The novel role of endosomal protein APPL1 in restricting NLRP3 inflammasome activation via mitophagy

Abstract

Nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain containing 3 (NLRP3) forms a multiprotein platform known as NLRP3 inflammasome for the maturation of potent pro-inflammatory cytokines IL-1β and IL-18 to promote inflammatory responses. Uncontrolled activation of NLRP3 inflammasome contributes to the pathogenesis of various diseases, such as gouty arthritis, neurodegenerative diseases and insulin resistance. However, due to the complex nature of the unique two-step activation model of NLRP3 inflammasome, the underlying regulatory mechanisms remain obscure. Mitophagy is a form of autophagy specialized in the degradation of mitochondria. It also negatively regulates NLRP3 inflammasome by the elimination of mitochondrial danger signals that are recognized as significant activators of NLRP3 inflammasome. Recent studies highlight the importance of early endosomes in mitophagy, but the interconnectivity of endosomes network and NLRP3 inflammasome regulation has not been investigated before. In this project, the effects of early endosome disruption on NLRP3 inflammasome regulation in macrophages were examined using in vitro and in vivo models with deficiency of the early endosome protein adaptor protein, phosphotyrosine interaction, pleckstrin homology domain and leucine zipper containing 1 (APPL1). Key findings: 1. Ablation of APPL1 in primary mouse macrophages led to excessive NLRP3 inflammasome activation and the secretion of its endproducts IL-1β and IL-18 specifically, in combination with the accumulation of damaged mitochondria and loss of mitochondrial membrane potential (MMP). 2. Increased mitochondrial damage in APPL1 deficient macrophages was coupled with the upregulation of mitochondrial reactive oxygen species and DNA release, which served as activation signals of NLRP3 inflammasome. Pharmacological inhibition of these danger signals reversed NLRP3 inflammasome overactivation in APPL1-null macrophages. 3. In response to NLRP3 agonist, APPL1 and the small GTPase Rab5 were recruited to damaged mitochondria for their delivery to mitophagosomes and lysosomes for degradation, but these translocations were blocked in APPL1 knockout macrophages. 4. APPL1-mediated mitophagy and NLRP3 inflammasome regulation required its endosomal localization and interaction with Rab5. Adenovirus-mediated expression of full-length wild-type APPL1 and constitutively active Rab5 were sufficient to rescue defective mitophagy and NLRP3 inflammasome overactivation caused by abrogation of APPL1. However, mutant forms of APPL1 that lack endosomal localization or Rab5 binding did not. 5. Hematopoietic-cell specific APPL1 knockout (BMT-KO) promoted insulin resistance in diet-induced obese mice and caspase-1 expression in adipose tissue macrophages, along with increased IL-1β in circulation, visceral adipose tissue and liver. BMT-KO also aggravated endotoxin-induced septic shock, which was associated with elevation of NLRP3 inflammasome products in circulation and peritoneal cavity. Conclusions: Collectively, the novel link and crosstalk between the endosome network and NLRP3 inflammasome are established in this project. The current findings indicate that APPL1 modulates an early endosome-mediated mitophagy pathway for the regulation of NLRP3 inflammasome in macrophages. Deletion of APPL1 impairs this pathway and, in consequence, exaggerates IL-1β and IL-18 production by NLRP3 inflammasome, which promotes obesity-induced insulin resistance and septic shock in vivo.