TMAO enhanced M1 polarization via activating the NLRP3 inflammasome. (A) Representative immunofluorescence staining of NLRP3 (green, Alexa Fluor 488), ASC (red, Alexa Fluor 594), DAPI (blue) on BMDMs after TMAO (300 μM) stimulation for 24 hours. Scale bar, 5 μm. (B) Western blotting analysis of NLRP3, IL-1β, and cleaved IL-1β of BMDMs cultured with TMAO (300 μM) or choline (300 μM) for 24 hours. (C-G) BMDMs were cultured with TMAO (300 μM) in the presence or absence of CY-09 (1 μM) for 24 hours. (C) Casapse-1 activity (N = 5 in each group; **P < .01). (D) Representative image of FLICA Casapse-1 that binds only to activated caspase-1 (green, FAM FLICA; blue, DAPI). Scale bar, 5 μm. (E) Flow cytometry analysis of FLICA Casapse-1 was determined. (F) The concentrations of IL-1β in the supernatant of BMDMs cultured with TAMO or TMAO+CY-09 were detected by ELISA. N = 4 in each group. **P < .01. (G) Expression of IL-1β (left to right column, n = 4, 3, 4), IL-6 (left to right column, n = 3, 4, 3), TNF-α (left to right column, n = 3, 3, 5), CXCL9 (left to right column, n = 4, 3, 3), and CXCL10 (left to right column, n = 3, 3, 6) in BMDMs stimulated with TMAO or TMAO+CY-09 were determined by RT-PCR. *P < .05, **P < .01. (H) Expression of IL-1β, IL-6, TNF-α, CXCL9, and CXCL10 in Nlrp3^−/− BMDMs or WT BMDMs was determined. N = 3 in each group.