Original Contribution
Origin of the phagocytic respiratory burst and its role in gut epithelial phagocytosis in a basal chordate

https://doi.org/10.1016/j.freeradbiomed.2014.02.007Get rights and content

Highlights

  • We discover the phagocytic respiratory burst (PRB) pathway in the epithelial cells of an invertebrate (the amphioxus).

  • The PRB participates in epithelial immunity and epithelial phagocytosis in amphioxus.

  • This study illuminates the origin and functional evolution of the PRB.

Abstract

The vertebrate phagocytic respiratory burst (PRB) is a highly specific and efficient mechanism for reactive oxygen species (ROS) production. This mechanism is mediated by NADPH oxidase 2 (NOX2) and used by vertebrate phagocytic leukocytes to destroy internalized microbes. Here we demonstrate the presence of the PRB in a basal chordate, the amphioxus Branchiostoma belcheri tsingtauense (bbt). We show that using the antioxidant NAC to scavenge the production of ROS significantly decreased the survival rates of infected amphioxus, indicating that ROS are indispensable for efficient antibacterial responses. Amphioxus NOX enzymes and cytosolic factors were found to colocalize in the epithelial cells of the gill, intestine, and hepatic cecum and could be upregulated after exposure to microbial pathogens. The ROS production in epithelial cell lysates could be reconstructed by supplementing recombinant cytosolic factors, including bbt-p47phox, bbt-p67phox, bbt-p47phox, and bbt-Rac; the restored ROS production could be inhibited by anti-bbt-NOX2 and anti-bbt-p67phox antibodies. We also reveal that the gut epithelial lining cells of the amphioxus are competent at bacterial phagocytosis, and there is evidence that the PRB machinery could participate in the initiation of this phagocytic process. In conclusion, we report the presence of the classical PRB machinery in nonvertebrates and provide the first evidence for the possible role of PRB in epithelial cell immunity and phagocytosis.

Section snippets

Animal experimentation ethics statement

The experimental use of amphioxus (B. belcheri tsingtauense) in this research had been approved and supervised by the Animal Care and Ethics Committee (ACEC) of the School of Life Sciences (SOLS) at Sun Yat-Sen University since 2010 (track: SYSU-SOLS-ACEC2010B0022). SOLS ACEC ensures that animal experiments abide by local laws and the International Guiding Principles for Biomedical Research Involving Animals, as issued by the Council for the International Organizations of Medical Sciences.

Animal, bacterial strains, and cells

Comparative analysis of the NOX gene systems

Using NCBI-BLAST and human sequences as seeds, we performed an exhaustive search for NOX-related proteins in the amphioxus genome and other genomes (Drosophila, sea urchin, zebrafish, mouse, and human). Amphioxus has two DUOX proteins that form a co-ortholog pair to vertebrate DUOXs 1 and 2 and a single NOX2 that corresponds to vertebrate NOXs 1, 2, and 3 (Figs. 1B and C and Supplementary Fig. S1). Vertebrate NOXs 1, 2, and 3 are close paralogs, which were possibly created by two rounds of

Discussion

Here we demonstrate that a full-fledged PRB pathway is presented in amphioxus and is required by the gut epithelial immunity. To our knowledge, this is the first report of a functional PRB machinery in nonvertebrates. Our data have shown that oral bacterial infection leads to induction of oxidative stress and an increased level of ROS in amphioxus digestive tract epithelial cells, whereas the reduction of ROS by antioxidant NAC significantly decreases the survival rates of infected amphioxus.

Acknowledgments

This work was supported by Project 2013CB835305 (973), 31171193 (NNSF), 2011CB946101 (973) and 2008AA092601 (863); and projects from the Commission of Science and Technology of Guangdong Province and Guangzhou City, and from Sun Yat-sen University Science Foundation. Data analysis was also supported by the Guangdong Province Key Laboratory of Computational Science and the Guangdong Province Computational Science Innovative Research Team.

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    These authors contributed equally to this work.

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