Background
Due to its regulatory properties, adenosine has been recognized as a guardian molecule with many protective functions in and out of the brain. For example, HIV silencing in T-cells has been associated with upregulating the adenosine-producing ecto-5’-nucleotidase Nt5e (known as CD73). We hypothesized that under HIV-induced inflammatory conditions in the brain extracellular ATP/adenosine would increase and it will lead to reduce microglial activation and silence HIV in infected microglia cells.
Methods
Our studies were performed using iPSC-derived astrocytes (iA), neurons (iN), microglia iMG, and the immortalized microglia cell line HC69, a clone latently infected with a single round HIV-1 containing a short-lived GFP. Commercially available iPSC-microglia from Tempo (iMGT), which lack expression of the adenosine receptor 3 (A3), were infected with the single round reporter and used as control. HC69 monoculture and iA+HC69 coculture were used in the initial evaluation of A3 agonists and antagonists. iMG were added to the co-/tri-cultures on day 12 of differentiation, allowing them to differentiate fully. The cultures were then infected with the macrophage-tropic HIV-1 AD8 virus on day 26. The cultures were treated 24 h post-infection with 2 mM NECA (A3 agonist) or adenosine receptor antagonists (1 mM) for four days. Untreated cells were used as control. RNA and DNA were extracted and analyzed by qPCR. A decrease in the RNA-to-DNA ratio was used to measure the extent of viral suppression. Flow cytometry and microscopy were also used to evaluate HIV expression and latency.
Results
Co- and tri-cultures with HIV-infected iMG showed a significant reduction of HIV expression compared to iMG/HIV monocultures. Cocultures of iA + control iMGT/HIV did not decrease HIV expression. NECA potently induced HIV latency in all cultures of HC69 and iMG/HIV but not in control iMGT/HIV. As expected, when co-cultures of iA+iMG/HIV were treated with adenosine receptor antagonists, HIV replication was not reduced. Staining of the iA+HC69 cocultures showed increased expression of CD73 in HC69 cells.
Conclusions
The potent suppression of HIV expression by NECA strongly suggests that the anti-inflammatory properties of adenosine can be exploited to reduce HIV expression in the brain. This approach, which mimics the natural regulatory/homeostatic mechanisms that play a role in reducing inflammation and HIV expression in microglia cells, suggests new avenues for HIV treatment in the CNS.
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