Cathepsin B Regulate Non-canonical NLRP3 Inflammasome Pathway By Modulating Activation Of Caspase-11 In Kupffer Cells

Background/Aims:The inflammasomes are multimeric protein complexes triggered by a range of substances that emerge during infections,and serve as activation platforms for caspase-1,which proteolytically activates the pro-inflammatory cytokines interleukin-1β（IL-1β）and IL-18.Recently,a non-canonical inflammasome pathway was described which engages caspase-11 to mediate pyroptosis and the subsequent release of IL-1α,IL-1β,and IL-18 in a Toll-like receptor 4（TLR4）-independent way.Caspase-11 activation is required for both IL-1 family cytokines secretion and cell death in response to the cytoplasmic lipopolysaccharide（LPS）.Cathepsin B,the lysosomal protease,is capable of activating caspase-11under cell-free conditions which may regulate non-canonical NLRP3inflammasome pathway.In this study,we aimed to further investigate cathepsin B as potential subcellular localized activators of proinflammatory caspases which may be released upon proinflammatory stimuli and regulate non-canonical NLRP3 inflammasome pathway by moudulating the activaty of caspase-11.Methods:Pharmacological and gene silencing approaches were used to evaluate the impact of cathepsin B on regulating non-canonical NLRP3inflammasome pathway in Kupffer cells（KCs）which were isolated separately from wild-type（WT）and TLR4^–/–C57BL/6 mice.A sepsis model was also created to investigate the effect of cathepsin B on survival.Meanwhile cathepsin B activity and the expression level of caspase-4 were detected in human peripheral blood mononuclear cells（PBMC）which were separated from patients suffered from SIRS or sepsis and healthy volunteers.Results:KCs loaded with LPS activate caspase-11 independently of the LPS receptor TLR4.Simultaneously,LPS stimulation caused cathepsin B activity and caspase-11 expression increase in TLR4^–/–mice.Cathepsin B activity inhibition reduced the activation of caspase-11 and inflammasome and benefited survival in TLR4^–/–mice.Upregulation of cathepsin B activity and caspase-4 activation were found in peripheral blood mononuclear cells（PBMC）of patients with SIRS or sepsis.Conclusion:The anomalous distribution and activation of Cathepsin B of KCs were found in endotoxemia.Cathepsin B can activate caspase-11Which leads to the activation of non-classical NLRP3 inflammasome in both animal experiment and cell experiment.Down-regulated expression of Cathepsin B or inhibition of Cathepsin B activity can reduce the toxicity of large dose of LPS to KCs,and benefit survival in WT and TLR4^–/–mice surffered from endotoxemia.Meanwhile,the increase of Cathepsin B activity and abnormal distribution of Cathepsin B were confirmed in PBMC of patients with SIRS or sepsis.Our results suggest a critical role for cathepsin B as activators of proinflammatory caspases-11 and the regulatory effect in LPS-induced caspases-11-dependent necrosis.