Impact of antidepressants on viability of primary astrocytes
Isolated primary astrocytes (Fig. 1A) were treated with different concentrations of antidepressants from 0-160 µM. Data from the MTT exclusion test indicated that after 24 h paroxetine, fluoxetine and sertraline led to apparent reduction in cell viability starting at 20 µM [paroxetine: F(7,16) = 78.743, p < 0.001; post hoc (only the first listed if there were two or more significant values, e.g.), p < 0.001 (20 µM vs 0 µM),; sertraline: F(7,16) = 40.654, p < 0.001; post hoc, e.g. p < 0.001 (20 µM vs 0 µM); fluoxetine: F(7,16) = 72.540, p < 0.001; post hoc, e.g. p < 0.001 (20 µM vs 0 µM); Fig. 1B-D]. Treatment of astrocytes with citalopram and fluvoxamine resulted in a reduction in cell viability at 160 µM [fluvoxamine: F(7, 16) = 4.631, p = 0.005; post hoc, p = 0.003 (160 µM vs 0 µM); citalopram: F(7, 16) = 8.371, p < 0.001; post hoc, p = 0.002 (160 µM vs 0 µM); Fig. 1E and F]. No significant change in cell viability was observed with the treatments of venlafaxine (Fig. 1G). Non-toxic dosages were selected for following experiments.
Effects of antidepressants on the CytoM-induced iNOS expression and NO production in primary astrocytes
The antidepressants alone did not result in iNOS induction in primary astrocytes (data now shown). CytoM treatment induced significant expression of iNOS (Fig. 2). The expression was further increased when cells were pretreated with paroxetine, fluoxetine and sertraline compared to the CytoM alone group [paroxetine: F(3, 8) = 28.426, p < 0.001; post hoc, p = 0.003 (CytoM + 10 µM vs CytoM); fluoxetine: F(3, 8) = 28.737, p < 0.001, post hoc, p = 0.013 (CytoM + 10 µM vs CytoM); sertraline: F(3, 12) = 36.813, p < 0.001, post hoc, e.g. p = 0.001 (CytoM + 5 µM vs CytoM); Fig. 2A-C; full blots were all in supplemental Fig. 1]. Treatment of citalopram or fluvoxamine led to a gradual upward trend of iNOS expression with significant changes being observed at higher concentrations [citalopram: F(6, 14) = 26.437, p < 0.001; post hoc, e.g. p = 0.012 (CytoM + 40 µM vs CytoM); fluvoxamine: F(6, 14) = 31.368, p < 0.001; post hoc, p < 0.001 (CytoM + 80 µM vs CytoM); Fig. 2D and E]. Venlafaxine had no significant effect on iNOS expression (Fig. 2F).
NO production was indicated by measurement of medium nitrite. In line with the iNOS expression, CytoM induced significant production of NO and the production was further increased by pretreatments with paroxetine, sertraline and fluoxetine [paroxetine: F(5, 18) = 24.005, p < 0.001; post hoc, e.g. p = 0.005 (CytoM + 2.5 µM vs CytoM); fluoxetine: F(5, 12) = 36.773, p < 0.001; post hoc, e.g. p = 0.004 (CytoM + 1.25 µM vs CytoM); sertraline: F(5,18) = 33.797, p < 0.001; post hoc, p = 0.032 (CytoM + 10 µM vs CytoM); Fig. 3A-C]. Treatments of citalopram, fluvoxamine and venlafaxine showed no significant effect on the NO production (Fig. 3D-F). The non-elevated nitrite levels with 10 µM of paroxetine and fluoxetine, 40 or 80 µM of citalopram and fluvoxamine were discordant with the corresponding iNOS expression.
Effects of antidepressants on the CytoM-induced production of IL-6 and IL-1β in astrocytes
Levels of both IL-6 and IL-1β were significantly elevated upon CytoM stimulation. Different from their impact on iNOS and NO, the six antidepressants all led to inhibition on CytoM-induced IL-6 expression [Figure 4; paroxetine: F(5, 12) = 6.037, p = 0.005; post hoc, e.g. p = 0.026 (CytoM + 5 µM vs CytoM); fluoxetine: F(5, 12) = 11.647, p < 0.001; post hoc, e.g. p = 0.025 (CytoM + 5 µM vs CytoM); sertraline: F(5, 12) = 7.201, p = 0.002; post hoc, p = 0.007 (CytoM + 10 µM vs CytoM); citalopram: F(8, 18) = 5.535, p = 0.001; post hoc, e.g. p = 0.018 (CytoM + 5 µM vs CytoM); fluvoxamine: F(8, 18) = 10.140, p < 0.001; post hoc, e.g. p = 0.018 (CytoM + 1.25 µM vs CytoM); venlafaxine: F(9, 20) = 7.000, p < 0.001; post hoc, e.g. p = 0.021 (CytoM + 40 µM vs CytoM)].
The CytoM-induced expression of IL-1β was also suppressed by pretreatments of paroxetine, fluoxetine, sertraline, fluvoxamine and venlafaxine [Figure 4A-C, E and F; paroxetine: F(5, 18) = 13.822, p < 0.001; post hoc, p = 0.023 (CytoM + 10 µM vs CytoM); fluoxetine: F(5, 18) = 10.938, p < 0.001; post hoc, p = 0.005 (CytoM + 10 µM vs CytoM); sertraline: F(5, 18) = 10.449, p < 0.001; post hoc, p = 0.023 (CytoM + 10 µM vs CytoM); fluvoxamine: F(8, 27) = 5.749, p < 0.001; post hoc, e.g. p = 0.015 (CytoM + 10 µM vs CytoM); venlafaxine: F(9, 30) = 9.792, p < 0.001; post hoc, e.g. p = 0.03 (CytoM + 2.5 µM vs CytoM)]. Interestingly, citalopram had no effect on the CytoM-induced IL-1β expression (Fig. 4D).
Effects Of Antidepressants On The Cytom-induced Signaling Activation
To understand the underlying mechanisms of the differential effects of the antidepressants on CytoM-induced inflammatory responses, we analyzed the MAPKs, STAT3 and NFκB signaling pathways (Fig. 5). CytoM treatment activated p38, JNK1 and p65/NFκB, inhibited STAT3, but had no effect on JNK2 and ERK1/2 in primary astrocytes (Fig. 5A and E). Pretreatments of the antidepressants did not affect the CytoM-induced activation of p38 and p65 (Fig. 5D and E). In contrast, the antidepressants per se blunted the baseline STAT3 activity [Figure 5B; left panel: time, F(2, 24) = 50.821, p < 0.001; antidepressant, F(3, 24) = 3.769, p = 0.024; right panel: time, F(2, 24) = 28.562, p < 0.001; antidepressant, F(3, 24) = 28.534, p < 0.001]. Amidst, citalopram, fluvoxamine and venlafaxine further reduced the STAT3 phosphorylation in addition to the CytoM-induced inhibition [Post hoc for 30 min, p = 0.013 (CytoM + citalopram vs CytoM, p = 0.004 (CytoM + fluvoxamine vs CytoM), p = 0.003 (CytoM + venlafaxine vs CytoM); for 60 min, p = 0.013 (CytoM + venlafaxine vs CytoM)). Meanwhile, the antidepressants inhibited the CytoM-induced JNK1 activation [Figure 5C; left panel: time, F(2, 24) = 72.213, p < 0.001; antidepressant, F(3, 24) = 10.481, p < 0.001; right panel: time, F(2, 24) = 93.802, p < 0.001; antidepressant, F(3, 24) = 19.089, p < 0.001; post doc for 30 min, p < 0.001 (CytoM + any antidepressant vs CytoM)].
Blockage of JNK and STAT3 signaling on the CytoM-induced inflammatory responses
Compared with the group that treated with CytoM alone, the JNK inhibitor SP600125 promoted the CytoM-induced expression of iNOS, and inhibited the production of IL-1β with no effect on the IL-6 production [Figure 6A; iNOS: inhibitor, F(2, 12) = 17.362, p < 0.001; post hoc, p < 0.001 (CytoM + SP600125 vs CytoM); IL-1β: inhibitor, F(2, 12) = 8.734, p = 0.005; post hoc, p < 0.001 (CytoM + SP600125 vs CytoM)]. The STAT3 inhibitor AG490 did not change the expression of iNOS and IL-1β, but elevated the CytoM-induced production of IL-6 [IL-6: inhibitor, F(2, 12) = 2.891, p = 0.094; post hoc, p = 0.002 (CytoM + AG490 vs CytoM)]. Effects of the inhibitors on kinase activity were confirmed as in Fig. 6B. Interestingly, results also showed that the baseline STAT3 phosphorylation was inhibited by the JNK inhibitor SP600125 [Inhibitor, F(2, 18) = 8.945, p = 0.002; post hoc, p = 0.001], while the CytoM-induced JNK phosphorylation was promoted by the STAT3 inhibitor AG490 [Inhibitor, F(2, 18) = 68.066, p < 0.001; post hoc, p < 0.001].
Antidepressants Show No Effect On The Astrocyte Phenotype Polarization
It has been reported that the CytoM as composed of C1q, TNF-α and IL-1α are necessary and sufficient to induce A1 astrocytes [12]. Indeed, the treatment of CytoM led to a conversion of astrocytes from resting (non-reactive) to A1 type as indicated by the elevated expression of C3 (Fig. 7A). In contrast, the expression of S100A10 was not changed by the CytoM treatment (Fig. 7B), suggesting no conversion of the resting cells to A2 type. However, none of the six antidepressants displayed an effect on the expression of C3 or S100A10 (Fig. 7). We also investigated effects of the JNK and STAT3 inhibitors, SP600125 and AG490, on the phenotype polarization of astrocytes. Similarly, no change was observed in the expression of C3 and S100A10 with treatment of either inhibitor (supplemental Fig. 2).