Extracellular Vesicles Released by <em>Leishmania</em>: Impact on Disease Development and Immune System Cells

Leishmania spp. release extracellular vesicles (EVs) containing parasite molecules, including several antigens and virulence factors. These EVs can interact with the host cells, such as immune cells, contributing to the parasite–host relationship. Studies have demonstrated that Leishmania-EVs can promote infection in experimental models and modulate the immune response. Although the immunomodulatory effect has been demonstrated, Leishmania-EVs can deliver parasite antigens and therefore have the potential for use as a new diagnostic tool and development of new therapeutic and vaccine approaches. This review aims to bring significant advances in the field of extracellular vesicles and Leishmania, focusing on their role in the cells of the immune system

Extracellular Vesicles Released by Leishmania (Leishmania) amazonensis Promote Disease Progression and Induce the Production of Different Cytokines in Macrophages and B-1 Cells

The extracellular vesicles (EVs) released by Leishmania can contribute to the establishment of infection and host immunomodulation. In this study, we characterized the shedding of EVs from Leishmania (Leishmania) amazonensis promastigotes. This species is the causative agent of cutaneous leishmaniasis, and its role during interactions with bone marrow-derived macrophages (BMDMs) and peritoneal B-1 cells was evaluated. Leishmania amazonensis promastigotes cultivated in vitro at different times and temperatures spontaneously released EVs. EVs were purified using size-exclusion chromatography (SEC) and quantitated by nanoparticle tracking analysis (NTA). NTA revealed that the average size of the EVs was approximately 180 nm, with concentrations ranging from 1.8 × 108 to 2.4 × 109 vesicles/mL. In addition, the presence of LPG and GP63 were detected in EVs obtained at different temperatures. Naïve BMDMs stimulated with EVs exhibited increased IL-10 and IL-6 expression. However, incubating B-1 cells with parasite EVs did not stimulate IL-10 expression but led to an increase in the expression of IL-6 and TNFα. After 7 weeks post-infection, animals infected with L. amazonensis promastigotes in the presence of parasite EVs had significant higher parasite load and a polarization to Th2 response, as compared to the group infected with the parasite alone. This work demonstrated that EVs isolated from L. amazonensis promastigotes were able to stimulate macrophages and B-1 cells to express different types of cytokines. Moreover, the immunomodulatory properties of EVs probably contributed to an increase in parasite burden in mice. These findings suggest that the functionality of L. amazonensis EVs on immune system favor of parasite survival and disease progression

Avaliação do efeito de vesículas extracelulares liberadas por Leishmania (Leishmania) amazonensis na imunização de camundongos BALB/c

Leishmanioses são um grupo de doenças causadas pelo protozoário do gênero Leishmania, ordem Kinetoplastida e da família Trypanosomatidae. A doença é endêmica em 97 países, sendo que 18 destes estão no continente Americano, com o Brasil apresentando 90% dos casos reportados nas Américas. Estudos recentemente demonstraram que algumas espécies do gênero Leishmania são capazes de liberar vesículas extracelulares (VEs) contendo antígenos, fatores de virulência, RNA, DNA e lipídeos do parasita. VEs têm papel importante na relação parasita-hospedeiro, na sobrevivência do patógeno em diferentes níveis, na facilitação da infecção em modelos experimentais, na imunomodulação e na adaptação do parasita ao ambiente do hospedeiro. Este trabalho teve como objetivo caracterizar e avaliar o papel protetor de vesículas extracelulares liberadas por promastigotas de Leishmania (Leishmania) amazonensis com distintos perfis de virulência em protocolos diferentes de imunização. Foram utilizadas promastigotas de L.amazonensis virulenta (recém recuperada de lesões em animais) e atenuada por cultivo em longo período em cultura (100 passagens em cultura in vitro). Animais infectados com o parasita atenuado desenvolveram menor lesão na pata e com menor carga parasitária comparado ao grupo infectado com promastigotas do parasita virulento, comprovando a atenuação. Analisando as VEs liberadas pelo parasita atenuado e virulento foi observado diferenças na maneira em que os parasitas liberam suas vesículas e na sua composição com diferenças na expressão de LPG e gp63, fatores de virulência presentes no parasita que também foram detectadas nas VEs dos parasitas virulentos e atenuado. Para avaliar o papel protetor grupos de camundongos foram imunizados com VEs do parasita na presença ou não de adjuvante Alum e em protocolos de duas e três doses e posteriormente foram desafiados com promastigotas do parasita virulento. Análise da carga parasitária mostraram diminuição na carga em camundongos imunizados com VE emulsificadas em adjuvante, em comparação aos grupos controles. Em relação a modulação do sistema imune vimos um mix de resposta com a presença de citocinas e anticorpos tanto do perfil Th1 como do perfil Th2 sendo produzidas. Esses resultados sugerem que VEs liberadas por L. amazonensis foram capazes de induzir um efeito protetor em modelo de imunização e após desafio com o parasita virulento.Leishmaniasis is a group of diseases caused by the protozoan of the genus Leishmania, order Kinetoplastida and the family Trypanosomatidae. The disease is endemic in 97 countries, which 18 are in the American continent, with Brazil having 90% of cases reported in the Americas. Studies have recently shown that some species of the genus Leishmania are able to release extracellular vesicles (EVs) containing antigens, virulence factors, RNA, DNA and parasite lipids. EVs play an important role in the host-parasite relationship, in the survival of the pathogen at different levels, in facilitating infection in experimental models, in immunomodulation and in the adaptation of the parasite to the host's environment. This work aimed to characterize and evaluate the protective role of extracellular vesicles (EVs) released by Leishmania (Leishmania) amazonensis promastigotes with distinct virulence profiles in different immunization protocols. Promastigotes of L. amazonensis virulent (recently recovered from in vivo) and attenuated by long-term culture were used (100 passages in vitro culture). Animals infected with the attenuated parasite developed less lesion in the paw and with a lower parasite load compared to the group infected with promastigotes of the virulent parasite, confirming the attenuation. Analyzing the VEs released by the attenuated and virulent parasite, a difference was observed in the way in which the parasites release their vesicles and in their composition with differences in the expression of LPG and gp63, virulence factors present in the parasite that were also detected in VEs released by virulent parasites. To evaluate the protective role, groups of mice were immunized with EVs of the parasite in the presence or not of Alum adjuvant and in a protocol of two and three doses and were subsequently challenged with promastigotes of the virulent parasite. Analysis of the parasite load showed a decreased in the load in mice immunized with VE emulsified in adjuvant, compared to control groups. Regarding the modulation of the immune system, we saw a mix of responses with the presence of cytokines and antibodys of both Th1 and Th2 profile being produced. These results suggest that EVs released by L. amazonensis were able to induce a protective effect in a model of immunization and challenge with the virulent parasite.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)FAPESP: 2019/21614-