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Immunology

Unexpected allies

Without bacteria, mammals do not produce the necessary inflammatory reaction to deal with injuries

Laura DaviñaMice visibly feel pain when their paws are injected with an irritating substance. They lick the swollen limbs and avoid using them to scurry about, a reaction that does not surprise anyone who has suffered inflammation because of a cut on the hand or foot. But things are not like this always. At the lab of immunologist Mauro Teixeira, from the Immunology and Biochemistry Department at the Federal University of Minas Gerais/UFMG, some mice feel nothing when they get these injections, and feel very little pain when their feet are poked with a sharp object. Apparently, these super-mice are immune to pain. But what is special about these mice is the absence of microbes that live in the bodies of all mammals – including human beings. These microbes are essential for the immune system to function properly. The group from Minas Gerais discovered that, in addition to being less sensitive to pain, the germ-free mice did not have the means to fight infections caused by harmful micro-organisms nor the means to protect an injured foot.

Maintaining these rodents in a micro biota free from bacteria that normally live in mammals, demands a lot of caution and a complex apparatus. These mice live in plastic bubbles; everything they consume – air, water, food – is sterilized and delivered through special passages. The mice, monitored 24 hours a day, cannot have any contact with the outside world. Instead of causing diseases, these bacteria species that are part of the micro-biota provide help for a number of life-sustaining functions, such as the digestion of food, the production of vitamin K, which is necessary for blood coagulation, and the control of fat storage. In addition, the micro-biota competes for space that otherwise would be available for invaders that are harmful to the body. Every person normally has ten times more bacteria than human cells – most of these bacteria are found in the intestines, where they live in harmony with the body.

Germ-free mice have existed for many decades in laboratories that research the action of bacteria on the body. The innovative approach used by Teixeira’s group was to unravel – instead of studying diseases – how the micro-biota helps the immune system mature. An article published this year in Proceedings of the National Academy of Sciences (PNAS) explains the secret of the super-mice. When they are injected with irritating substances such as carrageen, extracted from red seaweed, their bodies produce a protein called interleukin -10 (IL-10), with a powerful anti-inflammatory action. This is contrary to what happens to normal mice, in which the carrageen triggers a cascade of biochemical reactions that provoke the inflammation, which is essential to draw the animal’s attention to the injury and recruit defense cells to the injury site. When harmful bacteria take advantage of an injury to invade the body, these defense cells eliminate the bacteria, thus preventing further damage.

Laura DaviñaUntil 2004, this anti-inflammatory reaction of the animals without the micro-biota was unknown. That year, Teixeira’s group published the findings of an experiment in the Journal of Immunology. In the experiment, Teixeira and his team momentarily interrupted the blood flow of a major intestinal artery in two groups of mice – one group was germ-free and the other group had the normal micro-biota. When the blood flows again strongly, it generally causes a lesion in the intestine. In the normal mice, this lesion caused a general inflammation that killed all the animals. “The response to the intestinal inflammation was so intense that it attacked all of the bodies’ healthy cells and tissues,” explains Flávio Almeida Amaral, a member of the research team from Minas Gerais, who is currently working on research studies for his doctorate thesis at England?s William Harvey Research Institute. Amaral points out that the germ-free mice had a different response: they overcame the inflammation and survived.

What happened to the normal mice was that a standard process had gone out of control. Inflammation, which generally causes swelling, increase of local temperature and pain at the place of injury, induces the body to produce chemical substances called mediators. These molecules send off messages to the immune system, which in turn sends defense cells such as macrophages and leukocytes to the injured site, where they battle the harmful micro organisms. In the normal mice being tested in the experiments conducted by Teixeira, this response was so intense that it was not restricted to the intestine. Defense cells in large quantities flowed into the blood stream, fighting the bacteria, and, much like stray bullets, also injuring the cells of healthy tissues. In sensitive organs such as the lungs, this process causes more damage and may lead to death.

The group from UFMG discovered that animals that had never had any contact with bacteria react to the injury by producing interleukin-10, the natural anti-inflammatory substance. But they lose this anti-inflammatory capacity when they acquire a micro-biota – to this end, it is enough for them to have contact with the feces of normal mice. “It is as if harmless bacteria maintained the mammal’s body in a state of alert, ready to fight the invaders with inflammation,” Teixeira explains.

The discovery was a major advance in the understanding of how immunity is developed; but it was not enough. It was necessary to understand in detail how the germ-free mice produce interleukin and perhaps discover a strategy that would be useful in the treatment of acute and chronic inflammatory diseases, such as rheumatoid arthritis, asthma, and multiple sclerosis. In 2007, the group published more details again in the Journal of Immunology. The details now included annexins and lipoxins, the proteins that activate the immune system. After observing the lesion in the intestine, the researchers detected three times higher lipoxin levels in the germ-free mice. These mice also had higher levels of annexins in comparison to those detected in the normal mice. This was the first time that research demonstrated that lipoxins and annexins are essential to stimulate the production of interleukin-10. As these are smaller molecules, they might be good candidates to be used as the basis for anti-inflammatory drugs. The expectation of the group from the UFMG is that compounds derived from these proteins might substitute the existing, cortisone-based anti-inflammatory drugs, which produce serious side effects such as weight gain, loss of muscular mass and tendency to diabetes and osteoporosis.

Laura DaviñaIndeed, the team from UFMG showed that injecting lipoxin and annexin into the blood of mice with micro-biota protects the body from lesions in the intestine caused by a temporary blocking of the blood flow. Treatment is efficient in terms of significantly reducing local hemorrhaging and fighting inflammation, retrieving the characteristic properties and the proper functioning of the animals intestinal tissue. Lipoxin and annexin also reduce the mortality rate of these animals by 50%. These benefits result from the actions of these proteins, which act directly and independently on the site of the lesion, preventing the tissues from accumulating liquid in excess and forming the edema that is typical of inflammation.

However, avoiding inflammation is not always a good thing. Danielle da Gloria de Souza, a colleague of Teixeira’s at UFMG, warns that anti-inflammation mechanisms can sometimes jeopardize the situation when the inflammation comes together with an invasion of bacteria, that is, infection. Anti-inflammatory substances block the communication between the injured site with leucocytes, the defense cells that surround and destroy harmful bacteria. If leucocytes are not recruited, the body becomes vulnerable and fertile ground for infections. “This is a drawback. Protection drops by half,” says Danielle. “On the other hand, this novelty has to be studied further, as it opens up a promising horizon given the efficient and timely response to battle inflammation,” she adds.

The researchers from UFMG state that it is necessary to understand how bacteria influence the production of specific mediators and why, when these mediators are absent, the body secretes other mediators. A possible explanation must involve the so-called gene programming: the active micro-biota in the body that hosts genes linked to the stimulation of the immune system; the absence of the referred bacteria obliges the body to organize other defense strategies.

The study’s latest finding reveals that the germ-free mice have nearly 50% higher resistance to pain in comparison to the mice with the micro-biota. “It is not normal pain,” Teixeira points out; “if we prick their foot, they feel pain. We’re talking about the over-sensitivity caused by inflammation.” The researchers identified interleukyn-10 as being responsible for the pain-killing effect, but there is still a need to detail the pain-resistant processes to understand how the pain-killing effect occurs. “For the time being, these are just guesses and hypotheses. Based on the available data, we will re-investigate which chemical signals and mediators are directly involved in the pain producing and pain-resistant process,” says Amaral, who believes that some day this knowledge will be useful for the arsenal of pain killers available in pharmacies.

“The relationship between micro-biota and the protection of the body was already known,’ says Teixeira. “We took a step forward and refined the knowledge on the collaboration that occurs between bacteria and the defense system by providing more detailed information on the stages and substances that help organize this relationship.” In his opinion, the scope of these discoveries is much more extensive than the practical results they might have on the development of pharmaceutical products. The research study shows that a peaceful co-existence with bacteria is what actually allows us to survive in a world where these tiny organisms are becoming increasingly numerous and powerful.

Scientific Articles
SOUZA, D. G. et al. The essential role of the intestinal micro-biota in facilitating acute inflammatory response. The Journal of Immunology. v. 173, n. 6, p. 4137-4146. Sept. 2004.
SOUZA, D. G. et al. The required role of endogenously produced lipoxin A4 and annexin-1 for the production of IL-10 and inflammatory hypo responsiveness in mice. The Journal of Immunology. v. 179, n. 12, p. 8533-8543. Dec. 2007.
AMARAL, F. A. et al. Commensal micro-biota is fundamental for the development of inflammatory pain. PNAS. v. 105, n. 6, p. 2193-2197. Feb. 2008.

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