Fig. 4
Development of tolerance is partially impaired in Q140/Q140 microglia. A Schematic experimental design. Microglia were pre-treated with LPS (100 ng/ml) for 12 h, washed and incubated in serum free medium for an additional 24 h (recovery). A second stimulation with LPS (100 ng/ml) was performed for 6 h. Control groups were stimulated only once with LPS for 6 h. B Expression of Il-1b, Il-6 and Tnf after the second stimulation with LPS is reported as fold-change compared to cells stimulated only once (baseline represented by the horizontal dotted line in each graph). mRNA levels were normalized over the geometric mean of three housekeeping genes. Ratio t-test. Asterisks show significant differences compared to the baseline of cells stimulated only once. The symbol # shows a statistically significant difference between genotypes. N ≥ 5. C Levels of IL-6 (N ≥ 4) and TNF (N ≥ 5) released by microglia in the culture medium. D Quantification of LDH released in the medium during 24 h recovery period following the first LPS stimulation. Similar levels of LDH released in the medium by Q7/7 and Q140/140 cells indicate microglia were healthy and had similar viability at the time of the second stimulation with LPS. Two-tailed paired t-test. N = 3. E Expression of the non-tolerizeable gene Fpr1 is increased to a similar extent in Q7/7 and Q140/140 microglia stimulated for a second time with LPS, indicating gene priming. Two-way ANOVA and Sidak’s multiple comparisons post-test. F Il-10 and Tgfb mRNA expression in naïve microglia, at the end of the first stimulation with LPS and immediately prior exposure to the second dose of LPS (24 h recovery). mRNA levels were normalized over cyclophilin A levels. N ≥ 4 One-way ANOVA and Tukey’s multiple comparison post-test were used to compare gene expression changes across time points for each genotype. Comparisons between genotypes at each time point were performed with the unpaired two-tail t-test. Bars are mean values ± STDEV. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001