Experimental therapy with lectin is capable of stimulating the immune system

Protein binds to existing sugars on the surface of defense cells and stimulates the production of substances that combat pathogens.

By Karina Toledo

Agência FAPESP – An experimental therapy utilizing a type of protein known as a lectin is capable of stimulating the immune system and increasing resistance to such diseases as leishmaniasis, toxoplasmosis and paracoccidioidomicosis. Researchers believe that the same method could be used to combat other infectious diseases and tumors.

The study is being conducted at the Universidade de São Paulo Ribeirão Preto School of Medicine (FMRP-USP) under the coordination of Professor Maria Cristina Roque Antunes Barreira and funded by FAPESP. Roque-Barreira coordinates the Immunochemical and Glycobiology Laboratory, which has been investigating the role of lectins – proteins capable of recognizing the information (patterns) contained the layer of sugar that coats cells – in immunity.

All animal and plant cells possess a layer, called the glycocalyx, on the plasma membrane that contains sugars of very diverse structures. In addition to protecting the cell, the glycocalyx participates in the activation of a series of intracellular signals.

“Lectin molecules have a specialized region that binds to a specific type of sugar, and this binding initiates certain responses in the cell, which could be result in proliferation, migration, cell death or the production of chemical mediators,” explained Roque-Barreira. The group coordinated by Roque-Barreira is dedicated to studying lectins capable of inducing defense cells to produce cytokines so that a more efficient immune response against certain types of microorganisms results.

To uncover the mechanisms of sugar recognition on the surface of defense cells, the researchers tested a lectin – called ArtinM – extracted from jackfruit seeds in a line of human immune cells.

“Lectin from jackfruit, in addition to other plants, has been used as a tool in many laboratories because of its capacity to induce cells in culture to proliferate. However, this has always been performed empirically, and no one knew exactly what interactions occurred,” commented Roque-Barreira.

The FMRP-USP researchers observed that the action of ArtinM on immune cells was related to the production of interleukin 12, a cytokine capable of activating a type of defense cell called a lymphocyte T helper (TH1) cell.

“These lymphocytes secrete large concentrations of another cytokine called interferon-gamma (IFN-γ), which increases the microbicidal activity of macrophages to fight against intercellular parasitism,” explained Roque-Barreira.

The researchers then verified the effect of the protein in two animal models. In the first model, mice were infected with the protozoan Leishmania major, which causes cutaneous leishmaniasis.

“We used a line of mice that were highly susceptible to L. major. In these animals, ArtinM induced interleukin 12 production, making the animals more resistant to infection,” stated Roque-Barreira.

The mice in the second model of the study were infected with Paracoccidioides brasiliensis, which causes paracoccidioidomicosis – a disease endemic to São Paulo State that causes pulmonary fibrosis and can attack other organs. In this model, the administration of ArtinM also made the animals more resistant to infection.

The next step was to discover what molecule was interacting with the lectin. According to Roque-Barreira, “We found that the lectin binds to the sugars of Toll-like receptor 2 (TLR2), which are found in large quantities on the surface of phagocytes (a group of leukocytes that include neutrophils, macrophages and dendritic cells). This interaction initiates a signal that stimulates the cell to produce interleukin 12.”

To prove their findings, the researchers worked with mice that had the gene that codes for the TLR2 receptor knocked out. “We observed that, without TLR2, ArtinM, in fact, no longer induced the production of interleukin 12,” Roque-Barreira stated.

Recombinant proteins

However, according to Roque-Barreira, the therapeutic application of lectin extracted from jackfruit seeds is limited for humans because the protein, which is foreign to the organism, could set off an undesired immune reaction. The scientists, therefore, decided to investigate whether the pathogens that cause the diseases also express lectins capable of stimulating interleukin 12 production.

In the fungus P. brasiliensis, the researchers found paracoccin, a lectin capable of binding to N-acetylglucosamine.

In Toxoplasma gondii, the protozoan that causes toxoplasmosis, the researchers found that microneme proteins, molecules that bind to sugars, possess properties that are very similar to ArtinM. “Two of these proteins, MIC1 and MIC4, have the ability to recognize sialic acid and galactose – sugars that can be expressed on the surface of defense cells,” explained Roque-Barreira.

In vitro tests showed that paracoccin and MIC1 and MIC4 also interact with TLR2 receptors and induce the production of interleukin 12. The next stage was to test the effect in vivo. To this end, the researchers used two experimental models: mice infected with P. brasiliensis and mice infected with T. gondii.  

Twelve days after infection with T. gondii, 100% of the group of untreated animals had died. However, in the group that received MIC1 and MIC4, 80% of the mice were still alive after inoculation of the parasite.

“We also evaluated the number of protozoan cysts that had formed on the animals’ brains. Among the treated mice, we found an average 300 cysts, whereas we found more than 1,000 cysts in the untreated mice. This shows that the therapy made the macrophages more efficient in eliminating the parasites,” said Roque-Barreira.

In addition to verifying the effect of the treatment with paracoccin in the mice infected with P. brasiliensis, the scientists quantified the number of fungal colonies that formed in the lungs of the mice. They found an average of 300,000 colonies among the untreated animals, though the number fell to 50,000 in the animals that received the lectin.

“Using a microscope, we also observed that the lungs of the animals treated with paracoccin presented six times fewer inflammatory lesions than the control group,” commented Roque-Barreira. The results were published in the technical journal Medical Mycology at (http://informahealthcare.com/doi/abs/10.3109/13693780903501671).

In the two models used in the study, the effect of the therapy was tested before and after infection. According to Roque-Barreira, the lectins were capable of protecting the animals both when administered in a prophylactic manner in addition to therapeutically.

“This approach can also be used in humans because the infected patient is in contact with the antigens contained in the parasite. Administering a protein from the pathogen itself will not create additional hypersensitivity in the organism but will protect against the rapid progression of the disease,” she said.

The FMRP-USP group developed recombinant forms of these proteins, i.e., proteins that can be produced in large quantities using genetically modified organisms as tools to express the genes for paracoccin, MIC1 and MIC4.

“Activation of the TH1 immune response could be beneficial not only against infectious disease but also against cancer. In the liver, for example, we have evidence that the administration of lectin prevents carcinogenesis,” Roque-Barreira added.

The scientists also showed that ArtinM was capable of inducing the death of leukemic tumor cells. The results of this study were published in PLoS One at (www.plosone.org/article/info:doi/10.1371/journal.pone.0027892).

The article “Protection against Paracoccidioides brasiliensis infection conferred by the prophylactic administration of native and recombinant ArtinM” (doi:10.3109/13693780903501671) can be read by subscribers at informahealthcare.com/doi/abs/10.3109/13693780903501671.

The article “The Recognition of N-Glycans by the Lectin ArtinM Mediates Cell Death of a Human Myeloid Leukemia Cell Line” (doi:10.1371/journal.pone.0027892) can be read at www.plosone.org/article/info:doi/10.1371/journal.pone.0027892.

The article “ArtinM offers new perspectives in the development of antifungal therapy” (doi: 10.3389/fmicb.2012.00218) can be read at www.frontiersin.org/fungi_and_their_interactions/10.3389/fmicb.2012.00218/abstract.

The article “The immunomodulatory effect of plant lectins: a review with emphasis on ArtinM properties” (doi: DOI 10.1007/s10719-012-9464-4) can be read at link.springer.com/article/10.1007%2Fs10719-012-9464-4.