Molecular Mechanism Of SIV Invasion Of Blood - Brain Barrier

Acquired immune deficiency syndrome (AIDS) is characterized by serious damage to cellular immune functions, repeated opportunistic infections, malignancies and degeneration of the central nervous system. HIV not only attacks the immune system, but induces pathological changes including hyperplasia of stellate glial cells, which is accompanied by perivascular infiltration of lymphocytes and macrophages and AIDS is clinically characterized by acute encephalitis and dementia. In animal models, central nervous system (CNS) symptoms are associated with direct infection of parenchymal cells, particularly glial cells, astrocytes and microglial cells.We sought to investigate how HIV/SIV can penetrate the blood-brain barrier (BBB) and invade the CNS during the early stage of infection. Several mechanisms of neuroinvasion have been proposed including direct infection of endothelial cells (ECs), invasion of infected monocytes, macrophages or T cells, or fluid-phase endocytosis hypothesis. In brief, neuroinvasion may be achieved by either infected cells or cell-free virus passing through ECs or disrupted tight junctions.It remains to be determined which route is most relevant to SIV neuroinvasion, and which molecules play the significant role of promoting SIV infection of EC.PART 1:SIV infection of ECs via cell-free virus and virus infected cellsIt was recently reported that infection of uninfected T cells with CD4+T cells requires cell-cell contact in the form of a structure termed the virological synapse which promotes transfer of HIV from infected to uninfected cells. T cells mediated infection can be 100 times as effective as cell-free virus infection. However whether contact between infected T cells and ECs promotes higher efficiency SIV infection remains to be seen.We thus compared infection of ECs with cell-free S1Vmac239 and SIVmac239-infected T cell line, CEMxl74. SIV infection of ECs was assessed using nested-PCR, indirect immunofluorescence assay, Western blotting and ELISA.We found the provirus DNA in ECs infected using both methods, but ECs infected with infected cells contained higher levels of provirus DNA than ECs infected with cell-free virus and the supernatant of those cultures contained higher levels of SIV P27.We conclude that T cell-mediated infection of ECs is more efficient than cell-free virus infection of ECs, and thus may be responsible for SIV neuroinvasion.PART 2:The influence of CD44 and hyaluronic acid (HA) on T cell-mediated SIV infection of ECs.CD44 molecules on the surface of lymphocytes interact with hyaluronic acid molecules on the surface of the ECs, contributing to facilitate rolling of lymphoid cells in vessels in vitro. Since binding of CD44 to HA promotes lymphocytes homing, we suspected that this protein may be involved in transmission of SIV from infected T cells to ECs. To investigate this, we incubated lymphocytes with exogenous CD44 antibodies or hyaluronic acid to block CD44, and incubated ECs with hyaluronidase to reduce hyaluronic acid. We used IFA, Western blotting, and FCM to establish that these treatments effectively reduced T-cell mediated SIV infection of ECs.PART 3:Immortalization of rat mesenchymal stem cellsLacking a rhesus monkey brain capillary model for SIV neuroinvasion, we used the immortalized monkey endothelial cell lines, CD4-RF/6A cells to model brain capillary ECs.We used the whole bone method to separate rat mesenchymal stem cells. Adherent cells were collected and were found to be 95.6% CD90+. We used recombinant adenovirus mediated transfection to express the hTERT gene in rat bone marrow mesenchymal stem cells, which induced telomerase and maintained the cell chromosome karyotype stability, significantly prolonging the life of these cells.This culture method provides a basis for establishment of a rhesus monkey brain capillary model which could be differentiated by rhesus monkeys MSC cells.Overall, we found that while cell-free SIV infected ECs, T cell-mediated infection of ECs is more efficient than cell-free SIV infection of ECs, and thus may be responsible for SIV neuroinvasion. The effective transfer of virus from the large number of infected mononuclear macrophages and T cells in the bloodstream to ECs, may promote SIV neuroinvasion.T cell-mediated infection of ECs is dependent upon the binding of CD44 and hyaluronic acid, and inhibition of this interaction reduced T cell-mediated SIV infection of ECs.