Mass Spectrometry Identification of Biomarkers in Extracellular Vesicles From Plasmodium vivax Liver Hypnozoite Infections.

Latent liver stages termed hypnozoites cause relapsing Plasmodium vivax malaria infection and represent a major obstacle in the goal of malaria elimination. Hypnozoites are clinically undetectable, and presently, there are no biomarkers of this persistent parasite reservoir in the human liver. Here, we have identified parasite and human proteins associated with extracellular vesicles (EVs) secreted from in vivo infections exclusively containing hypnozoites. We used P. vivax-infected human liver-chimeric (huHEP) FRG KO mice treated with the schizonticidal experimental drug MMV048 as hypnozoite infection model. Immunofluorescence-based quantification of P. vivax liver forms showed that MMV048 removed schizonts from chimeric mice livers. Proteomic analysis of EVs derived from FRG huHEP mice showed that human EV cargo from infected FRG huHEP mice contain inflammation markers associated with active schizont replication and identified 66 P. vivax proteins. To identify hypnozoite-specific proteins associated with EVs, we mined the proteome data from MMV048-treated mice and performed an analysis involving intragroup and intergroup comparisons across all experimental conditions followed by a peptide compatibility analysis with predicted spectra to warrant robust identification. Only one protein fulfilled this stringent top-down selection, a putative filamin domain-containing protein. This study sets the stage to unveil biological features of human liver infections and identify biomarkers of hypnozoite infection associated with EVs.

The different impact of drug-resistant Leishmania on the transcription programs activated in neutrophils.

Drug resistance threatens the effective control of infections, including parasitic diseases such as leishmaniases. Neutrophils are essential players in antimicrobial control, but their role in drug-resistant infections is poorly understood. Here, we evaluated human neutrophil response to clinical parasite strains having distinct natural drug susceptibility. We found that Leishmania antimony drug resistance significantly altered the expression of neutrophil genes, some of them transcribed by specific neutrophil subsets. Infection with drug-resistant parasites increased the expression of detoxification pathways and reduced the production of cytokines. Among these, the chemokine CCL3 was predominantly impacted, which resulted in an impaired ability of neutrophils to attract myeloid cells. Moreover, decreased myeloid recruitment when CCL3 levels are reduced was confirmed by blocking CCL3 in a mouse model. Collectively, these findings reveal that the interplay between naturally drug-resistant parasites and neutrophils modulates the infected skin immune microenvironment, revealing a key role of neutrophils in drug resistance.

Natural resistance to meglumine antimoniate is associated with treatment failure in cutaneous leishmaniasis caused by Leishmania (Viannia) panamensis.

The multifactorial basis of therapeutic response can obscure the relation between antimicrobial drug susceptibility and clinical outcome. To discern the relationship between parasite susceptibility to meglumine antimoniate (SbV) and therapeutic outcome of cutaneous leishmaniasis, risk factors for treatment failure were considered in evaluating this relationship in ninety-one cutaneous leishmaniasis patients and corresponding clinical strains of Leishmania (Viannia) panamensis. Parasite susceptibility to 32 µg SbV/mL (plasma Cmax) was evaluated in primary human macrophages, PBMCs, and U937 macrophages. Early parasitological response to treatment was determined in lesions of a subgroup of patients, and pathogenicity of Sb-resistant and sensitive clinical strains was compared in BALB/c mice. Parasite survival in cell models and patient lesions was determined by qRT-PCR of Leishmania 7SLRNA transcript. Parasite loads in BALB/c mice were quantified by limiting dilution analysis. The disparate Sb-susceptibility of parasite subpopulations distinguished by isoenzyme profiles (zymodemes) was manifest in all cell models. Notably, Sb-resistance defined by parasite survival, was most effectively discerned in U937 macrophages compared with primary human host cells, significantly higher among strains from patients who failed treatment than cured and, significantly associated with treatment failure. Each unit increase in transformed survival rate corresponded to a 10.6-fold rise in the odds of treatment failure. Furthermore, treatment failure was significantly associated with naturally Sb-resistant zymodeme 2.3 strains, which also produced larger lesions and parasite burdens in BALB/c mice than Sb-sensitive zymodeme 2.2 strains. The confounding effect of host risk factors for treatment failure in discerning this association was evidenced in comparing strains from patients with and without the defined risk factors for treatment failure. These results establish the association of natural resistance to meglumine antimoniate with treatment failure, the importance of host risk factors in evaluating drug susceptibility and treatment outcome, and the clinical and epidemiological relevance of natural Sb-resistance in L. (V.) panamensis subpopulations.

Neutrophil Activation: Influence of Antimony Tolerant and Susceptible Clinical Strains of L. (V.) panamensis and Meglumine Antimoniate.

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.

Multiparameter Flow Cytometry Analysis of the Human Spleen Applied to Studies of Plasma-Derived EVs From Plasmodium vivax Patients.

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.

Plasma-derived extracellular vesicles from Plasmodium vivax patients signal spleen fibroblasts via NF-kB facilitating parasite cytoadherence.

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.

Proteomics study of human cord blood reticulocyte-derived exosomes.

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.

Corrigendum: Spleen-Dependent Immune Protection Elicited by CpG Adjuvanted Reticulocyte-Derived Exosomes from Malaria Infection Is Associated with Changes in T Cell Subsets' Distribution.

[This corrects the article on p. 131 in vol. 4, PMID: 27900319.].

Spleen-Dependent Immune Protection Elicited by CpG Adjuvanted Reticulocyte-Derived Exosomes from Malaria Infection Is Associated with Changes in T cell Subsets' Distribution.

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.