Researchers say exosomes shed from reticulocytes in malaria-infected mice protect uninfected animals against lethal challenge.

Exosomes released by reticulocytes in malaria-infected mice could provide a springboard for the development of new immunomodulating malaria treatments, researchers claim. A team at the Barcelona Centre for International Health Research (BCIHR) in Spain, the University of Texas at El Paso, and Barcelona’s Institució Catalana de Recerca i Estudis Avançats (ICREA), has found that the reticulocytes in mice infected with a nonlethal strain of Plasmodium yoelii package a diverse range of parasite antigens into exosomes that can be isolated from peripheral blood.

More importantly, they subsequently found that immunizing noninfected mice with these exosomes triggered the formation of antiparasite antibodies. An immunization strategy that combined the antigen-containing exosomes with the immunostimulator cytosinephosphate-guanosine oligodeoxynucleotides (CpG-ODN ), in addition provided the vast majority of treated animals with long-term protection against subsequent, and repeated challenge with a fatal strain of the P. yoelii species.

The BCIHR’s Hernando A. Del Portillo, M.D., and colleagues, describe their research in PLoS One, in a paper titled “Exosomes from Plasmodium yoelii-Infected Reticulocytes Protect Mice from Lethal Infections.” They claim their work is the first to demonstrate that reticulocyte exosomes from infected animals can play a role in modulating immune responses.

Exosomes are 30–100 nm membrane vesicles that are released by cells into the extracellular environment upon fusion of multivesicular bodies (MVBs) with the plasma membrane, the researchers explain. Initially described as system that enables developing reticulocytes to dispose of unwanted cellular cargo, exosomes have since been shown to be secreted by a range of different cell types including dendritic cells, macrophages, B cells and tumor cells.

Microparticles (MPs) are another type of vesicle found circulating in blood, but in contrast with exosomes, which originate from the fusion of MVBs with the plasma membrane, MPs originate by budding or shedding from the plasma membrane, and are bigger in size (100–1,000 nm). MPs have in addition been associated with malaria pathology both in human and rodent models, and production of MPs by parasitized red blood cells has also been indicated in malaria immune responses and inflammation.

Given that reticulocytes are the cells preferentially invaded by malaria parasites such as Plasmodium vivax and the reticulocyte-prone nonlethal Plasmodium yoelii 17X strain, the BCIHR team hypothesized that reticulocyte-derived exomsomes (rex) secreted from malaria-infected cells may contain parasite proteins as well as their designated cargo for disposal. They purified exosomes from peripheral blood of BALB/c mice infected with P. yoelii 17X at about two weeks post-infection (p.i.), when reticulocytosis reached 60–90%. The exosomes were then coated on latex beads, stained with a panel of FITC- or PE-labeled antibodies, and analyzed by flow cytometry. The presence of specific markers indicated that the exosomes were derived from an internal cellular compartment and weren’t plasma membrane fragments, and had indeed originated from reticulocytes (this was confirmed in part by the presence of CD71 and Itga4, which have both previously been identified in reticulocyte-derived exosomes).

Subsequent LC-MS/MS evaluation of the proteomes of exosomes purified from the plasma of infected BALB/c mice identified many of the proteins most frequently found in exosomes, including those previously described in rex. There were no markers known to be associated with MPs or apoptotic vesicles. What was most notable, the authors comment, was that proteomic analysis also picked up several P. yoelii antigens, including serine-repeat antigens, merozoite surface proteins 1 and 9, enzymes, proteases, and heat-shock proteins. This provided a strong indication that peripheral blood exosomes obtained from BALB/c mice with P. yoelii 17X infections contain parasite proteins that are probably derived from infected reticulocytes.

To see whether the peripheral blood exosomes from Plasmodium-infected mice could induce parasite-specific immune responses, the team then immunized mice with the exosomes derived from the P. yoelii–infected animals (exPy), and from noninfected animals (exC). The animals were immunized twice, 20 days apart, and then 20 days after the second administration sera were tested by immunofluorescence (IFA). This confirmed that immunization with exPy resulted in the production of IgG antibodies capable of specifically recognizing infected red blood cells, the team notes.

They then investigated the effects of immunizing mice with either exC or exPY, and then subjecting the animals to challenge with P. yoelii 17XL lethal parasites. This showed that immunization with exPy attenuated the course of parasitaemia, increased survival time, up to a 65% (1–2% to 65%) increase in reticulocytosis, as well as a change in the parasite’s cell tropism to reticulocytes: in fact some 90% of infected cells in the infected, exPy-immunized animals were reticulocytes. Importantly, the authors stress, “these results demonstrated that peripheral blood exosomes in this rodent malaria model induced immune responses which modulated the course of infection.”

To exclude the possibility that exosomes from other cells capable of antigen presentation were the source of the exosomes obtained from the blood of BALB/c mice infected with P. yoelii 17X, the researchers cultured isolated reticulocytes from the blood of P. yoelii-infected mice for 24 hours, and purified the exosomes from supernatants. Electron microscopy confirmed their purity and showed the vesicles had a size and morphology compatible with exosomes, and then flow cytometry analysis of rex bound to beads revealed the surface expression of Lamp1, Tfrc, and Itga4 markers.

These in vitro-purified rex from infections (rexPy) or from anemic mice (rexC) were then used to immunize new groups of animals at 20-day intervals. The animals were again then infected 20 days later with P. yoelii 17XL lethal parasites. Encouragingly, the results again matched those observed when exosomes from the peripheral blood of infected animals were used for immunization, including higher survival times, increase in reticulocytosis (from 1–2% up to 60%), and infection preference for reticulocytes.

In a final set of experiments the BCIHR-led investigators tested the effects of rex immunization in combination with CpG oligodeoxynucleotide (CPG-ODN), a potent immunostimulator that has been shown to help provide protection against rodent malaria. BALB/c mice were first immunized subcutaneously with CPG-ODN combined with either rexPy or rexC, and then given an exosome booster 20 days later. Subsequent IFA and ELISA analyses showed that the rexPy-immunized mice (but not the rexC-immunized mice) had generated predominantly IgG2a and IgG2b antibodies that recognized infected RBCs. This result was of particular interest because antibody responses to the lethal strain of P. yoelii are generally suppressed during infection.

Critically, 83% of the animals that had received the dual CPG-ODN/rexPy immunization survived a subsequent challenge with P. yoelii 17XL, completely cleared the infection, and remained immunoprotected over the course of three subsequent challenges with P. yoelii 17XL during the following 156 days. These animals again showed concomitant reticulocytosis and a change of cell tropism to reticulocytes.

“To our knowledge, the results presented here provide the first description of exosomes, not MPs, containing malaria proteins…And that in addition to their cargo-disposable role in differentiation to erythrocytes, rex have a role in modulating immune responses,” the authors conclude. “Moreover, when combined with CpG-ODN, immunizations with rexPy obtained from reticulocyte in vitro culture elicited the same responses as above in addition to conferring complete and long lasting protection in close to 85% of the mice tested…While further experimentation is required to determine the exact role of rex during infections, rex represents a new player in immune response to malaria that could also be used as a vaccine tool against infections.”

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