Discussion
Meth abuse is a well-established risk factor for HIV/AIDS (5, 57). The drug can enhance virus replication and clinical progression, often promoting poor adherence to anti-retroviral therapies. Meth has also been shown to modulate inflammatory cytokine expression (10–12). Specifically, Meth has been reported to enhance IL-1β expression in dendritic cells and the rat hypothalamus (13, 14). The effects of Meth on host defenses and the miRNAs that regulate these processes, are not well-defined (3–5, 9, 18, 23, 58). In addition, miRNA targets are highly sensitive to changes in their expression (23, 24). IL-1β has been shown to participate in an auto-regulatory loop, and stimulate NFκB-dependent miR-146a to disrupt key inflammatory responses (31, 43). Several direct targets of miR-146a have been implicated in HIV-1 pathobiology (47, 48, 59). Notably, miR-146a targets both TRAF6 and IRAK1, signaling molecules that facilitate innate immune responses (26, 27, 48, 60). HIV-1 can induce expression of IL-1β, which is associated with progression of HIV/AIDS, and in microglial cells HIV-1 infection resulted in overexpression of miR-146a (16, 61).
Here, we demonstrate that Meth can induce expression of IL-1β and miR-146a in CD4+ T-cells, with overexpression of the cytokine leading to increased expression of miR-146a. Meth mediated miR-146a overexpression targeted TRAF6 to modulate innate immune signaling pathways. Based on our findings, we hypothesized that IL-1β signaling results in increased IL-1β mRNA levels and increased miR-146a expression. We further hypothesized that IL-1β-dependent induction of miR-146a would target key innate immune pathways by decreasing TRAF6 expression (Figure S2). Although there are several known targets of miR-146a, only TRAF6 was significantly affected during Meth treatment. We also observed that induction of an IL-1β auto-regulatory loop contributed to Meth mediated increases in HIV-1 replication.
Meth treatment of CD4+ T-cells augmented Caspase-1 activation and enhanced IL-1β release. Subsequently, both IL-1β mRNA and miR-146a levels rose, indicating that IL-1β increased expression of these transcripts. We also explored changes in TRAF6 and IRAK1 proteins, both direct targets of miR-146a. While we found no change in IRAK1 protein expression, there was decreased TRAF6 expression, indicating selective inhibition of TRAF6 by Meth via miR-146a.
To further elucidate the role of Meth in IL-1β and miR-146a overexpression, we blocked IL-1β signaling at two steps. Cells were treated with either IL-1RA to block binding of IL-1β to its receptor, or exogenous IFNα, a type I IFN known to inhibit Caspase-1 activation and antagonize mature IL-1β release. We observed decreased IL-1β release after exogenous IFNα treatment, along with decreased Caspase-1 activation. Blocking either the IL-1 receptor with IL-1RA, or release of IL-1β with IFNα, abrogated Meth mediated effects on miR-146a and IL-1β mRNA. Our data demonstrate that Caspase-1 activation, and increased IL-1β release and signaling are critical for Meth mediated miR-146a overexpression and enhanced IL-1β mRNA expression. Notably, Caspase-1 and IL-1β are associated with pryoptosis, or inflammation mediated apoptosis; pryoptosis is linked to progression of HIV-1 infection via CD4+ T-cell depletion (62–64).
At day 1 of HIV-1 infection, we observed enhanced release of IL-1β, followed by increased IL-1β mRNA and miR-146a expression at day two. Decreased TRAF6 expression, a known miR-146a target, is associated with increased HIV-1 replication (47). We observed decreased TRAF6 expression upon infection with HIV-1 at the gene expression level. IRAK1 protein expression levels were also decreased following HIV-1 infection. These findings are consistent with previous reports that HIV-1 decreases IRAK1 and TRAF6 expression (47, 55).
In sum, we found that Meth enhanced HIV-1 replication through an IL-1β positive auto-regulatory loop. By blocking IL-1 signaling using IL-1RA, we observed dose dependent decreased HIV-1 replication after Meth treatment. We also observed that Meth enhanced IL-1β mRNA levels were abrogated in a dose dependent manner upon treatment with IL-1RA. These results support the involvement of an IL-1β auto-regulatory loop in Meth mediated enhanced HIV-1 replication. Alternatively, when HIV-1 replication was inhibited using IFNα, we found decreased IL-1β and miR-146a expression. Our results indicate that increased levels of IL-1β directly contribute to Meth mediated increased HIV-1 replication in CD4+ T-cells. Based on our results, it appears that Meth mediated increased IL-1β expression acts to prime cells to be more susceptible to infection with HIV-1.
These studies unveil unique effects of Meth on IL-1β to dysregulate innate immune signaling pathways and enhance HIV-1 infection. This novel mechanism of action of Meth points to potential therapeutic targets relevant to drug abusing hosts.