Figure 2.

Increased capacity for pyroptosis in GSDME‐expressing peripheral blood monocytes from RA patients. CD14+ monocytes were purified from healthy controls and RA patients, and cultured for 4 hours in vitro. A, Left, Brightfield and fluorescence microscopy images of peripheral blood monocytes stained with Hoechst and propidium iodide (PI; red) (positive staining indicates lytic cell death). Bottom panels show higher‐magnification views (original magnification × 1600) of the boxed areas in the top panels (original magnification × 400). Arrows indicate pyroptotic cell bubbles. Right, Percentage of PI‐positive cells in 5 randomly chosen fields in peripheral blood monocytes from healthy controls (n = 7) and RA patients (n = 7). B, Lactate dehydrogenase (LDH) release from peripheral blood monocytes from healthy controls (n = 4) and RA patients (n = 4). C, Flow cytometric analysis (left) and percentage (right) of PI‐positive monocytes from healthy controls (n = 3) and RA patients (n = 3). D, Western blot of the expression of GSDME‐FL and GSDME‐N (left) and quantification of the expression of GSDME‐N (right) in monocytes from RA patients (n = 6), left untreated or treated with tumor necrosis factor (TNF) for 24 hours. Values are the protein level relative to β‐actin. E, Left, Phase‐contrast and fluorescence microscopy images of RA peripheral blood monocytes stained with Hoechst (blue) and PI (red). Phase‐contrast microscopy and PI staining images were merged. Bottom panels show higher‐magnification views (original magnification × 1600) of the boxed areas in the top panels (original magnification × 400). Arrows indicate pyroptotic cell bubbles. Right, Percentage of PI‐positive cells among untreated RA monocytes (n = 7) and RA monocytes treated with TNF (n = 7). Symbols represent individual subjects; bars show the mean ± SD. See Figure 1 for other definitions. Color figure can be viewed in the online issue, which is available at http://onlinelibrary.wiley.com/doi/10.1002/art.41963/abstract.