Fig. 4

Pro-inflammatory cytokines in cultured microglia from TLR2^-/- and TLR4^-/- mice after in vitro NA stimulation. Microglia cultures isolated from WT, TLR2^-/-, and TLR4^-/- mice strains were stimulated in vitro with LPS (a TLR4 agonist), the synthetic lipoprotein P3C (a TLR2 agonist), or neuraminidase (NA) for 6 h. Total RNA was then isolated, and the mRNA levels of IL-1β (a), TNFα (b), and IL-6 (c) were quantified by qPCR. mRNA levels are expressed relative to GAPDH using the Pfaffl method. While NA was able to stimulate cytokine production in WT and TLR2^-/- microglial cells, it was unable to do so in TLR4^-/- cells. As expected, and similarly to NA, LPS induced cytokine production in WT and TLR2^-/- microglia, but not in TLR4^-/- cells. On the contrary, and also as expected, P3C was able to induce cytokine overexpression in WT and TLR4^-/- microglia, but not in TLR2^-/- cells. The histograms show means + SD of n = 3–5 independent cultures. Two-way ANOVA was used to compare treatments. Letters (a–c) above each bar indicate the absence (same letter) or presence (different letters) of a statistically significant difference (P < 0.001) between the compared groups