Rabbit Anti-Human NLRP3 Polyclonal Antibody Knockout Validated

Apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC) is a component of inflammasome, which plays crucial roles in the inflammatory response. In mammals, ASC regulates caspase-1 activation, thereby inducing pyroptosis and producing activated inflammatory cytokines. In addition, ASC also interacts with receptor-interacting protein kinase 2 (RIPK2) and induces nuclear factor-kappa B (NF-kappa B) activation. However, the role of ASC remains poorly understood in fish. In this study, we focused on elucidating the role of ASC in fish that were infected with Aeromonas hydrophila using Japanese medaka (Oryzias latipes) as fish model, and ASC-knockout (KO) medaka was established using CRISPR-Cas9 system. ASC-KO and wild type (WT) medakas were infected with A. hydrophila, and mortality was observed. ASC-KO medaka demonstrated higher mortality than WT. Moreover, the expression of immune-related genes in the kidney and intestine of the ASC-KO and WT medakas challenged with A. hydrophila were analyzed. Following A. hydrophila infection, the kidney of ASC-KO medaka exhibited significantly lower expression of NF-kappa B regulated genes (e.g., IL-1 beta, IL-6, IL-8 and TNF-alpha) and RIPK2 gene than in WT kidney. Moreover, to investigate the immune response against A. hydrophila via ASC in the medaka, bacterial burden, superoxide anion production, and lactate dehydmgenase release in the kidney cells of ASC-KO medaka were measured. After infection, these responses in ASC-KO medaka were significantly decreased compared to those in WT. These results suggest that the medaka ASC plays a critical role against A. hydrophila infection by inducing inflammatory responses and cell death for bacterial clearance.

The role of corticosteroids in acute lung injury (ALI) remains uncertain. This study aims to determine the underlying mechanisms of corticosteroid treatment for lipopolysaccharide (LPS)-induced inflammation and ALI. We used corticosteroid treatment for LPS-induced murine ALI model to investigate the effect of corticosteroid on ALI in vivo. Moreover, LPS-stimulated macrophages were used to explore the specific anti-inflammatory effects of corticosteroids on NLRP3-inflammasome in vitro. We found corticosteroids attenuated LPS-induced ALI, which manifested in reduction of the alveolar structure destruction, the infiltration of neutrophils and the inflammatory cytokines release of interleukin-1 beta (IL-1 beta) and interleukin-18 (IL-18) in Lung. In vitro, when NLRP3-inflammasome was knocked out, inflammatory response of caspase-1 activation and IL-1 beta secretion was obviously declined. Further exploration, our results showed that when corticosteroid preprocessed macrophages before LPS primed, it obviously inhibited the activation of caspase-1 and the maturation of IL-1 beta, which depended on inhibiting the nuclear factor-kappa B (NF-kappa B) signal pathway activation. However, when corticosteroids intervened the LPS-primed macrophages, it also negatively regulated NLRP3-inflammasome activation through suppressing mitochondrial reactive oxygen species (mtROS) production. Our results revealed that corticosteroids played a protection role in LPS-induced inflammation and ALI by suppressing both NF-kappa B signal pathway and mtROS-dependent NLRP3 inflammasome activation.