Meth Enhances IL-1β Expression and Caspase-1 Activation in CD4+ T-Cells
Meth has been shown to alter inflammatory cytokine expression in several murine and human models, both in the periphery and the CNS (10–12, 39). In particular, Meth has been linked to enhanced IL-1β expression in dendritic cells and in the rat hypothalamus (13, 14). Thus, we first sought to study the effects of Meth treatment on IL-1β expression in CD4+ T-cells.
Healthy donor CD4+ T-cells were treated daily with 100 μM Meth, and culture supernatants were harvested on days 1 and 3. We observed significantly increased release of IL-1β on days 1 and 3 of Meth treatment (Figure 1A). These results suggested that IL-1β may be a key cytokine released during Meth exposure.
Figure 1  Meth enhances IL-1β expression and Caspase-1 activation in CD4+ T-cells. CD4+ T-cells were treated daily with or without Meth. (A) Expression of IL-1β was determined from cell culture supernatants by ELISA analysis. Relative expression was calculated by normalizing Meth treated 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). (B) CD4+ T-cells were untreated, treated with Meth, or treated with Nigericin. 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). Two steps are required for IL-1β to become its mature, released form. First, the IL-1β gene is translated to a precursor protein, known as pro-IL-1β (40). Pro-IL-1β undergoes post-translational processing by the NLRP3 Inflammasome and Caspase-1 to yield its mature form (40, 41). Interestingly, Mahajan et al. found that Meth increased expression of IL-1β in dendritic cells, and in microglial cells Meth has been shown to induce activation of the NLRP3 Inflammasome (13, 42). To assess induction of IL-1β processing in Meth treated CD4+ T-cells, we analyzed Caspase-1 activation relative to untreated cells 24 h after Meth treatment. Nigericin, a potent microbial toxin known to induce activation of Caspase-1 and the NLRP3 Inflammasome, was used as a positive control. We found that Meth treatment significantly increased the activation of Caspase-1 relative to untreated controls, concordant with increased IL-1β expression (Figure 1B).