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The pathogen Shigella is the most common cause of bacterial diarrhoeal deaths globally. It is estimated to have caused , with 63% of these deaths in children under five years old.

There have been many efforts to develop a vaccine against Shigella but most candidates fail to demonstrate long-lasting effects in clinical trials. As with meningococcal disease, it is also a challenge to protect against all the different Shigella serotypes. 

Researchers from the London School of Hygiene & Tropical Medicine have been examining the problem of Shigella from another angle: studying if it is possible to train immune cells not normally stimulated by vaccines to destroy this pathogen.

They have found an unusual ally in the fight against human disease: the zebrafish. We asked Margarida and Serge about their new research published in and how it might one day help to protect humans from deadly pathogens.

Why are zebrafish useful for studying diseases that affect humans?

Although zebrafish and humans look very different, they share more than 70% of their genes. Most importantly in our case, the key components of zebrafish and human immune systems have changed very little since they first evolved which makes the zebrafish model suitable for studying infectious diseases that affect humans. 

The zebrafish has key advantages over other vertebrate models, for example, we can study innate immunity in isolation (the innate immune system is present from 1-day post-fertilisation, whereas adaptive immunity is only fully developed from 3 to 4 weeks post-fertilisation), and at the larval stage zebrafish are fully transparent – which means that we can infect zebrafish with fluorescently-labelled bacteria and study their interactions with zebrafish immune cells under the microscope non-invasively.

Also, the zebrafish genome is easily amenable to modification, and using newly developed tools such as CRISPR, we can manipulate the immune system for our work.

What is the difference between innate and adaptive immunity?

Our immune system has two arms: the innate and the adaptive. The adaptive system is well known to develop a memory of prior encounters with pathogens and to produce specific antibodies to fight new infections. This is the fundamental mechanism underlying many successful vaccines. 

On the other hand, the innate immune system was traditionally not recognised as having a memory of past infections. Only in recent years has scientific evidence suggested that innate immune cells have better and more robust responses to infection if the host has been previously exposed to an infection. This is the exciting concept of trained immunity, and the memory is preserved in the genome of cells by epigenetic changes. 

These epigenetic modifications alter the expression of genes without changing the DNA sequence. The epigenetic memory is generated in hematopoietic stem cells (primitive cells that can differentiate into all blood cells), and during differentiation into blood cells (such as macrophages and neutrophils) the information is passed on.

What are neutrophils and what do we know about the role they play in fighting infection?

Neutrophils and macrophages are white blood cells and a first line of defence of our immune system. These cells are usually the first to reach an infection site where they phagocytose (eat) the invading microbes. Both cell types have mechanisms to kill ingested microbes, but the killing activity of neutrophils is more potent. For example, neutrophils can produce neutrophil extracellular traps (NETs) to capture pathogens and reactive oxygen species (ROS, highly reactive molecules) to degrade pathogens.

Unlike macrophages, neutrophils are very difficult to study in the lab because they do not proliferate, so they cannot be expanded in vitro (i.e. in a petri dish), and can only be maintained for a short period of time. There are a few immortalised neutrophil lines (similar to the immortalised epithelial cell line called HeLa) that are stable in vitro and can be used to manipulate their genome. However, studies have shown that they do not fully behave like freshly isolated neutrophils from donors. Therefore, for our work to better understand the role of neutrophils in host defence, we turned to zebrafish. 

How did you train the zebrafish immune system to combat Shigella?

To train the zebrafish immune system, we exposed innate immune cells to a small dose of Shigella (that neutrophils and macrophages could easily clear) by injecting bacteria into zebrafish larvae. The immune system was left to rest for two days, and we then injected a higher dose (normally lethal) of Shigella to observe if there were differences in host survival. 

We were amazed to observe that zebrafish larvae first exposed to Shigella were protected against secondary infection, showing improved bacterial clearance. Because of the key role of neutrophils in both zebrafish and human Shigella infection, we hypothesised that this improved protection could be due to trained neutrophils. This is a very exciting and novel research avenue since there is very little literature reporting training in neutrophils.

What difference did the training make in how the zebrafish immune system reacted to a Shigella infection?

It was incredible. We compared the immune responses from Shigella-trained zebrafish larvae to naïve larvae (i.e. those that had not been exposed to any pathogen) upon a lethal infection of Shigella. We found that training significantly contributed to host protection that was linked to 1) lower bacterial burdens (because immune-trained neutrophils can better control Shigella infection), and 2) faster resolution of inflammation. This is the first time that trained immunity with Shigella was demonstrated in zebrafish neutrophils.

What would you hope to learn by expanding this research into adult zebrafish?

For most of the work we have done, the interval between training and reinfection was two days. This interval was long enough to see resolution of the first inflammatory stimulus (i.e. first infection with Shigella) and the impact of trained responses. However, to understand if the epigenetic changes that occurred in neutrophils could be maintained throughout the life of the zebrafish, we plan to infect immune-trained zebrafish at different intervals. This could also inform how trained innate immune cells interact with adaptive immune cells (such as B- and T-cells) to control infections.

If these epigenetic changes are maintained and long-lasting, one exciting outcome would be to observe the transmission of innate immune memory to the offspring of immune-trained zebrafish (a novel concept in the field called ‘transgenerational immunity’).
 
Could we potentially train the human immune system to fight Shigella?

We think there is great potential in using these findings to train the human immune system. To understand epigenetic changes in zebrafish, we performed a technique called ChIPseq (chromatin immunoprecipitation sequencing) in trained neutrophils. It will be interesting to perform the same analysis on human neutrophils trained with Shigella and to search for a similar epigenetic signature. From ChIPseq analysis, we discovered that mitochondrial ROS plays a key role in the enhanced protective mechanism of Shigella-trained neutrophils. Future therapeutic strategies in humans could manipulate the production of mitochondrial ROS in neutrophils by using pro-oxidants (such as SkQN or MitoK3). 

However, there remain significant hurdles. For example, oral whole-cell vaccines have been tested against Shigella, but they were not very effective and often using either killed or attenuated bacteria – and we demonstrated that for the immune system to be efficiently trained, we need live and virulent bacteria. The other problem concerns our current knowledge of trained immunity because the enhanced pro-inflammatory responses upon the new infection may not return to basal levels and remain chronically activated. This can cause other serious diseases beyond infection.

Could ‘re-programmed’ neutrophils be used to fight other pathogens?

Yes. A key aspect of trained immunity is that protection induced by immune training is non-specific, meaning that innate immune cells can better control a wide variety of infections with microbes that resemble the training agent or that are completely different (e.g. training with a bacterium to protect against a pathogenic fungi or virus). In our study, we show that Shigella immune training can protect against bacterial infections other than Shigella, including Pseudomonas aeruginosa and Staphylococcus aureus (also important human pathogens and antimicrobial-resistant threats).

What sort of new treatments or therapies for humans do you hope your work will ultimately lead to?

First we aim to resolve fundamental understanding of mechanisms underlying innate immune training to then inform therapeutic strategies on how trained cells can be used to better protect against Shigella, especially in children. 

We hope that this work can inform the development of more effective vaccines against shigellosis, possibly by creating an adjuvant (substance formulated as part of a vaccine) to be used in a Shigella vaccine to boost innate immune responses. In this way, vaccinated people would trigger both innate and adaptive immune responses to more effectively combat Shigella infections.