Impact of Antipsychotic Drugs on Il-6 Secretion by Microglia
Based on the hypothesis that IL-6 plays a central role in neuroinflammation, we assessed the effect of various antipsychotics drugs on microglia cells in the neuroinflammatory microenvironment by activating the cells with LPS together with selected drugs. Although an overall significant difference was found for risperidone (F(5,11) = 4.07, P = 0.025), post-hoc analyses did not reveal any significant differences in IL-6 levels for specific concentrations of the drug (Table 1, Fig. 1A). In contrast, both haloperidol and clozapine significantly lowered IL-6 levels (F(5,11) = 14.30, P < 0.001 and F(5,12) = 13.99, P < 0.001 respectively). Both caused a significant decrease of IL-6 secretion by the N9 cells at a dose of 10− 5M (equivalent to 375.9 × 10− 5 mg/ml for haloperidol and 326.8 × 10− 5 mg/ml for clozapine), an effect that disappeared in lower doses (57.27 ± 9.02 vs. 103.15 ± 20.77, P = 0.012 for haloperidol and 67.27 ± 0.67 vs. 103.15 ± 20.77, P = 0.020 for clozapine. Table 1, Fig. 1B and 1C).
Table 1
– Interleukin 6 Secretion Mediated by Drugs
| 10− 5 | 10− 6 | 10− 8 | 10− 10 | 10− 12 | 0 |
Risperidone (pg/ml ± SD)! | 106.11 ± 6.67 | 124.73 ± 3.05 p = 0.556 | 138.1 ± 7.92 p = 0.165 | 127.70 ± 13.04 p = 0.408 | 128.80 ± 2.36 p = 0.359 | 103.15 ± 20.77 p = 1.000 |
Haloperidol, (pg/ml ± SD)! | 57.27 ± 9.02 | 115.60 ± 7.21 p = 0.002 | 114.63 ± 1.58 p = 0.002 | 130.00 ± 3.74 p < 0.001 | 115.57 ± 2.90 p = 0.002 | 103.15 ± 20.77 p = 0.012 |
Clozapine (pg/ml ± SD)! | 67.27 ± 0.67 | 109.53 ± 8.92 p = 0.006 | 117.83 ± 2.11 p = 0.002 | 124.93 ± 5.81 p < 0.001 | 120.43 ± 6.49 p = 0.001 | 103.15 ± 20.77 p = 0.020 |
LPS | | | | | | 103.15 ± 20.77 |
!P Compared to a concentration of 10− 5M of the same drug. |
The Impact of Neurotransmitters on IL-6 Secretion by Microglia
Next, we assessed the effect on microglia-secreted IL-6 of neurotransmitters that are attenuated by antipsychotics drugs. Dopamine had an overall significant effect on IL-6 secretion that was also evident in the post-hoc analysis (F(3,9) = 41.17, P < 0.001, Fig. 2). IL-6 levels were significantly higher compared to LPS control at the lowest dopamine concentration of 1 ∝M (427.90 ± 42.24 vs. 295.03 ± 23.05, P = 0.001). No difference was found for dopamine at a concentration of 10 ∝M. Interestingly, we found decreased IL-6 levels at a dopamine concentration of 100 ∝M (218.13 ± 5.47 vs. 295.03 ± 23.05, P = 0.023). Notably, the rise in IL-6 levels induced by dopamine 1 ∝M was also significantly higher than dopamine 10 ∝M and 100 ∝M (P < 0.001 for both comparisons). ANOVA was significant for the overall serotonin results (F(3,11) = 3.76, P = 0.044), but the post-hoc analysis did not reveal any significant differences. Finally, the ANOVA finding for acetylcholine was not significant (F(3,8) = 0.73, P = 0.564).
Antipsychotics and Neurotransmitters’ Impact on IL-6 Secretion
We then sought to determine whether different concentrations of dopamine influence the effect of clozapine on microglial IL-6 secretion. The results showed that IL-6 secretion was significantly lower compared to the LPS control for most dopamine concentrations (0.001, 0.1 and 10 ∝M. F(6,12) = 11.47, P < 0.001) at a high dose of clozapine (10− 5M or 326.8 × 10− 5 mg/ml), while the 1 ∝M and 100 ∝M doses yielded no significant differences (Table 2 and Fig. 3A).
Table 2
– Interleukin 6 Secretion Mediated by Antipsychotic Drugs and Dopamine
| Dopamine Concentration |
| 0∝M | 0.001∝M | 0.1∝M | 1∝M | 10∝M | 100∝M |
Clozapine 10− 5M (pg/ml ± SD) | 30.96 ± 9.88 | 42.59 ± 11.70 | 52.14 ± 29.98 | 72.83 ± 13.32 | 39.92 ± 13.48 | 78.81 ± 13.28 |
P compared to LPS only | 0.001 | 0.005 | 0.032 | 0.181 | 0.008 | 0.355 |
Clozapine 10− 7 M (pg/ml ± SD) | 113.63 ± 4.95 | 134.63 ± 8.34 | 132.00 ± 18.79 | 166.13 ± 24.56 | 149.87 ± 24.57 | 172.80 ± 12.72 |
P compared to LPS only | 1.000 | 0.736 | 0.819 | 0.047 | 0.250 | 0.047 |
Haloperidol 10− 5 M (pg/ml ± SD) | 0.21 ± 0.36 | 7.15 ± 7.24 | 16.91 ± 2.59 | 69.23 ± 13.59 | 28.57 ± 8.72 | 20.01 ± 6.38 |
P compared to LPS only | < 0.001 | < 0.001 | < 0.001 | 0.001 | < 0.001 | < 0.001 |
P compared to Dopamine 1∝M | < 0.001 | < 0.001 | < 0.001 | NA | 0.001 | < 0.001 |
Haloperidol 10− 8 M (pg/ml ± SD) | 94.93 ± 14.09 | 126.23 ± 9.10 | 151.93 ± 4.98 | 195.13 ± 13.17 | 145.23 ± 12.04 | 118.03 ± 6.18 |
P compared to LPS only | 0.773 | 0.650 | 0.009 | < 0.001 | 0.033 | 0.977 |
P compared to Dopamine 1∝M | < 0.001 | < 0.001 | 0.008 | NA | 0.002 | < 0.001 |
LPS only (pg/ml ± SD) | 109.50 ± 3.96 | | | | | |
When we tested the impact of low-dose clozapine (10− 7M equivalent to 326.8 × 10− 7 mg/ml) on IL-6 secretion, it emerged that IL-6 levels were significantly higher compared to the LPS control (F(6,13) = 5.95, P = 0.004) at dopamine concentrations of 1 ∝M and 100 ∝M (166.13 ± 24.56 vs 109.50 ± 3.96, P = 0.047 and 172.80 ± 12.72 vs. 109.50 ± 3.96, P = 0.047, respectively). Notably, IL-6 levels for the N9 cells treated with low-dose clozapine were higher than the LPS control for all dopamine concentrations, although not always to a level of significance (Table 2 and Fig. 3A).
Testing of high-dose haloperidol (10− 5M or 375.9 × 10− 5 mg/ml) revealed that all IL-6 measurements were significantly lower compared to the LPS control (F(6,14) = 86.60, P < 0.001) and that they spread in a bell-shaped curve (Table 2, Fig. 3B). Interestingly, IL-6 levels for the dopamine 1∝M-treated cells were significantly higher compared to the cells treated at other dopamine concentrations (Table 2 and Fig. 3B).
Finally, we tested low-dose haloperidol (10− 8M equivalent to 375.9 × 10− 8 mg/ml) and found that three dopamine concentrations showed significant differences compared to the LPS control (0.1, 1 and 10 ∝M), and that IL-6 levels were higher than the control (F(6,14) = 33.45, P < 0.001. Table 2 and Fig. 3B). Again, the cells treated with dopamine 1 ∝M exhibited significantly higher levels of IL-6 compared to the other treated cells (Table 2, Fig. 3B).
The Effects of Antipsychotics and Neurotransmitters on Microglial Viability
We assessed the effect of antipsychotics and neurotransmitters on the viability of microglia cells to rule out confounding effects. The results for the MTT assay are detailed in the supplementary table. The ANOVA analysis of the antipsychotics alone (in all concentrations used) was significant only for clozapine (F(5,12) = 3.62, P = 0.032), but the differences were not significant in the post-hoc analysis. For the combination of clozapine and dopamine, clozapine at a high dose (10− 5M) produced an increase in microglial viability as observed in the MTT signal (F(6,14) = 4.550, P = 0.009), specifically, between dopamine 0.001 ∝M and dopamine 0.1 ∝M (0.61 ± 0.09 vs. 0.77 ± 0.06, P = 0.02). There were no significant differences compared to LPS alone control. ANOVA was significant overall for low-dose clozapine (10− 8M) (F(6,14) = 4.511, P = 0.0 l), but no comparable significance was seen on the post-hoc analyses. The ANOVAs for haloperidol were not significant for both tested concentrations (F(6,14) = 1.713, P = 0.190 and F(6,14) = 1.220, P = 0.353).