IFNα Treatment Inhibits HIV-1 Replication and Disrupts IL-1β and miR-146a Expression
IFNα has been shown to inhibit both HIV-1 replication and IL-1β expression (15, 56). Thus, we next sought to probe the expression of IL-1β and miR-146a expression in IFNα treated HIV-1 infected CD4+ T-cells.
Healthy donor CD4+ T-cells were infected with HIV-1 alone (HIV+), infected with HIV-1 and treated with Meth (HIV+Meth), or infected with HIV-1 and treated with IFNα (HIV+IFNα). These experimental conditions allowed us to assess changes in IL-1β expression in baseline, enhanced, or diminished HIV-1 infection, respectively.
We first confirmed that HIV-1 replication was diminished in the presence of IFNα (Figure 6A). Next, we assessed IL-1β mRNA and miR-146a expression in CD4+ T-cells treated under each condition. We observed, relative to HIV+ cells, significantly decreased miR-146a and IL-1β mRNA expression with IFNα treatment (Figure 6B). There was significantly increased IL-1β release in HIV+Meth treated cells, and significantly decreased IL-1β release in HIV+IFNα cells relative to HIV+ cells (Figure 6C). TRAF6 and IRAK1 mRNA expression were unchanged in HIV+ and HIV+Meth conditions, but significantly increased expression of IRAK1 mRNA was observed in cells treated with HIV+IFNα (Figure 6D). Finally, TRAF6 protein was maintained at low levels of expression among HIV+, HIV+Meth and HIV+IFNα samples (Figure 6E). However, IRAK1 protein showed increased expression in cells treated with HIV+IFNα compared to both HIV+ and HIV+Meth treated cells (Figure 6E).
Figure 6  IFNα treatment inhibits HIV-1 replication and disrupts IL-1β and miR-146a expression. 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 IFNα daily. (A) Culture supernatants were analyzed for HIV-1 replication by p24 ELISA, and p-values were calculated relative to HIV+ controls (*p < 0.05, **p < 0.01, ***p < 0.001). (B) Cells were harvested at 2 days P.I. miR-146a and IL-1β mRNA expression was analyzed by RT-qPCR. Fold change was calculated by normalizing HIV+Meth or HIV+IFNα to HIV+ samples. Data represent the mean ± SD of 3 independent experiments, and p-values were calculated relative to HIV+ controls (**p < 0.01). (C) Culture supernatants were collected 2 days P.I. and analyzed for IL-1β concentration by ELISA. (*p < 0.05, ***p < 0.001). (D) Cells harvested at 2 days P.I. were analyzed for TRAF6 and IRAK1 mRNA expression by RT-qPCR. Fold change was calculated by normalizing HIV+Meth or HIV+IFNα to HIV+ samples. Data represent the mean ± SD of 3 independent experiments, and p-values were calculated relative to HIV+ controls (***p < 0.001). (E) Protein extracts from cells harvested at 2 days P.I. were analyzed for TRAF6 and IRAK1 expression by Western Blot analysis. GAPDH was used as a loading control. Relative band intensity was calculated using ImageJ software, and p-values were calculated relative to HIV+ controls (***p < 0.001). Taken together, these results demonstrate that immunomodulation by IFNα inhibited HIV-1 replication concordantly with decreased IL-1β and miR-146a expression in CD4+ T-cells. Interestingly, TRAF6 protein and mRNA levels were unchanged among HIV+, HIV+Meth, and HIV+IFNα samples. Moreover, IFNα significantly increased the expression of IRAK1 at RNA and protein levels concordant with decreased miR-146a expression.