Using zebrafish to model phage therapy

Last week, we introduced a new paper describing use of a cystic fibrosis zebrafish model to test phage therapy outcomes. This week, we dive (pun intended) deeper into this paper to highlight this useful new animal model for the phage therapy community.

Why zebrafish?

Zebrafish are becoming recognized as a promising animal model, especially for human biology studies, for a variety of reasons. Long story short, they're cheaper than mice, they reproduce more rapidly, you can easily manipulate their genes, they're transparent (so you can fluorescently label and then follow what happens to their cells, live, under the microscope), and mutation of many of their genes leads to similar disease phenotypes as we see in humans.

The authors of the paper we're profiling here (Cafora et al. 2019) wanted to test whether phages could treat Pseudomonas aeruginosa infections in a cystic fibrosis (CF) zebrafish model. Zebrafish have a similar chloride ion pump (CFTR, cystic fibrosis transmembrane regulator) to humans, and it can be mutated in zebrafish, thus mimicking human CF.

A bit of background on CF

CF patients have mutations in CFTR, so they have overly sticky mucus in their lungs, and are highly prone to infection throughout their lives. P. aeruginosa is a common culprit. CF patients end up receiving antibiotics extremely frequently, leading to antibiotic resistance, and so phage therapy may be a good option for CF patients.

But wait, do zebrafish have lungs?

No, they do not, which complicates use of zebrafish as a model for CF, since the disease primarily leads to lung infections. But zebrafish have mucins, which are the sugar-rich proteins that make up mucous, and zebrafish mucins are similar to human mucins. Also, CF affects other organs, not just lungs (e.g. the pancreas). Zebrafish do have a pancreas, and its function IS affected by the CFTR mutation, so the authors make the point that the fish is still a reasonably good model for studying CF.

What did the authors do?

Infection of CF zebrafish with P. aeruginosa

They generated zebrafish embryos with CFTR mutations and infected them with a commonly studied P. aeruginosa strain called PAO1. They checked, and P. aeruginosa did indeed infect the CFTR mutant fish more effectively than wild type fish, just like P. aeruginosa infects people with CF more effectively than people without CF.

Phage treatment of the P. aeruginosa infections in the CF zebrafish

They used phages (and antibiotics, as a control, and also in combination with the phage treatment) to see if they could reduce the zebrafish mortality. They chose four phages, all able to infect the P. aeruginosa strain (which they GFP-labeled so they could follow its spread and growth in vivo), and used them together in a cocktail. They found that the phages did improve survival of the zebrafish, both in the wild type and CF embryos, and they concluded that combining phages with antibiotics led to the best results.

Looking at the immune response to phage therapy

There hasn't been a lot of research into the immune response to phages during phage therapy nor under natural conditions. This is likely to become a more active area of research, especially as phage therapy becomes more commonly administered.

What kind of immune system does a zebrafish have?

Within days, zebrafish embryos have a fully developed innate immune system, and for their first 30 days, they ONLY have an innate immune system. So if you study them during this period, you don't have to worry about the adaptive immune system, and can study the innate immune response in isolation. This is useful, because the innate immune system is the critical immune system at play when it comes to human lung infections.

What did the authors find?

Cafora et al. looked at the zebrafish immune response during their phage therapy experiments. They measured gene expression changes in two immune cytokines: IL-β and TNF-α, which are both indicators of inflammation.

• P. aeruginosa + / - phage: In both wild type and CF fish, they saw that P. aeruginosa provoked an immune response, and that the phage cocktail reduced this immune response.

• Naive fish: When neither the P. aeruginosa nor the phage was there, the CF fish appeared to exhibit a constitutive inflammatory state.

• Phage alone: Phages administered alone (without P. aeruginosa) reduced the constitutive inflammatory state in the CF fish.

Conclusions

These authors validated a new model for testing phage therapy in a CF animal (and while they were at it, they also generated some interesting new leads on how their phages might interact with the innate immune system).

What makes this paper unique?

Other studies have previously evaluated phage therapy (against Vibrio anguillarum and Aeromonas hydrophila) within zebrafish, but this is the first to evaluate P. aeruginosa/phage pairs in zebrafish, and the first to do so in a CF-mimicking context in any animal model.

Importance of work like this

The zebrafish model is cheap, genetically tractable, and well-understood. It allows real time visualization of cells (including infecting bacterial strains!), and phages are able to infect cells while inside the fish, making it particularly useful for phage therapy experiments.

An added bonus with zebrafish is the ability to genetically manipulate it to mimic genetic diseases in humans, so we can test phage therapy in those contexts, as has been done here for CF.

At a time where we need to start collecting as much data as possible about how phages infect their hosts in vivo and how they impact immune responses (and not just in one or two phages, but in as many types as possible), using the zebrafish model could represent a great opportunity to start doing these experiments without spending a lot of money.

So you're thinking of using zebrafish to evaluate your phages?

Here are some extra resources:

• A great blog post from Elizabeth Burke at the NIH that explains why zebrafish make good animal models for human diseases.

• A methods paper showing how to infect zebrafish embryos with Pseudomonas, and how to observe them under the microscope.

• A paper showing successful phage treatment of Aeromonas hydrophila in zebrafish

• A paper showing successful phage treatment of Vibrio anguillarum in zebrafish

• The NIH Zebrafish Core: A place to get help with your zebrafish experiments: "The core's goal is to help researchers of any expertise perform zebrafish experiments aimed at illuminating basic biology and human disease mechanisms."

• ZFIN: A database full of information and ways to connect with zebrafish researches, get your questions answered, find mutant strains, etc. (Also check out the ZFIN forum!)

• A comparison of the zebrafish genome and the human genome (sneak preview: around 70% of human genes have an obvious zebrafish counterpart)

Main source for this post:

Cafora, M., Deflorian, G., Forti, F., Ferrari, L., Binelli, G., Briani, F., ... & Pistocchi, A. (2019). Phage therapy against Pseudomonas aeruginosa infections in a cystic fibrosis zebrafish model. Scientific Reports, 9, 1-10. https://doi.org/10.1038/s41598-018-37636-x