Inhibition Of Pyroptosis And Phagocytosis Of Macrophages Induced By Acinetobacter Baumannii By HCMV-IE86 Protein

Objective: Acinetobacter baumannii(A.baumannii)is one of the main pathogens causing community and hospital-acquired pneumonia,with a fatality rate of 35%.With the explosive prevalence of multidrug-resistant A.baumannii(MDR-AB)and pan-drug-resistant A.baumannii(XDR-AB),anti-infection treatment of A.baumannii has become the focus of clinical attention.After A.baumannii infects the host,macrophages through pattern recognition receptors(PRR)and regulatory receptors mediate macrophages to recognize pathogen-related molecular patterns(PAMP)on the surface of microorganisms.NLRP3 inflammasome,as a major component of natural immunity,plays an important role in regulating immune response and inflammatory response.Pyroptosis is an important immune response of the body to pathogens.NLRP3 inflammasome mediated pyroptosis amplifies inflammatory responses by activating Caspase-1,releasing interleukin 1β(IL-1β),inflammatory factors and adhesion factors.In the initial stage of host cell infection with pathogens,the activation of inflammasome promotes the occurrence of Pyroptosis,inhibits the replication of pathogens,and plays an important role in antagonizing pathogen infection and invasion.More and more studies have focused on the role of pyroptosis and activation of inflammasome in the process of A.baumannii infection.Human Cytomegalovirus(HCMV)is a viral disease with a high infection rate in the population,and nearly 100% of the general populations are carriers of the virus in developing countries.The IE86 protein encoded by ie2 gene expressed by HCMV infection is the earliest and most active cellular regulatory protein after HCMV infection,which plays an important role in the replication and proliferation of HCMV.Studies have found that HCMV infection of monocytes leads to inflammatory phenotype,which greatly increases the probability of host infection with pathogenic bacteria.In the case of immune deficiency or low immune function,including developmental combined immunodeficiency newborns,HCMV virus activation,leading to many clinical complications and opportunistic pathogen infection and even death.Among them,recurrent refractory infantile pneumonia is the highest complication of child mortality.In recent years,researchers have found that cell death plays a two-way regulatory role in the process of host anti-viral infection,and begin to pay attention to the role of cell death in the process of HCMV replication infection.This study will focus on the effects of HCMV-IE86 protein on the pyroptosis and phagocytosis of host cells induced by A.baumannii,and the effect of A.baumannii on the prognosis of pneumonia induced by A.baumannii,which provides a new idea for the development of targeted therapy and is of great significance to provide treatment direction for A.baumannii and HCMV co-infection.Methods: In this study,Phorbol Myristate acetate(PMA),IFN-γ and LPS-induced human mononuclear cell line U937 were used as the main u937-derived macrophage model.After A.baumannii and HCMV were infected,the phagocytosis capacity of macrophages and the expression level of IE86 protein were observed to determine the best infection time of A.baumannii and HCMV.Immunofluorescence and Westernblot were used to investigate the effects of HCMV infection on cell pyroptosis and macrophage function.To further verify the effects of HCMV-IE86 protein on cell pyroptosis and macrophage function,MND was used to inhibit HCMV-IE86 protein expression level,and immunofluorescence assay was used to detect the phagocytosis of U937-derived macrophages.Westernblot was used to detect the expression level of pyroptosis protein.Meanwhile,in order to confirm the effect of HCMV-IE86 protein on phagocytosis and pyroptosis of U937-derived macrophages,we carried out the overexpression of HCMV-IE86 protein expression in U937-derived macrophages through pc DNA3.1(+)-ie2 plasmid transfection experiment in order to verify the effect of HCMV-IE86 protein overexpression on the phagocytosis of U937-derived macrophages at the cellular level.In addition,we also constructed ie2 transgenic mice to explore the effect of HCMV-IE86 protein on the prognosis of pneumonia induced by A.baumannii by regulating cell death by HE staining,blood routine examination and Westernblot detection in lung tissue.Results:(1)U937 cells were successfully induced into M1-type macrophages.In the induction and differentiation process,24 h after PMA induction and stimulation,all U937 cells had adhered to the wall,most of the cells were still round or quasi-round,and the clustering growth was obvious.At this time,U937 cells had been induced into M0-type macrophages.After the induction of LPS and IFN-γ for 12 h,U937 cells were induced to differentiate into m1-type macrophages.The cell morphology was more irregular,and some cells showed a long spindle shapes and pseudopodia appeared on the cell surface.The results showed that U937 cells could be induced to differentiate into M1-type macrophages by PMA,LPS and IFN-γ.(2)A.baumannii infected U937 macrophages and induced pyroptosis and phagocytic bacteria.Heat inactivation of A.baumannii infected u937-derived pyroptosis macrophages suggested that A.baumannii infection induced pyroptosis of U937-derived macrophages,and the degree of pyroptosis increased with time.Meanwhile,the amount of A.baumannii phagocytosis of U937-derived macrophages increased with the increase of stimulation time.(3)HCMV infection can inhibit pyroptosis and phagocytosis of U937-derived macrophages.After 72 h of HCMV infection in U937 macrophages,the expression of coke death related proteins was significantly lower than that in HCMV/ A.baumannii group,suggesting that HCMV infection inhibited coke death in U937 cells.At the same time,HCMV can inhibit the phagocytosis of U937-derived macrophages to A.baumannii.After MND inhibited HCMV-IE86 protein expression,the expression of pyroptosis related protein and phagocytosis rate in A.baumannii /HCMV/MND group was slightly higher than that in A.baumannii /HCMV group,but still lower than that in A.baumannii group.These results suggest that HCMV-IE86 protein expression inhibition can significantly promote pyroptosis and phagocytosis of U937-derived macrophages.(4)In vitro and in vivo experiments confirmed that HCMV-IE86 protein overexpression could inhibit pyroptosis of U937-derived macrophages and phagocytosis of U937-derived macrophages to A.baumannii.In vitro experiments,HCMV-IE86 protein overexpressed by pc DNA3.1(+)-ie2 plasmid for 72 h could significantly inhibit the phagocytic function of U937-derived macrophages.while HCMV-IE86 protein overexpressed can significantly inhibit the pyroptosis of u937-derived macrophages.This is consistent with the result that HCMV infection inhibits pyroptosis of U937-derived macrophages.In addition,we successfully constructed ie2 transgenic mice that can stably express IE86 protein and infected with A.baumannii to construct the model of pulmonary infection.The results showed that there was no obvious abnormality in eating activity of mice in WT/PBS group.However,the condition of mice infected with A.baumannii was significantly worse than that of WT/PBS group.In WT/ A.baumannii group,lung tissues were swollen,interstitial capillaries were obviously congested and dark red,and the surface of lung tissues were uneven in color,with needle-like hemorrhagic spots and spots diffuse in both lung lobes.Compared with WT/ A.baumannii group,the injury of ie2/A.baumannii group was more serious,alveolar structure disappeared,alveolar wall was broken,alveolar wall thickened,edema,part of alveolar structure disappeared,and patchy necrosis and congestion appeared on the surface of lung tissue in ie2/A.baumannii group.The results of HE staining results were consistent with the pathological changes of lungs.Conclusion: In this study,Inhibition of pyroptosis of U937-derived macrophages by HCMV-IE86 protein at cellular level,and the phagocytosis of U937-derived macrophages against A.baumannii was also inhibited.At the same time,our laboratory creatively constructed an ie2 transgenic mouse model,which broke the barrier that HCMV has strict species specificity and could stably express HCMV-IE86 protein.Animal level experiments in vivo also show that HCMV-IE86 protein can aggravate the pathological damage of lung tissue of A.baumannii infected mice,which is not conducive to the prognosis of pneumonia.