In the brain, HIV establishes latent or active infection primarily in astrocytes and microglia cells where viral proteins are produced and shed (Kaul et al, 2001
). Viral proteins such as Tat and gp120, and the immune response to viral proteins are thought to be primary contributors to HIV-related neurodegeneration (Kaul and Lipton, 2006
). Importantly, it is now widely accepted that METH abuse by HIV-infected individuals leads to increased neurological deficits and neuronal dysfunction (Nath et al, 2001
; Rippeth et al, 2004
). In the current study, we examined the effects of METH on activation of the HIV LTR, which controls viral replication in the human host cells, including microglia. We have reported here a dose-dependent activation of the HIV LTR by METH that was independent of the presence of the HIV protein Tat in CHME-5 microglial cells transfected with an LTR-containing vector. METH also activated NF-κB, a cellular transcription factor and well established regulator of the HIV LTR. Disruption of NF-κB signaling blocked the induction of HIV LTR and consequently HIV gene expression. In addition, we have demonstrated here, using CHME-5 cells infected with HIV, that METH caused increased transcription of the HIV genome integrated as part of the human host genome, more closely recreating in vivo conditions. Under these conditions, METH was also capable of inducing NF-κB nuclear translocation, allowing transcription. Collectively, our data suggest that METH directly induces HIV expression in human microglia cells through activation of NF-κB.
We found that METH, applied at concentrations lower that those reported to happen in the brain of users, increased the activation of NF-κB signaling in human microglia cells. We detected significant effects in the 300–700 µM range in transfected cells and even below 50 µM in infected microglial cells. Estimated level in the spleen of single users is between 100 to 400 µM and binge users between 240 to 1144 µM (Tallóczy et al, 2008
). Based on distribution and pharmacokinetic studies of METH in the human body, METH concentration in the spleen reaches its peak in 3.5 minutes and in the brain in 9 minutes; peak concentration in the spleen is twice that in brain (Volkow et al, 2010
). However, they found the brain showed one of the slowest clearance rates. Taken together, it is feasible that METH doses in the human brain may occur in the 30–500 µM range, where we observed an effect of METH on HIV LTR in microglial cells.
The HIV LTR controls the expression of the viral genes, and the ability of METH to increase the activity of the HIV LTR in microglial cells would increase the production of viral proteins and possibly viral particles in the brain. The viral proteins gp120 and Tat have been shown to promote neurodegeneration (King et al, 2006
; Mocchetti et al, 2007
), and the combined exposure of METH and Tat and/or gp120 acts synergistically to promote neurotoxicity (Cadet and Krasnova, 2007
). It is therefore plausible that one of the mechanisms that METH abuse in HIV-infected individuals increases neurodegeneration is through the increase viral gene expression. Our data supports the possibility that METH can act directly on human microglial cells and increase the activity of the HIV LTR, increasing viral protein production without necessarily increasing viral load.
Our findings are consistent with a previous study showing that METH can increase HIV replication in peripheral monocytes through an NF-κB-related mechanism (Toussi et al, 2009
). The importance of NF-κB in regulating HIV gene expression is well documented, and other studies suggest METH can increase NF-κB toxicity in the brain (Asanuma and Cadet, 1998
). Our data show that NF-κB is essential for the METH-related increase in HIV LTR activity, however, we also found a more modest but significant increase in transcriptional activity of NF-AT (results not shown), a cellular transcription factor that can influence the HIV LTR. This finding suggests that other cellular transcription factors may be altered by METH and influence viral transcription from the HIV LTR. We are presently investigating the role of METH in inducing epigenetic changes at the level HIV promoter leading to HIV reactivation as well as its specificity for particular cellular signaling pathways. Also, additional studies examining METH-related changes to the microglia transfactome are currently underway.
“Microglial activation” is a general term for the phenotypic changes of microglial cells in response to perturbations to the brain such as those that occur following injury or pathological conditions e.g. stroke, seizures, and viral infection. Activated microglia exhibit increased proliferation, hypertrophy, increased migration, increased phagocytosis, and increased production of pro-inflammatory molecules such as cytokines and chemokines (Tambuyzer et al, 2009
). METH has previously been shown to activate microglia in rodent brain (Guilarte et al, 2003
; LaVoie et al, 2004
; Thomas et al, 2004
), and humans who abuse METH have increased binding of a radiotracer for activated microglia compared to control subjects (Sekine et al, 2008
). The mechanisms of microglial activation by METH are not known, but it is thought to involve glutamate receptor signaling (Thomas and Kuhn, 2005
). We found that METH increases activation of rat primary microglial cells (Supplemental Figure 2
), which suggests that microglia, in the absence of neurons capable of both releasing and responding to glutamate, can be activated by METH. In human microglial cells, we found that METH increased nuclear NF-κB and activation of a NF-κB-dependent reporter. In addition to being a primary cellular transcription factor for the HIV LTR, NF-κB contributes to the production of pro-inflammatory cytokines in activated microglia cells (Mattson, 2005
). Therefore, it is plausible that HIV-infected microglial cells exposed to METH may contribute to local neurodegeneration by both the production of viral proteins and promoting local neuroinflammation through NF-κB activity and/or, more importantly, by the synergistic interaction between METH and the viral proteins. Additional studies are needed to examine role of NF-κB in METH and HIV-related neurodegeneration, and whether METH is capable to interact, directly or indirectly, with viral proteins in ways that may explain the increased HIV-related neurologic disorders observed in HIV patients consuming psychostimulants such as METH.
In conclusions, we show that METH can directly increase HIV LTR activity in human microglial cells in vitro through an NF-κB-dependent mechanism. These findings support a role of METH in promoting HIV gene expression in the progression of HIV-associated neurodegeneration.