1985) and the presence of xenobiotic-metabolising enzymes being found in particularly sensitive species (Stegeman 1978). The issue with these responses is that they are too specific to the substance that triggers them and thus cannot be used as a generalised indicator. MMC were identified by several authors as being apart of many disease processes and the alterations that coincide with them including starvation (Agius and Roberts, 1980) or chemical exposure (Meinelt, Kruger, Pietrock, Osten & Steinberg 1997) indicates that these centres can be used as a sensitive indicator of stressful conditions in aquatic environments (Agius and Roberts, 2003; Wolke, Murchelano, Dickstein and George, 1985). They are found in close to all fish species, easily measured and able to be compared statistically. Studies comparing MMC prevalence in different organs in fish extracted from water polluted with toxic chemicals have seen contradicting results. Some authors observed an increase in number and size (Wolke, Murchelano, Dickstein and George, 1985) while others have recorded the opposite occurring (Kranz, 1989). The latter should be noted that at low levels of pollution, an increase was observed and was attributed to the cellular defence system to remove debris through an increase in phagocytic activity which was followed by an increase in MMCs. Further Research In order to determine whether MMCs function in the same manner as GCs, further studies are required. A possible method of this would be to determine whether the MMC is capable of specific B cell clonal expansion and high-antibody affinity production, both of which are the results of the GCs B cell proliferation, differentiation and SHM. While the presence of AID-expressing cells in MMCs has led to the implication of that antibody affinity maturation occurs (Saunders et al., 2010), more study into whether the action of antibody SHM gene within and is dependent on the MMC is required. In the GC, the response is Tcell dependent as the development of B-cells requires the assistance of TFC. T-cell dependent antigens are proteins that are required to induce the GC response whereas T-cell independent antigens are polysaccharide based and therefore cannot be presented in TFC or induce the GC response (Fagarasan, 2000). In order to determine the degree of similarity between the MMC and GC response, a study into the effect of both T cell dependent and independent antigens on the MMC response would be useful. Another avenue of research could be a comparative analysis of MMCs and GCs to determine the similarities and differences in gene expression. By observing these comparisons, one would be able to provide more clarity as to whether the GC is evolutionary connected to the MMC. Conclusions The MMC shares several similarities with the mammalian GC including structural, cellular and molecular, implying an evolutionary link between the two and the MMCs role in the adaptive immune system. 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