The Role Of TNF-β In Pneumocystis-mediated Acute Lung Injury

Pneumocystis jirovecii pneumonia(PJP)often occurs in the immunocompromised patients.The incidence of PJP is about 5%-15%.Patients often present with fever,cough,tachypnea and other clinical symptoms.The death rate in severe cases can be 60%at the highest.A large amount of inflammatory factors released after pneumocystis infection can lead to inflammatory storm,resulting in acute lung injury.However,the details of inflammatory cytokine changes after PJP remains unclear,their roles in the disease mechanism are not clear,and the intervention measures are lacking.1,3-β-D-glucan(BDG)is the main component of pneumocystis cell wall.Alveolar macrophages can recognize pneumocystis through BDG receptor and play phagocytosis and clearance when activated.A large amount of BDG may cause excessive activation of macrophages,leading to a release of a large number of inflammatory cytokines and finally immune damage.However,how inflammatory cytokines are involved in the regulation of macrophage immune response remains unclear.Nuclear factor-kappa B(NF-κB)is a key signaling pathway in the inflammation process,which is widely involved in macrophages and other immune responses.NLR Pyrin domain containing 3(NLRP3)is the most characteristic inflammasome in macrophages,which can lead to inflammatory reaction and pyroptosis of the macrophages.As the number of inflammatory macrophages in the lung increases dramatically after PJP,we hypothesized that the acute lung injury mediated by pneumocystis may be related to NF-κB and NLRP3,but no relevant reports have been reported so far.In this paper,the role of inflammatory cytokines in acute lung injury inpneumocystis was explored.Part I:Expression profile of inflammatory cytokines in renal transplant recipients after PJPBackgroundCytokines,which include proinflammatory and anti-inflammatory factors,normally maintain a balance.However,when the immune system is over-activated,an immune damage would occur due to the release of a large number of inflammatory factors.The excessive activation of immune system and the release of a large number of inflammatory factors after pneumocystis infection are considered to be important reasons for the aggravation of PJP.Therefore,early identification of highly expressed inflammatory factors and intervention of them may be an important mean to prevent PJP from progressing to severe after pneumocystis infection.However,the profile of changes of inflammatory cytokines after PJP remains unclear.Purposes1.To detect the cytokines in patients with PJP after renal transplantation and screen out the highly expressed cytokines.2.To further verify the proinflammatory cytokines by expanding the sample size,and understand the inflammatory factors highly expressed in the body after the occurrence of PJP.Methods1.According to the inclusion and exclusion criteria,renal transplant recipients were divided into PJP group and healthy control group.4 patients in each group were randomly selected.The expressions of cytokines in peripheral blood mononuclear cell(PBMC)were detected by protein chip method.The highly expressed cytokines were screened out and further bioinformatics analysis was performed.2.ELISA was used to detect the high expression of inflammatory factors in peripheral blood in all patients.Results1.A total of 40 patients with PJP after renal transplantation and 40 healthy recipients were enrolled in this study.Randomly selected from two groups,4 cases were chosen for testing the cytokines changes using the protein chip method.A total of 19 cytokines were found increased(>1.2 times).Bioinformatics analysis revealed that TNF-α(tumor necroses factor alpha),TNF-β(tumor necrosis factor-beta).Interleukin-1β(IL-1β)had a proinflammatory effect,and TNF-β was significantly upregulated(P<0.05).2.The level of TNF-β in peripheral blood of PJP patients was significantly higher(P<0.01).There was no significant difference in TNF-α and IL-1β.Conclusions1.In PJP after renal transplantation,the expressions of most cytokines tend to be up-regulated.Bioinformatics analysis suggested that the differential cytokines could participate in the regulation of the activation of immune cells and promote inflammation.2.TNF-β was up-regulated in peripheral blood mononuclear cells and increased in plasma after pneumocystis infection,suggesting that TNF-β may be involved in pneumocystis mediated immune injury.Part II:The role of TNF-β in BDG-induced macrophage inflammationBackgroundBDG is a major component of Pneumocystis cell wall.Alveolar macrophages can recognize pneumocystis through glucan receptors,and play phagocytosis and clearance roles when activated.A large amount of BDG may lead to an excessive activation of alveolar macrophages,which may release proinflammatory factors and cause immune damage.TNF-β is one of the common proinflammatory factor,the first part of our study found that TNF-β expression in peripheral blood mononuclear cells was elevated,the concentration in plasma was also significantly increased,implying that TNF-β is likely to be involved in the pneumocystis mediated immune injury,but how it is involved is not clear,and its role on macrophages changes induced by BDG is unclear.Purposes1.To analyze the concentration level of BDG in peripheral blood of PJP patients and explore its application value,so as to provide a theoretical basis for further research on cell model construction using BDG.2.To establish cell models,to explore the activation effect of BDG on macrophages,to investigate the changes of inflammatory factors secreted by macrophages induced by BDG and study the specific ways of TNF-β promoting inflammatory response.Methods1.The clinical characteristics of PJP patients and the level of BDG in peripheral blood were retrospectively analyzed,and the sensitivity,specificity and diagnostic efficiency were calculated.2.BDG was used to induce and activate macrophages,and the levels of NO and TNF-α,IL-6 and IL-1β in the supernatant of macrophages were detected to establish a BDG-induced macrophage model.3.The levels of NO and the concentrations of TNF-α,IL-6 and IL-1β in the supernatant of macrophages after TNF-β stimulation were studied.Results1.Retrospective analysis showed that up to 83.6%of PJP patients had hypoxia symptoms,and the level of BDG was significantly increased.The diagnostic sensitivity of BDG was 74.5%,the specificity was 89.6%,and the area under the ROC curve was 0.845.2.It was found that with the increase of BDG concentration,the concentration levels of TNF-α,IL-6 and IL-1β and the content of NO in macrophage supernatant were correspondingly increased.After TNF-β stimulation,the concentration levels of TNF-α,IL-6 and IL-1β and the content of NO were further increased.Conclusions1.The level of BDG in peripheral blood of PJP patients is significantly increased,which might have high application value in the diagnosis of PJP.2.TNF-β can further stimulate macrophages to secrete inflammatory factors TNF-α,IL-6 and IL-1β,leading to a significant increase in inflammation levels.Part III:The molecular mechanism of TNF-β in Pneumocystis induced acute lung injuryBackgroundNF-κB is widely involved in the activation of macrophages and other immune responses,and is a key signaling pathway in inflammation process.NLRP3 is the most characteristic inflammasome in macrophages,and its high expression can cause pyroptosis of macrophages.As the inflammatory macrophages increase remarkably in the lung after pneumocystis infection,we hypothesized that the acute lung injury mediated by pneumocystis may be related to NF-κB and NLRP3,though there were no such reports.In this part,we will focus on the molecular mechanism of TNF-βinduced immune injury.Purposes1.To observe the morphological changes of alveolar macrophages in patients with PJP and whether the NF-κB pathway was activated and NLRP3 expression increased.2.To investigate whether TNF-β can activate NF-κB pathway and promote NLRP3 expression in BDG-induced macrophages,and the interaction between NF-κB and NLRP3.3.To investigate whether TNF-β can up-regulate NLRP3 inflammasome pathway protein and to explore the molecular mechanism of TNF-β in promoting inflammatory response in immune injury.Methods1.Scanning electron microscopy was used to observe the morphological changes of alveolar macrophages in patients with PJP.Immunofluorescence was used to detect the nuclear entry of NF-κB P65 and the expression of NLRP3.2.Cell model was constructed and the expression of inflammatory factors was detected by ELISA,and the phosphorylation level of NF-κB P65 and NLRP3 expression were detected by Western blot.Nuclear NF-κB P65 and NLRP3 expression were detected by immunofluorescence assay.3.The cell model was constructed,TNF-β was stimulated,LDH expression was detected by ELISA,and cleaved-GSDMD was detected by Western blotting.Activedcaspasel,pro-IL-1β and IL-1β expression were detected by immunofluorescence.The changes of NLRP3 function were observed.Results1.The study found that the macrophages in PJP patients had obvious morphological changes such as enlargement and aperture.Immunofluorescence showed that the nucleus entry of NF-κB P65 was increased,and the expression of NLRP3 was up-regulated.2.Studies found that TNF-β could further promote the activation of NF-κB P65 pathway and expression of NLRP3 on the basis of BDG stimulation.By interfering with the function of NF-κB and NLRP3,the levels of inflammatory factors TNF-α,IL-6 and IL-1β can be down-regulated.Intervention of NF-κB pathway can downregulate the expression of NLRP3,and intervention of NLRP3 can inhibit the activation of NF-κB pathway.3.It was found that TNF-β could further up-regulate the expression of NLRP3/caspase-1/IL-1β inflammasome pathway protein on the basis of BDG stimulation,and the content of LDH(lactate dehydrogenase)in macrophage supernatant was significantly increased.Knockdown of NLRP3 can down-regulate the expression of inflammasome pathway proteins and LDH content.Conclusion1.Swelling and hiatus of alveolar macrophages were observed in PJP patients,accompanied by the activation of NF-κB pathway and NLRP3 expression.2.TNF-β promotes macrophage inflammation through NF-κB/NLRP3 dual pathway,and intervention of NF-κB and NLRP3 can reduce inflammation,and NFκB and NLRP3 have a mutual regulatory effect.3.TNF-β can activate NLRP3 inflammasome pathway,which may lead to pyroptosis of macrophages.By interfering with NLRP3 protein activity,pyroptosis can be reduced.