M cells hold the key to gut immunity
18 June 2010
Pioneering work on the immune system in our gut reveals the important role of intestinal ‘microfold’ cells in the functioning of our immune system.
Laboratory for Epithelial Immunobiology
RIKEN Research Center for Allergy and Immunology
Each of us has more than 100 trillion bacteria residing in our gut. In addition to these gut-resident microbiota, other potentially harmful bacteria such as Salmonella sometimes enter our bodies by ingestion. The gut provides these bacteria with an ideal environment to proliferate, but the gut has a special immune system that constantly monitors the state of the intestines and eliminates problematic populations of bacteria, attacking invading bacteria that have broken through the intestinal epithelial layer. The gut immune system, however, remains a mystery to scientists. Hiroshi Ohno of the Laboratory for Epithelial Immunobiology at the RIKEN Research Center for Allergy and Immunology (RCAI) is solving the mysteries of the gut immune system by focusing on the microfold cells (M cells) located on the surface of the bowel. His research could improve our understanding of the effects of intestinal bacteria on our health.
What is gut immunity?
Figure 1. Microfold cells (M cells) in the human intestine. M cells are found on part of the follicular epithelial cells covering Peyer's patches — concentrations of immune cells (T cells, B cells and dendritic cells).
(1) GP2 proteins on the surfaces of M cells bind to and ingest E. coli or Salmonella bacteria for transfer to lower sites.
(2) Dendritic cells receive and degrade bacteria, then present pieces of the antigens to T cells.
(3) T cells are activated and send a command to B cells to start producing antibodies.
(4) B cells undergo differentiation and maturation to produce lgA antibodies.
(5) lgA antibodies are secreted into the intestines where they patrol for bacteria.
“The surface of the small intestine is covered with protrusions called ‘villi’, which themselves are covered with absorptive epithelial cells that ingest nutrients produced as a result of the digestion of food,” says Ohno. The apical plasma membrane of epithelial cells forms ‘microvilli', providing a large surface area to increase the efficiency of the intestines for absorbing nutrients (Fig. 1).
Scanning electron microscopy depicts the dome-shaped epithelial region covering the gut lymphoid tissue surrounded by villi (Fig. 2, left). The epithelial cells in this region also possess microvilli. Yet detailed observation reveals that not all epithelial cells have microvilli (Fig. 2, right). “These are the M cells I am studying. M cells play a very important role in gut immunity.”
The immune system, present in some form in virtually all organisms, eliminates invading foreign matter, such as bacteria, through the action of various immune cells (e.g. T cells, B cells and dendritic cells) that in humans are produced by the thymus and bone marrow. T and B cells are stored in the lymph nodes or spleen, whereas dendritic cells are dispersed throughout the body on ‘patrol’. When a dendritic cell encounters a foreign body such as bacteria, called an antigen, the dendritic cell engulfs and degrades the antigen, producing signals that trigger T cells which in turn help B cells to start creating specific antibodies. In this ‘systemic’ immune system, B cells produce antibodies such as immunoglobulin G (lgG) to attack antigens.
However, 60–70% of all immune cells in our bodies are located in the intestines, not in the lymph nodes or the spleen, to constitute the unique ‘intestinal’ immune system. The intestines are home to a range of bacteria, as well as foreign matter including pathogenic microbes brought in by ingestion. The specific immune tissue in the gut, such Peyer's patches, is equipped to protect ourselves from those microbes by secreting immunoglobulin A (IgA) instead of IgG as described below. “Immunology has developed centering around the thymus gland, bone marrow and systemic immunity. Since these subjects are now well understood, over the past ten years attention has also begun to focus specifically on gut immunity,” says Ohno. In 2002, he established the Laboratory for Epithelial Immunobiology at the RIKEN RCAI, where he launched a full-scale study of gut immunity. “Having studied immunology focusing on T cells, I began studying the mechanism of how epithelial cells transport proteins intracellularly. This is because I thought that the combination of immunology and epithelial cell biology would lead to a unique research project on gut immunity.”
Figure 2. Electron microscopy image of the small intestine of a mouse. (Left) Dome-shaped follicule-associated epithelial cells surrounded by villi. The Peyer's patch immune cells is located below the dome-shaped eptihelium. (Right) Enlargement of the follicule-associated epithelial cells to show individual cells. Epithelial cells bear microvilli of 0.5 μm in length and 0.1 μm in diameter (not discernable even in this enlarged picture). M cells do not have microvilli, making them look like a tiny ‘dent’ surrounded by peripheral cells.enlarge image
Discovery of a M-cell receptor that binds with bacteria for uptake
M cells, discovered in 1974, are thought to ingest bacteria and induce immune responses, but the mechanism remains a mystery. “I decided to find the genes specifically expressed in M cells so that I could clarify the cells’ functions,” says Ohno.
However, this posed a considerable challenge. “The number of M cells in the small intestine is only around one ten-millionth of the total number of epithelial cells. Epithelial cells are also difficult to culture,” says Ohno. He eventually gave up on the idea of isolating M cells for analysis, and instead attempted to analyze the genes expressed in M-Cell-containing Peyer’s patch epithelial cells by RNA microarray analysis. He compared the results with those for villous epithelial cells devoid of M cells to identify the genes specifically expressed by M cells. RNA microarray analysis allows analyses to be performed with a tiny amount of sample, and indicates the presence or absence of particular gene expression.
Ohno finally identified the presence of a gene called GP2 specifically expressed in M cells among intestinal epithelial cells. “The GP2 gene produces GP2 proteins, and these proteins are present on the surface of M cells.”
It is known that pancreatic fluid secreted by the pancreas contains large amounts of GP2, but its functions are still unknown. How do GP2 molecules on the surface of M cells function? “It was found that they serve as receptors that bind with Escherichia coli (E. coli)and Salmonella bacteria, which are then ingested into M cells and transferred to immune cells underneath . The underside of the M cells is in contact with the dendritic cells in the Peyer's patch. The dendritic cells accept bacteria from the M cells, degrade them, and present pieces of the bacteria as antigens to the T cells. The T cells are then activated and give instructions to B cells, commanding them to create antibodies against the antigens. Thus, the B cells start to create IgA.
“M cells have a very interesting shape because each cell has a pocket-like hollowed part on its underside.” The dendritic cells extend their dendrites into the pocket (Fig. 1). “I think the bacteria ingested by M cells need to be transferred to the dendritic cells as soon as possible so that immune responses are initiated immediately.”
IgA is secreted into the intestine, where it binds with bacteria to eliminate them. Mice that cannot produce lgA are known to have about 100 times the number of normal intestinal bacteria, which not only increases the risk of intestinal inflammation due to invasion of the intestinal mucosa, but also leads to the production of the harmful metabolic substances that cause, for example, colorectal cancer. “M cells initiate gut immune responses to produce lgA to contain the intestinal bacteria not to increase excessively, and also to prevent bacteria from invading the body at the mucous boundary."
These research results were published in Nature in 2009. “We may be able to control the gut immune response because we have understood the mechanism of how M cells ingest bacteria and how they they initiate gut immune responses.” Enhancing gut protective immunity will lead to the prevention and treatment of inflammatory bowel diseases and enteric infections. Furthermore, controlling gut immune responses can contribute to preventing food allergies by suppressing abnormal immune responses to particular food components.
Figure 3. Expression of GP2 proteins in small intestine epithelial cells. GP2 proteins (green) and UEA-1 lectin (red) used as a marker for detecting M cells in mice. GP2 is more specific for M cells than UEA-1 because GP2 exists only in M cells. Areas in yellow indicate cells bearing both GP2 and UEA-1.enlarge image
Additionally, GP2 proteins can be used effectively as a reliable marker to identify M cells (Fig. 3). Markers, often a gene or molecule that exists only in specific cells, serve as landmarks showing the presence of certain cells. “Mice and humans have GP2 proteins in common. The GP2 marker will significantly promote research on gut immunity now that scientists have obtained a marker for M cells that can be used across the boundaries between mice and humans.” One of the issues that remains to be clarified is how the gut immune system distinguishes pathogenic bacteria from avirulent bacteria.
Development of oral vaccines
Ohno’s research results have yet another importance. “We are anticipating the development of oral vaccines that are administered by mouth,” he says. Most vaccines are currently administered by injection, producing IgG through the systemic immune response. The antibodies thus produced circulate in the bloodstream and start attacking antigens such as bacteria and viruses only after they have invaded the body. However, the invasion of antigens can be prevented even before they break into the body if the immune response is initiated through M cells to produce IgA. IgG is also produced when using M cells, however, when the dendritic cells that have ingested antigens through M cells move to the lymph nodes and the spleen, where they present the antigens to T cells. “We are now exploring a low-molecular weight compound that can bind to GP2 proteins. If the compound is combined with a vaccine, the vaccine will readily bind to GP2. Then the vaccine will be ingested into M cells, and mucosal immune responses will be initiated,” says Ohno.
Oral vaccines, however, have been difficult to realize because the vaccine must pass through the stomach, where they are subject to degradation, before reaching the intestines. “A clue to solving the problem lies in the existence of M cells in the tonsils. GP2 is found on the M-cell surfaces in this area. If the M cells in the tonsils can initiate immunel responses through GP2 and produce IgA, we will be able to develop more effective oral vaccines based on simpler processes.”
Cells interconnect for quick information transmission
Another important discovery that has come out of the process of exploring genes specifically expressed in M cells is related to M-Sec gene, which is specifically expressed in M cells, as well as macrophages and dendritic cells. “It is well known that dendritic cells or macrophages connect each other via a nanotube that extends as part of the cell membrane and over which the cells exchange information. We have found that the proteins created by M-Sec gene initiate the formation of nanotubes,” says Ohno.
Figure 4. Creation of nanotubes and transmission of information by M-Sec genes. HeLa cells (widely used experimental human cells) with M-Sec genes extend their cell membranes toward each other and form a nanotube (indicated by arrows, left). Calcium signals activated in the right-hand cell are transmitted to the left-hand cell.enlarge image
When two cells expressing M-Sec are put into culture, their plasma membranes connect by forming a nanotube (Fig. 4), and calcium flux stimulus applied to one cell is transmitted to another. “Information can be transmitted faster and more reliably when two cells are directly connected than when messenger molecules are secreted,” says Ohno. He believes that the nanotubes among M cells form a fine mesh that could also function as an efficient net for capturing bacteria.
Ohno’s research results on M-cell nanotubes were published in Nature Cell Biology in 2009. “A short time before the publication of our paper, another research group published a paper on macrophage nanotubes.” The paper describes how HIV-infected macrophages extend their nanotubes to bind to B cells and force HIV-derived proteins, called Nef, into the B cells to inhibit their function to produce antibodies such as IgG. “We have confirmed that cells fail to form nanotubes when the functions of the M-Sec proteins are inhibited. This could provide a means to prevent the immunodeficiency caused by HIV infection. We are advancing our research with the aim of developing drugs that work by a mechanism that is different to that of the current treatments for HIV.”
Understanding how intestinal bacteria promote health
More than 100 trillion intestinal bacteria of more than 500 kinds live in our intestines. “It is certain that intestinal bacteria are closely related to health, disease prevention and disease development,” says Ohno. The consumption of foods containing lactic acid bacteria such as bifidobacteria and lactobacilli is now popular because these bacteria are believed to be effective in promoting health and preventing disease. However, the health-promotion mechanism behind consumption of these bacteria is not well known. “‘Bad’ bacteria and ‘good' bacteria are mostly distinguished on the basis of experiments in a test tube. It is therefore necessary to look a little harder at how bacteria affect our health in the intestinal environment.”
Ohno is conducting experiments using germ-free mice to examine the effects of these bacteria on living organisms. He has found that when germ-free mice are infected with O157, a pathogenic strain of E. coli, their intestinal epithelial cells die due to the toxins produced by the O157 strain. However, he also found that these epithelial cells survive when the intestines host a substantial population of bifidobacteria. It seems that the substances produced by the bifidobacteria serve to protect epithelial cells. “I want to bring together the results of these experiments to help understand the effects of intestinal bacteria on epithelial cells, gut immunity and human health.”
In another area of research, Ohno has established a joint project with Masahira Hattori from the Graduate School of Frontier Sciences at the University of Tokyo with the aim of analyzing intestinal bacteria as a whole at all levels, including DNA, RNA, metabolic substances and individual bacteria. In this project, the researchers are looking into intestinal bacteria in people in various states of health, including healthy people, sick people, and people who have and have not ingested bifidobacteria or lactobacilli. “I imagine a time when I can use the bathroom in the morning and have my intestinal bacteria automatically analyzed and I can receive scientific advice about what food or medication to take to return to the ideal healthy state.”
M cells, however, still form the core of Ohno’s research. “We do not know yet how M cells are differentiated. I really want to dig out all the facts about M cells,” he says. Recently, Ohno’s laboratory discovered a factor that induces differentiation in M cells. Further research progress is therefore expected in the near future. “I hope that the Laboratory for Epithelia Immunobiology at the RIKEN RCAI becomes known as the key research site for M cells.”