Mycoplasma Gallisepticum Mediates NF-κB/NLRP3 Activation And Induces Immunosuppression In Chickens By Regulating Autophagy

Mycoplasma gallisepticum（MG）also known as Mycoplasma septicum.MG infection caused chronic respiratory disease in chickens with decreased immunity and compromised immune systems,resulting in secondary infection that worsened symptoms and increased mortality,causing significant financial losses for the poultry industry.Autophagy is one of the most important cellular self-protection mechanisms that ensured cell survival by eliminating invading pathogens and death-inducing signals,which plays an important role in the regulation of the host immune response.However the role of autophagy in the immunosuppression of MG-infection is unclear.In this study,we established an in vitro model of MG-infected chicken-like macrophages（HD11）and examined the changes in the expression levels of intracellular autophagy and ROS/NF-кB/NLRP3 pathway-related factors at different time points.The present study investigated the effect of MG infection on autophagy,analyze the relationship between autophagy and immunosuppression.Moreover,the mechanism of immunosuppression in chickens caused by MG-infection through both in vitro and in vivo models and provided a theoretical basis for the prevention and treatment of MG-infection.In this study,HD11 cells were selected for in vitro experiments and infected with MG R_lowstrain solution（1×10⁹ CCU/m L）by different MOI and the optimal dose of MG infection was determined to be 400 MOI by CCK-8 assay,and 1,4,8 and 12 hours were selected as the time points for evaluating the MG infection model of HD11 cells.ELISA results showed that MG infection led to a decrease in antioxidant enzyme activity and anti-inflammatory factors,an increase in oxidative stress products and inflammatory factors in HD11 cell supernatants.JC-1 and DAFH-DA fluorescent probes treated cells showed that the intracellular mitochondrial membrane potential（ΔΨm）was significantly reduced（p<0.05）and the ROS content was significantly increased（p<0.05）in the MG-infection group compared to the control group.The ultrastructural observations showed that MG-infection caused changes in the ultrastructure of HD11 cells,resulting in pathological damage such as broken mitochondrial cristae,mitochondrial vacuolation,cytoplasmic vacuolation and loss of nuclear membrane.The above results indicated that MG-infection leads to an increase in damaged mitochondria in HD11 cells,excessive accumulation of ROS,oxidative stress and results in inflammatory damage.RT-q PCR and Western blot assays showed that the expression levels of autophagy-related factors increased at 1,4 and 8 hours in the MG-infection group compared to the control group.While the expression levels of m TOR and p62 decreased,and the expression levels of autophagy-related factors decreased in cells at 12 hours,while the expression levels of m TOR and p62 increased.The expression levels of TLR2-NF-κB and NLRP3-related factors in HD11 cells of MG-infection group increased at 4 hours and the highest expression level was observed at 12hours.The results indicated that the level of autophagy in HD11 cells increased at 1,4 and 8 hours of infection and decreased at 12 hours,and MG-infection activated the NF-κB and NLRP3 signaling pathways.In this study,the in vivo model of MG-infection was constructed using White Leghorns chickens based on pre-laboratory results and the thymus was collected at 1,3 and 7 day after infection.ELISA results showed that the antioxidant enzyme activity was reduced and oxidative stress increased in the thymus of chickens in the MG-infection group at the all three time points compared to the control group.Histopathological and ultrastructural observations showed that MG-infection caused inflammatory cell infiltration,vasodilatation,congestion and increased cell gaps in the chicken thymus.Mitochondrial cristae were broken and vacuolated,perinuclear gaps were increased,chromatin was marginalized and nucleoli were ruptured in the thymus lymphocytes,indicating that MG-infection damaged the histological structure of the chicken thymus and caused pathological damage.RT-q PCR assay showed that compared to the control group,the m RNA expression levels of autophagy-related factors decreased,while the m RNA expression levels of m TOR increased,and the m RNA expression levels of TLR2-NF-κB and NLRP3-related factors increased in the MG-infection group at all the three time points.Western blot results showed that compared to the control group,the protein expression levels of autophagy-related factors decreased,while the protein expression levels of m TOR increased significantly（p<0.05）,and the expression levels of TLR2-NF-κB and NLRP3-related factors increased in the MG-infection group.These results indicated that MG-infection leads to a decrease in autophagy levels and activation of NF-κB and NLRP3 signaling pathways in the thymus.In summary,MG-infection caused significant pathological damage to the chicken thymus,with massive inflammatory cell infiltration and capillary dilation,and induced changes in the ultrastructure of HD11 cells,a decrease in mitochondrial membrane potential,an increase in the number of damaged mitochondria and contents of ROS,indicating that MG-infection could damage chicken immune organs and immune cells.In the early stages of MG infection（1,4 and 8 hours）,HD11 cells recognized pathogen invasion and activated cellular autophagy,and MG-infection inhibited HD11 cell autophagy as the duration of infection increased.Similarly,in vivo study revealed that the autophagy level in thymus tissue decreased at 1,3 and 7 days of MG-infection,and the NF-кB/NLRP3 signaling pathway was abnormally activated in HD11 cells and thymus tissue,indicating that MG-infection mediates inflammatory damage to immune cells and immune organs by increasing the number of damaged mitochondria through the inhibition of autophagic lysosome formation.This study provides theoretical basis and research evidence for immunosuppression caused by MG-infection and provides new ideas for the treatment and drug development against MG-infection.