Cheng-Cheng Hou1, Hai-Fen Ma1, Jing-Fen Ye1, Dan Luo1, Hua-Fang Bao1, Jian-Long Guan2. 1. Department of Rheumatology and Immunology, Huadong Hospital Affiliated to Fudan University, Shanghai, China. 2. Department of Rheumatology and Immunology, Huadong Hospital Affiliated to Fudan University, Shanghai, China. jianlong_guan@126.com.
Abstract
OBJECTIVES: Intestinal Behçet's syndrome (IBS) has high morbidity and mortality rates with serious complications. The purpose of this study was to investigate the expression of pyroptosis-related proteins in the intestinal tissues of IBS patients and explore the role of plasma exosomes derived from IBS patients in the pyroptosis of intestinal epithelial cells. METHOD: Immunohistochemistry was used to investigate the expression of nucleotide-binding domain-like receptor protein 3 (NLRP3), caspase-1, and gasdermin D (GSDMD). Quantitative real-time PCR was employed to measure the mRNA levels of IL-1β and IL-18 in the intestinal tissues. Plasma exosomes were isolated and observed by transmission electron microscopy. The exosomes were co-cultured with intestinal epithelial cells in vitro. Western blot was used to measure the expression of pyroptosis-related proteins including NLRP3, full-length GSDMD, N-terminal GSDMD, pro-caspase-1, and cleaved caspase-1. The levels of IL-1β and IL-18 were detected by enzyme-linked immunosorbent assay. Cell death was measured by using the lactate dehydrogenase (LDH) release assay. RESULTS: Expression of NLRP3 (12.2% ± 1.2%, 8.1% ± 0.9%, t = 4.692, p = 0.009), caspase-1 (24.6% ± 2.1%, 4.2% ± 1.8%, t = 12.842, p = 0.000), and GSDMD (16.6% ± 1.9%, 9.8% ± 1.3%, t = 5.194, p = 0.007) were significantly increased in the intestinal tissues of patients with IBS compared with normal control (NC) group, respectively. The relative mRNA levels of IL-1β (t = 4.308, p = 0.005) and IL-18 (t = 3.096, p = 0.021) in the intestinal tissues were significantly higher in IBS patients than in NC group, while the protein levels of IL-1β (t = 3.873, p = 0.018) and IL-18 (t = 4.389, p = 0.012) were also significantly increased, which was consistent with the results of the relative mRNA levels. Moreover, we found that exosomes from IBS patients significantly induced pyroptosis of intestinal epithelial cells via the activation of NLRP3 inflammasome in vitro experiments. CONCLUSIONS: Plasma exosomes derived from IBS patients may induce pyroptosis of intestinal epithelial cells via the activation of NLRP3 inflammasome. Key Points •The role of exosomes in IBS is first reported in this study. • In this study, we explored the mechanism that plasma exosomes derived from IBS patients may induce pyroptosis of intestinal epithelial cells via the activation of NLRP3 inflammasome.
OBJECTIVES: Intestinal Behçet's syndrome (IBS) has high morbidity and mortality rates with serious complications. The purpose of this study was to investigate the expression of pyroptosis-related proteins in the intestinal tissues of IBSpatients and explore the role of plasma exosomes derived from IBSpatients in the pyroptosis of intestinal epithelial cells. METHOD: Immunohistochemistry was used to investigate the expression of nucleotide-binding domain-like receptor protein 3 (NLRP3), caspase-1, and gasdermin D (GSDMD). Quantitative real-time PCR was employed to measure the mRNA levels of IL-1β and IL-18 in the intestinal tissues. Plasma exosomes were isolated and observed by transmission electron microscopy. The exosomes were co-cultured with intestinal epithelial cells in vitro. Western blot was used to measure the expression of pyroptosis-related proteins including NLRP3, full-length GSDMD, N-terminal GSDMD, pro-caspase-1, and cleaved caspase-1. The levels of IL-1β and IL-18 were detected by enzyme-linked immunosorbent assay. Cell death was measured by using the lactate dehydrogenase (LDH) release assay. RESULTS: Expression of NLRP3 (12.2% ± 1.2%, 8.1% ± 0.9%, t = 4.692, p = 0.009), caspase-1 (24.6% ± 2.1%, 4.2% ± 1.8%, t = 12.842, p = 0.000), and GSDMD (16.6% ± 1.9%, 9.8% ± 1.3%, t = 5.194, p = 0.007) were significantly increased in the intestinal tissues of patients with IBS compared with normal control (NC) group, respectively. The relative mRNA levels of IL-1β (t = 4.308, p = 0.005) and IL-18 (t = 3.096, p = 0.021) in the intestinal tissues were significantly higher in IBSpatients than in NC group, while the protein levels of IL-1β (t = 3.873, p = 0.018) and IL-18 (t = 4.389, p = 0.012) were also significantly increased, which was consistent with the results of the relative mRNA levels. Moreover, we found that exosomes from IBSpatients significantly induced pyroptosis of intestinal epithelial cells via the activation of NLRP3 inflammasome in vitro experiments. CONCLUSIONS: Plasma exosomes derived from IBSpatients may induce pyroptosis of intestinal epithelial cells via the activation of NLRP3 inflammasome. Key Points •The role of exosomes in IBS is first reported in this study. • In this study, we explored the mechanism that plasma exosomes derived from IBSpatients may induce pyroptosis of intestinal epithelial cells via the activation of NLRP3 inflammasome.