The tetracycline COL-3 reduces the inflammatory response of microglial cells challenged with either LPS or αSa
Figure 2A confirms that amyloid fibrils were present in αS shaken samples for 96h (lower image), whereas non-shaken samples did not produce fibrilar αSa species.
To evaluate the anti-inflammatory potential of the non-antibiotic tetracycline COL-3, we used microglial cells activated with the bacterial cell wall component LPS (10 ng/ml), a prototypical agonist for Toll-like receptor-4 (TLR4) [36]. After 24 hours of treatment, LPS strongly stimulated the production/release of the proinflammatory cytokine TNF-α. When the cultures were exposed to either 10 or 20 μM COL-3, the production of TNF-α induced by LPS was significantly reduced by more than 30 and 75%, respectively (Figure 2B). No significant effect was observed, however, at 1 µM, indicating that the suppressive action of the tetracycline derivative was concentration-dependent and only significant after 10 µM. COL-3 at 20 µM appeared as effective as the reference tetracycline DOX, at 50µM. The reference anti-inflammatory drug DEX (2.5 µM) wasmore effective than either 20 µM COL-3 or 50 µM DOX to reduce TNF-α release.
Cellular expression of the phenotypic activation marker Iba-1 was also estimated under the same experimental conditions. We established that COL-3 suppressed the LPS-induction of microglial Iba-1 expression only at 10 or 20 µM (Figure 2B). At 50 µM, DOX also caused a robust decrease of the Iba-1 immunosignal. Note that 2.5µM DEX reduced Iba-1 expression with an efficacy that was similar to that of COL-3 and DOX, at 20 and 50µM, respectively. Photomicrographs illustrated LPS-treated microglial cell cultures, exposed or not to COL-3 (20 µM) or DOX (50 µM) and then processed sequentially for CD11b and Iba-1 fluorescence immunodetection, binding proteins that are constitutively expressed in microglia (Figure 2C).
To model inflammatory reactions as they may occur in PD we used αSa to trigger an inflammogenic response in microglial cell cultures. As expected, a 24-hour exposure to αSa caused a robust increase of TNF-α release whereas the monomeric form of αS (αSm) does not induce a significant effect compared to control levels (Figure 2D). At 10 or 20 µM, COL-3 significant reduced TNF-α release induced by αSa. DOX exerted a comparable repressive effect at 50µM. DEX at 2.5 µM was as effective as COL-3 and DOX at 20 and 50 µM, respectively.
Coherent with previous observations, we also demonstrated that the induction of Iba-1 expression induced by αSa was efficiently curtailed by COL-3 (10 or 20 µM), DOX (50 µM) and DEX (2.5 µM) (Figure 2E). Micrographs illustrate microglial cultures treated with αSa in the presence or not of COL-3 (20µM) or DOX (50 µM) and then processed sequentially for CD11b and Iba-1 fluorescence immunodetection (Figure 2F). It is interesting to point out that αSm does not induce any effect.
The tetracycline COL-3 reduces glucose accumulation in microglial cells challenged with either LPS or αSa
Recent studies have demonstrated that glucose is an essential fuel for microglial inflammatory processes [32,37]. Accordingly, we found that a 24-h treatment with 10 ng/ml LPS leads to an almost 2-fold increase in [3H]-2-DG uptake in microglial cultures (Figure 3A). [3H]-2-DG uptake was significantly reduced when LPS-treated cultures were exposed to either 10 or 20 µM COL-3, whereas a lower COL-3 concentration (1 µM) was not effective (Figure 3A). A similar effect was obtained with DOX used at a concentration of 50 µM. Surprisingly, DEX (2.5 µM) failed to alter [3H]-2-DG uptake, indicating that the anti-inflammatory effects of the glucocorticoid and the non-antibiotic tetracycline derivative may occur through distinct mechanisms. The uptake of [3H]-2-DG was also strongly reduced by inhibiting NADPH oxidase activity with 300µM APO or by exposing the cultures to 500 µM unlabeled 2-DG, a concentration of the glucose analog susceptible to restrain glucose utilization by these cells without affecting their survival (dos-Santos Pereira et al, 2020). As expected, glucose uptake was also efficiently prevented when 50 mM glucose was added acutely to the cultures to compete with [3H]-2-DG during uptake measurement (Figure 3B).
Interestingly, αSa also robustly increased [3H]-2-DG in microglial cell cultures (Figure 3C), suggesting that the greater demand of glucose by microglial cells was not circumscribed to LPS-induced inflammatory events. In contrast, αSm did not induce statistical effects. Noticeably, COL-3 (10 or 20 µM) reduced substantially glucose uptake when applied concomitantly to αSa. This effect was mimicked by DOX at 50 µM. A co-treatment of αSa-treated cultures with 300 µM APO or 500 µM 2-DG also led to a significant reduction in [3H]-2-DG uptake. As expected [3H]-2-DG uptake was also prevented when glucose was added in excess to compete with the radioligand when performing uptake measurements (Figure 3D).
COL-3 prevents the accumulation of NADPH in microglial cells challenged with either LPS or αSa
Glucose may have a key impact on microglial inflammatory processes by stimulating the pentose phosphate pathway and consequently the synthesis of NADPH, the requisite substrate for the superoxide producing enzyme NADPH oxidase [32,37]. Coherent with this view, we found that NADPH levels were increased substantially in microglial cells activated by either 10 ng/ml LPS or 70 µg/ml αSa but not with αSm (Figure 4A,B). Of interest, COL-3 (20 µM) caused a significant reduction of NADPH levels in both inflammation paradigms (Figure 4A,B). As expected, the suppressive effect of COL-3 was mimicked by DOX at a concentration of 50 µM.
Both the inactive glucose analog 2DG (500 µM) and the NADPH oxidase inhibitor APO (300 µM) mimicked the inhibitory effect of COL-3 on NADPH production in microglia activated by LPS or αSa, indicating that the non-antibiotic tetracycline may interfere with a glucose-dependent mechanism that promotes NADPH synthesis and as a consequence ROS production via the NADPH oxidase enzyme.