Background
Cyclic adenosine 3′,5′-monophosphate (cAMP) is a secondary messenger involved in the regulation of diverse cellular, metabolic, and immunologic processes. Ex vivo and in vitro studies have shown that cAMP levels are elevated in the setting of HIV infection, with T cells from PLWH harboring twice the cAMP level as uninfected individuals. In our previous studies, we identified the adenosine signaling pathway which leads to production of cAMP as a host determinant of HIV latency. Here, we used forskolin, a cell-permeable diterpene that activates adenylyl cyclase, and SCH-58261, an adenosine receptor antagonist, to investigate the impact of cAMP modulation on the establishment, maintenance, and reversal of HIV latency.
Methods
Intracellular cAMP in primary CD4+ T cells and cell lines was quantified using a colorimetric enzyme-linked immunosorbent assay (ELISA). To determine the impact of forskolin and SCH-58261 on latency, HIV latently-infected J-Lat 5A8 cells, J-Lat NFkB.RFP cells (reporting NFkB activation via RFP), and HC69.5 microglia were pretreated with varying drug doses followed by phytohaemagglutinin (PHA) or PMA/ionomycin treatment; GFP-positive cells (reflecting viral expression) were enumerated by flow cytometry. To determine the impact of cAMP modulation on initial establishment of latency, Jurkat cells were infected with a dual-reporter virus construct, Duo-Fluo HIV BFP/GFP, which enables enumeration and isolation of HIV latently-infected, productively-infected, and uninfected cells by flow cytometry. Data were analyzed using unpaired t tests.
Results
Forskolin treatment increased cAMP levels in J-Lat (p=0.0071) and primary CD4+ T cells (p=0.0071) (Fig.1A). Treatment with SCH-58261 reversed the phenotype (p=0.0011). Forskolin exerted a dose-dependent inhibitory impact on PHA- (p=0.0286) and PMA/Ionomycin- (p=0.0011) mediated latency reversal in J-Lat cells (Fig.1B), and NFkB activation was suppressed in parallel (p<0.0001). Forskolin suppressed HIV transcription in HC69.5 microglia (p=0.0046). Forskolin also inhibited the initial establishment of latency in Jurkat cells, increasing the ratio of productively-infected to latently-infected cells as compared to DMSO negative controls (p=0.0036).
Conclusions
Elevated cAMP levels block HIV transcription and prevent viral reactivation in T cells and microglia, in an NFkB-dependent manner. cAMP induction also prevents initial establishment of HIV latency. cAMP modulators including forskolin should be explored in HIV cure strategies.