Effect of Rab27a on exosome secretion and HIV release in macrophages

CABRINI, MERCEDES; JANCIC, CAROLINA; CEBALLOS, ANA; SABATTÉ, JUAN; REMES-LENICOV, FEDERICO; RODRIGUEZ RODRIGUES, CHRISTIAN; THERY, CLOTILDE; AMIGORENA, SEBASTIAN; GEFFNER, JORGE;; OSTROWSKI, MATIAS

Buenos Aires

Congreso; First French-Argentine Immunology Congress; 2010

Exosomes are secreted membrane vesicles potentially involved in intercellular communication and in the pathogenesis of infectious diseases. Exosomes are formed as intraluminal vesicles of multivesicular endosomes, and their secretion in HeLa cells is regulated by the GTPase Rab27a. It has been proposed that HIV can use the exosomal machinery as a dissemination pathway in macrophages. Herein, we determined the involvement of Rab27a in exosome secretion in the model human monocytic cellline THP-1, and its eventual implication in HIV release. The silencing of Rab27a was done with a short hairpin RNA and quantified by quantitative PCR. Exosomes were then purified from cell-culture supernatant by ultracentrifugation. Whereas the protein composition of exosomes secreted by Rab27a-silenced cells was not modified as compared with control cells, their amount was reduced by more than 70%, indicating a secretion defect. HIV production and release was subsequently analyzed. The release and intracellular distribution of HIV exhibited a high variability between experiments, probably reflecting differences in the status of the cells at the moment of infection. The difficulty in interpreting these data led us to study the role of Rab27a in HIV replication in primary monocyte-derived macrophages from Rab27a-deficient patients. Deficiency of Rab27a resulted in the perinuclear accumulation of the HIV-positive compartments observed by confocal microscopy, indicating a defect in the traffic of these organelles. The nature of these compartments and their contribution to the extracellular release of HIV is currently under study. Overall, these data indicate that Rab27a regulates exosome secretion in a cell line broadly used as a macrophage model. Moreover, Rab27a might be involved in the traffic of HIV-positive intracellular compartments in primary macrophages. These finding will allow detemnining the level of convergence between the exosomal and the HIV-release pathway in macrophages.