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Microglial Cells That Are Latently Infected By HIV Are Activated
By Inflammatory Stimuli and Induce Neuronal Damage
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Reported by Jules Levin
9th IAS Conference on HIV Science (IAS 2017), July 23-26, 2017, Paris
Alvarez-Carbonell, David1; Garcia-Mesa, Yoelvis1; Dobrowolski, Curtis1; and Karn, Jonathan1
Department of Molecular Biology and Microbiology, Case Western Reserve University Medical School, Cleveland, Ohio 44106 USA
ABSTRACT
Background: The major reservoirs for HIV in the CNS are in the microglia, perivascular macrophages, and to a lesser extent, astrocytes. We hypothesized that toll-like receptor (TLR) responses to inflammatory conditions by microglial cells induce latent HIV proviruses and that these activated microglial cells, in turn, are responsible for the neuronal damage seen in HIV-associated neurodegeneration.
Methods: Primary human cells, including cells obtained from adult brain tissue, were transformed with lentiviral vectors expressing a combination of SV40 T antigen and hTERT. These newly developed human microglial cell lines (hμglia) were used to generate latently infected cells using a single-round HIV virus carrying a green fluorescence protein (GFP) reporter (hμglia/HIV, clones HC01 and HC69). Neuronal viability was assessed through time-lapse digital images recording of live cells, MAP-2 immunohistochemistry, and Western blot analysis.
Results: The immortalized cells have microglia-like morphology, demonstrate the expected migratory and phagocytic activity, express key microglial surface markers (CD11b, TGFβR, and P2RY12), and have RNA expression profiles characteristic of primary microglial cells. The inflammatory cytokines TNF-α and IL-1β stimulated HIV transcription. Surprisingly, two TLR3 agonists, poly (I:C) and bacterial ribosomal RNA (rRNA) also potently reactivated HIV in hμglia/HIV cells using a previously unreported mechanism mediated by IRF3. The selective induction of IRF3 by poly (I:C) was confirmed by chromatin immunoprecipitation (ChIP) analysis. LPS (TLR4 agonist), flagellin (TLR5 agonist), and FSL-1 (TLR6 agonist) reactivated HIV to a lesser extent through transient NF-κB induction. Pam3CSK4 (TLR2/1 agonist) and HKLM (TLR2 agonist) only weakly reversed HIV latency. When LUHMES cells or mouse primary neurons were co-cultured with HIV infected microglia, extensive neurodegeneration was observed as shown by the significantly reduced MAP-2 staining of neuronal axons (dendritic damage), decreased level of synaptophysin, GluR1 AMPA and synaptophysin (synaptic damage), and increased levels of phosphor-Tau (neuronal microtubules damage).
Conclusions: The cell lines developed and rigorously characterized will provide an invaluable resource for the study of HIV infection in microglial cells. Since HIV patients characteristically have chronic inflammation due to the release of microbial components into the circulation, the TLR responses that we have documented are likely to contribute to CNS-disease progression.
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