When the human immune system learns of the presence of a parasitic infection, such as the causative agent of Chagas disease, leishmaniasis and toxoplasmosis, a highly efficient operation prevents the microorganisms from spreading inside cells and throughout the body. The attack mechanism against these intracellular parasites has just been revealed, according to an article published in January 2016 on the website of the journal Nature Medicine. “This knowledge can help us to discover more efficient vaccines in the induction of CD8 T lymphocytes, a problem in immunology,” says immunologist Ricardo Gazzinelli of the René Rachou Research Center, an arm of the Oswaldo Cruz Foundation in Minas Gerais State, and coordinator of the National Institute of Science and Technology for Vaccines (INCTV).
It is known that cytotoxic lymphocytes detect and combat the pathogens that reside in cells, while antibodies guard the environment outside them. But until now it was not known that the CD8 T lymphocytes were also active in combating protozoa and bacteria. “It is important against viruses,” says Gazzinelli, “because it destroys the infected cell on which they depend to proliferate.” It turns out that bacteria and parasites are autonomous organisms and, if released into the extracellular medium, once the host cell dies, they are able to infect other cells. Gazzinelli specializes in the relationship between the human immune system and the parasites that cause diseases (see Pesquisa FAPESP Issues nº 160, 164 and 221). He spent the 2013/2014 academic year in the United States, thanks to a visiting professor research internship at the Capes Institute/David Rockefeller Center for Latin American Studies at Harvard University, for which he was selected. Early on, after presenting his work at a lecture, he was recruited by Judy Lieberman, a physicist and doctor whose laboratory investigates the molecular mechanisms by which cytotoxic lymphocytes destroy cells infected with viruses and bacteria. “Does it work the same way for protozoa?” she asked.
And so a dynamic partnership began, which also included Dr. Farokh Dotiwala, Lieberman’s laboratory colleague, and biologist Rafael Polidoro, at the time a doctoral student who followed Gazzinelli to Harvard. The first result was a description of the destructive mechanism of intracellular parasites: the microptose. The term is inspired by apoptosis, cell death, apparently because they are very similar: they form bubbles in the cell membrane, mitochondria become dilated, the DNA is damaged and the chromatin, which contains the genetic material, appears condensed. What differentiates the two types of deaths are the actors. In the case of apoptosis, lymphocytes recognize the infected cells through markers provided by the immune system and release tiny sacs, or granules, containing a protein, perforin, that perforates the membrane, and an enzyme called granzyme. This enters the cell and degrades the proteins needed for homeostasis, which leads to cell death.
Gazzinelli, Lieberman and their colleagues showed that the CD8 T lymphocytes of humans (but not those of rodents) also release an antimicrobial substance called granulysin, which enters the infected cell and is attracted by membranes with a low cholesterol content: the parasites. The granulysin pierces the membrane of the parasites and invading bacteria and allows the entry of granzyme, which generates compounds very reactive to the oxygen base (free radicals) and deactivates the defense mechanisms of the organism against oxidative stress. It is these oxidative processes that, in most cases, eliminate the parasite. When this attack is examined under a microscope, it appears to be very similar to apoptosis. But chemical distinctions inherent in the body—apoptosis depends on enzymes called caspases, which do not exist in protozoa—and different actors (granulysin and microorganisms) justified the creation of a new term, microptose.
“An amazing thing is that the death of the parasites is faster than the death of the host cell, although it is triggered afterwards,” notes Gazzinelli. This prevents protozoa from escaping and invading other cells, a situation that needs to be combated by the immune system through macrophages patrolling the area between the cells and devouring the invaders. It was, until now, what was thought to occur. “This explains why the CD8 T are so efficient in fighting infection by intracellular protozoa,” says Gazzinelli. It may be why many cases of Chagas disease are asymptomatic, for example.
The discovery that granulysin does not exist in all species is also important. “The usefulness of doing studies of certain diseases using mice has to be reviewed,” says Gazzinelli, although he does not discard them as test animals. His group repeated the discovery producing transgenic rodents capable of expressing the protein, and they were much more resistant to protozoan infections.
The fruitful partnership between the Minas Gerais group and Harvard will continue in the coming years. “We plan to dissect the role of CD8 T lymphocytes in combating these infections,” says Gazzinelli. Why doesn’t it always work? Why are there patients who develop Chagas disease? It was within this context that Rafael Polidoro defended his thesis in 2014 and the following year moved to Lieberman’s laboratory for a postdoctoral fellowship.
In terms of vaccines, Gazzinelli also plans to review tests for a therapeutic vaccine against Chagas disease proposed in 2015 based on a study published in PLOS Pathogens, led by immunologists Joseli Lannes-Vieira of the Oswaldo Cruz Foundation, and Maurício Rodrigues, a professor at the Federal University of São Paulo (Unifesp), who died last year and co-coordinated INCTV. “We want to use the vaccine in transgenic mice expressing granulysin to see if it is more efficient,” says Gazzinelli.
DOTI WALA, F. et al. Killer lymphocytes use granulysin, perforin and granzymes to kill intracellular parasites. Nature Medicine. Online. January 11, 2016.
PEREIRA, I.R. et al. A human type 5 adenovirus-based Trypanosoma cruzi therapeutic vaccine re-programs immune response and reverses chronic cardiomyopathy. PLOS Pathogens. V. 11, No. 1, e1004594. January 24, 2015.