Fig 2: Effects of CAP agonists on M1 macrophage polarization. (a) The mRNA expression of M1 type marker genes (i.e., TNFa, IL-1ß, and IL-6) were suppressed by the treatment of Ach and GTS-21 in INF? induced BMDM. Data are presented as means ± SD (n = 3 biological replicates). ** p < 0.01, *** p < 0.001 as compared to INF? induced cells. (b) Representative immunoblot images and quantitative analysis of inflammatory-related genes in LPS induced RAW264.7 cell treated with Ach and GTS-21. Data are presented as means ± SD (n = 3 biological replicates). ** p < 0.01 as compared to LPS-induced cells, ns indicates not significant. (c) Representative immunoblot images and quantitative analysis of inflammatory-related genes in INF? induced BMDM treated with Ach and GTS-21. Data are presented as means ± SD (n = 3 biological replicates). * p < 0.05, ** p < 0.01 as compared to INF? induced cells, ns indicates not significant. (d) The mRNA expression of TNFAIP3 was upregulated by the treatment of Ach and GTS-21 in LPS/INF? induced RAW264.7 and BMDM. Data are presented as means ± SD. ** p < 0.01 as compared to LPS/INF? induced cells. (e,f) Representative immunoblot images and quantitative analysis of TNFAIP3 in RAW264.7 (e) and BMDM (f) treated with Ach and GTS-21. Data are presented as means ± SD (n = 3 biological replicates). * p < 0.05 as compared to INF? induced cells. (g) GTS-21 inhibited expression of TNFa was restored by siRNA interference of TNFAIP3. Data are presented as means ± SD (n = 3 biological replicates). ** p < 0.01, ns indicates not significant.

Fig 3: A20 is a functional miR-21 target for regulation of NF-?B and NLRP3 inflammasomes.a–e A20 (a), Nlrp3 (b), Asc (c), Casp1 (d), and IL-1b (e) mRNA levels in LPS-treated WT and miR-21 KO BMDMs. f A20, IL-1b and NLRP3 inflammasome protein levels in LPS-treated WT and miR-21 KO BMDMs after 4 h. g Sequence alignment of the A20 3'-UTR with miR-21 across multiple species. h Activity of luciferase reporters containing WT or mutant A20 3'-UTRs that were used with miR-21 mimics or respective controls to cotransfect HEK-293T cells. i BMDMs were treated with 500 ng/ml LPS for different lengths of time. Cell extracts were immunoblotted for IKBa and p-IKBa. j, k WT and miR-21 KO BMDMs were left unstimulated (control) or stimulated with LPS (500 ng/ml), ATP (5 mM), or nigericin (20 mM) alone or stimulated with LPS (500 ng/ml) for 4 h and then treated with 5 mM ATP or 20 mM nigericin for 30 min. Culture supernatants were analyzed for IL-6 secretion (j) and TNF secretion (k). The data represent the mean ± SD of one among three biological replicates, with three technical replicates each (*P < 0.05; **P < 0.01; Student’s t test)

Fig 4: miR-21 regulates NF-?B and NLRP3 inflammasomes through A20.a–d A20 (a), Nlrp3 (b), Asc (c), and Casp1 (d) mRNA levels in LPS-treated miR-21 KO BMDMs transfected with control or A20 siRNA. e Protein levels of NLRP3 inflammasomes in LPS-treated miR-21 KO BMDMs transfected with control or A20 siRNA. f Activation of caspase-1 in LPS-primed and ATP- or nigericin-treated miR-21 KO BMDMs transfected with control or A20 siRNA. g Supernatants were analyzed for IL-1ß in (f). h BMDMs were treated with 500 ng/ml LPS for different lengths of time in miR-21 KO BMDMs transfected with control or A20 siRNA. Cell extracts were immunoblotted for IKBa and p-IKBa. The data represent the mean ± SD of one among three biological replicates, with three technical replicates each (*P < 0.05; **P < 0.01; Student’s t test)

Fig 5: miR-21 deficiency inhibits pyroptosis by preventing cleavage of GSDMD.a Imaging assay of pyroptosis in WT and miR-21 KO BMDMs treated as indicated. Scale bars: 100 µm. b Immunoblotting demonstrates cleavage of GSDMD. WT and miR-21 KO BMDMs stably expressing Flag–GSDMD were stimulated as indicated. c Immunoblotting with mouse antibody for GSDMD demonstrates cleavage of GSDMD in WT and miR-21 KO BMDMs treated with ATP or nigericin. d Immunoblotting demonstrates cleavage of GSDMD in WT and miR-21 KO BMDMs treated with A20 siRNA. GSDMD-FL full-length GSDMD, GSDMD-N N-terminal cleavage products of GSDMD. All data are representative of three independent experiments