Study On The Activation Of NLRP3 Inflammasome By Pathogenic Fowl Adenovirus 4 Infection

Since 2015,chicken hepatitis and hydropericardium syndrome(HHS)caused by the new genotype of fowl adenovirus serotype 4(FAdV-4)has been spread in chicken farms of China.At present,the pathogenicity of FAdV-4 remains unknown.Existing studies indicated that the expression of interleukin 1β(IL-1β)in livers of chicken infected with pathogenic FAdV-4 is significantly increased and accompanied by a strong inflammatory reaction.The expression of IL-1β and excessively inflammation are closely related to the activation of NLRP3 inflammasome.Therefore,investigation of the effect of FAdV-4 infection on the activation of NLRP3 inflammasome is significant for the study of the pathogenicity of FAdV-4.The present paper established a SYBR Green I real-time fluorescent quantitative PCR assay for detecting chicken NLRP3 and Caspase-1 genes,and evaluated the activation of NLRP3 inflammasomes in chicken tissues and chicken macrophages infected with pathogenic FAdV-4.The thesis includes the following aspects:1.Establishment and application of SYBR Green I fluorescence quantitative PCR detection method for chicken NLRP3 and Caspase-1 genesAlthough NLRP3 inflammasome plays an important role in natural immunity and disease development,there is no report on the real-time fluorescent quantitative PCR detection method of chicken NLRP3 and Caspase-1 at home and abroad.In this study,a SYBR Green I fluorescent quantitative PCR detection method for chicken NLRP3 and Caspase-1 was successfully established by optimizing the reaction conditions and specificity,sensitivity,and repeatability tests.The test results shown that the established method has good specificity with a detection limit of 1.0×102 copies/μL,which is 100 times more sensitive than the conventional PCR;the intra-assay and inter-assay coefficients of variation are both less than 5%which indicated a with good repeatability.In this study,the established chicken NLRP3 and Caspase-1 fluorescent quantitative PCR method was used to detect the transcription levels of NLRP3 and Caspase-1 genes in different tissues of chickens infected with the pathogenic FAdV-4 strain and healthy chickens.The results shown that compared with the control group,the transcription level of NLRP3 and Caspase-1 gene in the heart,liver,and spleen of the infected chickens were extremely significantly higher than those in the control group,and the transcription levels in the cecal tonsils and bursa of Fabricius were significantly higher than the control group,suggesting that these organs are the main target organs of pathogenic FAdV-4.The chicken NLRP3 and Caspase-1 SYBR Green Ⅰ fluorescence quantitative PCR detection method established in this research can be used to detect the transcription levels of chicken NLRP3 and Caspase-1 in different tissues.It will provide a specific and sensitive quantification method to study the role of NLRP3 inflammasome in regulating natural immune response after virus infection.2.Detection of NLRP3 inflammasome activation in chicken tissues infected by pathogenic FAdV-4In this part,an animal test was designed to verify whether the pathogenic FAdV-4 infection can cause the activation of NLRP3 inflammasomes in chicken tissues.Blood serum was collected at different time points post-infection and samples of heart,liver,lung,spleen,kidney,proventriculus,bursa of Fabricius,and cecal tonsils were collected.Fluorescent quantitative PCR,immunohistochemistry and ELISA were used to detect NLRP3 inflammasome activation in the collected serum and tissue samples.Comparing to the control group,a higher level of IL-1β secretion was detected in serum and pericardial effusion;immunohistochemistry results illustrated that chicken NLRP3,cleaved-Caspase-1 and mature-IL-1β protein have tissue-specific distribution.The NLRP3,Cleaved-Caspase-1 and mature-IL-1β mainly distributed in the cytoplasm.A medium positive staining was observed in the heart and liver,and a strong positive staining was observed in the spleen,lung and cecal tonsil.This result also corresponded to the histopathological changes in those organs after FAdV-4 infection;the above results indicated that FAdV-4 maninly infected chicken heart,liver,spleen,lung and cecal tonsil with a up-regulated expression of NLRP3,CleavedCaspase-1 and mature-IL-1β.FAdV-4 infection activates the NLRP3 inflammasome to regulate the expression of the pro-inflammatory cytokine IL-1β in chickens,and this effect is more significant in the immune organs.3.Detection of NLRP3 inflammasome activation induced by pathogenic FAdV-4 in HD11 cellsThe release of pro-inflammatory cytokine in macrophages is a key regulator of host immune response and plays an important role in many inflammatory diseases.In order to explore whether the activation of NLRP3 inflammasome is induced by FAdV-4 infection in macrophages,chicken bone marrow-derived macrophages HD11 was stimulated with ATP for chicken NLRP3 and Caspase-1 gene siRNA test,chicken NLRP3 and Caspase-1 gene overexpression test and Caspase-1 activity inhibition test.The results shown that ATP participated in the FAdV-4 induced NLRP3 inflammasome activation in vitro;compared with the pathogenic FAdV-4 stimulation group,IL-1β secretion in the si-NC group was extremely significantly decreased,and the expression of NLRP3 protein and IL-1β secretion level decreased in the si-NLRP3 group.Similarly,the expression of Caspase-1 and IL-1β decreased in the si-Caspase-1 group;Compared with si-NLRP3 and si-Caspase-1 groups,it was found that overexpression of NLRP3 and caspase-1 led to the increased expression level of IL-1β,however,compared with the group stimulated by pathogenic FAdV-4,the secretion level of IL-1β was not significantly increased;Caspase-1 activity inhibitor Ac-YVAD-CHO treatment of HD11 cells resulted in the decrease of IL-1β secretion level.The results shown that FAdV4 can induce the activation of NLRP3 inflammasome in HD11 cells and regulate the secretion of IL-1β.