Macrophage, a vital part of the immune system, plays a crucial role in the innate immune response . LPS is an essential molecule of the extracellular membrane of gram-negative bacteria, which besides maintaining the structural integrity of the cells, elicits the pathogen-induced inflammation . The LPS/ATP-stimulated J774A.1 macrophage cells are widely accepted in-vitro model of-inflammatory cell NLRP3 inflammasome-employed for the investigation of NLRP3 inflammasome activation mechanism . Hence, in the current study, we used the LPS/ATP-stimulated J774A.1 macrophage cells as an inflammatory cell NLRP3 inflammasome activation model. Accumulating pieces of evidence states that the aberrant activation or dysregulation of NLRP3 inflammasome manifests the most prevalent inflammatory conditions . An in-depth investigation of the NLRP3 inflammasome activation signaling pathway can enable the development of more efficient drug targets for effective management of the resulting inflammatory disorders. Our study reveals the involvement of ASM in LPS/ATP-induced ceramide production in J774A.1 cells, which in turn elicits the NLRP3 inflammasome activation by mediating the TXNIP overexpression. We herein reported for the first time that the ASM/Cer/TXNIP signaling module braces the NLRP3 inflammasome activation.
Sphingolipid metabolism constitutes a significant part of lipid metabolism, which generates an array of active cellular lipids, imparts structural integrity to the cell and regulates a large number of crucial cellular functions [19, 20]. A plethora of stress stimuli generate excessive ceramide through sphingolipid metabolism. As a secondary signaling molecule, it activates the signal transduction required for the occurrence of biological processes such as inflammation, apoptosis, and cellular differentiation [21, 22]. Accumulating evidence has validated that the ceramide accumulation triggers the assembly and activation of NLRP3 inflammasome in various pathological conditions [7, 8]. Two major routes of ceramide synthesis, i.e., de novo synthesis of ceramide via ceramide synthase serine palmitoyltransferase (SPT) and sphingomyelinases-neutral sphingomyelinase (NSM)/ASM-mediated hydrolysis of sphingomyelin membrane have been reported for the accumulation of intracellular ceramide [21, 23]. However, recent studies suggest that the elevated level of ceramide in stress response is an outcome of ASM-mediated sphingomyelin hydrolysis [9, 10]. Our previous findings have demonstrated the participation of imipramine in the amelioration of LPS-induced pulmonary inflammation in mice mediated by suppression of ceramide levels . Imipramine is a well known ASM inhibitor, which interferes with the ASM and lysosomal membrane interaction and elicits lysosomal destruction of ASM .
In this study, we found that ASM activity and the content of Cer significantly increased after LPS/ATP treatment, whilst the ASM inhibitor imipramine significantly attenuated the increase in ASM activity and Cer content in response to LPS/ATP. This outcome indicates a positive correlation between the LPS/ATP-induced Cer accumulation and ASM activation in J774A.1 cells. Additionally, imipramine suppressed LPS/ATP-induced TXNIP/NLRP3 inflammasome activation. However, its role in ASM/Cer mediated TXNIP/NLRP3 inflammasome activation remains unexplored. This prompted us to investigate if the ASM/Cer signaling pathway involves TXNIP/NLRP3 inflammasome activation as a downstream component.
TXNIP contributes significantly to biological processe such as the regulation of inflammation, oxidative stress, cell apoptosis, glucose and lipid metabolism . An escalated level of TXNIP negatively impacts these biological processes, which majorly contribute to the onset of inflammatory disorders [26, 27]. As per the previous findings, TXNIP serves the crucial task of NLRP3 assembly by directly interacting with NLRP3 [15, 27]. Txnip inhibitor verapamil has been widely employed by the researchers to suppress TXNIP expression and the associated NLRP3 inflammasome activation with an aim to manage the inflammatory conditions . In this study, verapamil suppressed LPS/ATP-induced TXNIP/NLRP3 inflammasome activation. However, it did not affect LPS/ATP-induced ASM activity and ceramide production. We further stimulated J774A.1 cells with C2-Ceramide to elucidate that the TXNIP/NLRP3 inflammasome activation is a downstream event of the ASM/Cer signaling pathway. The outcome of this study indicated that C2-Ceramide could remarkably enhance the TXNIP, NLRP3, and Caspase-1 protein expression and IL-1β, IL-18 secretion. Furthermore, verapamil inhibited C2-Ceramide mediated TXNIP overexpression and NLRP3 inflammasome activation. Therefore, our findings suggest that ceramide leads to TXNIP overexpression and subsequent NLRP3 inflammasome activation.