To investigate the potential of WNT-3A and WNT-5A as pro- and anti-inflammatory regulators, we stimulated naïve and LPS-pretreated mouse primary microglia with increasing doses (0, 30, 100, 300, 1000 ng/mL) of WNTs for 6 h. Cells were then lysed and COX2 expression, which served here as a generic marker of pro-inflammatory transformation (Mitchell et al. 1995), was analyzed in whole cell lysates by immunoblotting. In line with previously published data (Halleskog et al. 2011, 2012), WNT-3A (Fig. 1) and WNT-5A (Fig. 2) increased the level of COX2 expression, supporting the pro-inflammatory action of these two WNTs. LPS, a bacterial cell wall component, which generally promotes microglia activation through action at Toll-like receptor 4 (TLR4) (Lehnardt 2010), exceeded the elevation of COX2 levels upon WNT stimulation substantially. However, in combination, increasing doses of WNT-3A (Fig. 1) and WNT-5A (Fig. 2) reduced the LPS-induced expression of COX2 dose-dependently. WNT-3A led to 66% (300 ng/mL) and 82% reduction (1000 ng/mL), whereas WNT-5A diminished LPS effects on COX2 expression by 62% and 67% at the respective concentrations. The WNT-induced down-regulation of protein levels of COX2 could be achieved through translational or transcriptional regulation. To shed more light on underlying mechanisms, we analyzed mRNA expression of COX2 and other candidate genes as markers of the cell's pro-inflammatory transformation: Quantification of COX2, interleukin 6 (IL-6), and TNFα mRNA by quantitative PCR was performed in cells that were control treated, stimulated with LPS (100 ng/mL) or with LPS and WNT-3A (300 ng/mL) or WNT-5A (300 ng/mL) for 6 h (Fig. 3). WNT-3A led to a reduction of COX2, IL-6, and TNFα mRNA expression to 25%, 35%, and 35% of LPS-induced gene expression, respectively. WNT-5A diminished LPS effects to 36%, 46%, and 57%, respectively. The data indicate on one hand that WNTs can act anti-inflammatory by reducing LPS-induced expression of COX2 and proinflammatory cytokines. On the other hand they argue that WNTs affect LPS-induced gene expression on the transcriptional level.
Figure 3. Wingless/int1 (WNT)-3A and WNT-5A reduce the LPS-induced cyclooxygenase 2 (COX2), IL-6, and TNFα mRNA levels in mouse primary microglia. cDNA from microglia cells stimulated with ctrl, 100 ng/mL LPS, or costimulated with LPS and 300 ng/mL WNT-3A or WNT-5A for 6 h, was analyzed with QPCR for mRNA expression of COX2, IL-6 and TNFα. mRNA levels are presented as arbitrary units normalized to LPS (100%). Normalization is described in materials & methods section. At least four independent experiments are summarized (error bars – SEM; ***p < 0.001). Compared to the LPS-induced response mRNA expression in unstimulated control was very low: WNT-3A ctrl values (arbitrary units ± SEM) for COX2: 0.0653 ± 0.0154; IL-6: 0.0486 ± 0.0457; TNFα: 0.2317 ± 0.0307. WNT-5A ctrl values for COX2: 0.0483 ± 0.0125; IL-6: 0.0005 ± 0.0001; TNFα: 0.4972 ± 0.2542.
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