Liver cancer and microflora: details
Intestinal microbiota may suppress liver anti-cancer immunity
Vyacheslav Kalinin, "Elements"
Scientists have figured out how the intestinal microbiota affects the anti-cancer immunity of the liver. Previously, it was already known that bile acids, which are produced in the liver and then enter the digestive tract, are modified there by microorganisms. After that, they can return to the liver and affect a special type of immune lymphocytes – NKT cells, which normally prevent the formation of liver tumors.
Fig. 1. Intestinal-hepatic circulation of bile acids in the body and the mechanism of their influence on oncogenesis in the liver. The liver secretes bile acids, which then enter the intestine, where they can be modified by the microbiota. Some of the acids then return through the intestinal epithelium (gut epithelium; see Intestinal epithelium) and portal vein (portal vein) to the liver (secondary acids, secondary acids). Some modifications allow secondary acids to influence the level of CLCX16 chemokine, a large amount of which suppresses the activity of a special type of immune cells – NKT cells (Natural killer T cell, NKT) that resist the formation of tumors in the liver. A drawing from the discussed article in Science.
It has now been established that harmful modification is carried out by bacteria of the genus Clostridium, and the molecular details of this process have been revealed. The obtained data can not only be used in further cancer therapy studies, but also show the relationship of the microbiota and its metabolites with immune responses in the liver.
Since 1980, the number of cases of liver cancer and metastases of cancers of other organs to the liver has increased 3 times. In developed countries, it is becoming one of the main causes of death from cancer (R. L. Siegel et al., 2018. Cancer statistics, 2018). This is due, on the one hand, to factors that provoke liver cancer (excessive alcohol consumption and subsequent cirrhosis of the liver, harmful foods, various chemical compounds in the environment, hepatitis B and C viruses), and on the other hand, to a decrease in mortality from a number of other cancers.
It has recently been shown that the effectiveness of various cancer treatment methods, as well as oncogenesis as such, can be influenced by the intestinal microbiota – a collection of microorganisms in the digestive tract (L. Zitvogel et al., 2018. The microbiome in cancer immunotherapy: Diagnostic tools and therapeutic strategies). At the moment, it is not completely clear exactly how microorganisms get into the digestive tract and populate it. Now the prevailing opinion is that initially babies are born "clean", and the microbiota gets to them with food, from the air and other ways and is formed in the first couple of years of life. However, there are studies showing that, for example, some lactobacilli occur in the placenta during pregnancy.
The microbiota includes a wide variety of microorganisms – both anaerobic and aerobic (and in total, an adult healthy person may have several tens of trillions of microorganisms in the intestine – their number usually exceeds the number of the body's own cells). The relations of most of them with the host organism are mutualistic, the main useful function of the microbiota is the assimilation of food. But the processes of interaction between the microbiota and various organs are very diverse. Thus, microbiota microorganisms can stimulate the immune system to produce cytokines, immunoglobulins, and the products of their metabolism can also affect the central nervous system.
And, as it turned out, the microbiota can stimulate or, conversely, suppress the anti-cancer immune response in the liver. And this is the main means of resisting cancer in the body: the main function of neutralizing cells that are out of control and forming tumors is performed by lymphocytes of the immune system. The liver has its own specific set of immune cells, consisting of T- and B-lymphocytes, macrophages. Unlike the general immune system, there are a lot of NKT cells in the liver (see Natural killer T cell, NKT), which make up to 40% of the number of T-lymphocytes.
The products of microbiota metabolism enter the liver from the digestive tract through the intestinal epithelium and the portal vein, which delivers 75% of all blood entering the liver. They have been shown to influence the development of hepatocellular carcinoma (S. Yoshimoto et al., 2013. Obesity-induced gut microbial metabolite promotes liver cancer through senescence secretome), but the mechanisms of this effect remained unknown. Hepatocellular carcinoma is the most common primary liver cancer resulting from the degeneration of hepatocytes, which make up approximately 80% of the total liver mass. This cancer is difficult to treat. In the early stages, when the tumor is less than 5 cm, it can be removed or a liver transplant can be performed. In the later stages, treatment is almost never successful.
A large team of scientists from scientific institutions in the USA, Germany and Thailand, led by Tim F. Greten, investigated the role of intestinal microbiota in the formation of liver immunity to hepatocellular carcinoma (GC) on model mice, as well as the effect of microbiota on metastases of cancers of other organs, since such metastases occur most often in the liver. In accordance with the early results of other scientists, the suppression of the microbiota by injecting animals with a mixture of antibiotics in drinking water led to a sharp suppression of HA caused by the removal of the MYC oncogene repression by doxycycline (Fig. 2).
Fig. 2. Suppression of bacteria of the digestive tract suppresses the development of liver cancer. In transgenic mice, hepatocellular carcinoma was caused at 4 weeks of life, after which they were given either pure drinking water (H2O) or water with a mixture of antibiotics (AVX). After another 10 weeks, the mice were slaughtered, the liver was extracted and the nodes of cancerous tumors were counted on it – the graph clearly shows that the tumors developed much more strongly in mice treated with plain water. The livers of mice from each group are shown at the bottom left for example. A drawing from the discussed article in Science.
Suppression of the microbiota did not affect the formation of primary tumors after injection of cancer cells of other organs under the skin of mice, but the number of liver metastases was greatly reduced. At the same time, there was no slowing down of metastasis to the lungs. Consequently, the effect of microbiota on metastasis is specific to the liver.
To understand how the antitumor effect of microbiota suppression occurs, the authors analyzed its effect on the set of immune cells in the liver. They found a noticeable increase in the number of NKT cells, while the number of other immune cells did not change significantly. This was also observed in healthy mice regardless of their age and gender (Fig. 3). The accumulation of NKT cells turned out to be specific to the liver (for example, the set of immune cells in the spleen did not change).
Fig. 3. Suppression of the intestinal microbiota stimulates the formation of natural killers T (NKT) in the liver. Mice who were given water or water with antibiotics (AVX) were transplanted EL4 cancer cells under the skin and, after 2.5 weeks, the content of immune lymphocytes of various types in their liver was determined. A drawing from the discussed article in Science.
It has previously been shown that the type 6 chemokine receptor CXCR6 is important for the accumulation and survival of NKT cells in the liver (F. Geissmann et al., 2005. Intravascular immune surveillance by CXCR6+ NKT cells patrolling liver sinusoids). Indeed, all NKT liver cells of experimental mice expressed CXCR6. Also, NKT cells of the liver turned out to be active producers of interferon γ – more active than other lymphocytes.
The natural assumption that it is with NKT cells that they fight tumors and liver metastases was confirmed in in vivo experiments on mice: in CXCR6 knockout mice (in which the activity of NKT cells in the liver was greatly reduced), metastasis did not depend on the suppression of the microbiota.
The only ligand of the CXCR6 receptor is the chemokine CXCL16, so scientists traced the expression of these two proteins. It turned out that in the liver (more precisely, in the endothelium of the sinuses, but not in the hepatocytes) of mice taking antibiotics, the expression level of Cxcl16 mRNA was increased, which was accompanied by an increase in the number of NKT cells.
Next, it was necessary to establish a link between the microbiota and the expression of hepatic CXCL16 and CXCR6. It is well known that symbiont bacteria in the digestive tract regulate the composition of bile acids, which are synthesized in the liver. Through the gallbladder, they enter the intestine, where they participate in the absorption of fats. Bile acids make a kind of cycle: some of them enter the venous blood from the intestine and return to the liver (Fig. 1). There are so–called primary acids synthesized in the liver, and secondary acids that have passed through the intestine (perhaps more than once) and are modified there by microbiota enzymes. There are quite a lot of both. It is not known in detail which of the various microorganisms of the microbiota produce which modifications of bile acids and which of the modified bile acids are "useful", which are "harmful". In mice, the primary bile acid is, for example, β-muricholic acid (β-MCA), and the secondary is ω-muricholic acid (ω-MCA).
Scientists focused on the connection of bile acids with the expression of CXCL16. They found that colestyramine (a drug that lowers the level of bile acids) increased the level of NKT cells in the liver of mice and that the expression of Cxcl16 mRNA increased in parallel. This suggested that bile acids affect the accumulation of NKT cells by regulating the expression of CXCL16.
We checked it like this. Firstly, the suppression of the microbiota by antibiotics was expected to lead to an increase in the proportion of primary and a decrease in the proportion of secondary bile acids. Secondly, in cultures of liver sinus endothelial cells in vitro, when treated with ω-MCA, the expression of Cxcl16 mRNA was suppressed, and under the action of β-MCA, it was enhanced. Thirdly, when secondary acids were added to the mice's food, the accumulation of NKT cells was suppressed, and primary acids in the food stimulated it. And, accordingly, secondary acids stimulated the formation of cancerous tumors, and primary ones suppressed it.
The next question that the authors resolved concerned the identification of microbiota bacteria responsible for the "harmful" modification of bile acids. The authors evaluated the effect of antibiotics of different action spectra on the production of NKT cells. The addition of vancomycin or cefoperazone (cefoperazone), suppressing gram-positive bacteria, to the food of mice, reduced the content of secondary acids (and increased the level of NKT cells) in the liver, and neomycin showed only a weak effect. It has already been shown that the most important reactions of conversion of primary acids into secondary acids are mainly associated with a limited set of microbiota bacteria, which includes clostridia (J. M. Ridlon et al., 2006. Bile salt biotransformations by human intestinal bacteria). Indeed, vancomycin strongly inhibits the growth of clostridium, and neomycin has little effect on them. But after the mouse microbiota was suppressed and the digestive tract was repopulated with C. scindens, NKT cells accumulated worse in the liver and the growth of cancerous tumors was activated in it.
Thus, scientists have more or less fully figured out how the bacteria of the digestive tract, capable of converting primary bile acids into secondary ones, can affect the growth of liver cancer tumors.
The results obtained in mice were tested and confirmed on human liver tissues. It is also known that in addition to antitumor action, NKT cells are an important regulator of autoimmune responses. Therefore, the results obtained suggest that microbiota and bile acids may become a potential target for the treatment of autoimmune liver diseases.
Sources:
1) C. Ma et al. Gut microbiome–mediated bile acid metabolism regulates liver cancer via NKT cells // Science. 2018. DOI: 10.1126/science.aan5931.
2) N. Hartmann, M. Kronenberg. Cancer immunity thwarted by the microbiome // Science. 2018. (Popular synopsis to the article under discussion).
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