Plasmodium vivax is the most widely distributed human malaria parasite. Previous studies have shown that circulating microparticles during P. vivax acute attacks are indirectly associated with severity. Extracellular vesicles (EVs) are therefore major components of circulating plasma holding insights into pathological processes. Here, we demonstrate that plasma-derived EVs from Plasmodium vivax patients (PvEVs) are preferentially uptaken by human spleen fibroblasts (hSFs) as compared to the uptake of EVs from healthy individuals. Moreover, this uptake induces specific upregulation of ICAM-1 associated with the translocation of NF-kB to the nucleus. After this uptake, P. vivax-infected reticulocytes obtained from patients show specific adhesion properties to hSFs, reversed by inhibiting NF-kB translocation to the nucleus. Together, these data provide physiological EV-based insights into the mechanisms of human malaria pathology and support the existence of P. vivax-adherent parasite subpopulations in the microvasculature of the human spleen.
Reticulocyte-derived exosomes (Rex), extracellular vesicles of endocytic origin, were initially discovered as a cargo-disposal mechanism of obsolete proteins in the maturation of reticulocytes into erythrocytes. In this work, we present the first mass spectrometry-based proteomics of human Rex (HuRex). HuRex were isolated from cultures of human reticulocyte-enriched cord blood using different culture conditions and exosome isolation methods. The newly described proteome consists of 367 proteins, most of them related to exosomes as revealed by gene ontology over-representation analysis and include multiple transporters as well as proteins involved in exosome biogenesis and erythrocytic disorders. Immunoelectron microscopy validated the presence of the transferrin receptor. Moreover, functional assays demonstrated active capture of HuRex by mature dendritic cells. As only seven proteins have been previously associated with HuRex, this resource will facilitate studies on the role of human reticulocyte-derived exosomes in normal and pathological conditions affecting erythropoiesis.
Reticulocyte-derived exosomes (rex) are 30–100 nm membrane vesicles of endocytic origin released during the maturation of reticulocytes to erythrocytes upon fusion of multivesicular bodies with the plasma membrane. Combination of CpG-ODN with rex obtained from BALB/c mice infected with the reticulocyte-prone non-lethal P. yoelii 17X malaria strain (rexPy), had been shown to induce survival and long lasting protection. Here, we show that splenectomized mice are not protected upon rexPy+CpG inmunizations and that protection is restored upon passive transfer of splenocytes obtained from animals immunized with rexPy+CpG. Notably, rexPy immunization of mice induced changes in PD1− memory T cells with effector phenotype. Proteomics analysis of rexPy confirmed their reticulocyte origin and demonstrated the presence of parasite antigens. Our studies thus prove, for what we believe is the first time, that rex from reticulocyte-prone malarial infections are associated with splenic long-lasting memory responses. To try extrapolating these data to human infections, in vitro experiments with spleen cells of human transplantation donors were performed. Plasma-derived exosomes from vivax malaria patients (exPv) were actively uptaken by human splenocytes and stimulated spleen cells leading to changes in T cell subsets.
BackgroundTelomere maintenance in Drosophila relies on the targeted transposition of three very special non-LTR retrotransposons, HeT-A, TART, and TAHRE (HTT). The sequences of the retrotransposon array build up the telomere chromatin in this organism. We have recently reported the role of the chromosomal protein Putzig/Z4 in maintaining a proper chromatin structure at the telomere domain of Drosophila. Because the Putzig protein has been found in different cellular complexes related with cell proliferation, development, and immunity, we decided to investigate whether the previously described Putzig partners, DREF/TRF2 and KEN, could also be involved in the telomere function in this organism.ResultsWe have found that mutant alleles for Dref/Trf2 and Ken show alterations in HeT-A and TART expression, suggesting a possible role of these protein complexes in the regulation of the telomere retrotransposons. In agreement, both HeT-A and TART contain the specific DNA binding sequences for the DREF and the KEN protein proteins.ConclusionsWe have identified three new negative regulators involved in the control of the expression of the telomeric retrotransposons, Dref, Trf2, and Ken. Our results offer some clues on which other chromatin-related proteins might be involved in telomere regulation and retrotransposon control.
The spleen is a secondary lymphoid organ with multiple functions including the removal of senescent red blood cells and the coordination of immune responses against blood-borne pathogens, such as malaria parasites. Despite the major role of the spleen, the study of its function in humans is limited by ethical implications to access human tissues. Here, we employed multiparameter flow cytometry combined with cell purification techniques to determine human spleen cell populations from transplantation donors. Spleen immuno-phenotyping showed that CD45+ cells included B (30%), CD4+ T (16%), CD8+ T (10%), NK (6%) and NKT (2%) lymphocytes. Myeloid cells comprised neutrophils (16%), monocytes (2%) and DCs (0.3%). Erythrocytes represented 70%, reticulocytes 0.7% and hematopoietic stem cells 0.02%. Extracellular vesicles (EVs) are membrane-bound nanoparticles involved in intercellular communication and secreted by almost all cell types. EVs play several roles in malaria that range from modulation of immune responses to vascular alterations. To investigate interactions of plasma-derived EVs from Plasmodium vivax infected patients (PvEVs) with human spleen cells, we used size-exclusion chromatography (SEC) to separate EVs from the bulk of soluble plasma proteins and stained isolated EVs with fluorescent lipophilic dyes. The integrated cellular analysis of the human spleen and the methodology employed here allowed in vitro interaction studies of human spleen cells and EVs that showed an increased proportion of T cells (CD4+ 3 fold and CD8+ 4 fold), monocytes (1.51 fold), B cells (2.3 fold) and erythrocytes (3 fold) interacting with PvEVs as compared to plasma-derived EVs from healthy volunteers (hEVs). Future functional studies of these interactions can contribute to unveil pathophysiological processes involving the spleen in vivax malaria.
Emerging evidence indicates that innate host response contributes to the therapeutic effect of antimicrobial medications. Recent studies have shown that Leishmania parasites derived by in vitro selection for resistance to pentavalent antimony (SbV) as meglumine antimoniate (MA) modulate the activation of neutrophils. However, whether modulation of neutrophil activation extends to natural resistance to this antileishmanial drug has not been established. We have evaluated the influence of clinical strains of L. (V.) panamensis having intrinsic tolerance/resistance to SbV, on the inflammatory response of neutrophils during ex vivo exposure to MA. Accordingly, neutrophils obtained from healthy donors were infected with clinical strains that are sensitive (n = 10) or intrinsically tolerant/resistant to SbV (n = 10) and exposed to a concentration approximating the maximal plasma concentration (Cmax) of SbV (32 µg/ml). The activation profile of neutrophils was evaluated as the expression of the surface membrane markers CD66b, CD18, and CD62L by flow cytometry, measurement of reactive oxygen species (ROS) by luminometry, and NET formation using Picogreen to measure dsDNA release and quantification of NETs by confocal microscopy. These parameters of activation were analyzed in relation with parasite susceptibility to SbV and exposure to MA. Here, we show that clinical strains presenting intrinsic tolerance/resistance to SbV induced significantly lower ROS production compared to drug-sensitive clinical strains, both in the presence and in the absence of MA. Likewise, analyses of surface membrane activation markers revealed significantly higher expression of CD62L on cells infected with intrinsically SbV tolerant/resistant L. (V.) panamensis than cells infected with drug-sensitive strains. Expression of other activation markers (CD18 and CD66b) and NET formation were similar for neutrophils infected with SbV sensitive and tolerant clinical strains under the conditions evaluated. Exposure to MA broadly impacted the activation of neutrophils, diminishing NET formation and the expression of CD62L, while augmenting ROS production and CD66b expression, independently of the parasite susceptibility phenotype. These results demonstrated that activation of human neutrophils ex vivo is differentially modulated by infection with clinical strains of L. (V.) panamensis having intrinsic tolerance/resistance to SbV compared to sensitive strains, and by exposure to antimonial drug.
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