With over half the cells in the human body being bacterial rather than human, and over 99% of the genes in the human body being microbial encoded, medical microbiology is an essential component of future healthcare – and two of the principal areas of study are the gut and the lung.
Clinical trials from Atlantia support human immunity by investigating the impact that microbes have on future health and immune regulation. Bacterial-induced immune regulation has significant indications for potential developments in healthcare, including new treatment and diet possibilities for the regulation of afflictions such as allergies and asthma.
Three experts - Dr. Liam O’Mahony, Dr. John MacSharry and Dr. Barry Skillington – were interviewed in conjunction with Atlantia Food Clinical Trials to discuss microbial interactions with host immune systems, specifically relating to the gut microbiome, the lung microbiome, and an overview of clinical trials within the immunity among other areas.
Please could you introduce yourselves and outline your roles?
BS: My name is Barry Skillington, and I am the Chief Commercial Officer here with Atlantia Food Clinical Trials.
LOM: I am Liam O’Mahony, and I am the Professor of Immunology at the Department of Medicine and Microbiology, as well as the Principal Investigator at APC Ireland.
JM: Hello. My name is John MacSharry, and I lecture medical microbiology at UCC. I am also a member of the APC Microbiome Ireland Research Team and a Research Partner of Atlantia Food and Clinical Trials.
What is Atlantia’s research focus, and why is it so important?
LOM: As most people know, human beings have a great deal of bacteria living both in us and on us. In fact, at least half the cells in the human body are not human at all but bacterial. From a gene-content point of view, we know that of the several million genes present within the human host, of or slightly less than 1% is human-encoded.
This means that over 99% of the genes in the human body are microbially encoded. These genes encode for a great deal of enzymatic machinery that generates a whole variety of different metabolites. Many of these metabolites can be detected by the immune system and interact with the immune system as well as other systems in the body.
In order for the immune system to tolerate the presence of this wide variety of foreign antigens in the body, it must have extremely potent tolerance mechanisms. It is our clinical opinion that many of the signals coming from conventional bacteria are what drive these tolerance signals. That is a short explanation as to where our research focus is situated: simply trying to understand what these tolerance signals are that derive from bacteria. Furthermore, in cases of inflammatory disease, we need to understand if some of these tolerance signals are missing - and, if they are, how they can be replaced.
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What are the components involved in the tolerance response process, and why are these important to analyze?
LOM: There are many cells involved in the tolerance response, both from the innate immune system and also from the adaptive immune system. Ultimately, it is important to drive and promote a lot of B regulatory cells and T regulatory cells. We also know that microbiota-derived signals can have an influence on all the different cell types that are involved in this process: the epithelial cells, the macrophages, the ILCs, the dendritic cells, and so on.
The disease area in which I personally am most interested is allergies and asthma: it is widely known that on sites like the skin, the gut, and the lungs, the composition of the microbiota is very different than that which is associated with topic dermatitis, asthma, or with food allergies. In some cases, these differences are present before the diagnosis of the allergy or asthma. Therefore, it is possible that the missing microbes, or the changes in microbes at these body sites, may be contributing to the induction of allergies or asthma.
This may be particularly true early in life when we know many factors are important for the acquisition and development of a full adult-like community structure of the infant microbiome over the first two years of life. Many of these exposures that we know are important for microbiome development and are important risk factors for the development of allergies and asthma. One particular area is the influence of diet.
Why is diet so significant, and what impact does it have on the composition or the metabolic activity of microbes within the gut?
LOM: Diet has a hugely significant effect on both the composition and the metabolic activity of microbes within the gut. One of the more dramatic changes in the gut microbiome, from a dietary point of view, accompanies the introduction of non-digestible fibers. These are fibers that we cannot digest but that microbes in the gut can digest.
This digestion or fermentation results in the generation of short-chain fatty acids like butyrate, propionate, and acetate. In this study, we looked at one-year-old fecal samples from the PASTURE/Efraim birth cohort study. It was found that children who had the highest levels of butyrate or propionate at one year of age had the lowest levels of asthma amongst a whole range of different allergies and allergy sensitization by the time they were six years of age.
This was able to be correlated with early-life consumption of yogurts and vegetables. Perhaps, therefore, feeding a baby with the right microbes and vegetables offers the fibers for fermentation to generate these short-chain fatty acids in later life. Using mechanistic and experimental models, it could be shown that at least part of this protective effect was due to the induction of these regulatory T cells, partially through G-protein-coupled receptors.
Why is histamine an important metabolite to study?
LOM: The microbiome produces many different types of metabolites, of which a particularly interesting example is histamine. As may be widely recognized from the name, histamine is important within an allergic response, but in reality, there are four different receptors in the body that detect histamine – each of which can produce a very different outcome when activated. The pro-allergic receptors are histamine 1 and 4 receptors, whereas the histamine 2 receptor, for instance, is a regulatory receptor.
We have therefore been investigating whether bacteria can make histamine – and, if so, whether they can influence this system.
What we have found is that a significant number of bacteria within the human gut produce histamine, and patients have higher levels of histamine-secreting bacteria.
One of the bacterial strains that we found with the highest secrete of histamine was a Morganella morganii species. It was found that patients with severe asthma (defined as those on high-dose corticosteroids but still symptomatic) had the highest levels of this histamine-secreting bacterium, therefore suggesting that - at least in some asthma patients – some of the symptomatology could be related to the high levels of this histamine-secreting microbe in the gut.
Having created many experiment models to look at the mechanisms, the consensus today is that histamine deriving from gut microbes can have either protective or damaging effects, depending on both the metabolic activity of the histamine degrading enzymes within the gut of the host and the expression of the different histamine receptors on different host immune cells. It is, therefore, not solely the production of histamine that is important for the end effects but also how the immune system interprets the microbial-derived histamine.
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Are there any other factors changing the microbiomes of these patients?
LOM: In the study of patients with severe asthma, it was discovered that a microbe named Akkermansia muciniphila is significantly reduced in the gut microbiome. Again, these are patients who typically are on high dose corticosteroids but are either not improving or at least still symptomatic.
Once again, by using experimental models, we wanted to investigate if Akkermansia in the gut could have any effect on the lung. What we discovered was that Akkermansia in the gut has a very significant effect on lung function: with increasing doses of Methacholine, the airway hyperreactive response is attenuated.
The flow cytometric assessment of the type of eosinophils that are coming into the lungs was also performed. The Siglec-F(high) eosinophils or the IL-5 dependent eosinophils are the ‘dangerous’ eosinophils within the lungs of asthma patients. These are dramatically reduced in the experimental models in which Akkermansia was consumed. Therefore, as a new microbe for the investigation and possible treatment of asthma, Akkermansia is certainly one worth investigating further.
Is there any impact seen in patients who additionally suffer from a second disease?
LOM: If we were to consider the influence of obesity, as some asthma patients are also obese, we know that obesity can trigger an inflammatory response associated with obesity and, consequently, a change in the microbiome. Additionally, obesity is an immune-mediated disease. It is important to investigate what occurs in people who are both obese and who have asthma: do they have changes representative of both diseases, or do these signatures cancel each other out?
In this case, obese asthma patients have both signatures. This means that they have evidence of both being obese and of having asthma. The obese asthmatic person has many more microbiome changes than the non-obese asthmatic person and the non-asthmatic obese person. From an inflammatory-response point of view, when the RNA sequencing of peripheral blood from these individuals was examined, it could be seen that the obese asthmatic had a much more exaggerated inflammatory response regarding enrichments of different innate or antibacterial inflammatory responses.
What are the future areas of focus likely to be regarding bacterial-induced immune regulation?
LOM: Essentially, microbes are very important for immune regulation. Our particular focus is on the induction of immune tolerance: we believe that there is a non-redundant role in the bacterial-induced immune regulation I have discussed. Certain inflammatory responses or disorders may be related to a lack of tolerogenic signals due to either missing microbes or microbes which are not generating the right metabolites due to a change in diet - for example, a lack of fibers that would drive short-chain fatty acid production.
We believe that many of these signaling systems have developed over millennia and are well-integrated. The microbiome is extremely important, particularly in allergy and asthma. As for the future, there are a number of options to assist sufferers of such illnesses – whether that is through individual microbes, their components, metabolites such as short-chain fatty acids or even dietary interventions associated with some of these microbial interventions.
What is the difference between sampling the biomass of microbes in the gut and sampling those in the lung?
JM: The lung is slightly different from the gut in that there is much lesser biomass of the microbes. When the lung is sampled, a Bronchoalveolar Lavage must be performed to directly sample the lung, which is a fairly invasive procedure. This means it can be very difficult to obtain healthy controls in this procedure. There is also the occasional possibility of getting salivary microbes aspirating into the lung; however, there is a distinct lung microbial flora, and we do the sampling by Bronchoalveolar Lavage.
We have performed several lavages in collaboration with Dr. Des Murphy at Cork University Hospital. Typically, when we do a lavage, we examine the immune cells, such as the macrophages, eosinophils, and neutrophils, to check what is happening in the lung.
In a patient who has a lack of information or lung, the majority of the cells will be macrophages. However, when there is inflammation – be it a Th1, which is a neutral mediation inflammation, or a Th2, which is an eosinophil-mediated inflammation – we tend to find that there is an increase of these cells in the lung wash. This increase results in a reduction of the macrophage counts and suggests an immune activation in the lung, especially in the peripheral lung.
When the BALs are sampled and immune cells analyzed, we begin examining the immune cells initially, but, much like van Leeuwenhoek or Elie Metchnikoff, we follow the approach of using microscopes to search and look through tissues.
Many microbes are found inside the lung, including fungal hyphae, engulfed in mucus and, inside the macrophages. There is, therefore, a need for the constant processing of the lung. That makes sense, given that the lung is dead-ended, so it is necessary to have a mucociliary escalator and a macrophage dedicated to constantly sampling, monitoring, and controlling these microbes.
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What notable results have this examination of lung microbes elicited?
JM: Within one patient cohort, which was mainly from the Munster region in the south of Ireland, it was found that many of the samples have very high levels of bacteria, fungi, and viruses. When we began examining biopsies, it was found that there was also a great deal of bacteria adhered to the epithelial microbe as well. Such bacteria are clearly evading the mucociliary escalator and surviving inside the lung.
To reference one of the shotgun sequencing readouts that we use - many types of microbes were present. Using this shotgun sequencing - or whole-genome sequencing – to look through the asthma-patient population, we discovered that certain patients had blooms of haemophilus influenza: a fairly pathogenic gram negative usually resident in the upper respiratory tract. This can cause a great deal of aggravation when it gets into the lower respiratory tract.
Within these asthmatic populations, it was observed that the haemophilus influenza was high in several patients. When correlation analysis was performed on some of the metadata from these patients, we were able to see that this haemophilus correlated with high levels of TNF-α, IL-1β, IL-8, and the neutrophils themselves: the cellular factors of the Th1 response.
Other microbes were seen as well: such as Bradyrhizobium, a lactobacillus, and two lactobacillus species also changing in profile. This is likely due to disruption of the microbiome in the lung as a result of the haemophilus inflammation. In addition, where haemophilus is found, the lab will always find a haemophilus virus or an haemophilus phage HP1 present. Currently, it is being investigated to see whether the lung microbiome can be modulated by giving monoclonal antibodies to anti IL-5 and anti IgE, (which is the allergies or the antibody) and to see how we can change the lung microbiome in this way.
Shotgun sequencing is also very useful because, apart from examining bacteria, it also looks at viruses and fungi - and even protozoa. When several of these patient samples are looked through, we can find lots of viruses present: mainly bacterial viruses, such as phages, but also human viruses, such as Epstein-Barr viruses like Lymphocryptovirus and fungal sequences present in many of the patients.
It must be noted, however, that the fungal sequence databases are not useful for the fungus; you must move up to the species level to achieve this.
When the fungi present in the lung of these asthmatic patients are analyzed, many cytokines can be found that are associated with just the fungi present – notably with one particular pathogen: aspergillus fumigatus. Aspergillus fumigatus is associated with increased IL-6 and IL-13. It is also true that the decrease in macrophage levels is also associated with aspergillus because the neutrophils are invading to try and fight the aspergillus.
This lung infection with such fungi is also associated with increases in the systemic or circulating cytokines: IL-4, IL-6, IL-10, -13, -17, and TNF-α. Fungi are therefore playing a key role in aggravating asthma and the lung microbiome.
The lung microbiome is therefore very important from the perspective of those suffering or analyzing lung diseases, such as asthma and COPD.
Currently, the effects of COVID-19 on the lung and long-term immunity are unknown – but it is not unreasonable to state that there will likely be resulting changes to the microbiome causing aggravation, especially in asthmatics. At present, we are currently doing many studies on the lung microbiome. We hope this will help us establish and know what is happening in these diseases. It would also be worthwhile to establish a proven link between the gut and the lung, noting how each can affect the other, both beneficially and detrimentally.
It is also important to note with this microbial analysis that microbial changes can be strain-dependent, rather than just being a Bifidobacterium. Certain species such as Bifidobacterium longum or infantis may have to be examined. Once again, this is why we have targeted therapies and why we investigate which species are changing.
Much of the research in the APC has led to anti-inflammatory therapies but also to phage therapies. The next likely stage is to culture all these microbes that we have identified. Some are quite rare and may not be easy to culture, so we can develop microbiome diagnostics. We may also develop designer phage therapy and metabolic supplementation to disrupt bacteria like haemophilus influenzae, and perhaps even develop sublingual immunotherapy towards the microbiome to prevent the same blooms.
Why do you think it is so important to study gut and lung microbiomes?
JM: Within the research environment, it is important to have a firm grasp of why we are studying such microbiomes. Lung microbiome and gut microbiome are very important for health and wellbeing and, therefore, in the development of sustainable ways of living. For instance, we all live in sustainable cities and communities because, as a direct result of COVID isolation, we have all had to adapt and change our common areas: making sure our air quality is at a certain level.
Interestingly, economic growth has been directly affected by these necessary changes to prevent humans from inhaling such microbes into our lungs. The lung microbiome - and particularly the gut microbiome – are therefore very important in understanding our immune systems but also essential to fight not only allergy- and asthma-style diseases but also for tackling an infectious disease such as COVID-19.
IBD is also studied because the gut microbiome is extremely large and complex. Additionally, asthma is on the rise worldwide, and therefore we believe that analyzing and understanding the lung microbiome in asthma is only becoming more important.
Could you provide a brief overview of the clinical trial process in this area and in human health more generally?
BS: At Atlantia, trials are run from concept to completion. This means that the entire process is kept in-house, from start to finish. That includes the design, protocol, preliminary research and documentation, as well as regulation, ethics applications, recruitment of the volunteers from our database, the conduct of the study visits, the collection of all bio samples, the analysis of those samples through third party accredited labs, the statistical analysis, and finally, the reporting. That reporting can be a clinical study report, or Atlantia can help you with manuscripts for publications if needed.
Atlantia has a wealth of experience in many different areas of human health, but in terms of immunity, Atlantia has conducted studies on allergies and on lung function using spirometry and challenge tests. We have also performed studies on inflammatory diseases - like IBD and osteoarthritis - using inflammatory biomarkers, such as IL-6, IL-8, TNF-α, highly sensitive CRP, WOMAC questionnaires, and disease activity indexes like CDAI and CAI. This is alongside studies in immunity infection, such as URTI, in which influenza titre, the WURSS-24 questionnaire, Jackson Score, and the CARIFS questionnaire were all used. Finally, we have also studied H. pylori using UBTs and antibiotic-associate diarrhea – so the team has a real range of studies under their belts.
How should interested customers go about reaching you?
BS: If a customer has any queries, any particular projects that they would like advice on, or any projects for which they would like a price proposal, please do reach out to the team, who are more than happy to help. We recommend that they reach out directly to the sales team at [email protected].
Dr. John Macsharry | Prof. Of Medical Microbiology And Researcher At Apc Microbiome Ireland
Prof. MacSharry research is focused on the interactions of the host mucosal epithelium and microbes in the lung, gut, and urinary tract. Currently encompasses the Asthmatic lung microbiome, Gut sensing of commensal Bifidobacterium, COVID-19 immune responses, Microbial survival and Immune evasion during Urinary Tract Infection.
Dr. Liam O’mahony | Prof. Of Immunology At The Departments Of Medicine And Microbiology, Researcher At Apc Microbiome Ireland
Prof. O’Mahony's research interests are focused on the molecular basis for microbe and metabolite modulation of mucosal inflammatory responses, the basic mechanisms by which microbes influence allergic sensitization within the gut, skin and lungs.
Barry Skillington CCO At Atlantia Clinical Trials
Barry has over 25 years’ experience working in the food sector in both product and business development, in Ireland and the USA. He has spent the last seven years at Atlantia as CCO, where it is today as one of the leading Food Clinical Trials companies.
About Atlantia Clinical Trials
Atlantia Clinical Trials Ltd is a CRO that specializes in conducting studies on foods, beverages, and supplements for companies worldwide that want to scientifically validate their functional ingredients to support an: EFSA (European Food Safety Authority) Health Claim; FDA (Food & Drug Administration) Structure Function Claim; or General Product Marketing Claim.
Atlantia works with world-leading scientists (among the top cited 1% internationally, in the areas of digestive health and functional foods) at the: APC Microbiome Institute in University College Cork, Ireland; Teagasc, Moorepark, Ireland, and recognized centers of excellence globally.
Atlantia runs and operates its own clinic sites and conducts all studies to ICH-GCP standard (International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use - Good Clinical Practice). Its team includes physician experts in digestive health, mental health (psychological stress and cognition), cardiovascular health, sports performance, metabolic disease, bone health, immune health, and healthy ageing. The clinical team also includes project managers, research nurses, nutritionists, certified sports trainers and lab researchers.
Atlantia manages all elements from protocol design, placebo manufacture, recruitment, and study execution, to sample and data analysis, statistics, and report/dossier preparation to provide a service that is technically, scientifically, and clinically superior.
The clinical studies cover a broad spectrum of functional food and beverage categories, such as dairy, cereal, probiotics, different protein forms, infant-specific foods, vitamins/minerals, plant or marine extracts, and medical foods.
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