Cisplatin Toxicity Causes Neutrophil-Mediated Inflammation in Zebrafish Larvae.

Cisplatin is an antineoplastic agent used to treat various tumors. In mammals, it can cause nephrotoxicity, tissue damage, and inflammation. The release of inflammatory mediators leads to the recruitment and infiltration of immune cells, particularly neutrophils, at the site of inflammation. Cisplatin is often used as an inducer of acute kidney injury (AKI) in experimental models, including zebrafish (Danio rerio), due to its accumulation in kidney cells. Current protocols in larval zebrafish focus on studying its effect as an AKI inducer but ignore other systematic outcomes. In this study, cisplatin was added directly to the embryonic medium to assess its toxicity and impact on systemic inflammation using locomotor activity analysis, qPCR, microscopy, and flow cytometry. Our data showed that larvae exposed to cisplatin at 7 days post-fertilization (dpf) displayed dose-dependent mortality and morphological changes, leading to a decrease in locomotion speed at 9 dpf. The expression of pro-inflammatory cytokines such as interleukin (il)-12, il6, and il8 increased after 48 h of cisplatin exposure. Furthermore, while a decrease in the number of neutrophils was observed in the glomerular region of the pronephros, there was an increase in neutrophils throughout the entire animal after 48 h of cisplatin exposure. We demonstrate that cisplatin can have systemic effects in zebrafish larvae, including morphological and locomotory defects, increased inflammatory cytokines, and migration of neutrophils from the hematopoietic niche to other parts of the body. Therefore, this protocol can be used to induce systemic inflammation in zebrafish larvae for studying new therapies or mechanisms of action involving neutrophils.

Distinct macrophage phenotypes and redox environment during the fin fold regenerative process in zebrafish.

In contrast to mammals, zebrafish (Danio rerio) has the ability to regenerate injured sites such as different tissues present in the fin. It is known that cells of the innate immune system play essential roles in regeneration; however, some aspects of the molecular mechanisms by which these cells orchestrate regeneration remain unknown. This study aimed to evaluate the infiltration dynamics of neutrophils and macrophages in the regenerative process of fin fold in regard to the influence of the redox environment and oxidative pathways. Fin fold amputation was performed on transgenic larvae for macrophage-expressed gene 1 (mpeg1), lysozyme (lyz), myeloperoxidase (mpo) and tumour necrosis factor alpha (TNFα) at 3 days post-fertilization, followed by confocal microscopy imaging and measurement of the activities of oxidant and antioxidant enzymes. We observed initially an increase in the number of neutrophils (lyz:DsRed+/mpx:GFP+) and then macrophages (mpeg1+) in the injury site followed by a decrease in neutrophils at 7 days post-amputation (dpa). Moreover, macrophages switch from a pro-inflammatory to an anti-inflammatory profile throughout the process, while the activity of superoxide dismutase (SOD) increased at 1 dpa and catalase (CAT) at 5 dpa. Higher levels of lipid peroxidation were also detected during regeneration. Despite oxidative stress, there is, therefore, an antioxidant response throughout the regeneration of the caudal fin. The present work can contribute to future studies on the development of cell therapies, achieving greater effectiveness in the treatment of diseases related to the formation of fibrotic tissue.

Long-term dexamethasone treatment increases the engraftment efficiency of human breast cancer cells in adult zebrafish.

The host immune system tends to reject xenogenic-implanted cells making tumor development in adult host animal models difficult. Immune system suppression is used for successful xenotransplantation of human cancer cells in many animal models. The studies of cancer development processes in vivo offer opportunities to understand cancer biology and discover new therapeutic strategies. In this context, zebrafish is a model that has been widely applied in the study of human diseases, such as cancer. However, the long-term immunosuppression of these adult zebrafish is still under study as a xenograft animal model for human cancer. This work aimed to evaluate the effects of 21 days of (long-term) exposure of dexamethasone in zebrafish-transplanted with MGSO-3 cells, human breast tumor cell line. Our results show that the animals, while kept on dexamethasone treatment, remained with a 50% reduction in the number of peripheral lymphocytes. In vitro data demonstrated that up to 7 days of dexamethasone treatment did not alter the morphology, proliferation, or viability of MGSO-3 cells. The animals that received a prolonged dexamethasone treatment allowed the engraftment of tumor cells in 100% of the zebrafish tested. These animals also showed tumor progression over 21 days. The experimental group that received only previous exposure to dexamethasone had their tumors regressed after 14 days. In conclusion, the prolonged use of dexamethasone in zebrafish showed a potential strategy for in vivo monitoring of xenograft tumor growth for development studies, as well as in anticancer drug discovery.

Mitochondrial connections with immune system in Zebrafish.

Mitochondria are organelles commonly associated with adenosine triphosphate (ATP) formation through the oxidative phosphorylation (OXPHOS) process. However, mitochondria are also responsible for functions such as calcium homeostasis, apoptosis, autophagy, and production of reactive oxygen species (ROS) that, in conjunction, can lead to different cell fate decisions. Mitochondrial morphology changes rely on nutrients' availability and the bioenergetics demands of the cells, in a process known as mitochondrial dynamics, which includes both fusion and fission. This organelle senses the microenvironment and can modify the cells to either a pro or anti-inflammatory profile. The zebrafish has been increasingly used to research mitochondrial dynamics and its connection with the immune system since the pathways and molecules involved in these processes are conserved on this fish. Several genetic tools and technologies are currently available to analyze the behavior of mitochondria in zebrafish. However, even though zebrafish presents several similar processes known in mammals, the effect of the mitochondria in the immune system has not been so broadly studied in this model. In this review, we summarize the current knowledge in zebrafish studies regarding mitochondrial function and immuno metabolism.

Short chain fatty acids (SCFAs) improves TNBS-induced colitis in zebrafish.

The short-chain fatty acids (SCFAs) are metabolites originated from the fermentation of dietary fibers and amino acids produced by the bacteria of the intestinal microbiota. The most abundant SCFAs, acetate, propionate, and butyrate, have been proposed as a treatment for inflammatory bowel diseases (IBDs) due to their anti-inflammatory properties. This work aimed to analyze the effects of the treatment of three combined SCFAs in TNBS-induced intestinal inflammation in zebrafish larvae. Here, we demonstrated that SCFAs significantly increased the survival of TNBS-exposed larvae, preserved the intestinal endocytic function, reduced the expression of inflammatory cytokines and the intestinal recruitment of neutrophils caused by TNBS. However, SCFAs treatment did not appear to avoid TNBS-induced tissue damage in the intestinal wall and did not restore the number of mucus-producing goblet cells. Finally, exposure to TNBS induced dysbiosis of the microbiota with an increase in Betaproteobacteria and Actinobacteria, while the treatment with SCFAs maintained these population levels similar to control. Thus, we demonstrate that the treatment of three combined SCFAs presented anti-inflammatory properties previously seen in mammals, opening an opportunity to use zebrafish to explore the potential benefit of these and other metabolites to treat inflammation.

Short chain fatty acids (SCFAs) improves TNBS-induced colitis in zebrafish

The short-chain fatty acids (SCFAs) are metabolites originated from the fermentation of dietary fibers and amino acids produced by the bacteria of the intestinal microbiota. The most abundant SCFAs, acetate, propionate, and butyrate, have been proposed as a treatment for inflammatory bowel diseases (IBDs) due to their anti-inflammatory properties. This work aimed to analyze the effects of the treatment of three combined SCFAs in TNBS-induced intestinal inflammation in zebrafish larvae. Here, we demonstrated that SCFAs significantly increased the survival of TNBS-exposed larvae, preserved the intestinal endocytic function, reduced the expression of inflammatory cytokines and the intestinal recruitment of neutrophils caused by TNBS. However, SCFAs treatment did not appear to avoid TNBS-induced tissue damage in the intestinal wall and did not restore the number of mucus-producing goblet cells. Finally, exposure to TNBS induced dysbiosis of the microbiota with an increase in Betaproteobacteria and Actinobacteria, while the treatment with SCFAs maintained these population levels similar to control. Thus, we demonstrate that the treatment of three combined SCFAs presented anti-inflammatory properties previously seen in mammals, opening an opportunity to use zebrafish to explore the potential benefit of these and other metabolites to treat inflammation.