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Transmigration of Tetraspanin 2 (Tspan2) siRNA Via Microglia Derived Exosomes across the Blood Brain Barrier Modifies the Production of Immune Mediators by Microglia Cells.

ABSTRACT: Microglia are implicated in the neuropathogenesis of HIV. Tetraspanin 2 (Tspan2) is closely related to CD9 and CD81 proteins, and are expressed on microglia cells. They have been implicated in cell fusion and adhesion and in the immune response, and neuroinflammation. Developing therapeutics that target microglia remains a challenge as these therapeutics must cross the Blood-Brain Barrier (BBB). Our goal was to use microglia derived exosomes as a vehicle to deliver siRNA across the BBB to target human telomerase reverse transcriptase immortalized human microglial cells (HTHU) latently infected by HIV (HTHU-HIV) and to evaluate if the knockdown of Tspan2 gene expression in changes the activation state of microglia cells, thereby modulating the neuroinflammatory response. A blood brain barrier (BBB) model that closely mimics and accurately reflects the characteristics and functional properties of the in vivo BBB was used to examine HTHU microglia exosome effects on BBB permeability, and their ability to migrate across the and delivery small interfering RNA (siRNA) to cells on the CNS side of the BBB model. Exosomes were loaded with Texas-Red control siRNA (20 pmol) or Cy5-Tspan2 siRNA and then placed in the apical side of the BBB model, 24 h after incubation, HTHU-HIV cells microglial cells on the lower chamber were either imaged for siRNA uptake or analyzed for gene expression induced modifications. HTHU exosomes transmigrate from the apical side of the BBB to deliver Texas-Red control siRNA or Cy5-Tspan2 siRNA to HTHU-HIV microglia cells on the CNS side of the BBB model. A dose dependent (5-40 pmol) increase in Cy5-Tspan2 uptake with a corresponding decrease in gene expression for Tspan2 occurred in HTHU-HIV microglia. A decrease in Tspan2 gene expression as a consequence of knockdown with Tspan2 siRNA at both 20 and 40 pmol concentrations resulted in a significant decrease in C-X-C motif chemokine 12 (CXCL12) and C-X-C chemokine receptor type 4 (CXCR4) gene expression in HTHU-HIV microglia. Furthermore, a decrease in the gene expression levels of the Interleukins, IL-13 and IL-10 and an increase in the gene expression levels for the Fc gamma receptor 2A(FCGR2A) and TNF-α occurred in HTHU-HIV microglial cells These data demonstrate that HTHU exosomes cross the BBB and are efficient delivery vehicles to the CNS. Moreover, modifying the expression levels of Tspan2, has downstream consequences that includes alterations in cytokines and microglia biomarkers. Graphical Abstract Microglia-derived exosomes loaded with Tspan2 siRNA transmigrate across the BBB and knockdown Tspan2 gene expression in human microglial cells latently infected by HIV. This knockdown increases CXCL12, CXCR4, FCGR2A and TNF-α while decreasing IL-13 and IL-10 gene expression in HTHU-HIV microglial cells. Modulating Tspan2 modulates microglia cytokines and phenotype biomarkers.

SUBMITTER: Reynolds JL

PROVIDER: S-EPMC7282939 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Cladribine (CdA), an oral prodrug approved for the treatment of relapsing multiple sclerosis, selectively depletes lymphocytes. CdA passes the blood-brain barrier, suggesting a potential effect on central nervous system (CNS) resident cells. We examined if CdA modifies the phenotype and function of naive and activated primary mouse microglia, when applied in the concentrations 0·1-1 μM that putatively overlap human cerebrospinal fluid (CSF) concentrations. Primary microglia cultures without stimulation or in the presence of proinflammatory lipopolysaccharide (LPS) or anti-inflammatory interleukin (IL)-4 were treated with different concentrations of CdA for 24 h. Viability was assessed by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. Phagocytotic ability and morphology were examined by flow cytometry and random migration using IncuCyte Zoom and TrackMate. Change in gene expression was examined by quantitative polymerase chain reaction (qPCR) and protein secretion by Meso Scale Discovery. We found that LPS and IL-4 up-regulated deoxycytidine kinase (DCK) expression. Only activated microglia were affected by CdA, and this was unrelated to viability. CdA 0·1-1 μM significantly reduced granularity, phagocytotic ability and random migration of activated microglia. CdA 10 μM increased the IL-4-induced gene expression of arginase 1 (Arg1) and LPS-induced expression of IL-1β, tumor necrosis factor (TNF), inducible nitric oxide synthase (iNOS) and Arg1, but protein secretion remained unaffected. CdA 10 μM potentiated the increased expression of anti-inflammatory TNF receptor 2 (TNF-R2) but not TNF-R1 induced by LPS. This suggests that microglia acquire a less activated phenotype when treated with 0·1-1 μM CdA that putatively overlaps human CSF concentrations. This may be related to the up-regulated gene expression of DCK upon activation, and suggests a potential alternative mechanism of CdA with direct effect on CNS resident cells.

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Transmigration of Tetraspanin 2 (Tspan2) siRNA Via Microglia Derived Exosomes across the Blood Brain Barrier Modifies the Production of Immune Mediators by Microglia Cells.

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