Skin microbiome of all EB types (2023)

Wound infections affect people with all types of EB. When the skin is injured and damaged, bacteria that usually live in balance with our skin and immune system become part of the problem and contribute to EB symptoms. This research aims to catalogue the types of bacteria and identify patterns in different EB types that may hold clues to treating EB.

Prof Iain Chapple, from the Birmingham Dental School and Hospital, UK, works with the Department of Dermatology, Solihul Hospital, on the bacteria that naturally live on our skin. The different types of bacteria living on, and in, our bodies are called the ‘microbiome’. There are more of them than our own human cells and, when skin is healthy, these bacteria form a balance with each other and the cells of our immune system so no harm occurs. When skin is injured, some bacteria may take advantage of the wound to multiply faster than others and cause more damage. Immune cells that were in balance with the bacteria when skin was healthy are triggered to respond and cause inflammation that may harm the skin further. This project will look at immune cells called neutrophils and how they might regulate the balance between healthy bacteria on undamaged skin and the unhealthy bacteria that cause harm in all types of EB.

About our funding:

Research leader	Prof Iain Chapple
Institution	Birmingham Dental School and Hospital, UK
Type of EB	All types of EB
Patient involvement	At least 8 people each with DEB, JEB and EBS
Funding amount	£296,289
Project length	3 years (extended due to Covid)
Start date	June 2018
DEBRA internal ID	Chapple1

Final progress summary 2023:

The new knowledge uncovered in this project describes which microbes (bacteria, fungi and viruses) live on skin. The number and type make up the ‘microbiome’ of skin. Comparing changes in the microbiome of blistered and unblistered skin of people with and without different types of EB identified patterns that changed during wound healing.

The immune cells (neutrophils) from blood samples were found to be more active, especially in JEB, so medications that calm down the behaviour of these cells could be explored as potential treatments.

A specific protein found in blister fluid from people without EB that was missing from people with EB could also be a new target for therapies.

In December 2023, Prof Chapple published results from work funded by DEBRA UK titled Genotype-phenotype correlation in Junctional Epidermolysis Bullosa: signposts to severity. This was also reported in an article for a general audience.

Our research has advanced the current understanding of the roles of skin microbiome, blister protein content and neutrophil functional behaviour in EB wound healing. This can lead to development of novel dressing and rebalancing therapies such as the development of pre- and probiotics.

Prof Iain Chapple

Progress summary 2022:

94 skin swab samples have been collected and microbiome analysis has begun. Initial results show a difference in types of bacteria living in different parts of the body and a difference between those on the skin of people with DEB and JEB when compared to people without EB.

16 samples of EB blister fluid have been collected and studied to determine which immune system proteins (cytokines) are increased or reduced. This has provided some evidence that the immune response may be causing damage rather than healing in some EB types.

A poster summarising the progress to date was presented at the Society of Investigative Dermatologists in May 2022.

About our researchers:

Lead researcher: Prof Iain Chapple is Professor of Periodontology and Head of the School of Dentistry at the University of Birmingham UK and Consultant in Restorative Dentistry.

He leads a strong team as part of Birmingham’s Periodontal Research Group and is Director of Research for the Institute of Clinical Sciences at the University of Birmingham. Iain runs a national clinical oral and dental service for adult EB patients in close collaboration with Prof Adrian Heagerty, a Consultant Dermatologist and EB expert. Iain was awarded the Charles Tomes medal by the Royal College of Surgeons in 2012 for his research and also the International Association of Dental Research Distinguished Scientist for Periodontal Research in 2018.

Co-researchers: Dr Sarah Kuehne, Dr Josefine Hirschfeld, Dr Melissa M Grant and Prof Adrian Heagerty

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Why this research is important:

We are extremely excited by the DEBRA funding for this project, especially given the strong support and interest from our EB patients and delegates at the 2019 DEBRA Members' Weekend. This will enable us to answer fundamental questions about what bacteria live on the skin and colonise wounds, and what effect they may have on how our immune system responds to them and affects how blister sites heal. Ultimately, we hope this will help in the future to develop new approaches to therapy.

To find out more information on this project, you can view the DEBRA Members' Weekend 2018 presentation here.

Researcher's abstract:

Grant Title: Characterisation of the skin microbiome and investigation of neutrophil function in epidermolysis bullosa patients.

What is being investigated?
This research will investigate the different bacteria that are present on the skin of people with epidermolysis bullosa (EB). The human body has twice the number of bacterial cells compared with human cells hence we are actually a complex mix of human and bacterial elements, and health requires our immune system to live in harmony with our bacteria. Most of these bacteria are friendly. However, in EB wounds, bacteria may change and cause infections, delay wound healing, resulting in scarring. Currently, the bacteria living on the skin of people with EB are not well known. This group plan to investigate which bacteria are present in the skin of people with EB and how they behave.

Why is this being investigated?
The human body has specialized immune cells to protect it against infections. Their role is to locate and destroy any foreign cells or bacteria that can make us unwell. In health, we have health-promoting bacteria, that live quite happily with our immune system, however, if the environment changes (by for example trauma that causes a blister), different bacteria can start to grow and this can also upset our immune system. In some diseases, when this happens, the immune cells do not function as they should and can over-react to certain bacteria, in a way that also damages our tissues and can delay wound healing.

Why is this important?
This research is key to understanding whether particular immune cells, called neutrophils, work properly in epidermolysis bullosa (EB). Many people affected by EB often suffer from a range of infections and this is a hint that their immune system may not be working efficiently. Investigation of how neutrophils work may provide evidence to design treatment options that can improve their ability to clear disruptive bacteria, allowing the healthy bacteria to come back and re-establish that important balance between our “healthy” bacteria and our immune system.
These two aspects will be studied because in other diseases it is known that bacteria and the immune responses to them are closely linked. Both bacteria and immune cells release signals that influence skin healing, which can cause harm to the skin and can make us more susceptible to other infections. There will also be signals or “molecular” messages that help to heal the skin and a better understanding of these will facilitate development of treatments that stop the harmful signals but enhance the helpful ones. By bringing these areas of research together, we hope to advance the development of effective treatment options for wound healing in people with EB in the future.

Researcher's final progress update:

The outer surface of the skin is inhabited by many microbes, such as bacteria, fungi, and viruses, most of which are harmless and friendly. However, when there is trauma that causes a blister, these microbes may behave differently, causing infections, compromising wound healing, and creating scars. The human body has specialised immune cells that protect us from infections and help us to fight them, detecting the microbes and destroying them, so the wounds can heal.

In some diseases, the immune cells do not behave as they should and can over-react to certain microbes, causing a delay in wound healing and damaging our own tissues as a side effect. Our project aimed to discover which microbes inhabit the skin in EB, if they change during wound healing, and whether a specific type of immune cell, the neutrophil, works correctly in people with EB. EB has traditionally been thought of as simply an inherited condition that results in skin blistering with mild trauma. However, we propose that delayed healing of skin wounds in EB may result, in part from these key immune cells not working properly.

The study of the skin microbes can be complex. First of all, due to the low numbers of microbes on the skin, it is technically challenging sampling them to conduct any analysis. Secondly, the microbial communities change in composition throughout the body: the composition of the skin microbiome on the hands is different from the one of the feet. Hence, we had to be sure we used the right sampling method, such as cotton swabs, and we were able to identify the microbes, before taking samples from EB patients.

Once we proved our methods worked, we provided swabbing kits and asked people with and without EB to take skin swabs when a blister formed so they can transfer the microbes from their skin to the swab. By looking at the DNA, which works as a barcode, we were able to say which organisms were present and what they were doing.

We not only made observations on the unaffected skin, but we also have observed differences on the skin of blisters, and how these communities change during wound healing. We have found that the skin of EB patients has more microbes belonging to a specific group called “bacillales”, and that the composition of this group was specific for each EB subtype. We also saw differences in the fungi that inhabit the skin of people with EB and how these change during wound-healing.

Another important aspect of our project is the understanding of what happens in the blisters and how the immune system of people with EB responds to microbes. We asked our volunteers to collect fluid from blisters and we analysed the blister fluid to explore and quantify proteins found in them. We have shown that there are potential differences in these proteins that may tell us about how the blisters are forming or healing. Specifically, we identified an important protein that was not found in people with EB but was found in people without the condition. We now aim to understand the role of this protein and if it could be the focus of a novel treatment target. We also detected protein signatures for all the different types of blister fluid analysed, allowing us to explain differences between EB subtypes.

We noted differences in the behaviour neutrophils, a specific type of immune cell found in the blood. These cells are the first responders during infection and wound healing, and we observed how they responded to bacterial stimuli in patients with EB. We found heightened neutrophil responses, especially in people affected by junctional EB compared to people without the condition. This exaggerated behaviour is associated with tissue damage in the host and may also explain or contribute to reduced wound healing. These effects can be mitigated with natural products that prevent such damage, which may represent another novel treatment modality.

Taken together our findings have uncovered which microbes inhabit the skin of EB patients, how they change during wound healing, and how the body responds to the bacteria. Thanks to this new knowledge, it may be possible to develop novel therapies or wound-dressings using pre- and probiotics and/or antioxidant micronutrients, or protein replacement to restore a healthy and functional skin flora, limiting the risk of infection in people living with EB and helping promote wound healing. (From 2023 final progress report.)

Researcher's progress update 2022:

As soon as pandemic measures were eased, enrolment of patients with EB progressed to completion.
Variables that may influence and affect DNA extraction for the EB microbiome were identified and tested and the optimal protocol for the analysis of EB patients was defined.
The defined protocol was used to collect and extract 94 skin swab samples.
44 samples underwent sequencing and bioinformatic analyses. Partial analysis of these demonstrated a difference between the microbiome of people affected by junctional EB or dystrophic EB and healthy individuals.
Body sites need to be considered in order for a complete insight into the skin microbiome in EB.
The remaining samples were successfully processed and sequenced and are currently undergoing bioinformatics analyses.
We have analysed 16 blister fluid samples for cytokine content and protease activity.

The human skin is inhabited by many microbes such as bacteria, fungi, and viruses, most of which are harmless and friendly. However, when there is trauma that causes a skin blister, these microbes may behave differently, causing infections, compromising wound healing, and creating scars.

The human body has specialised immune cells that protect us from infections and help us to fight them, detecting the microbes and destroying them. In some diseases, the immune cells do not behave as they should and can over-react to certain microbes, causing a delay in wound healing and damaging our own tissues as a side effect.

Our project aims to discover which microbes inhabit the skin in EB, whether a specific type of immune cell, the neutrophil, works correctly in people with EB, and how proteins found in blisters change in EB.

In the reported study, we have optimised the method to look at the microbes that live on the skin and have used it to collect skin swabs from people with and without EB. We provided swabbing kits and asked people take skin swabs when a blister formed so they can transfer the microbes from their skin to the swab. By looking at the DNA, which works as a barcode, we will be able to say which organisms are present and what they are doing.

We have 94 samples that need to be analysed, and we are more than halfway through this long and complex analysis. From a first look into the data, we observed that people affected by dystrophic EB have a different microbiome than people without the condition. In particular, a group of bacteria, the proteobacteria, is more abundant in people with the skin condition. We observed a similar trend in people affected by junctional EB. By looking in more detail at which organisms inhabit the skin of people affected by JEB, we also highlighted the importance of considering at which body site the blister formed. In fact, the microbiome of the ankle looked very different from the one of the arm, even in people without EB. Hence, it is crucial to compare samples from the same body site.

As we are proceeding with our analysis, we will obtain greater insight into the microbiome of people with EB, and we will be able to identify the potential functions of bacteria and therefore also potential novel therapeutic targets.

Additionally, we have explored proteins found in the blisters of patients with EB. We have shown that there are potentially differences in these proteins that may tell us about how the blisters are forming or healing. More work is on-going to explore this further. (From 2022 progress report).