Enhanced NLRP3 inflammasome and IL-6 signaling pathways in TET2-deficient myeloid cells. (A) Systemic IL-6 and IL-6Ra levels were quantified in both BM plasma and PB of CH macaques (ZL26, ZL39, and ZH63) and age-matched transplanted control macaques (ZL19, ZL33, and ZJ52). (B) CD14⁺CD163⁺ macrophages were purified from PB collected from the 3 CH-edited macaques and age-matched controls. The macrophages were incubated with LPS, followed by addition of ATP at the end of culture to activate the NLRP3 inflammasome. RNA from unstimulated or stimulated macrophages was analyzed by quantitative reverse transcription polymerase chain reaction. Fold changes of NLRP3 inflammasome-related genes (NLRP3, PYCARD, IL1B, and IL18) and IL-6-signaling genes (IL6, IL-6R, and TRAF6) calculated by δ-deltaCt relative to the unstimulated control are shown. (C) Concentrations of IL-1β and IL-6 were measured in the supernatants of macrophages cultured with or without specific stimulants (LPS and ATP for IL-1β; LPS for IL-6). Results from at least 3 independent experiments are graphed as mean plus or minus standard error of the mean. Statistical analysis was conducted using an unpaired t test between CH and controls group, and P values are directly marked on each panel. (D) Caspase-1 activity was measured by flow cytometry in unstimulated and LPS/ATP-activated macrophages from CH macaques (ZL26 and ZH63) and transplanted control macaques (ZL38 and ZL33). Representative histograms are shown on the left, and the results from the 3 independent experiments on the right. (E) The representative confocal microscopic images of cytoplasmic ASC specks (indicated by the arrow) in naïve macrophages from CH macaques and controls (left: scale bar, 25 μM). The percentage of nuclei with ASC specks from at least 10 different fields (right). FITC, fluorescein isothiocyanate.