Mechanism Of NLRP3 Inflammasome Mediated IL-1? Secretion And Pyroptosis Induced By Classical Swine Fever Virus

Classical swine fever(CSF),an acute or chronic,febrile and highly contagious infectious disease of swine caused by classical swine fever virus(CSFV),and is a worldwide distribution disease which has a high degree of harm and can be resulted in huge economic losses of pig industry.The feature of CSF is hemorrhagic fever and immunosuppression.The basic pathological damage induced by CSF including vascular endothelial injury and a significant reduction in lymphocytes.The vascular endothelium damage can lead to vascular permeability increasing,and then cause a series of syndromes;the significant reduction of lymphocytes can cause immune suppression.All of these phenomena suggest that the occurrence and development of CSF are closely related with a series of pathology,physiological processes of the inflammatory response and cell death.Inflammasomes,as a polyprotein complex,was found in recent years which can lead to activation of caspase-1,secretion of cytokine IL-1?/IL-18,and then induced inflammatory response.Pyroptosis is a new form of programmed cell death that relied on the activation of caspase-1 and accompanied by the release of a large number of proinflammatory cytokines.The current studies show that inflammasomes and pyroptosis are widely involved in the development of infectious diseases,and play a critical role during these processes.However,there is no relevant studies have been reported yet to show whether there is an association between inflammasomes,pyroptosis and CSFV infection.Therefore,this study mainly focus on exploring the interrelationship and induction mechanism between CSFV infection,inflammasomes and pyroptosis to further understand the pathogenesis of CSFV from a new angle,and provide the necessary scientific basis for prevention and control of CSF.The study consists of four parts.Part I:Research of caspase-1 activation and IL-1?secretion in pig peripheral immune organs with CSFV infected.To investigate the association between CSFV infection and inflammatory response,healthy piglets which CSFV antibodies and antigens were negative,were challenged with CSFV.The results suggested that CSFV infection can cause pigs showed fever(?40.5?),tissue bleeding,swelling and necrosis,lymphocyte hyperplasia and infiltration,lymphoid nodule and white pulp lymphocytes reducing.The results of CSFV tissue tropism study showed that CSFV had higher tropism with peripheral immune organs(spleen,lymph nodes and tonsils).Further study found that CSFV infection can promote the increase in the mRNA expression level of IL-1?and caspase-1 in heart,liver,spleen,lung,kidney,lymph node,tonsil and bladder,in which the expression in peripheral immune organs and lung increased more significantly.Based on the above results,in order to explore relationship between CSFV infection and caspase-1 activation and IL-1?secretion,the expression and mature of IL-1?in peripheral immune organs of pig were detected.The results showed that CSFV infection can promote the expression and mature of IL-1?in pig peripheral immune organs.The protein expression level,cleavage and enzymatic activity of caspase-1 in peripheral immune organs of pigs were also detected.The results showed that CSFV infection can promote the enzymatic activity,expression and cleavage of caspase-1in peripheral immune organs.The research also found that CSFV infection inhibitd the mRNA expression of NLRP3 and ASC in heart,liver,spleen,lung,kidney,lymph nodes,tonsils and bladder from infected pigs.Part II:Mechanism of NLRP3 inflammasome mediated IL-1?secretion in peripheral blood monocyte with CSFV infected.Based on the results of the first part of this study,in order to further understand the relationship between CSFV infection and caspase-1activation and IL-1?secretion,we studied the mechanism of IL-1?secretion induced by CSFV infection.First of all,peripheral monocyte of pig were selected and infected with CSFV to establish the CSFV infection induced IL-1?secretion cell model.The results showed that CSFV infection can promote the secretion and mature of IL-1?,cleavage and activation of caspase-1 in peripheral monocyte.Further study found that,the mature and secretion of IL-1?in peripheral monocyte induced by CSFV infection was depended on the activation of caspase-1.The related studies showed that the activation of caspase-1 was related to the activation of inflammasome.Thus,the lentivirus,which can target knockdown the NLRP3 gene,was used to infect the peripheral monocyte,and then infected with CSFV.The secretion level of IL-1?in supernatant was detected.The results suggested that,the secretion of IL-1?were significantly suppression when NLRP3 was knockdown.At the same time,Co-IP was used to detect the formation of NLRP3inflammasome in CSFV infected peripheral monocyte.The results showed that CSFV infection can induce the formation of NLRP3 inflammasome.For the further study,the Glibenclamide(ATP-dependent K~+ion channels inhibitor),was used to treat peripheral monocyte,and then the cells were infected with CSFV.The secretion level of IL-1?in supernatant was detected.The results showed that the formation of NLRP3 inflammasom in peripheral monocyte which induced by CSFV infection was depended on the activation of K~+ion channels.The study also found that,the CSFV infection can inhibit the expression of NLRP3 and ASC in porcine peripheral monocyte.Part III:The NLRP3 inflammasome effects on the replication of CSFV in porcine peripheral blood monocyte.The results of above two parts studies suggested that,the infection of CSFV induced the activation of NLRP3 inflammasome.In order to further understand the relationship between NLRP3 inflammasome and CSFV infection,we investigated the effect of NLRP3 inflammasome on CSFV replication.The lentivirus mediated NLRP3 gene knockdown and NLRP3 inhibitor treated porcine peripheral monocyte,and then infected with CSFV,the qRT-PCR and IFA were used to detect the CSFV copy numbers and virus titer.The results showed that both knockdown NLRP3 and inhibition of NLRP3 activation could promote CSFV replication in peripheral monocyte.Part IV:Research about pyroptosis induced by caspase-1 activation in CSFV-infected porcine peripheral lymphoid organs and porcine peripheral monocyte.Previous studies have shown that CSFV infection induces activation of caspase-1 and inflammation.Pyroptosis is a new programmed cell death dependent on caspase-1,and associated with inflammation.Thus,we investigated the relationship between CSFV infection and pyroptosis.First of all,we performed TUNEL staining of paraffin embedded sections of peripheral immune organs of pigs from CSFV challenge experiments.The results showed that CSFV infection can increase the proportion of TUNEL positive cells in peripheral lymphoid organs of pigs.Further studies showed that CSFV infection promotes the cleavage of GSDMD in peripheral immune organs of pigs.In order to further analyze the relationship between CSFV infection and pyroptisis,calcein AM/EthD-III staining were used to detect the cell membrane damage in peripheral blood monocytes with CSFV infected.The results showed that CSFV infection increased the proportion of cell membrane damage in peripheral blood monocytes.Further studies have shown that cell membrane damage of peripheral blood monocytes induced by CSFV depends on the activation of caspase-1.We measured the GSDMD cleavage in the peripheral blood monocytes of CSFV infected pigs.The results showed that CSFV infection could promote the cleavage of GSDMD in the peripheral blood monocytes.In conclusion,CSFV infected porcine peripheral blood monocytes cause the activation of the ATP-dependent K+ion channel and active NLRP3 inflammasome assembly,which leads to the activation of caspase-1,the caspase-1 induced mature and secretion of IL-1?,resulting in inflammatory response.Inhibiting the activation of NLRP3 inflammasome can promote the replication of CSFV.At the same time,CSFV infection can induce pyroptosis.These studies suggested that NLRP3 is an immune defense mechanism against CSFV invasion,and pyroptosis was involved in the process of CSFV infection.This study provides a new perspective to understand the immune mechanism induced by CSFV infection,and provides a scientific basis for the new strategies to prevent and control CSF.