Figure 1.
Impact of Wt αSyn-priming on microglial cytokine release after TLR stimulation.
After treating the microglial cells either with Wt αSyn at 1 µg/mL (‘priming’ or pre-conditioning) or with ‘mock’ solution (no pre-conditioning) for 6 hrs, the TLR agonists were added to their specified final concentrations (see Materials and Methods), and incubated for further 18 hrs at 37 °C. The culture supernatants were harvested and used to measure (A) the levels of the interleukins IL-6, IL-10, IL-13, and IL-17, or (B) of the chemokines IP-10/CXCL10, RANTES/CCL5, MCP-1/CCL2, by ELISA. Values are the fold-change calculated as the signal ratio of αSyn-primed, TLR-stimulated cells (‘αSyn+TLR ligand’) relative to non-primed, TLR-stimulated cells (‘TLR ligand’). The results shown (mean ± SEM) are the average of several independent experiments (IL-6: N=5-7; IL-10: N=4-6; IL-13: N=3; IL-17: N=2-3; IP-10, RANTES, MCP-1, and MIP-1α: N=3-5), each containing duplicate samples. Statistically significant differences (* p<0.05) were calculated by applying the Student t test between the two sets of results, for all the TLR ligands tested. (#) denotes a result that is significantly different from that obtained after treatment of cells with Wt αSyn alone (p<0.05).
Figure 2.
Comparison of TLR gene expression levels of Wt αSyn-primed vs.
non-primed microgia, after TLR stimulation.
After treating cells as before (see legend to Figure 1), cells were lysed and the total RNA was extracted. Relative TLR gene expression levels were then examined by qRT-PCR and the hprt gene was used as the internal control to calculate ∆Ct values. The ∆∆Ct values were calculated by subtracting ∆Ct values of non-primed samples upon TLR stimulation (‘TLR ligand’) from ∆Ct values of samples treated with αSyn-priming upon TLR stimulation (‘Wt+TLR ligand’), to give the fold-change in TLR expression in cells with ‘αSyn+TLR ligand’ relative to ‘TLR ligand’ treatments. In all cases the TLR gene analysed (indicated inside bars) corresponded to the TLR agonist used in that particular sample. Fold-changes represent the average of three independent experiments (N=3), each one performed with duplicate samples. Bars correspond to SEM. Statistically significant differences (* p<0.05) were calculated by applying the Student t test between the two sets of results, for all the TLR ligands tested.
Figure 3.
Relative phagocytic capacity of WtαSyn-primed vs. non-primed microglia after TLR stimulation.
After treatment of the primary microglial cell cultures and incubation for a total of 24 hrs as described in the legend to Figure 1, cells were incubated with fluorescent microspheres for 1 hr. After fixing the cells, phagocytosis was assessed by fluorescence microscopy and calculation of the number of spheres/cell as described in the Methods section. Four images were analysed for each sample in each independent experiment. The ‘relative phagocytic capacity’ corresponds to the ratio (fold-change) of the number of spheres/cell of αSyn-primed cells followed by TLR stimulation (‘αSyn+TLR ligand’) relative to the number of spheres/cell of non-primed, TLR-stimulated cells (‘TLR ligand’). The values shown are an average of three (for ssRNA and PGN) or four (for all the others) independent experiments (N=3 or 4), and error bars represent the SEM. Statistically significant differences (* p<0.05) were calculated by applying the Student t test between the two sets of results, for all the TLR ligands tested. (#) denotes a result that is significantly different from that obtained after treatment of cells with Wt αSyn alone (p<0.05).
Figure 4.
Arg1 and iNOS PCR gene expression assays of αSyn-preconditioned microglia stimulated with Pam3 or ssRNA.
Primary microglial cells were either treated with Wt αSyn (Wt), A30P αSyn (A30P), or oligomeric Wt αSyn (oligomers) at 1 µg/mL, or with culture medium alone, for 6 hrs at 37 °C. Subsequently, the TLR agonists or medium alone were added accordingly, and cells were incubated for a further 18 hrs as described before. After treatment, RNA was extracted and reverse transcribed for PCR analysis of arginase-1 (Arg1) and iNOS gene expression. Actin expression was used as a reference and the positive controls for the PCR assays (PCR+) were from bone marrow-derived macrophages stimulated (for Arg1) or not (for iNOS) for 24 hrs with IL-4 (10 ng/mL).
Figure 5.
Quantitation of activated caspase-3 levels in treated microglial cells, by ELISA and immunofluorescence.
(A) After treatment of the primary microglial cell cultures and incubation for a total of 24 hrs as described in the legend to Figure 1, cells were lysed and tested by a specific ELISA assay for cleaved caspase-3 levels quantitation. The results shown (ng/mL cleaved casp-3 per µg of total protein) correspond to the mean of four independent experiments (N=4), each one performed with duplicate samples. Bars correspond to SEM. A discontinuous line represents the mean value obtained for untreated cells. Statistically significant differences were calculated by applying the Student’s t test in relation to the values obtained with the corresponding TLR ligand in the absence of αSyn-preconditioning (* p<0.05) and with Wt aSyn alone (# p<0.05). Treatment with staurosporine from Streptomyces sp. (5 µM) for 6 hrs was used as a positive control. (B) Cells were cultured in appropriate culture plates and treated as explained above (see legend to Figure 1) for subsequent labelling of cleaved caspase-3 and nuclear Hoechst 33342 staining for IF analysis, as described in the Methods Section. Samples were analyzed under the fluorescence microscope and three images from random fields containing ca. 80-90 cells each, were recorded, and analyzed for fluorescence quantification. The total specific red fluorescence (RF) and blue fluoresce (BF) were measured and the RF/BF ratio was used as a quantitation method and is represented in this figure. The results shown (RF/BF ratio) correspond to the mean of three images analysed (N=3) within one representative experiment, and bars correspond to SEM. A discontinuous line represents the mean value obtained for images from untreated cells. Statistically significant differences were calculated by applying the Student’s t test in relation to the values obtained with the corresponding TLR ligand in the absence of αSyn-preconditioning (* p<0.05) and with Wt αSyn alone (# p<0.05). Treatment with staurosporine from Streptomyces sp. (5 µM) for 6 hrs was used as a positive control.
Figure 6.
Immunofluorescence analysis of cleaved caspase-3 levels in treated microglial cells.
Representative images taken from immunolabeled primary microglial cells treated as described in the legend to Figure 1, with or without preconditioning by Wt αSyn. The TLR ligands tested were Pam3 and ssRNA40 at the concentrations described before (see Materials and Methods). Cells treated with 5 µM staurosporine (Strsp) from Streptomyces sp. for 6 hrs served as a positive control. Specific anti-(Asp175) cleaved caspase-3 primary antibodies and Alexa Fluor 594 secondary antibodies were used to visualize activated caspase-3 (first column), and nuclei were counterstained with Hoescht (second column). Merged images are shown in the third column and phase-contrast images of the same cultures are shown in the right column. White scale bars: 50 µm; grey scale bar (inset): 5 µm.
Figure 7.
Proposed model of the impact of αSyn-priming and TLR stimulation on microglial phenotype and neuroinflammation.
Surveyling microglia undergo polarization towards an M1-like phenotype after exposure to (TLR7) ssRNA and Imiq, and (TLR2/1) Pam3, TLR agonists, characterized by a lack of expression of Arg1, expression of iNOS, and high IL-6 production (see Results). On the one hand, αSyn-preconditioning of microglia and subsequent stimulation with (TLR7) ssRNA (and probably Imiq) produces an Arg1-/iNOS- (double negative) mixed or intermediate phenotype, and causes an increase in IP-10 and TNFα secretion, and a reduction of IL-13 levels. In addition, this treatment leads to a reduction in activated caspase-3 levels accompanied with a change in its intracellular location from the cytosol towards the nucleus of the microglial cell. On the other hand, exposure of αSyn-primed microglia to (TLR2/1) Pam3 agonist induces a skewing towards a different M1/M2 mixed or intermediate phenotype, exhibiting an Arg1+/iNOS+ (double positive) expression pattern, together with higher IL-6 and MCP-1 secretion levels. Remarkably, this phenotype agrees with the one observed for microglia that have been exposed to oligomeric αSyn. In addition, the ‘αSyn + Pam3’ treatment causes increase in activated caspase-3 levels in microglial cells.