Interleukin 38 alleviates aortic valve calcification by inhibition of NLRP3

Figures

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  Fig. 1.. IL-38 suppresses the spontaneous inflammatory and osteogenic activities in human AVICs from CAVD valves. (A and B) IL-38 protein levels in lysates of human AVICs from CAVD and non-CAVD valves were analyzed by ELISA (A) and immunoblotting (B). Human AVICs from CAVD valves have much lower levels of IL-38. Values are mean ± SEM, n = 3 donors. *P < 0.05 and **P < 0.01 versus human AVICs from non-CAVD valves. (C) Human AVICs from CAVD valves were treated with recombinant IL-38 (10 ng/mL) or vehicle (normal saline) for 72 h. Representative immunoblots (Left) and densitometric data (Right) show that recombinant IL-38 reduced cellular levels of inflammatory (ICAM-1 and VCAM-1) and osteogenic (RUNX2 and ALP) mediators. Values are mean ± SEM, n = 4 donors. ***P < 0.001 and ****P < 0.0001 versus vehicle. (D) Representative images of Alizarin Red S staining (Left) and spectrophotometric data (Right) show that treatment for 14 d with recombinant IL-38 reduced calcium deposition in human AVICs from CAVD valves. Values are mean ± SEM, n = 4 donors. **P < 0.01 versus vehicle. (Scale bar, 100 µm).

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  Fig. 2.. IL-38 suppresses the inflammatory and osteogenic responses to proinflammatory stimulation in human AVICs from non-CAVD valves. (A) Human AVICs from non-CAVD valves were pretreated with recombinant IL-38 (10 ng/mL) for 2 h followed by matrilin-2 (2 µg/mL) treatment for 72 h. Representative immunoblots (Upper) and densitometric data (Lower) show that treatment with recombinant IL-38 reduced the inflammatory and osteogenic responses to matrilin-2. (B) ELISA data show that recombinant IL-38 suppressed the production of IL-6 and CCL2 in human AVICs from non-CAVD valves exposed to matrilin-2. (C) Representative images of Alizarin Red S staining and spectrophotometric data show that treatment for 14 d with recombinant IL-38 reduced calcium deposition in human AVICs from non-CAVD valves exposed matrilin-2. Values are mean ± SEM, n = 4 donors. **P < 0.01 and ***P < 0.001 versus vehicle; #P < 0.05 and ###P < 0.001 versus matrilin-2 alone. (Scale bar, 100 µm).

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  Fig. 3.. IL-38 deficiency exacerbates aortic valve lesions in mice. (A) Old mice (16 mo old) were fed a standard diet or HFD for 4 mo. Representative Von Kossa staining images show that IL-38 KO-HFD mice displayed greater aortic valve leaflet thickening and calcification compared to WT-HFD mice. Values are mean ± SEM, n = 3 mice per group. *P < 0.05 and **P < 0.01 versus WT-standard diet; #P < 0.05 and ##P < 0.01 versus WT-HFD; &P < 0.05 and &&&P < 0.001 versus IL-38 KO-standard diet. (Scale bar, 100 µm). (B) Mouse AVICs from WT or IL-38 KO mice were treated with vehicle or matrilin-2 (2 µg/mL) for 72 h. Representative immunoblots (Left) and densitometric data (Right) show that AVICs from IL-38 KO mice exhibited higher spontaneous inflammatory and osteogenic activities in comparison to AVICs from WT mice. In addition, AVICs from IL-38 KO mice had moderately greater responses to matrilin-2 stimulation, but the difference between AVICs from IL-38 KO mice and AVICs from WT mice did not reach statistical significance. Values are mean ± SEM, n = 4 mice per group. *P < 0.05, **P < 0.01 and ***P < 0.001 versus WT vehicle. (C) Human AVICs from non-CAVD valves were pretreated with either scrambled…

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  Fig. 4.. Effects of IL-1R9 deficiency on aortic valve. (A) Representative images of hematoxylin and eosin staining of aortic valves show that valve leaflet thickening was occurred in IL-1R9 KO mice (12 wk old) in the absence of any treatment. Values are mean ± SEM, n = 3 mice per group. *P < 0.05 versus WT. (Scale bar, 100 µm). (B) Representative images of Alizarin Red S staining (Left) and spectrophotometric data (Right) show that mouse AVICs from IL-1R9 KO mice displayed greater calcium deposition compared to mouse AVICs from WT mice. Values are mean ± SEM, n = 3 mice per group. *P < 0.05 versus WT. Scale bar, 100 µm. (C) Mouse AVICs from WT and IL-1R9 KO mice were treated with vehicle or matrilin-2 (2 µg/mL) for 72 h. Representative immunoblots (Left) and densitometric data (Right) show that mouse AVICs from IL-1R9 KO mice had greater inflammatory and osteogenic responses to matrilin-2 stimulation. Values are mean ± SEM, n = 4 mice per group. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001 versus WT vehicle; #P < 0.05 and ###P < 0.001 versus WT + matrilin-2. (D) Representative immunoblots (Left) and densitometric data (Right) show that IL-1R9 protein levels were slightly…

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  Fig. 5.. IL-38 inhibits NLRP3 activation. (A) Human AVICs from non-CAVD valves were pretreated with recombinant IL-38 (10 ng/mL) for 2 h prior to matrilin-2 (2 µg/mL) treatment for 16 and 24 h. Representative immunoblots (Upper) and densitometric data (Lower) show that recombinant IL-38 suppressed NLRP3 inflammasome and caspase-1 p20 induced by matrilin-2. Values are mean ± SEM, n = 4 donors. **P < 0.01 and ***P < 0.001 versus vehicle; #P < 0.05 and ##P < 0.01 matrilin-2 alone. (B) Mouse AVICs from WT and IL-38 KO mice were treated with vehicle or matrilin-2 (2 µg/mL) for 16 and 24 h. Representative immunoblots (Upper) and densitometric data (Lower) show higher levels of Nlrp3 inflammasome and caspase-1 p-20 activation in mouse AVICs from IL-38 KO mice compared to mouse AVICs from WT mice, especially in the absence of matrilin-2 stimulation. Values are mean ± SEM, n = 3 per group. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001 versus WT vehicle. (C) Human AVICs from non-CAVD valves were pretreated with recombinant IL-38 (10 ng/mL) or MCC950 (1 µM) for 2 h and then exposed to matrilin-2 (2 µg/mL) for 72 h. Representative immunoblots (Left) and densitometric data (Right) show that…