It is noteworthy that ATP, LPS, or ethanol (10 mM) did not promote NLRP3/caspase-1 activation and recruitment into the mitochondria in TLR4-KO astrocytes (Figure 6). Immunofluorescence studies with pro-caspase-1 and NLRP3 in untreated astrocytes and those treated with ATP, LPS and ethanol (10 mM) from WT and TLR4-KO mice also demonstrated that while stimulation with ATP, LPS or ethanol reduces the pro-caspase-1 levels and increases NLRP3 expression (Figure 2) and its co-localization in the WT cell cytoplasm, no changes in these parameters were observed in untreated or treated TLR4-KO astrocytes. In short, the results suggest that the by triggering pro-IL-1β production (Gross et al., 2011), TLR4 acts as a priming signal which, along with ROS production (second signal), promotes NLRP3 inflammasome activation. Lack of TLR4 signaling (TLR4-KO) was able to abolish the production of not only pro-IL-1, but also of other cytokines which might target to the mitochondria to initiate mROS production.
