NLRP3 exacerbates EAE severity through ROS-dependent NET formation in the mouse brain

Abstract

BackgroundNeutrophil extracellular trap (NET) has been implicated in the pathology of multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE). However, the specific contributions of NLRP3, a NET-associated molecule, to EAE pathogenesis and its regulatory role in NET formation remain unknown.MethodsTo investigate the detrimental effect of NETs supported by NLRP3 in MS pathogenesis, we induced EAE in WT and NLRP3 KO mice and monitored the disease severity. At the peak of the disease, NET formation was assessed by flow cytometry, immunoblotting, and immunofluorescence staining. To further identify the propensity of infiltrated neutrophils, NET-related chemokine receptors, degranulation, ROS production, and PAD4 expression levels were evaluated by flow cytometry. In some experiments, mice were injected with DNase-1 to eliminate the formed NETs.ResultsOur data revealed that neutrophils significantly infiltrate the brain and spinal cord and form NETs during EAE pathogenesis. NLRP3 significantly elevates NET formation, primarily in the brain. NLRP3 also modulated the phenotypes of brain-infiltrated and circulating neutrophils, augmenting CXCR2 and CXCR4 expression, thereby potentially enhancing NET formation. NLRP3 facilitates NET formation in a ROS-dependent and PAD4-independent manner in brain-infiltrated neutrophils. Finally, NLRP3-supported NET formation exacerbates disease severity, triggering Th1 and Th17 cells recruitment.ConclusionsCollectively, our findings suggest that NLRP3-supported NETs may be an etiological factor in EAE pathogenesis, primarily in the brain. This study provides evidence that targeting NLRP3 could be a potential therapeutic strategy for MS, specifically by attenuating NET formation.