An arginase 2 promoter transgenic illuminates anti-inflammatory signalling in zebrafish

Background:

Macrophages and neutrophils are key immune cells that coordinate the initial immune responses to infectious and inflammatory stimuli and promote healing and restoration of homeostatic conditions following such insults. It is generally thought that neutrophils migrate to the stimuli first, followed by macrophages, with both cell types adopting a pro-inflammatory phenotype to combat infection and/or inflammatory stimuli. However, a sustained pro-inflammatory response may become detrimental, leading to tissue damage. Careful regulation of the pro- and anti-inflammatory aspects of the immune response is therefore required. It is well established that macrophages may adapt throughout the immune response to take on an anti-inflammatory phenotype and therefore promote healing and homeostasis. Further, recent studies suggest that some neutrophils may also develop an anti-inflammatory phenotype.

The zebrafish model has been extensively and successfully used in infection and inflammatory research. Zebrafish reporter lines have been generated to visualise signalling of pro-inflammatory cytokines, such as TNFα and IL-1β. Until now, an anti-inflammatory zebrafish reporter line has not been produced. In this study, the authors examine the use of the promoter of arginase, an anti-inflammatory enzyme, to drive GFP fluorescence to enable the visualisation of anti-inflammatory signalling in macrophages and neutrophils during infection and wound-healing responses.

Key findings:

The authors first examined the expression of Arginase in zebrafish and determined that of two isozymes, arginase 2 (arg2) is the most highly expressed in zebrafish macrophages and neutrophils. Therefore, the arg2 promoter was used to create the transgenic zebrafish GFP reporter, whereby arg2 expression leads to production of GFP protein.

In healthy zebrafish, macrophages or neutrophils were not GFP positive, indicating that at rest arg2 is not expressed in these cells. In order to examine whether anti-inflammatory arg2 expression occurs in infection and inflammatory immune responses, the authors tested the reporter line in wound and infection models.

In response to tailfin wounds, unlike macrophages, a population of neutrophils were found to be GFP-positive (therefore expressing arg2) very early in the inflammatory response. Interestingly, a small number of macrophages were found to express arg2 at late stages of the immune response. This suggests that both neutrophils and macrophages may adopt an anti-inflammatory phenotype during immune responses at the site of a wound.

The authors next examined arg2 expression in macrophages and neutrophils in several zebrafish infection models; Mycobacterium marinum, Candida albicans and Cryptococcus neoformans were studied. Neutrophils expressing arg2 were observed in infections caused by all three pathogens, with a small number of arg2 macrophages observed in M. marinum and C. neoformans infections.

Together, these data suggest that anti-inflammatory signalling occurs early in immune responses to wounds or infection, primarily in neutrophils, but is also apparent in a small proportion of macrophages. The authors suggest that early anti-inflammatory signalling may help to regulate the pro- and anti- inflammatory aspects of the immune response.

Why I chose this Preprint:

I chose this preprint as I have a background in zebrafish infection and inflammation research. This study improves our understanding of the regulation of pro- and anti-inflammatory signalling in immune responses. The generation of a zebrafish reporter for anti-inflammatory signalling is a valuable new tool, and may be beneficial for further research in a wide range of studies. In addition, the authors discuss how pathogens may subvert the host immune responses to promote an anti-inflammatory environment for their own survival. Arginase may therefore be a useful therapeutic target in future infection studies.

Questions to the authors:

1. There were a limited number of macrophages expressing arg2 late in the tailfin wound assays. Do you think that these macrophages may have phagocytosed the GFP-positive neutrophils found at the wound site?
2. In mock-infected zebrafish, arg2 expression was not observed in immune cells. Was this based on a location where an infection is usually established? Were any arg2 positive neutrophils observed at the ‘wound’ of the injection site?
3. Which zebrafish infection and inflammation models, other than the ones tested, do you envisage the arg2 zebrafish reporter being most useful for?

 

doi: https://doi.org/10.1242/prelights.31775

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