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A new probiotic modulates gut microbiota against hepatocellular carcinoma

24th August 2016

Over the last years the beneficial effects of the human intestinal microbiota on various health markers have been displayed, such as inflammation, immune response, metabolic function and weight. The importance of these symbiotic bacteria of ours has been proved. You can see these other posts related with our microbiota: “The good clostridia avoid us from allergies“, “Gut bacteria controlling what we eat” or “Good bacteria of breast milk

At the same time it has been seen that probiotics can be a good solution for many diseases with affected gut microbiota. Indeed, the beneficial role of probiotics to reduce gastrointestinal inflammation and prevent colorectal cancer has been proven.

However, recently it has been found that probiotics may have beneficial effects in other parts of the body beyond the gastrointestinal tract, particularly with immunomodulatory effects on an hepatocellular carcinoma (HCC). In this way, researchers at the University of Hong Kong, along with other from University of Eastern Finland, have published a study (Li et al, PNAS, 2016), where they have seen reductions of 40% in weight and size of HCC liver tumours in mice which were administered with a new mixture of probiotics, “Prohep.”

Hepatocellular carcinoma (HCC) is the most common type of liver cancer is the 2nd most deadly cancers, and it is quite abundant in areas with high rates of hepatitis. In addition, sorafenib, the drug most widely used to reduce the proliferation of tumour, is very expensive. The cost of this multikinase inhibitor is €3400 for 112 tablets of 200 mg, the recommended treatment of four pills a day for a month. Instead, any treatment with probiotics that would proved to be effective and could replace this drug would be much cheaper.

The new probiotics mix Prohep consists of several bacteria: Lactobacillus rhamnosus GG (LGG), Escherichia coli Nissle 1917 (ECN) and the whole inactivated by heat VSL#3 (1: 1: 1) containing Streptococcus thermophilus, Bifidobacterium breve, Bf. longum, Bf. infantis, Lb. acidophilus, Lb. plantarum, Lb. paracasei and Lb. delbrueckii.

In the mentioned work, Li et al. (2016) fed mice with Prohep for a week before inoculating them with a liver tumour, and observed a 40% reduction in tumour weight and size in comparison to control animals. As shown in Figure 1, the effect was significant at 35 days, and also for those who were given the Prohep the same day of tumour inoculation. Obviously, the effect of tumour reduction was much more evident when the antitumour compound Cisplatin was administered.

These researchers saw that tumour reduction was due to the inhibition of angiogenesis. This is the process that generates new blood vessels from existing ones, something essential for tumour growth. In relation to the tumour reduction, high levels of GLUT-1 + hypoxic were found. That meant that there was hypoxia caused by the lower blood flow to the tumour, since this was 54% lower in comparison to controls.

 

Fig 1 Li-Fig1B tumor size - days tumor

Figure 1. Change in tumour size. ProPre: administration of Prohep one week before tumour inoculation; ProTreat: administration of Prohep the same day of tumour inoculation; Cisplatin: administration of this antitumoral. (Fig 1B from Li et al, 2016).

 

These authors also determined that there was a smaller amount of pro-inflammatory angiogenic factor IL-17 and of Th17 cells of the immune system, cells also associated with cancer. The lower inflammation and angiogenesis could limit the tumour growth.

Moreover, these researchers established that the beneficial effects of probiotics administration were associated with the abundance of beneficial bacteria in the mice gut microbiota, analysed by metagenomics. So, probiotics modulate microbiota, favouring some gut bacteria, which produce anti-inflammatory metabolites such as cytokine IL-10 and which suppress the Th17 cell differentiation.

 

Fig 2 gut microbiota Eye of Science

Figure 2. Bacteria of the human intestinal microbiota seen by scanning electron microscope (SEM) (coloured image of Eye of Science / Science Source)

 

Some of the bacteria identified by metagenomics in the microbiota of mice that were administered with Prohep were Prevotella and Oscillibacter. The first is a bacteroidal, gram-negative bacterium, which is abundant in the microbiota of rural African child with diets rich in carbohydrates. Oscillibacter is a gram-positive clostridial, known in humans as a producer of the neurotransmitter GABA. Both are an example of the importance of some clostridial and bacteroidals in the gut microbiota. In fact, they are majority there, and although they are not used as probiotics, are found increasingly more positive functions, such as avoiding allergies (see “The good clostridia avoid us from allergies“).

It is known that these bacteria produce anti-inflammatory metabolites and therefore they would be the main involved in regulating the activity of immune cells that cause tumour growth. The observed reduction of tumour in these experiments with mice would be the result of combined effect of these administered probiotic bacteria together with the microbiota itself favoured by them. We see a potential outline of these actions in Figure 3.

Fig 3 Sung fig 2

Figure 3. Simplified diagram of the possible mechanisms of gut bacteria influencing on the polarization of Th17 cells in the lamina propria of the intestinal mucosa. The microbiota bacteria activate dendritic cells, which secrete cytokines (IL-22, IL-23, IL-27). The bacteria can promote Th17 immunity inducing IL-23, which can be involved by means of TLR ligands signal or extracellular ATP or serum amyloid A (SAA). Meanwhile, some probiotic strains could inhibit the development of Th17 by means of the production of IL-12 and IL-27, in addition to promoting the growth and colonization of the bacteria that induce Th17 (Sung et al 2012, Fig. 2).

 

Although we know that the cancer progression is a very complex process and that in the tumour microenvironments there is an infiltration of many different types of immune system cells, such as T cells, neutrophils, killer cells, macrophages etc, the Th17 helper cell subpopulation appears to be prevailing in the tumour progression, and therefore these effects of probiotics and microbiota open good prospects.

It is still early to say whether these findings will contribute to the treatment of human liver cancer, and therefore research in humans is needed, in order to see if these probiotics could be used as such or in tandem with some drug, depending on the tumour stage and size. In any case, all this opens a new range of possibilities for research of the molecular mechanisms of the beneficial effects of probiotics beyond the intestinal tract.

 

Bibliography

El-Nezami H (2016 april 27) HKU develops novel probiotic mixture “Prohep” that may offer potential therapeutic effects on liver cancer. The University of Hong Kong (HKU) 27 Apr 2016

El-Nezamy H, Lee PY, Huang J, Sung YJ (2015) Method and compositions for treating cancer using probiotics. Patent WO 2015021936 A1

Li J, Sung CYJ, Lee N, Ni Y, Pihlajamäki J, Panagiotou G, El-Nezami H (2016) Probiotics modulated gut microbiota suppresses hepatocellular carcinoma growth in mice. PNAS E1306-E1315

Oelschlaeger TA (2010) Mechanisms of probiotic actions – A review. Int J Med Microbiol 300, 57-62

Packham C (2016) Probiotics dramatically modulate liver cancer growth in mice. Medical Press, Med Research 23 Feb 2016

Silgailis M (2016) Treating some cancers with probiotics in the future ? Probiotic Prohep. Lacto Bacto: Health, Microbes and More 23 Feb 2016

Sung CYJ, Lee NP, El-Nezami H (2012) Regulation of T helper by bacteria: an approach for the treatment of hepatocellular carcinoma. Int J Hepatology ID439024, doi:10.1155/2012/439024

UEF News and Events (2016) A novel probiotic mixture may offer potential therapeutic effects on hepatocellular carcinoma. University of Eastern Finland 1 Mar 2016

 

We have good clostridia in the gut and some of them prevent allergies

21st March 2015

Clostridia: who are they ?

The clostridia or Clostridiales, with Clostridium and other related genera, are Gram-positive sporulating bacteria. They are obligate anaerobes, and belong to the taxonomic phylum Firmicutes. This phylum includes clostridia, the aerobic sporulating Bacillales (Bacillus, Listeria, Staphylococcus and others) and also the anaerobic aero-tolerant Lactobacillales (id est, lactic acid bacteria: Lactobacillus, Leuconostoc, Oenococcus, Pediococcus, Lactococcus, Streptococcus, etc.). All Firmicutes have regular shapes of rod or coccus, and they are the evolutionary branch of gram-positive bacteria with low G + C content in their DNA. The other branch of evolutionary bacteria are gram-positive Actinobacteria, of high G + C and irregular shapes, which include Streptomyces, Corynebacterium, Propionibacterium, and Bifidobacterium, among others.

 

flora_cover

 

Being anaerobes, the clostridia have a fermentative metabolism of both carbohydrates and amino acids, being primarily responsible for the anaerobic decomposition of proteins, known as putrefaction. They can live in many different habitats, but especially in soil and on decaying plant and animal material. As we will see below, they are also part of the human intestinal microbiota and of other vertebrates.

The best known clostridia are the bad ones (Figure 1): a) C. botulinum, which produces botulin, the botulism toxin, although nowadays has medical and cosmetic applications (Botox); b) C. perfringens, the agent of gangrene; c) C. tetani, which causes tetanus; and d) C. difficile, which is the cause of hospital diarrhea and some postantibiotics colitis.

 

clostridium_bacteria

Figure 1. The four more pathogen species of Clostridium. Image from http://www.tabletsmanual.com/wiki/read/botulism

 

Clostridia in gut microbiota

As I mentioned in a previous post (Bacteria in the gut …..) of this blog, we have a complex ecosystem in our gastrointestinal tract, and diverse depending on each person and age, with a total of 1014 microorganisms. Most of these are bacteria, besides some archaea methanogens (0.1%) and some eukaryotic (yeasts and filamentous fungi). When classical microbiological methods were carried out from samples of colon, isolates from some 400 microbial species were obtained, belonging especially to proteobacteria (including Enterobacteriaceae, such as E. coli), Firmicutes as Lactobacillus and some Clostridium, some Actinobacteria as Bifidobacterium, and also some Bacteroides. Among all these isolates, some have been recognized with positive effect on health and are used as probiotics, such as Lactobacillus and Bifidobacterium, which are considered GRAS (Generally Recognized As Safe).

But 10 years ago culture-independent molecular tools began to be used, by sequencing of ribosomal RNA genes, and they have revealed many more gut microorganisms, around 1000 species. As shown in Figure 2, taken from the good review of Rajilic-Stojanovic et al (2007), there are clearly two groups that have many more representatives than thought before: Bacteroides and Clostridiales.

 

Rajilic 2007 Fig 1

Figure 2. Phylogenetic tree based on 16S rRNA gene sequences of various phylotypes found in the human gastrointestinal tract. The proportion of cultured or uncultured phylotypes for each group is represented by the colour from white (cultured) passing through grey to black (uncultured). For each phylogenetic group the number of different phylotypes is indicated (Rajilic-Stojanovic et al 2007)

 

In more recent studies related to diet such as Walker et al (2011) — a work done with faecal samples from volunteers –, population numbers of the various groups were estimated by quantitative PCR of 16S rRNA gene. The largest groups, with 30% each, were Bacteroides and clostridia. Among Clostridiales were included: Faecalibacterium prausnitzii (11%), Eubacterium rectale (7%) and Ruminococcus (6%). As we see the clostridial group includes many different genera besides the known Clostridium.

In fact, if we consider the population of each species present in the human gastrointestinal tract, the most abundant seems to be a clostridial: F. prausnitzii (Duncan et al 2013).

 

Benefits of some clostridia

These last years it has been discovered that clostridial genera of Faecalibacterium, Eubacterium, Roseburia and Anaerostipes (Duncan et al 2013) are those which contribute most to the production of short chain fatty acids (SCFA) in the colon. Clostridia ferment dietary carbohydrate that escape digestion producing SCFA, mainly acetate, propionate and butyrate, which are found in the stool (50-100 mM) and are absorbed in the intestine. Acetate is metabolized primarily by the peripheral tissues, propionate is gluconeogenic, and butyrate is the main energy source for the colonic epithelium. The SCFA become in total 10% of the energy obtained by the human host. Some of these clostridia as Eubacterium and Anaerostipes also use as a substrate the lactate produced by other bacteria such as Bifidobacterium and lactic acid bacteria, producing finally also the SCFA (Tiihonen et al 2010).

 

Clostridia of microbiota protect us against food allergen sensitization

This is the last found positive aspect of clostridia microbiota, that Stefka et al (2014) have shown in a recent excellent work. In administering allergens (“Ara h”) of peanut (Arachis hypogaea) to mice that had been treated with antibiotics or to mice without microbiota (Germ-free, sterile environment bred), these authors observed that there was a systemic allergic hyper reactivity with induction of specific immunoglobulins, id est., a sensitization.

In mice treated with antibiotics they observed a significant reduction in the number of bacterial microbiota (analysing the 16S rRNA gene) in the ileum and faeces, and also biodiversity was altered, so that the predominant Bacteroides and clostridia in normal conditions almost disappeared and instead lactobacilli were increased.

To view the role of these predominant groups in the microbiota, Stefka et al. colonized with Bacteroides and clostridia the gut of mice previously absent of microbiota. These animals are known as gnotobiotic, meaning animals where it is known exactly which types of microorganisms contain.

In this way, Stefka et al. have shown that selective colonization of gnotobiotic mice with clostridia confers protection against peanut allergens, which does not happen with Bacteroides. For colonization with clostridia, the authors used a spore suspension extracted from faecal samples of healthy mice and confirmed that the gene sequences of the extract corresponded to clostridial species.

So in effect, the mice colonized with clostridia had lower levels of allergen in the blood serum (Figure 3), had a lower content of immunoglobulins, there was no caecum inflammation, and body temperature was maintained. The mice treated with antibiotics which had presented the hyper allergic reaction when administered with antigens, also had a lower reaction when they were colonized with clostridia.

 

fig 4 skefta

Figure 3. Levels of “Ara h” peanut allergen in serum after ingestion of peanuts in mice without microbiota (Germ-free), colonized with Bacteroides (B. uniformis) and colonized with clostridia. From Stefka et al (2014).

 

In addition, in this work, Stefka et al. have conducted a transcriptomic analysis with microarrays of the intestinal epithelium cells of mice and they have found that the genes producing the cytokine IL-22 are induced in animals colonized with clostridia, and that this cytokine reduces the allergen uptake by the epithelium and thus prevents its entry into the systemic circulation, contributing to the protection against hypersensitivity. All these mechanisms, reviewed by Cao et al (2014), can be seen in the diagram of Figure 4.

In conclusion, this study opens new perspectives to prevent food allergies by modulating the composition of the intestinal microbiota. So, adding these anti-inflammatory qualities to the production of butyrate and other SCFA, and the lactate consumption, we must start thinking about the use of clostridia for candidates as probiotics, in addition to the known Lactobacillus and Bifidobacterium.

 

fig 4 Cao b

Figure 4. Induction of clostridia on cytokine production by epithelial cells of the intestine, as well as the production of short chain fatty acids (SCFA) by clostridia (Cao et al 2014).

 

References

Cao S, Feehley TJ, Nagler CR (2014) The role of commensal bacteria in the regulation of sensitization to food allergens. FEBS Lett 588, 4258-4266

Duncan SH, Flint HJ (2013) Probiotics and prebiotics and health in ageing populations. Maturitas 75, 44-50

Rajilic-Stojanovic M, Smidt H, de Vos WM (2007) Diversity of the human gastrointestinal tract microbiota revisited. Environ Microbiol 9, 2125-2136

Rosen M (2014) Gut bacteria may prevent food allergies. Science News 186, 7, 4 oct 2014

Russell SL, et al. (2012) Early life antibiotic-driven changes in microbiota enhance 
susceptibility to allergic asthma. EMBO Rep 13(5):440–447

Stefka AT et al (2014) Commensal bacteria protect against food allergen sensitization. Proc Nat Acad Sci 111, 13145-13150

Tiihonen K, Ouwehand AC, Rautonen N (2010) Human intestinal microbiota and healthy aging. Ageing Research Reviews 9:107–16

Walker AW et al (2011) Dominant and diet-responsive groups of bacteria within the human colonic microbiota. The ISME J 5, 220-230

 

 

Bacteria in the gut are controlling what we eat

It seems to be so: the microbes in our gastrointestinal tract (GIT) influence our choice of food. No wonder: microbes, primarily bacteria, are present in significant amounts in GIT, more than 10 bacterial cells for each of our cells, a total of 1014 (The human body has about 1013 cells). This amounts to about 1-1.5 kg. And these bacteria have lived with us always, since all mammals have them. So, they have evolved with our ancestors and therefore they are well suited to our internal environment. Being our bodies their habitat, much the better if they can control what reaches the intestine. And how can they do? Then giving orders to the brain to eat such a thing or that other, appropriate for them, the microbes.

Imagen1Figure 1.Command centre of the gastrointestinal tract” (own assembly,  Albert Bordons)

Well, gone seriously, there is some previous work in this direction. It seems there is a relationship between preferences for a particular diet and microbial composition of GIT (Norris et al 2013). In fact, it is a two-way interaction, one of the many aspects of symbiotic mutualism between us and our microbiota (Dethlefsen et al 2007).

There is much evidence that diet influences the microbiota. One of the most striking examples is that African children fed almost exclusively in sorghum have more cellulolytic microbes than other children (De Filippo et al 2010).

The brain can also indirectly influence the gut microbiota by changes in intestinal motility, secretion and permeability, or directly releasing specific molecules to the gut digestive lumen from the sub epithelial cells (neurons or from the immune system) (Rhee et al 2009).

The GIT is a complex ecosystem where different species of bacteria and other microorganisms must compete and cooperate among themselves and with the host cells. The food ingested by the host (human or other mammal) is an important factor in the continuous selection of these microbes and the nature of food is often determined by the preferences of the host. Those bacteria that are able to manipulate these preferences will have advantages over those that are not (Norris et al 2013).

Recently Alcock et al (2014) have reviewed the evidences of all this. Microbes can manipulate the feeding behaviour of the host in their own benefit through various possible strategies. We’ll see some examples in relation to the scheme of Figure 2.

 

Fig 2 human microbiome behaviour appetite

Figure 2. As if microbes were puppeteers and we humans were the puppets, microbes can control what we eat by a number of marked mechanisms. Adapted from Alcock et al 2014.

 

People who have “desires” of chocolate have different microbial metabolites in urine from people indifferent to chocolate, despite having the same diet.

Dysphoria, id est, human discomfort until we eat food which improve microbial “welfare”, may be due to the expression of bacterial virulence genes and perception of pain by the host. This is because the production of toxins is often triggered by a low concentration of nutrients limiting growth. The detection of sugars and other nutrients regulates virulence and growth of various microbes. These directly injure the intestinal epithelium when nutrients are absent. According to this hypothesis, it has been shown that bacterial virulence proteins activate pain receptors. It has been shown that fasting in mice increases the perception of pain by a mechanism of vagal nerve.

Microbes can also alter food preferences of guests changing the expression of taste receptors on the host. In this sense, for instance germ-free mice prefer more sweet food and have a greater number of sweet receptors on the tongue and intestine that mice with a normal microbiota.

The feeding behaviour of the host can also be manipulated by microbes through the nervous system, through the vagus nerve, which connects the 100 million neurons of the enteric nervous system from the gut to the brain via the medulla. Enteric nerves have receptors that react to the presence of certain bacteria and bacterial metabolites such as short chain fatty acids. The vagus nerve regulates eating behaviour and body weight. It has been seen that the activity of the vagus nerve of rats stimulated with norepinephrine causes that they keep eating despite being satiated. This suggests that GIT microbes produce neurotransmitters that can contribute to overeating.

Neurotransmitters produced by microbes are analogue compounds to mammalian hormones related to mood and behaviour. More than 50% of dopamine and most of serotonin in the body have an intestinal origin. Many persistent and transient inhabitants of the gut, including E. coli, several Bacillus, Staphylococcus and Proteus secrete dopamine. In Table 1 we can see the various neurotransmitters produced by GIT microbes. At the same time, it is known that host enzymes such as amine oxidase can degrade neurotransmitters produced by microorganisms, which demonstrates the evolutionary interactions between microbes and hosts.

 

Table 1. Diversity of neurotransmitters isolated from several microbial species (Roschchina 2010)

Neurotransmitter Genera
GABA (gamma-amino-butyric acid) Lactobacillus, Bifidobacterium
Norepinephrine Escherichia, Bacillus, Saccharomyces
Serotonin Candida, Streptococcus, Escherichia, Enterococcus
Dopamine Bacillus, Serratia
Acetylcholine Lactobacillus

 

Some bacteria induce hosts to provide their favourite nutrients. For example, Bacteroides thetaiotaomicron inhabits the intestinal mucus, where it feeds on oligosaccharides secreted by goblet cells of the intestine, and this bacterium induces its host mammal to increase the secretion of these oligosaccharides. Instead, Faecalibacterium prausnitzii, a not degrading mucus, which is associated with B. thetaiotaomicron, inhibits the mucus production. Therefore, this is an ecosystem with multiple agents that interact with each other and with the host.

As microbiota is easily manipulated by prebiotics, probiotics, antibiotics, faecal transplants, and changes in diet, controlling and altering our microbiota provides a viable method to the otherwise insoluble problems of obesity and poor diet.

 

References

Alcock J, Maley CC, Aktipis CA (2014) Is eating behavior manipulated by the gastrointestinal microbiota? Evolutionary pressures and potential mechanisms. BioEssays 36, DOI: 10.1002/bies.201400071

De Filippo C, Cavalieri D, Di Paola M, Ramazzotti M, et al (2010) Impact of diet in shaping gut microbiota revealed by a comparative study in children from Europe and rural Africa. Proc Natl Acad Sci USA 107:14691–6

Dethlefsen L, McFall-Ngai M, Relman DA (2007) An ecological and evolutionary perspective on human-microbe mutualism and disease. Nature 449:811-818

Lyte M (2011) Probiotics function mechanistically as delivery for neuroactive compounds: Microbial endocrinology in teh design and use of probiotics. BioEssays 33:574-581

Norris V, Molina F, Gewirtz AT (2013) Hypothesis: bacteria control host appetites. J Bacteriol 195:411–416

Rhee SH, Pothoulakis C, Mayer EA (2009) Principles and clinical implications of the brain–gut–enteric microbiota axis. Nature Reviews Gastroenterology and Hepatology 6:306-314

Roschchina VV (2010) Evolutionary considerations of neurotransmitters in microbial, plant, and animal cells. In Lyte M, Freestone PPE, eds; Microbial Endocrinology: Interkingdom Signaling in Infectious Disease and Health. New York: Springer. pp. 17–52

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