Meth Enhances HIV-1 Replication via IL-1 Signaling in CD4+ T-Cells
Since Meth was found to induce IL-1β and miR-146a expression, and these play important roles in immune regulation, we explored their effects on HIV-1 infection of CD4+ T-cells.
We first confirmed that Meth increases HIV-1 replication in CD4+ T-cells, as shown in previous studies (Figure 5A) (5, 54). Next, we assessed the effects of Meth on release of IL-1β under various conditions. Cells were pretreated for 24 h with Meth before exposure to HIV-1; Meth was then administered daily. At 1 day post infection, we observed significantly increased release of IL-1β in cells either exposed to HIV-1 or HIV+Meth (Figure 5B). However, at 2 days post infection, we observed decreased release of IL-1β.
Figure 5  Methamphetamine enhances HIV-1 replication via IL-1 signaling in CD4+ T-cells. CD4+ T-cells were uninfected, infected with HIV-1, treated with Meth alone, or treated with Meth and infected with HIV-1 concomitantly. Cells were harvested at 2 days post infection and supernatants were harvested at the time points indicated on graphs. (A) Culture supernatants were analyzed for HIV-1 replication by p24 ELISA, and p-values were calculated relative to untreated controls (**p < 0.01). (B) IL-1β ELISA was used to determine the concentration of IL-1β from culture supernatants harvested on days 1 and 2 P.I. Relative expression was calculated by normalizing HIV and HIV+Meth samples to untreated controls. Data represent the mean ± SD of 3 independent experiments, and p-values were calculated relative to untreated controls (**p < 0.01, ***p < 0.001). (C) miR-146a and IL-1β mRNA expression was determined by RT-qPCR. Fold change was calculated by normalizing Meth, HIV, or HIV+Meth samples to untreated controls. Data represent the mean ± SD of 3 independent experiments, and p-values were calculated relative to untreated controls (*p < 0.05, **p < 0.01). (D) RT-qPCR was performed to assess changes in TRAF6 and IRAK1 mRNA expression on day 2 P.I. Fold change was calculated by normalizing Meth, HIV or HIV+Meth samples to untreated control cells. Data represent the mean ± SD of 3 independent experiments, and p-values were calculated relative to untreated controls (*p < 0.05, **p < 0.01). (E) Cells were harvested and lysed on day 2 P.I. Protein extracts were analyzed for IRAK1 and TRAF6 by Western Blot. GAPDH was used as a loading control. Relative band intensity was calculated using ImageJ software, and p-values were calculated relative to untreated controls (*p < 0.05, **p < 0.01). (F) CD4+ T-cells were infected with HIV-1 alone, infected with HIV-1 and treated with Meth daily, or infected with HIV-1 and treated with Meth and IL-1RA (200 or 400 ng/mL) daily. Culture supernatants were analyzed for HIV-1 replication by p24 ELISA, and p-values were calculated relative to HIV+ for HIV+Meth samples, or relative to HIV+Meth for HIV+Meth+Il-1RA samples (*p < 0.05, **p < 0.01, ***p < 0.001). (G) Cells were harvested at 2 days P.I. IL-1β mRNA expression was analyzed by RT-qPCR. Fold change was calculated by normalizing HIV+Meth or HIV+IL-1RA to HIV+ samples. Data represent the mean ± SD of 3 independent experiments, and p values were calculated relative to HIV+ for HIV+Meth samples, and relative to HIV+Meth for HIV+Meth+IL-1RA samples (*p < 0.05, **p < 0.01). We then analyzed the expression of IL-1β mRNA and miR-146a in Meth, HIV+, and HIV+Meth treated cells. We observed significantly increased IL-1β mRNA levels as well as increased miR-146a expression across all treatments 2 days post infection (Figure 5C).
Next, we assessed the expression of TRAF6 and IRAK1. Interestingly, TRAF6 mRNA levels were unchanged in Meth treated cells, but showed significantly decreased expression in HIV+ and HIV+Meth samples (Figure 5D). In contrast, IRAK1 mRNA showed significantly increased expression in the presence of Meth alone, but significantly decreased expression in HIV+ samples (Figure 5D). When cells were treated with Meth and HIV-1 in combination, IRAK1 mRNA displayed baseline expression (Figure 5D). By Western Blot, we observed decreased TRAF6 protein levels in Meth, HIV+, and HIV+Meth samples (Figure 5E). IRAK1 protein levels showed no change in Meth treated vs. untreated cells, but decreased expression in HIV+ and HIV+Meth treated cells 2 days post infection (Figure 5E).
These data demonstrate that both HIV-1 and Meth increased IL-1β and miR-146a expression. We also observed that HIV-1 inhibited TRAF6 and IRAK1 expression. Unlike Meth treatment, HIV-1 inhibited TRAF6 at the RNA level. Moreover, IRAK1 was inhibited only in HIV-1 infected samples, consistent with previous reports (55). These findings suggest that HIV-1 inhibits expression of TRAF6 and IRAK1 independent of miR-146a expression.
Interestingly, HIV+ and HIV+Meth samples displayed increased extracellular IL-1β levels on day 1 post infection, while IL-1β mRNA levels were significantly increased at day 2 post infection. These data suggest an important role for a Meth mediated IL-1β auto-regulatory feedback loop, which may augment the inflammatory state triggered during HIV-1 infection.
To assess the involvement of Meth induced IL-1β in enhanced HIV-1 replication, we blocked IL-1 signaling using exogenous IL-1RA. Cells were pretreated with IL-1RA and/or Meth, for 24 h before exposure to HIV-1; IL-1RA and Meth were then administered daily. When HIV-1 infected CD4+ T-cells were co-treated with IL-1RA and Meth, the effect of Meth on enhancing HIV-1 replication was significantly attenuated in a dose dependent manner. We observed that while 200 ng/mL IL-1RA was sufficient to reduce the effect of Meth on HIV-1 replication, virus replication in these cells was still significantly higher than in HIV+ cells (Figure 5F). However, when cells were treated with higher concentrations of IL-1RA (400 ng/mL) prior to Meth treatment, HIV-1 replication was significantly inhibited (Figure 5F). Notably, when HIV-1 infected CD4+ T-cells were treated with IL-1RA alone, there was no change in HIV-1 replication (Figure S1A).
Next, we analyzed IL-1β mRNA expression in HIV-1 infected CD4+ T-cells that were untreated, or treated with Meth, or treated with Meth and IL-1RA together. We observed that HIV+Meth+IL-1RA200ng/mL samples showed only slightly increased IL-1β mRNA expression, while IL-1β mRNA expression remained unchanged in HIV+Meth+IL-1RA400ng/mL samples relative to HIV+ controls (Figure 5G). These results are consistent with the pattern we observed during HIV-1 replication, wherein Meth augments IL-1β mRNA and HIV-1 replication, but these effects are slightly reduced in the presence of 200 ng/mL IL-1RA, and completely abrogated in the presence of 400 ng/mL IL-1RA.
Taken together, these results demonstrate that IL-1 signaling is important for Meth mediated effects on HIV-1 replication. Specifically, our results showed that increased IL-1β expression during Meth treatment plays a role in enhanced HIV-1 replication. Further, we observed relatively unchanged expression of miR-146a, TRAF6, and IRAK1 among HIV+, HIV+Meth, HIV+Meth+IL1RA200ng/mL, and HIV+Meth+IL-1RA400ng/mL samples (Figures S1B,C), suggesting that an IL-1β positive feedback loop is central to enhanced HIV-1 replication in the presence of Meth.