Analysis Of The Role Of NLRP3 Inflammasome Complex Assembly In Inflammation Caused By CVA16 Infection

Coxsackievirus A16(CVA16)is a member of the Enterovirus genus of Picornaviridae,and its virus particles are non-enveloped symmetric spherical particles.It is one of the main pathogens of hand,foot and mouth disease.The main susceptible population of CVA16 are infants aged 0-5 years.The main clinical symptoms include the herpes in the hand,foot and mouth,fever,acute angina,etc.Systemic inflammatory response mainly in heart,lung and nervous system appeared in severe cases.Neurogenic pulmonary edema and pulmonary hemorrhage are the main causes of death in severe cases.Compared with the EVA71 virus,CVA16 infection has repetitive characteristics,and there is no vaccine that can prevent its infection.The inflammasome is a group of cytoplasmic protein complexes that act as a platform for the activation of caspase-1,which promotes the maturation and secretion of the inflammatory factors IL-1β and IL-18[1,2].The NLRP3 inflammasome is the most studied inflammasome,which are composed of the receptor protein NLRP3(NACHT,LRR and PYD domains-containing protein 3),ASC(Apoptosis-associated speck-like protein containing a CARD)and caspase-1.In the case of activation,the pyrin domain(PYD)in NLRP3 and ASC bonded to each other,the caspase recruitment domain(CARD)of ASC binds to the CARD domain on procaspase-1.Three components forming NLRP3 inflammasome[1,3,4].The inflammasome complex triggers procaspase-1 self-cleavage,producing an active caspase-1 pl0/p20 tetramer and inducing the transformation of the pro-inflammatory cytokines interleukin IL-1β and IL-18 from immature precursor to secreted form.Recent studies have shown that inflammasome play a role in identifying viral infections,initiating innate immunity and inflammatory responses[5].There are many types of viruses that can activate NLRP3 inflammasome,including many RNA viruses such as influenza viruses[6],hepatitis C virus[7],enterovirus A 71[8]and so on.This study was to investigate the activation of NLRP3 inflammasome by CVA16 virus infection and the possible mechanisms,as well as the role of activated NLRP3 inflammasome in infected rhesus monkeys’tissues.The main research contents include two aspects:First,in vitro experiments,we detected that CVA16 infection can activate NLRP3 inflammasome in the three cell lines of Vero,KMB17 and SHSY5Y.The assembly of NLRP3 inflammasome can be visually observed by confocal microscopy in all three cells.The assembled NLRP3 inflammasome activates Caspase-1 from the zymogen to the enzyme,thereby pro-IL1β is cleaved into EL-1β,exerting an inflammatory effect.However,due to differences between cell lines,the mechanisms of activation in the three cells may be different;In vivo,we first used the CVA16 virus to infect 1 day old BALB/c suckling mice.The performance of NLRP3 inflammasome activation was observed in mice lung tissue.Since rhesus monkeys are more suitable to be animal models of CVA16 than suckling mice,we further infected rhesus monkeys with the CVA16-EGFP virus.After infection,rhesus monkeys showed heavier inflammatory symptoms in the lungs,but no obvious central nervous system symptoms.In the peripheral blood lymphocytes of infected rhesus monkeys,the NLRP3 inflammasome was mobilized and the active inflammatory factor IL-1β was expressed.In the lungs with severe inflammatory lesions,the NLRP3 inflammasome activation was significant,while no activation was observed in the brain.Based on our results,CVA16 in vitro infected cells can activate NLRP3 inflammasome,but the specific activation mechanism is still unclear.In the animal model of CVA16 infection,the NLRP3 inflammasome is also activated,and its activation is closely related to the inflammatory symptoms caused by the infection.