Meth Increases miR-146a and IL-1β mRNA Expression via IL-1 Signaling
IL-1β has been shown to up-regulate miR-146a expression in THP-1 monocytes by activating its NFκB-dependent transcription (31). Meth treatment of CD4+ T-cells increased extracellular IL-1β levels followed by enhanced miR-146a and IL-1β mRNA expression, and decreased TRAF6 protein expression. These results suggested that Meth may modulate the innate immune response via IL-1β signaling to enhance miR-146a and IL-1β mRNA and decrease TRAF6. To address this hypothesis, we blocked IL-1 signaling by employing an IL-1 Receptor Antagonist (IL-1RA).
IL-1RA abrogated both Meth induced miR-146a overexpression and increased IL-1β mRNA levels (Figure 4A). Furthermore, TRAF6 protein expression levels, which decreased in the presence of Meth alone, were increased in Meth+IL-1RA treated samples relative to controls (Figure 4B). By ELISA analysis, we observed unchanged extracellular concentrations of IL-1β in samples treated with Meth+IL-1RA, whereas Meth alone resulted in significantly increased extracellular IL-1β levels (Figure 4C). These results support the hypothesis that IL-1 signaling mediates Meth induced miR-146a to target TRAF6. Furthermore, abrogated levels of IL-1β mRNA in cells treated with IL-1RA support the role of IL-1β signaling in a positive auto-regulatory loop.
Figure 4  Meth increases miR-146a and IL-1β mRNA expression via IL-1 signaling. (A) CD4+ T-cells treated with or without Meth for 3 days and IL-1RA were analyzed for miR-146a and IL-1β mRNA expression by RT-qPCR. Fold change was calculated by normalizing Meth treated and Meth+IL-1RA treated cells 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). (B) Protein extracts from cells treated for 3 days with or without Meth and IL-1RA were analyzed for TRAF6 by Western Blotting. 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). (C) Culture supernatants were harvested after 3 days of treatment and analyzed for IL-1β by ELISA. Relative expression was calculated by normalizing Meth and Meth+IL-1RA treated samples to untreated controls. Data represent the mean ± SD of 3 independent experiments, and p-values were calculated relativeto untreated controls (*p ≤ 0.05, **p ≤ 0.01). (D) CD4+ T-cells were untreated, treated with Meth, treated with Nigericin, or treated with IFNα and Meth for 24 h. Caspase-1 Activation was measured using fluorescent labeling with FAM-FLICA, and analyzed by Flow Cytometry. Data represent the mean ± SD of 3 independent experiments, and p values were calculated relative to untreated controls (***p < 0.001). (E) CD4+ T-cells were untreated, treated with Meth, or treated with IFNα and Meth, daily for 3 days. Culture supernatants were analyzed for IL-1β expression by ELISA. Relative expression was calculated by normalizing Meth treated 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, ***p < 0.001). (F) Cells were untreated, treated with Meth, or treated with IFNα and Meth, daily for 3 days. miR-146a and IL-1β mRNA expression were determined by RT-qPCR. Fold change was calculated by normalizing Meth treated and Meth+IFNα treated cells 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). (G) Cells were untreated, treated with Meth, or treated with IFNα and Meth, daily for 3 days. Protein extracts were analyzed for 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 < 0.01). IFNα, a member of the Type I IFN family, has been shown to negatively regulate IL-1β expression, resulting in a dynamic antagonistic relationship between these cytokines (15). This occurs because Type I IFNs can inhibit Caspase-1 and Inflammasome activation (52). We observed that Meth enhanced Caspase-1 activation in CD4+ T-cells, and thus explored the effects of exogenous IFNα on the activation of Caspase-1 in CD4+ T-cells.
CD4+ T-cells were untreated, treated with Nigericin, treated with Meth, or treated with Meth and IFNα concomitantly (Meth+IFNα) for 24 h. The cells treated with Meth alone showed increased Caspase-1 activation; IFNα abrogated this effect, consistent with the antagonistic relationship between IFNα and Caspase-1 (Figure 4D). Furthermore, by ELISA analysis, we examined IL-1β release under each condition. In the presence of IFNα, there was no change in extracellular IL-1β levels on day 1, but there was significantly decreased IL-1β release on day 3 (Figure 4E). These results show that IFNα inhibits release of IL-1β in Meth treated CD4+ T-cells by inhibiting Caspase-1 activation.
After establishing the inhibitory effects of exogenously added IFNα on Caspase-1 activation and IL-1β release, we evaluated its effects on Meth mediated IL-1β mRNA and miR-146a overexpression. While Meth alone significantly increased miR-146a and IL-1β mRNA expression, when cells were treated with IFNα+Meth, miR-146a expression was unchanged and IL-1β mRNA levels were significantly decreased (Figure 4F). Further, we analyzed the expression of TRAF6 protein. Consistent with our earlier results, we observed decreased expression of TRAF6 in Meth treated cells, but IFNα abrogated this decrease (Figure 4G).
Exogenous IFNα counteracted Meth induced Caspase-1 activation and overexpression of IL-1β, in agreement with previous reports that IFNα antagonizes IL-1β (15, 53). The abrogation of Meth mediated miR-146a overexpression by IFNα further supports the role of IL-1β in Meth mediated miR-146a overexpression.