Neuronal apoptosis by HIV-1 Vpr: contribution of proinflammatory molecular networks from infected target cells.
BACKGROUND
Human immunodeficiency virus type 1 (HIV-1) induces neuronal dysfunction through host cellular factors and viral proteins including viral protein R (Vpr) released from infected macrophages/microglia. Vpr is important for infection of terminally differentiated cells such as macrophages. The objective of this study was to assess the effect of Vpr in the context of infectious virus particles on neuronal death through proinflammatory cytokines released from macrophages.


METHODS
Monocyte-derived macrophages (MDM) were infected with either HIV-1 wild type (HIV-1wt), Vpr deleted mutant (HIV-1∆Vpr) or mock. Cell lysates and culture supernatants from MDMs were analyzed for the expression and release of proinflammatory cytokines by quantitative reverse transcription-PCR and enzyme-linked immunosorbent assay respectively. Mitogen-activated protein kinases (MAPK) were analyzed in activated MDMs by western blots. Further, the effect of Vpr on neuronal apoptosis was examined using primary neurons exposed to culture supernatants from HIV-1wt, HIV-1∆Vpr or mock-infected MDMs by Annexin-V staining, MTT and Caspase - Glo® 3/7 assays. The role of interleukin (IL)-1β, IL-8 and tumor necrosis factor (TNF)-α on neuronal apoptosis was also evaluated in the presence or absence of neutralizing antibodies against these cytokines.


RESULTS
HIV-1∆Vpr-infected MDMs exhibited reduced infection over time and specifically a significant downregulation of IL-1β, IL-8 and TNF-α at the transcriptional and/or protein levels compared to HIV-1wt-infected cultures. This downregulation was due to impaired activation of p38 and stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK) in HIV-1∆Vpr-infected MDMs. The association of SAPK/JNK and p38 to IL-1β and IL-8 production was confirmed by blocking MAPKs that prevented the elevation of IL-1β and IL-8 in HIV-1wt more than in HIV-1∆Vpr-infected cultures. Supernatants from HIV-1∆Vpr-infected MDMs containing lower concentrations of IL-1β, IL-8 and TNF-α as well as viral proteins showed a reduced neurotoxicity compared to HIV-1wt-infected MDM supernatants. Reduction of neuronal death in the presence of anti-IL-1β and anti-IL-8 antibodies only in HIV-1wt-infected culture implies that the effect of Vpr on neuronal death is in part mediated through released proinflammatory factors.


CONCLUSIONS
Collectively, these results demonstrate the ability of HIV-1∆Vpr to restrict neuronal apoptosis through dysregulation of multiple proinflammatory cytokines in the infected target cells either directly or indirectly by suppressing viral replication.
Department of Infectious Diseases & Microbiology, Graduate School of Public Health, University of Pittsburgh, 130 DeSoto Street, Pittsburgh, PA 15261, USA.
Debjani
D
Guha
Pruthvi
P
Nagilla
Carrie
C
Redinger
Alagarsamy
A
Srinivasan
Gerald P
GP
Schatten
Velpandi
V
Ayyavoo
D017209:Apoptosis / Q000502:physiology*; D002478:Cells, Cultured; D053263:Gene Regulatory Networks / Q000502:physiology*; D057809:HEK293 Cells; D015658:HIV Infections / Q000235:genetics / Q000378:metabolism* / Q000821:virology; D006801:Humans; D018836:Inflammation Mediators / Q000008:administration & dosage / Q000502:physiology*; D016209:Interleukin-8 / Q000037:antagonists & inhibitors / Q000096:biosynthesis; D009474:Neurons / Q000473:pathology / Q000502:physiology* / Q000821:virology; D038301:Virus Inactivation; D054325:vpr Gene Products, Human Immunodeficiency Virus / Q000008:administration & dosage / Q000502:physiology*
D018836:Inflammation Mediators; D016209:Interleukin-8; D054325:vpr Gene Products, Human Immunodeficiency Virus; C514912:vpr protein, Human immunodeficiency virus 1