Written by: Ken Estrellas
Written By: Rebecca Tweedell
Original Article: Barbosa et al. Frontiers in Microbiology 2019
The Gist of It:
With the warm weather of summer fading away, many people are happy to see the insects going away, too. In addition to buzzing around our food and potentially stinging or biting us, several insects can carry disease. One such disease that affects people throughout the Americas, the Mediterranean basin, the Middle East, and Central Asia is cutaneous leishmaniasis, which causes raw skin sores that can lead to permanent scarring and deformity. Leishmania amazonesis is one of the species of Leishmania parasites responsible for this disease, and they are transmitted from person to person through the bite of the sandfly. While there are treatments for leishmaniasis, the L. amazonesis species often has a natural resistance to these drugs. Scientists at the Universidad Federal de Sao Paulo in Brazil have been working to understand more about L. amazonesis in an effort to find new ways to treat these infections. They recently focused in on the extracellular vesicles released from these parasites. Extracellular vesicles are cell-derived structures that can bleb off and be released into the surrounding environment. Cells can use these extracellular vesicles to communicate with surrounding cells and even to deliver cargo, such as proteins that would make the receiving cell resistant to drugs. The researchers found that L. amazonesis parasites were releasing extracellular vesicles into their environment, and that these extracellular vesicles could perform different functions depending on what cell type they encountered. When two different types of immune cells, bone marrow-derived macrophages and B-1 cells, were exposed to the extracellular vesicles, they reacted differently. Importantly, though, they both reacted in a way that would favor the parasite’s survival. The researchers went on to show that in a mouse model of leishmaniasis, mice that were given extracellular vesicles in addition to their infection had worse skin sores and higher numbers of parasites than mice that were infected without the addition of extracellular vesicles. Understanding more about how L. amazonesis infects and how it uses extracellular vesicles to improve its pathogenicity will help identify new ways to fight these parasites.
The Nitty Gritty:
The researchers began by determining whether extracellular vesicles (EVs) were released from L. amazonesis promastigotes after various amounts of time at 26°C (the average temperature inside the sandfly), 34°C, and 37°C. Nanoparticle tracking analysis showed that EVs were released over time at all 3 temperatures, but the number of particles released at 26°C was significantly higher. EVs released at all 3 temperatures were similar in size, averaging approximately 180 nm. Dot blot and ELISA analysis of the EVs revealed that they contained proteins with known immunomodulatory properties, GP63 and LPG. Bone marrow-derived macrophages (BMDMs) and B-1 cells were exposed to EVs obtained at the 3 different temperatures, and cytokine secretion was measured by ELISA. In BMDMs, TNFα secretion was not affected, IL-6 secretion was increased in response to EVs from parasites grown at 26°C and 37°C, and IL-10 secretion was only increased in response to EVs from parasites grown at 26°C. Interestingly, in B-1 cells, secretion of IL-6, IL-10, and TNFα were decreased in response to EVs from all 3 temperatures, showing that EVs can have different effects depending on the type of cell they encounter. To determine the significance of these in vitro findings in vivo, BALB/c mice were infected with L. amazonesis either with or without the addition of EVs from parasites grown at 26°C (mimicking the EVs that would be present during transmission from the sandfly). Co-injection of EVs with the parasites increased the number of parasites present 7 weeks post-infection and enhanced the progression of lesions. Overall, these findings show that EVs from L. amazonesis can play a key role in the parasite’s pathogenesis.
Original Research Article: Barbosa, F.M.C., et al. “Extracellular vesicles released by Leishmania (Leishmania) amazonesis promote disease progression and induce the production of different cytokines in macrophages and B-1 cells.” Front Microbiol 9 (2018): 3056.
Written by: Kaitlyn Sadtler
Original Article: Colin-York et al. Journal of Cell Science 2019
The Gist of It:
There are many things that affect the way our immune system functions. One of those is the strength of the connection, or immunologic synapse, between two key immune cells, a T cell and an antigen presenting cell. Through this synapse, the T cell learns from the antigen presenting cell what pathogen it is facing by being presented (or shown) the antigen (which is a small piece of that pathogen). To improve our understanding of the immune system, it is important to use models that preserve this immunologic synapse. When studying our immune system, researchers can use a variety of models and tools, from looking at patient samples to considering animal models to using cells in a dish. When using cells in a dish, there are even more options, including cells freshly isolated from humans or animals, as well as so-called immortalized cells or cell lines. Immortalized cells were once normal cells living in a human or an animal but have been transformed so that they can live indefinitely outside the body in a tissue culture dish. One specific type of immortalized cell is the Jurkat. Jurkats were made using T cells collected from a 14 year old boy in the 1970’s. This boy had a disease called T-cell leukemia, which is a type of blood cancer. The leukemic T cells have a variety of mutations but still retain important T-cell functions. Recently, researchers looked at the formation of an immunologic synapse in primary T cells (cells directly collected from humans or animals) versus immortalized Jurkat cells. They focused specifically on a protein that is responsible for cell structure and movement known as actin. Actin can form long rods or filaments that help move the cell membrane (both on a large scale to move the cell, and on a smaller scale to make tiny protrusions like those used in immunologic synapses). Scientists discovered that there were differences between Jurkats and primary T cells. In Jurkats (which are bigger than their primary T cell counterparts) the actin looked different at the synapse; actin was more concentrated or tightly packed in primary cells and more diffuse in Jurkats. Additionally, the two cell types used different mechanisms for the structure and function of their actin filaments, with the Jurkats relying more heavily on actin turnover, in which individual pieces of actin hop on or hop off the filament on their own, and primary cells relying on a protein called myosin-II. Seeing these differences in primary versus immortalized cells shows us that we might need to re-think our assumptions about T cells and what conclusions we can draw from different models. Studies like these help scientists truly understand their data and what implications their experiments have on our understanding of human health and disease.
The Nitty Gritty:
Colin-York et al. analyzed the differences between (1) Jurkat T cells, (2) human CD4+ T cells, and (3) murine CD4+ T cells. Using total internal reflection (TIRF) and structured illumination microscopy (SIM) they noted distinct differences when cells were activated on a synthetic planar lipid bilayer system (dipalmitoylphosphatidylcholine [DOPC] + anti-CD3). Although calcium flux remained the same (determined by Fluo-4 AM labeling), actin dynamics were altered. In both human and mouse primary derived T cells, there was a distinct punctate actin staining that was absent in Jurkat T cells. Lamelipodial leading edge curvature and dynamics were greater in primary murine T cells when compared with Jurkat T cells, though this did not have a significant change upon the velocity of TCR (T cell receptor) microcluster movement. When modulating actin behavior with several compounds, researchers found that in primary T cells, the flow of F-actin was largely dependent upon myosin-II (determined by blocking with blebbistatin), whereas in Jurkats actin flow was largely dependent upon actin nucleation. These results suggest differences in the formation and dynamics of supramolecular activation clusters that may affect the interpretation of data gathered from Jurkat T cells.
Original Research Article: Colin-York, H., et al. “Distinct actin cytoskeleton behaviour in primary and immortalised T-cells.” J Cell Science (2019): jcs-232322.
Written By: Abel B. Cortinas