Spirulina promotes macrophages aggregation in zebrafish (Danio rerio) liver

Abstract The immune system of teleosts offers many ideas to deepen the immune mechanisms and cells in general. The use of zebrafish as an experimental model is increased in recent years, thanks to its genetic and anatomical characteristics. It is known that several natural compounds exert an action on the immune system, boosting it. Spirulina, a non-toxic blue-green alga, has been declared a superfood for its peculiar biological activities. In this study, we test the immunostimulant effect of spirulina on zebrafish liver macrophages by immunohistochemical analysis using optical and confocal microscopy. Our results have shown an increase in the number of macrophages after feeding with spirulina, furthermore, this natural ‘superfood’ can induce macrophages aggregation. These data not only provide information on the possible effect of this alga as a complementary feed on the immune cells of teleost, but also improve the knowledge of the immune mechanisms of vertebrates. Graphical Abstract


Introduction
To protect themselves from infections in their aquatic habitat, teleosts have evolved both innate and adaptive immunity that are represented by cell and humoral components (Biller-Takahashi and Urbinati 2014). Innate immunity involves the recruitment of immune cells, such as macrophages, to clean blood and parenchyma from toxic molecules, which may promote injury, aging, or necrosis (Var and Byrd-Jacobs 2020).
Bony fish have functional macrophage subsets that are similar to those found in mammals (Hodgkinson et al. 2015).
Macrophages and neutrophils constitute the majority of the zebrafish innate immune system (Varol et al. 2015). Macrophages can also be present as aggregates (MAs) (Schwindt et al. 2006). These are important cells in the adaptive immune response because they are involved in the removal and killing of degenerating cells as well as the recycling of some cellular components (Matsche et al. 2019).
In recent years, research work has focused on the biological impacts of natural and nutraceutical substances, which can be utilized to not only prevent but also treat the symptoms of many diseases Corsaro et al. 2015;Vadal a et al. 2016). In addition, these compounds are also able to boost the immune system, having a plethora of biological activities: antioxidants, antibacterial, antivirals, immunostimulants, antidepressants, antitumor, etc. Novellino 2017, 2018;Aghraz et al. 2018;Gervasi et al. 2018Gervasi et al. , 2020Daliu et al. 2019;Durazzo et al. 2020;Fumia et al. 2021;Alesci, Nicosia, et al. 2022).
Spirulina (Arthrospira platensis), belonging to the Cyanophyta, Oscillatoriaceae. is a filamentous, multicellular, photosynthetic prokaryote non-toxic blue-green alga composed of single or multiple cells with a length of 200-500 lm and a width of 5-10 lm (Lo Cascio et al. 2017). According to the World Health Organization and the United Nations Food and Agriculture Organization, spirulina is one of the best dietary supplements and human health foods in the twenty-first century because of its extraordinarily high nutritional content (Han et al. 2021). This alga is an excellent source of several macro and micronutrients, according to chemical analysis. In fact, spirulina has a high protein, vitamin, essential amino acid, dietary mineral, and essential fatty acid concentration, giving it a variety of health benefits (Hosseini et al. 2013). Therefore, this alga is named a 'superfood' (Jung et al. 2019). Spirulina has anti-oxidant, anti-inflammatory, antitumor, immunity-boosting, and organ-protecting properties (Calabr o et al. 2021;Han et al. 2021). This alga can alter the immune system by enhancing macrophage phagocytic activity (Matufi et al. 2020).
The purpose of this study is to perform an evaluation of the effect of spirulina on macrophages in zebrafish liver by immunohistochemical characterization. Because macrophages express Toll-like receptor (TLR)-2 (Lauriano, Pergolizzi, et al. 2016), phylogenetically preserved receptors from fish to mammals Lauriano, Pergolizzi, et al. 2016;Lauriano et al. 2021;), a-smooth muscle actin (a-SMA) (Ludin et al. 2012), and S100 ), a evolutionary conserved protein (Alesci et al. 2020), these antibodies were tested in our study to characterize these immune cells and their aggregates.

Results and discussion
The Mallory Trichrome histological staining shows homogeneous hepatic parenchyma characterized by well-organized hepatocytes in cords, which are arranged in a radius around well-defined centrilobular veins. The microscopic anatomy of the organ appears to be superimposed on the mammalian counterpart. The control group has some macrophages with a well-defined eccentric nucleus. The spirulina-fed group shows clear MAs ( Figure S1). Histochemical staining Periodic Acid Schiff (PAS) shows numerous macrophage aggregates in the treated group ( Figure S2). Histological results show in general a greater presence of macrophages, organized in aggregates, not present in the control group.
Immunohistochemical analysis with TLR-2 characterizes macrophage aggregates in the treated group ( Figure S3). Clustered macrophages are also immunopositive for a-SMA and S100 in the spirulina-fed group. Scattered macrophages are characterized in the control group ( Figures S4 and S5). The confocal colocalization of TLR-2 and a-SMA confirms data obtained by immunoperoxidase ( Figure S6). These results could support the thesis that spirulina stimulates recruitment and induces macrophage aggregation in zebrafish liver. Statistical analysis corroborates the results obtained with histological and immunohistochemical analysis (Table S2).
The liver of zebrafish is structurally comparable to that of mammals, and the expression and activities of marker genes in zebrafish macrophages are similar to those in mammals (Shwartz et al. 2019).
MAs can be found in a variety of fish organs, including the kidney, liver, and spleen, and can include pigments such as melanin, ceroid/lipofuscin, or hemosiderin (Schwindt et al. 2006).
Spirulina has anti-oxidant and immunostimulant properties, which are extremely effective on innate immune cells (Chei et al. 2020), reduces pro-inflammatory cytokine expression and release (Shariat et al. 2019) and promotes macrophage phagocytic activity (Matufi et al. 2020), maximizing their action mediated by TLR-2 (Balachandran et al. 2006). Spirulina supplementation can improve immunocompetent cell activity, enhancing immunological response (Choi et al. 2013). A study conducted by Iskandar et al. in 2015 showed that the number of macrophages and activated plasma cells increased after treatment with spirulina. Higher Spirulina concentrations result in a higher number of macrophages (Iskandar et al. 2015).
Our study performed a histological and immunohistochemical assessment of macrophage aggregates in spirulina-fed zebrafish liver. We noted a significant increase in the number of macrophages following the administration of spirulina, according to Iskandar et al. (2015). Immunohistochemical analysis by TLR2, a-SMA and S100 was performed to characterize individual and aggregated macrophages. Previous studies showed the presence of these antibodies in teleosts Lauriano, Pergolizzi, et al. 2016;Alessio et al. 2021;Alesci, Pergolizzi, Capillo, et al. 2022).
TLR-2 stimulates the generation of NO and inflammatory cytokines in macrophages (Kakutani et al. 2012). Ludin et al. (2012) discovered a population of bone marrow-resident macrophages and monocytes that express a-SMA and was responsible for maintaining the primitive phenotype of undifferentiated hemopoietic stem and progenitor cells (Ludin et al. 2012). In addition, to investigate the interaction between neutrophils and hepatic stellate cells in zebrafish a-SMA was performed (Yang et al. 2018). S100 proteins activate nuclear factor-kappa B (NF-jB), causing the production of pro-inflammatory cytokines and the migration of neutrophils, monocytes, and macrophages (Xia et al. 2017). S100 proteins are expressed in phagocytes (Foell et al. 2007) and have been linked to inflammatory and cancer-related processes in recent investigations. Neutrophils and macrophages express S100 at high levels in fish (Sado and Matushima 2008).
Increasing macrophage aggregates, immunopositive to the antibodies tested, were evident in the spirulina-fed group. Colocalization of a-SMA and TLR-2, using confocal light microscopy techniques Zaccone et al. 2015;Lauriano, _ Zuwała, et al., 2016;Pergolizzi et al. 2021) corroborated our results. Statistical analysis also confirmed that the number of macrophages in the treated group was significantly higher, corroborating that spirulina increases the recruitment of these immune cells, improving the innate immunity of the fish. In addition, the increased presence of macrophage aggregates could indicate that spirulina can induce the formation of macrophage clusters, enhancing the immune response.

Conclusions
In general, fish innate immunity appears to be highly evolved with potentially enhanced functionality when compared to mammals, however, fish adaptive immunity appears to be less sophisticated. Our data could therefore provide important contributions to better understand fish immunology and the immune system in vertebrates.