GSDME-dependent pyroptosis signaling pathway in diabetic nephropathy.

Diabetic nephropathy (DN) is one of the serious chronic microvascular complications of diabetes, and leads to the increased morbidity and mortality in diabetic patients. Gasdermin E (GSDME)-dependent pyroptosis signaling pathway plays important roles in a variety of physiological and pathological processes. However, its role and mechanism in DN are still unclear. In this study, we established a rat DN model by intraperitoneal injection of streptozotocin (STZ) successfully. Structural and functional disorders in the kidney were exhibited on the 12th week after STZ injection; the expressions of caspase-3 and GSDME at protein level in renal cortex were significantly up-regulated. At the 20th week, GSDME-N increased significantly, accompanied by the upregulation of caspase-1 in renal cortex and the release of mature IL-1ß (mIL-1ß) in serum. Furthermore, we found the protein levels of GSDME, caspase-3, caspase-1 and IL-1ß were all increased in HK2 and HBZY-1 cells under high-glucose conditions. We also found that the expression of GSDME-N significantly decreased when caspase-3 was knockdown. In contrast, knockdown of GSDME has no effect on caspase-3. Interestingly, either caspase-3, caspase-1 or GSDME knockdown reduced the release of mIL-1ß. Finally, injection of adeno-associated virus (AAV) 9-shGSDME into the rat kidney reduced kidney damage and renal cell pyroptosis in comparison with wild-type diabetic rats. These results indicated that the activation of caspase-1 induced IL-1ß maturation, and the activation of caspase-3 mediated cleavage of GSDME responsible for the formation of plasma membrane pore, followed by cytoplasmic release of mIL-1ß. Overall, we identified a pro-pyroptosis role for GSDME in DN, which does provide an important basis for clinical therapeutic studies.

NEK7-Mediated Activation of NLRP3 Inflammasome Is Coordinated by Potassium Efflux/Syk/JNK Signaling During Staphylococcus aureus Infection.

Staphylococcus aureus (S. aureus) is a foodborne pathogen that causes severe diseases, such as endocarditis, sepsis, and bacteremia. As an important component of innate immune system, the NLR family pyrin domain-containing 3 (NLRP3) inflammasome plays a critical role in defense against pathogen infection. However, the cellular mechanism of NLRP3 inflammasome activation during S. aureus infection remains unknown. In the present study, we found that spleen tyrosine kinase (Syk) and c-Jun N-terminal kinase (JNK) were rapidly phosphorylated during S. aureus infection. Moreover, a Syk/JNK inhibitor and Syk/JNK siRNA not only reduced NLRP3 inflammasome-associated molecule expression at the protein and mRNA levels, apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC) speck formation, and interleukin-1ß (IL-1ß), and IL-18 release but also rescued the decreased NIMA-related kinase 7 (NEK7) expression level following suppression of the NEK7-NLRP3 interaction in macrophages. Interestingly, Syk/JNK phosphorylation levels and NLRP3 inflammasome-associated molecule expression were decreased by blockade of K+ efflux. Furthermore, activation of the NLRP3 inflammasome and a lower NEK7 protein level were found in vivo upon S. aureus infection. Taken together, our data indicated that S. aureus infection induces a K+ efflux/Syk/JNK/NEK7-NLRP3 signaling pathway and the subsequent activation of the NLRP3 inflammasome for the release of proinflammatory cytokines. This study expands our understanding of the basic molecular mechanism regulating inflammation and provides potential value for anti-infective drug development against S. aureus infection.

Mst1/2-ALK promotes NLRP3 inflammasome activation and cell apoptosis during Listeria monocytogenes infection.

Listeria monocytogenes (L. monocytogenes) is a Gram-positive intracellular foodborne pathogen that causes severe diseases, such as meningitis and sepsis. The NLR family pyrin domain-containing 3 (NLRP3) inflammasome has been reported to participate in host defense against pathogen infection. However, the exact molecular mechanisms underlying NLRP3 inflammasome activation remain to be fully elucidated. In the present study, the roles of mammalian Ste20-like kinases 1/2 (Mst1/2) and Anaplastic Lymphoma Kinase (ALK) in the activation of the NLRP3 inflammasome induced by L. monocytogenes infection were investigated. The expression levels of Mst1/2, phospho (p)-ALK, p-JNK, Nek7, and NLRP3 downstream molecules including activated cas-pase-1 (p20) and mature interleukin (IL)-1ß (p17), were up-regulated in L. monocytogenes-infected macrophages. The ALK inhibitor significantly decreased the expression of p-JNK, Nek7, and NLRP3 downstream molecules in macrophages infected with L. monocytogenes. Furthermore, the Mst1/2 inhibitor markedly inhibited the L. monocytogenes-induced activation of ALK, subsequently downregulating the expression of p-JNK, Nek7, and NLRP3 downstream molecules. Therefore, our study demonstrated that Mst1/2-ALK mediated the activation of the NLRP3 inflammasome by promoting the interaction between Nek7 and NLRP3 via JNK during L. monocytogenes infection, which subsequently increased the maturation and release of proinflammatory cytokine to resist pathogen infection. Moreover, Listeriolysin O played a key role in the process. In addition, we also found that the L. monocytogenes-induced apoptosis of J774A.1 cells was reduced by the Mst1/2 or ALK inhibitor. The present study reported, for the first time, that the Mst1/2-ALK-JNK-NLRP3 signaling pathway plays a vital proinflammatory role during L. monocytogenes infection.