Could Our Gut Be the Source of a New Generation of Drugs?
The anti-inflammatory role of the bacteria Faecalibacterium prausnitizii, present in the gut, has now been identified. We created a start-up to test the effects of this bacteria in patients suffering from chronic inflammatory bowel disease. This microorganism could give rise to a new type of probiotic.
Photo: Epithelial cells (blue ovals) making up the intestinal mucous membranes are in contact with bacteria (blue and pink blobs). This mucous membrane comes from a patient suffering from an inflammatory bowel disease © INRA / Bertrand Nicolas
The food industry is constantly boasting about the health virtues of food supplements in the form of probiotics. Yet studies are rare and not always convincing. Sometimes however, analysis of gut flora can bring some interesting surprises. This is how we discovered that the bacteria Faecalibacterium prausnitzii, the most abundant in the gut flora - making up between 3 and 5% of bacteria - had anti-inflammatory properties, identified using human clinical data.
In 2008, with my gastroenterologist colleague Harry Sokol also at INRA, who is university professor at the Saint Antoine Hospital in Paris, we compared the composition of the microbiota from people suffering from an inflammatory bowel disease (Crohn’s), with that of healthy subjects. In this chronic illness, inflammation of the intestine wall causes lesions which give rise to fistulas or abnormal thickening of the wall, obstructing the gut and requiring surgery. We collected and analysed microbiota samples after such operations, and then six months later. After this period, one group of patients was in remission, while the health of the others had not improved. Was there a difference in the composition of their microbiota? To our huge surprise, those who relapsed had quantities of F. prausnitzii that were far lower than those in remission (0.5% compared to 3.5%). This observation led us to deduce the anti-inflammatory properties of this bacteria in humans, which we were able to verify in vitro on cultures of epithelial gut cells, and in vivo in mice with an inflamed colon.
This study marks the beginning of a new strategy to identify beneficial bacteria, using comparison of the composition of gut microbiota. With other scientists, we have since continued our research to better understand the mechanisms at play in the delicate symbiosis that we maintain with our microbiota.
In terms of health, the challenges are indeed immense. The microbiota is comprised of 1,014 microorganisms (several kilograms) and plays an essential role in immunity, digestion of food and protection against pathogenic agents. It is believed that the intestinal microbiota interacts with most of the organs in the body and that these exchanges help to maintain a balance in the various physiological conditions in a healthy person: homeostasis (*).
RESTORING BALANCE
The first human pathologies resulting from a break in homeostasis and imbalances in the composition of the intestinal microbiota (dysbiosis), are diarrhoea due to taking antibiotics, discovered in 2004. In 2007, the first dysbioses were detected in patients suffering from Crohn’s disease and ulcerative colitis. These pathologies fall under the term of chronic inflammatory bowel disease.
Since our founding work, the approach of comparing healthy and dysbiotic microbiota has been widely used. It is now considered applicable to many pathologies, mainly including chronic inflammatory bowel diseases. Others could also benefit however: diabetes, obesity, colorectal cancer, irritable bowel syndrome, and even neuropsychiatric disorders such as autism.
New strategies of prevention and therapy have appeared in human health: targeting the intestinal microbiota and restoring its balance. To restore homeostasis, it is possible to transfer gut microbiota. Faecal transplantation or faecal bacteriotherapy are approaches that started in the 1990s, but bacteria such as F. prausnitzii can also be administered. These bacteria are promising treatments against chronic bowel diseases and are considered as a new generation of probiotics.
For nearly ten years, we have been studying this bacterium and its interactions with its host. Our goal? To be able, in the near future, to test it in patients suffering from these chronic diseases, to correct anomalies in the dialogue between the microbiota and immunity.
SENSITIVE TO OXYGEN
What have we learned about the physiology and modes of action of F. prausnitzii? Firstly, it is extremely sensitive to oxygen, as exposure of just a few minutes at normal atmosphere is fatal. This makes it difficult to isolate. For its metabolism, this bacterium is able to ferment fructose, starch, inulin and acetate by releasing CO2. In the human intestinal microbiota, it is one of the most important producers of the molecule butyrate, one of the main nutrients for cells in the gut lining. In other words, it promotes the growth and renewal of the intestinal mucous membranes.
While F. prausnitzii is one of the most abundant bacteria in the microbiota, its implantation varies along the gasto-intestinal tract. The highest concentrations are found at the terminal ileum, part of the small bowel, and in the ascending and transverse colon. Distribution depends on several environmental factors, such as concentrations of oxygen and bile salts, the bacterial population, the mucus layer and the pH.
At what period of life does it appear? Initially, this bacterium is absent from new-born faeces. Its presence is then detected in babies aged 6 months and increases up to the age of 2 years. It becomes dominant in children from the age of 3. This evolution suggests that colonisation requires the prior implantation of other oxygen-consuming strains, so that an anaerobic atmosphere (free of oxygen), essential to its life, can be formed.
Its relation to health has been determined. Numerous clinical trials show that the quantity of F. prausnitzii is reduced in the faeces and intestinal mucus of patients suffering from chronic bowel disease. Its presence is also reduced with irritable bowel syndrome and coeliac disease, but also for metabolic disorders such as obesity and diabetes. It is also reduced in quantity in patients suffering from colorectal cancer. This imbalance in the composition of the microbiota is usually accompanied by local inflammation and lesser diversity in genes in the microbiota. Inversely, the presence of F. prausnitzii in the microbiota improves health. Recently, a Franco-American team showed that its abundance promotes the response to immunotherapy treatments in patients with melanoma.
IDENTIFIED MOLECULES
What molecular mechanism does F. prausnitzii use on our immune system and how does it reduce inflammation? Our initial work showed that the presence of the bacteria reduces the molecules produced by immune cells, to attract other immune cells as reinforcements. We also observed in vitro that F. prausnitzii induces low production of pro-inflammatory molecules by the lymphocytes and monocytes, and high production of anti-inflammatory molecules. This effect is also observed in intestinal epithelial cells. When these are attacked by pro-inflammatory molecules, we noted that putting them in contact with the products secreted by a culture of F. prausnitzii reduced the inflammation in these cells.
These observations in vitro were confirmed by in vivo data. In an animal model (mice with chemically-induced colon inflammation), the severity of the symptoms was reduced by administering the live bacteria, or just by administering the products they secrete, i.e. the culture supernatant. Subsequently, other research confirmed the beneficial effects of F. prausnitzii and its culture supernatant.
In order to understand the protective role of the bacteria, we tried to identify specifically the molecules produced by F. prausnitzii that are involved in this phenomenon. We know that they naturally produce butyrate. We started by testing its effects on mice with colon inflammation. But butyrate only partially protected the mice.
To identify other molecules secreted in the gut by F. prausnitzii, we conducted another experiment. We took mice without a microbiota; one group was colonised with two bacteria F. prausnitzii and Escherichia coli. A second only with E. coli. We chemically induced inflammation of the colon in both groups. Then, we analysed all the molecules present in the blood, the ileum, the colon and the faeces of animals in both groups.
We noted that the mice colonised with F. prausnitzii and E. coli had far fewer pro-inflammatory molecules than those colonised with E. coli alone. Of the numerous molecules identified in samples from the gastro-intestinal tract and the blood, shikimic acid and salicylic acid, two chemical compounds precursors of aspirin, were associated with the protective effect of F. prausnitzii. In vitro, we established the functional link between the salicylic acid and the anti-inflammatory effects. This molecule is therefore directly involved in the beneficial action of F. prausnitzii.
We also studied the molecules produced by F. prausnitzii in culture and identified a protein, we called MAM for microbial anti-inflammatory molecule. Its composition makes it very hydrophobic, hence difficult to purify and produce. We had to overcome this obstacle to verify its anti-inflammatory action. We introduced the gene coding for this protein directly into the nucleus of intestinal epithelial cells. These were cultivated in vitro and then stimulated with an inflammatory agent. We noted that the MAM protein synthesised by the epithelial cells reduced the activation of a significant molecular actor, the protein NF-B, involved in the production of inflammatory proteins. To confirm this result, we tested the protein in mice with intestinal inflammation. They ingested the bacteria Lactococcus lactis - contained in dairy products - in which we had introduced the gene coding for the MAM protein. This treatment protected the mice from the inflammation. The MAM protein produced by F. prausnitzii is therefore a new molecule able to inhibit the NF-B pathway in vitro and prevent intestinal inflammation in vivo.
How can this research be used to provide new preventive and therapeutic solutions to patients suffering from chronic bowel diseases? Given that a high level of population of F. prausnitzii needs to be maintained in the intestinal microbiota, we could imagine acting directly by administering the bacteria, or indirectly on the population of F. prausnitzii using probiotics or prebiotics.
We could also envisage using the MAM protein in the form of medication, as it seems able to prevent intestinal inflammation in vivo. Although this is an encouraging clinical and therapeutic picture, some essential steps remain (legal, industrial and proof of concept), before this bacterium can be used in human treatment.
Legally, F. prausnitzii does not figure on any list of bacteria presenting perfect hygienic innocuousness with a history of health use in humans. Its use in the form of a drug will have to be submitted to the European Medicines Agency (EMA). For them to rule, we need to provide conclusive trial results in humans.
VARIOUS PATENTS
Conducting these trials on a significant number of patients requires substantial financial investment. It also calls for F. prausnitzii (which is highly sensitive to oxygen) to be produced industrially. Luckily, we have the know-how, protected by patents. In December 2016, I jointly founded the biotechnology company NextBiotix, with my colleagues Harry Sokol and Patrick Gervais, professor at AgroSup Dijon and member of the food processing and microbiological unit in Dijon. Our primary goal is to implement a clinical trial to test the effects of F. prausnitzii in patients suffering from chronic bowel disease within the next two years.
(*) Homeostasis is a body’s capacity to maintain or restore its various physiological constants (temperature, blood flow, blood pressure, etc.) to levels within normal.
PROBIOTICS CHANGE STATUS
According to the World Health Organisation, probiotics are microorganisms which, when ingested in sufficient quantities, provide beneficial effects to human health. This long-accepted idea was developed by the Russian Élie Metchnikoff, Nobel Prize for Medicine in 1908. These yeasts and bacteria enhance the digestion of lactose, help to restore the intestinal barrier and modulate the immune system. They can also inhibit the action of pathogenic viruses and bacteria, giving them an important role in the prevention and treatment of diarrhoea associated with antibiotics. On the French market, probiotics can be found 90% in specific foods (usually fermented dairy products) and 10% in food supplements. The European Food Safety Authority controls the use of probiotics in Europe and has very strict requirements. It has rejected almost 200 health claim requests for probiotics. Only the effects of yoghurt bacteria on lactose intolerance were accepted. But a new generation of probiotics is coming, thanks to research on the microbiota. Bacteria from this work could be used to restore the intestinal balance. These new probiotics do not just concern healthy individuals, but also sick people. As well as F. prausnitzii, the best example of such a probiotic is Akkermansia muciniphila, a bacterium from the human microbiota which seems to play a major role in metabolic diseases, such as obesity and diabetes. A change in paradigm should be noted here, as these new-generation probiotics would be administered to patients in the form of medication and controlled by specific regulations.
Photo © MP / Leemage
OXYGEN-FREE CULTURE
In contact with molecular oxygen (O2), the bacterium Faecalibacterium prausnitzii dies rapidly. It is a challenge to culture it owing to its strictly anaerobic nature: the bacteria can only be grown in the absence of oxygen, not only for sampling and transport, but also for culture and isolation. These technical constraints explain the low isolation frequency for F. prausnitzii in the laboratories. Biologists work in special enclosures filled with an oxygen-free gas such as nitrogen (N2).
Photo © INRA / Bertrand Nicolas
> AUTHOR
Philippe Langella
Biologist
Philippe Langella is head of the laboratory for interactions between commensal bacteria and probiotics with their host, at the INRA Micalis Institute.
