Spermine alleviates EV71-induced inflammation by inhibiting GBP5 and NLRP3 activation in macrophages Free

Backgrounds Hand-foot-and-mouth disease (HFMD) is a common childhood infectious disease primarily caused by enteroviruses, with enterovirus 71 (EV71) being particularly associated with severe cases. Although most HFMD cases follow a self-limiting course, EV71 infection can lead to severe neurological complications and even death, posing a substantial burden on public health systems. In recent years, the widespread use of EV71 vaccines has helped curb the disease's prevalence; however, significant gaps remain in specific antiviral treatments and immunomodulatory strategies for severe HFMD. Current research indicates that EV71 infection triggers abnormal macrophage activation, driving excessive inflammatory responses—a key mechanism underlying disease progression to severe forms. Notably, spermine, an endogenous polyamine, has been shown to regulate viral-induced inflammation in various contexts, yet its precise role in severe HFMD remains unclear. Therefore, investigating the molecular mechanisms of EV71-induced macrophage-associated cytokine storms through the lens of arginine metabolism could not only elucidate the pathophysiological basis of severe HFMD but also provide novel therapeutic targets for safer and more effective clinical interventions.

Methods and materials THP-1 cells were cultured in RPMI-1640 with 10% FBS, while RAW264.7 cells were maintained in DMEM/10% FBS. Experimental groups included: (1) untreated controls; (2) EV71-infected cells (10³ TCID₅₀/mL, 24h); (3) EV71-infected cells treated with 10 μmol/L spermine; and (4) cells treated with 3 mmol/L DFMO (eflornithine hydrochloride). Post-infection treatments were administered following PBS washing after initial 24h viral exposure.qRT-PCR measured GBP5, NLRP3 and inflammatory cytokine mRNA in EV71-infected/spermine-treated macrophages. siRNA-mediated GBP5 knockdown evaluated NLRP3 inflammasome activation and cytokine secretion. Polyamine biosynthesis inhibitors assessed spermidine metabolism's role in GBP5-NLRP3 pathway regulation. Data were analyzed using SPSS 25.0.

Results The results showed that GBP5 knockdown suppressed NLRP3 inflammasome assembly , resulting in decreased secretion of CXCL10 and TNFSF10 in macrophages (P<0.0001). EV71 infection significantly upregulated the expression of GBP5, NLRP3, and inflammatory cytokines (CXCL10 and TNFSF10) in macrophages (P<0.0001). Spermine treatment effectively reduced their expression (P<0.0001). GBP5 knockdown in macrophages led to decreased expression of NLRP3, CXCL10, and TNFSF10 (P<0.0001). Conversely, inhibition of spermine biosynthesis elevated the expression of GBP5, NLRP3, CXCL10, and TNFSF10 (P<0.0001).

Conclusions This study demonstrates that inhibition of the M1 macrophage marker GBP5 reduces NLRP3 and pro-inflammatory cytokines (CXCL10 and TNFSF10) expression in macrophages. EV71 infection upregulates GBP5 and NLRP3 in macrophages, leading to excessive CXCL10/TNFSF10 secretion and hyperinflammation. Notably, spermine effectively suppresses EV71-induced GBP5/NLRP3 overexpression and subsequent cytokine release, exhibiting potent anti-inflammatory effects. Our research results indicate that the inhibition of polyamine synthesis can promote the activation of GBP5/NLRP3 inflammasomes and the production of inflammatory mediators in macrophages, confirming spermine's regulatory role. These findings elucidate the molecular mechanism underlying spermine's therapeutic potential against severe HFMD, providing a theoretical foundation for its clinical application in mitigating EV71-driven inflammatory storms. (Acknowledgements:This study was supported by Grant from School of Public Health of Southern Medical University, China , Grant No.GW202329; Corresponding author: Hong Cao, gzhcao@smu.edu.cn).