At early time points, MSU-induced γH2AX levels in Nlrp3−/− and casp-1−/− DCs were comparable with those detected in WT DCs (Fig. 3A).

In contrast, 24 h MSU stimulation in the absence of NLRP3 or caspase-1 resulted in markedly decreased γH2AX levels. These data are consistent with the comet assay results underlining the likelihood of the NLRP3 inflammasome being involved in cellular responses to DNA damage. To confirm whether NLRP3 inflammasome activators directly induce DNA breaks, we used rotenone to provoke robust ROS production by mitochondria in order to activate NLRP3 PLX3397 manufacturer indirectly [10]. Similarly to MSU, rotenone treatment markedly induced γH2AX levels, which are reduced in both Nlrp3−/− and casp-1−/− DCs compared with WT (Fig. 3B). We also used camptothecin (campto), a topoisomerase I inhibitor, to promote DNA damage independently CTLA-4 antibody inhibitor of ROS [11], and observed that the genotoxic effect induced by this drug was not lowered in either Nlrp3−/− or casp-1−/− DCs

(Fig. 3C). Finally, DNA damage induced by high-dose γ-radiation was also reduced in DCs lacking Nlrp3 and casp-1 after 24 h (Fig. 3D). Taken together, these results indicate that NLRP3 inflammasome may be involved in regulation of DDR. MSU, H2O2, rotenone, and γ-radiation all trigger the generation of ROS, which in turn react with DNA to cause base lesions. To clarify whether the DNA damage detected in DCs depended on ROS generation, we assessed ROS production following stimulation with MSU alone or in O-methylated flavonoid combination with LPS or H2O2 in DCs from WT and Nlrp3−/− mice. We did not observe any differences in the levels of MSU, LPS/MSU, or H2O2-induced ROS produced between WT and Nlrp3−/− DCs (Fig. 4A). However, after 8 h of MSU

exposure, ROS-mediated oxidative stress did induce upregulation of genes encoding the antioxidant proteins peroxiredoxin 1 and catalase more strongly in WT DCs than in Nlrp3−/− DCs (Fig. 4B). These data indicate that ROS generated by MSU treatment are equally abundant in WT and Nlrp3−/− DCs, but that they likely show a differential response in mediating redox and oxidative stress control. To test whether ROS did induce oxidative DNA damage following MSU stimulation, we assessed the formation of the DNA adduct 8-oxoguanine (8-oxoG), the major oxidation product and an important marker of free radical induced DNA lesions and oxidative stress [12]. We compared the proportion of 8-oxoG positive MSU-treated DCs prepared from WT and Nlrp3−/− mice. Following MSU treatment, the number of 8-oxoG positive nuclei was substantially increased in WT DCs compared with untreated controls (Fig. 4C and D). Importantly, the presence of 8-oxoG lesions was markedly lower in DCs deficient in Nlrp3, suggesting that the base excision repair system responsible for 8-oxoG repair in the DNA was more active in Nlrp3−/− cells than WT DCs.