As shown in Figure 4D and E, IL4 mRNA levels were not significant

As shown in Figure 4D and E, IL4 mRNA levels were not significantly changed after TGFB1 treatment for 24 hours. Using a mouse specific cytokine array, we revealed that primary microglia release very low levels of IL4 and treatment of primary microglia with TGFB1 did not result in any changes in IL4 release after 24 hours. These data suggest that the enhancement of Arg1 and Ym1 expression by TGFB in IL4 treated microglia might, at least partially, be mediated by increasing IL4R ex pression, thus, enhancing the microglial sensitivity to IL4 signals. Mitogen activated protein kinase mediates TGFB1 enhanced Arg1 expression in IL4 treated primary microglia To investigate the pathways involved in TGFB1 mediated enhancement of IL4 induced Arg1 expression, the TGFB Smad and the IL4 Stat6 signalling pathways were analysed by monitoring Smad2 3 nuclear accumu lation and phosphorylation of Smad2 and Stat6, respect ively.

Whereas treatment of primary microglia with TGFB1 resulted in increased nuclear accumulation of Smad2 3, IL4 treatment failed to induce nuclear accu mulation of Smad2 3. Immunoblotting against phospho Smad2 and phospho Stat6 revealed that TGFB1 exclusively increased Inhibitors,Modulators,Libraries the levels of phosphory lated Smad2 and failed to increase the levels of phos phorylated Stat6. Inhibitors,Modulators,Libraries Vice versa, IL4 treatment resulted Inhibitors,Modulators,Libraries in increased levels of phospho Stat6, whereas phosphoryl ation of Smad2 was not observed after treatment with IL4 for 1 and 2 hours. MAPK has been shown to be activated in microglia after TGFB1 treatment.

To analyse the role of MAPK signalling on TGFB1 mediated enhancement of IL4 induced Arg1 expression, BV2 cells Inhibitors,Modulators,Libraries and primary microglia were treated with IL4, TGFB1 and IL4 TGFB1 in the absence or presence of the MEK1 2 inhibitor PD98059 for 24 hours. Western blotting results from BV2 cells showed that IL4 treatment alone increased Arg1 protein levels, which was partially inhibited in the presence of PD98059. TGFB1 and IL4 co treatment increased IL4 induced Arg1 protein levels and the MEK1 2 inhibitor PD89059 partially blocked the TGFB1 mediated increase in Arg1 protein levels. Using primary microglia we confirmed the results obtained with BV2 cells. Treatment with IL4 sig nificantly increased the protein levels of Arg1. Interest ingly, in the presence of PD89059, IL4 failed to increase the protein levels of Arg1.

Combination of IL4 and Inhibitors,Modulators,Libraries TGFB1 dramatically increased the protein levels of Arg1 compared to IL4 treatment alone. However, in the pres ence of the MEK1 2 inhibitor PD89059, TGFB1 enhanced Arg1 up regulation was significantly impaired and the amount of Arg1 was similar to the levels after therefore treatment with IL4 alone. These data demonstrate that TGFB1 activated MAPK signalling is essential for TGFB1 mediated enhancement of IL4 induced Arg1 expression in microglia. Discussion In this study we demonstrate for the first time that TGFB enhances the IL4 induced alternative activation of microglia.

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