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

Frontiers in Cellular and Infection Microbiology

Marcus V. G. Lacerda¹  Laura Pedró-Cos²  Marco Antonio Fernández-Sanmartín²  Míriam Díaz-Varela³  Haruka Toda³  Iris Aparici-Herraiz³  Melisa Gualdrón-López⁴  Carmen Fernandez-Becerra⁴  Hernando A. del Portillo⁵  Ricardo Lauzurica⁶ 

[1] Fundaçao de Medicina Tropical Dr. Heitor Vieira Dourado, Manaus, Brazil;Instituto Leônidas & Maria Deane (ILMD), Fiocruz, Manaus, Brazil;IGTP: Germans Trias i Pujol Research Institute, Barcelona, Spain;ISGlobal, Hospital Clinic - Universitat de Barcelona, Barcelona, Spain;ISGlobal, Hospital Clinic - Universitat de Barcelona, Barcelona, Spain;IGTP: Germans Trias i Pujol Research Institute, Barcelona, Spain;ISGlobal, Hospital Clinic - Universitat de Barcelona, Barcelona, Spain;IGTP: Germans Trias i Pujol Research Institute, Barcelona, Spain;ICREA: Catalan Institution for Research and Advanced Studies, Barcelona, Spain;Nephrology Service, Germans Trias i Pujol University Hospital, Badalona, Spain;

关键词: Plasmodium vivax;    human spleen;    extracellular vesicles;    multiparameter flow cytometry;    interaction;   

DOI  :  10.3389/fcimb.2021.596104

来源: Frontiers

【 摘 要 】

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.

【 授权许可】

CC BY