Cystatin C is a potent cysteine protease inhibitor that plays an important role in various biological processes including cancer, cardiovascular diseases and neurodegenerative diseases. However, the role of CstC in inflammation is still unclear. In this study we demonstrated that cystatin C-deficient mice were significantly more sensitive to the lethal LPS-induced sepsis. We further showed increased caspase-11 gene expression and enhanced processing of pro-inflammatory cytokines IL-1β and IL-18 in CstC KO bone marrow-derived macrophages (BMDM) upon LPS and ATP stimulation. Pre-treatment of BMDMs with the cysteine cathepsin inhibitor E-64d did not reverse the effect of CstC deficiency on IL-1β processing and secretion, suggesting that the increased cysteine cathepsin activity determined in CstC KO BMDMs is not essential for NLRP3 inflammasome activation. The CstC deficiency had no effect on (mitochondrial) reactive oxygen species (ROS) generation, the MAPK signaling pathway or the secretion of anti-inflammatory cytokine IL-10. However, CstC-deficient BMDMs showed dysfunctional autophagy, as autophagy induction via mTOR and AMPK signaling pathways was suppressed and accumulation of SQSTM1/p62 indicated a reduced autophagic flux. Collectively, our study demonstrates that the excessive inflammatory response to the LPS-induced sepsis in CstC KO mice is dependent on increased caspase-11 expression and impaired autophagy, but is not associated with increased cysteine cathepsin activity.

Stefin B (cystatin B) is an inhibitor of lysosomal and nuclear cysteine cathepsins. The gene for stefin B is located on human chromosome 21 and its expression is upregulated in the brains of individuals with Down syndrome. Biallelic loss-of-function mutations in the stefin B gene lead to Unverricht–Lundborg disease-progressive myoclonus epilepsy type 1 (EPM1) in humans. In our past study, we demonstrated that mice lacking stefin B were significantly more sensitive to sepsis induced by lipopolysaccharide (LPS) and secreted higher levels of interleukin 1-β (IL-1β) due to increased inflammasome activation in bone marrow-derived macrophages. Here, we report lower interleukin 1-β processing and caspase-11 expression in bone marrow-derived macrophages prepared from mice that have an additional copy of the stefin B gene. Increased expression of stefin B downregulated mitochondrial reactive oxygen species (ROS) generation and lowered the NLR family pyrin domain containing 3 (NLRP3) inflammasome activation in macrophages. We determined higher AMP-activated kinase phosphorylation and downregulation of mTOR activity in stefin B trisomic macrophages—macrophages with increased stefin B expression. Our study showed that increased stefin B expression downregulated mitochondrial ROS generation and increased autophagy. The present work contributes to a better understanding of the role of stefin B in regulation of autophagy and inflammasome activation in macrophages and could help to develop new treatments.