Elsevier

Fish & Shellfish Immunology

Volume 112, May 2021, Pages 159-167
Fish & Shellfish Immunology

Full length article
Escherichia coli induced ferroptosis in red blood cells of grass carp (Ctenopharyngodon idella)

https://doi.org/10.1016/j.fsi.2020.09.036Get rights and content

Highlights

  • E. coli incubation induced 4099 DEGs in grass carp RBCs.

  • The mRNA expression of some cytokines were significantly increased after E. coli incubation.

  • E. coli can induce teleost RBCs cell death through an iron-mediated ferroptosis pathway.

Abstract

The red blood cells (RBCs) of fish make up around 95% of the total peripheral blood cells, and the long-held paradigm is that RBCs are mainly responsible for transporting oxygen. Previous studies have showed that the RBCs can be involved in the immune response against bacterial infection; however, this mechanism remains enigmatic. Here, we explored the structure of grass carp RBCs (GcRBCs). The results showed that the GcRBCs released a pseudopodia-like structure when grown in a 24-well plate, and the transmission electron microscopy (TEM) result showed that GcRBCs contained some organelle-like structures. To further verify the organelle-like structures might be the mitochondria and lysosome which similar to other immune cells, a fluorescent labeling assay was used to verify it. To decipher the antibacterial immunity of GcRBCs, transcriptomic profiling of grass carp RBCs after the incubation with E. coli was analyzed. The results showed that there were 4099 differently expressed genes (DEGs) of GcRBCs upon E. coli incubation, including 2041 up-regulated and 2058 down-regulated genes. In addition, to validate our transcriptomic data, we checked the expression of several cytokines, such as CCL4, CCL20, IL4, IL12 and IFN-α, and the results showed that all the selected gens were significantly up-regulated after E. coli incubation. Furthermore, E. coli incubation induced hemoglobin oxidation and increased the heme in GcRBCs, which further activated the expression of heme oxygenase 1 (HO-1), autophagy related genes 5 (ATG5), and ferritin. In contrast, E. coli incubation inhibited the expression of Ferroportin-1 (FPN1), which increased intracellular iron levels, induced Fenton reaction to release reactive oxygen species (ROS), and activated the ferroptosis signaling pathway in GcRBCs. Herein, we demonstrate that E. coli can induce teleost RBCs cell death through an iron-mediated ferroptosis pathway, which sheds new light on the interaction between bacteria and teleost RBCs.

Introduction

Grass carp (Ctenopharyngodon idella) is one of the most important freshwater fish in China [1]. However, high-density culture of grass carp has been accompanied by increasing incidence of disease outbreaks in recent years, such as hemorrhagic disease and bacterial septicemia, which has resulted in serious economic losses [2]. Therefore, it is important to develop effective strategies against pathogens. In previous studies, most researchers have focused on kidney [3], spleen and mucosal structures [4] that mediated immune responses against pathogens invasion. Blood is an extremely important tissue in animals, and is closely related to the metabolism, nutrition status, and diseases of the body. When the fish is affected by physiological or external pathological factors, the blood could be an important indicator [5,6]. Erythrocytes, as the most numerous cell-type in the blood, are generally accepted as having a primary function in respiration [[7], [8], [9]]. However, Nelson observed that human erythrocytes could also participate in immune responses in 1953 [10]. Erythrocytes are an extremely important, integral part of human blood cellular immunity [9].

A variety of molecular receptors are distributed on the surface of RBCs, which lead RBCs to widely involve in the immune response, such as in modulating T-cell proliferation [5,11], endothelial nitric oxide synthase (eNOS)-like protein activity [12], hormone binding [13], and complement receptor (CR1)-dependent immune complex clearance [14]. In contrast to mammalian erythrocytes, which lack a cell nucleus and organelles, teleost RBCs are nucleated and contain organelles in their cytoplasm, which indicates that the fish RBCs harbor immunity functions [[15], [16], [17]]. In recent years, a set of biological processes have been correlated to immunity in teleost RBCs, such as phagocytosis and presentation [18], interferons production [19,20], and cytokines production [21,22]. The result from rainbow trout suggested that nucleated RBCs are involved in several immune functions, such as leukocyte activation, antigen presentation, and immune cytokine production [23,24]. In our previous study, RBCs of grass carp showed antibacterial activity, which is associated with phagocytosis and ROS generation [18]. However, the exact mechanism remains enigmatic.

To better decipher the antibacterial mechanism of teleost RBCs, the transcriptomic profiling of after E. coli incubation was analyzed in this study. The differentially expressed genes (DEGs) between E. coli-infected and non-E. coli-infected were identified. Furthermore, transcriptomic profiling and qRT-PCR revealed that E. coli incubation could up-regulate the expressions of some cytokine genes, induce hemoglobin oxidation, and increase the heme in GcRBCs, which further increased the intracellular iron levels, finally activating the ferroptosis signaling pathways of GcRBCs.

Section snippets

Experimental animals

Juvenile grass carp (100–150 g) were supplied from a farm located in Guangdong Province, China. Before the experiments were carried out, these fish were maintained at 25–26 °C in a flow-through water supply system for at least two weeks acclimation to our laboratory conditions.

Isolation and purification of RBCs

The GcRBCs were isolated and purified as described previously with minor modifications [18]. Briefly, blood samples were collected from the caudal vein using heparinized syringes and mixed with a 0.7% buffer saline (0.7 g

GcRBCs contain a pseudopodia-like structure

RBCs are the most numerous cell-type in the blood, but unlike in mammalia RBCs, teleost RBCs are nucleated cells. In our previous study, we found that the GcRBCs showed extensive phagocytic activity against different bacteria. These results indicated that the GcRBCs may contain some similar functions to certain immune cells, such as macrophage. To further study the function of GcRBCs, the cells were isolated and cultured in 24-well plates to verify whether the GcRBCs were adherent. Under an

Discussion

Erythrocytes are highly abundant circulating cells in the blood, a major function of which is respiratory gas exchange. In mammals, mature RBCs are flexible, oval, and biconcave disks that lack cell nuclei, organelles and ribosomes [36]. Compared to mammals, teleost RBCs are oval, flattened, and biconvex disks with a cytoskeleton composed of a marginal band of microtubules, a cell nucleus, and organelles in their cytoplasm, which allow them to synthesize proteins and molecules de novo in

CRediT authorship contribution statement

Minxuan Yang: Performed the experiments, Wrote the paper, All authors read the manuscript and approved it in its final version. Zhijie Lu: Performed the experiments, All authors read the manuscript and approved it in its final version. Fenglin Li: Performed the experiments, All authors read the manuscript and approved it in its final version. Fei Shi: Performed the experiments, All authors read the manuscript and approved it in its final version. Fanbin Zhan: Analyzed the data, All authors read

Acknowledgments

This work was jointly supported by the National Natural Science Foundation of China (31902409, 31872606, 31572657, U1701233); Foundation of Guangdong Provincial Marine and Fisheries Bureau (GDME-2018C006, D21822202); Foundation of China-ASEAN Maritime Cooperation (CAMC-2018F); Guangdong Provincial Special Fund For Modern Agriculture Industry Technology Innovation Teams (2019KJ141). The Fund of Southern Marine Science and Engineering Guangdong Laboratory (Zhanjiang) (ZJW-2019-06) JL was

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