Mechanisms Of ESAT6 From Mycobacterium Tuberculosis In Regulation Of Macrophages Activation

Tuberculosis is an airborne infectious disease caused by Mycobacterium tuberculosis?MTB?,which kills more than 2 million people every year.The number of deaths worldwide caused by tuberculosis in 2015 has exceeded that of AIDS,making tuberculosis one of the top ten causes of death.China is one of the five countries with the world's largest tuberculosis epidemic and the economic burden.China's hospitalized expenditure on tuberculosis alone was more than 1.7 billion Yuan in 2012.Therefore,there is an urgent need for study of the pathogenesis of MTB,the development of effective new tuberculosis diagnosis and treatment.Macrophages are the first-line defense of the body's innate immune system against MTB infection and the important effector cells from the adaptive immune system which remove the MTB.MTBs are cleared by activated macrophages via enhancing phagocytosis,initiating respiratory bursts,releasing proinflammatory factors,acidizing phagocytic lysosomes and enhancing antigen processing and presentation ability.The effectively escape from the immune killing by macrophages,is essential to the pathogenesis of MTB.ESAT6?Early secreted antigenic target 6-kDa?is an important virulence factor of MTB,secreted by the ESX-1 system?ESAT6 secretory system-1?,and involved in the regulation of MTB on the innate immune function of macrophages.But the detailed mechanism remains unclear.Studies suggest that the secretion of ESAT6 in the phagosome helps MTB translocate from the phagocytic lysosome to the cytoplasm and escape the killing of the lysosomes.And extracellular ESAT6 stimulation can promote the secretion of IL6,TNF and other pro-inflammatory factors in macrophages;and can activate Caspases-induced macrophage apoptosis.However,these effects help macrophages limit MTB infection.So,how does ESAT6 affect the survival of macrophages form the intra-phagosome stimuli mode? And further,what is its difference with extracellular ESAT6 stimulation? In this project,we delivered ESAT6 to the phagosome of THP-1 macrophages by ESAT6 nanocapsules.It was found that the effects of ESAT6 on the survival,proinflammatory activation and glucose metabolism of THP-1 macrophages stimulated by the intra-phagosome stimulus mode was significantly different from that of extracellular ESAT6 stimulus mode.Why the effects of extracellular stimulus and intra-phagosome stimulus are so different? We used high-throughput RNA sequencing technology to systematically study and compare the two stimuli,and found that THP-1 macrophages had significant differences in proinflammatory activation between the two stimuli.And we proposed a possible mechanism of ESAT6 stimulation on THP-1 proinflammatory activation.Finally,we investigated the value of a variety molecules from the inflammatory cytokine pathway in identification of different tuberculosis infection populations.Objective1.To establish the treatment mode of ESAT6-stimulated macrophages via intraphagosome stimulus mode,and observe the different effects between intra-phagosome and extracellular ESAT6 stimuli modes on macrophage.2.To get a clear idea on the transcriptional response characteristics from macrophages treated with ESAT6 via extracellular stimuli mode and intra-phagosome stimuli mode.3.To analyze the similarities and differences in macrophage transcriptional response to intra-phagosome and extracellular ESAT6 stimuli modes.4.To explore the possible mechanism that ESAT6 regulates the proinflammatory activation of THP-1 macrophages.5.To observe the value of CMPK2,MALAT1 and inflammatory cytokine pathway molecules in the identification of different tuberculosis infection status.Methods1.The establishment the intra-phagosome model of ESAT6-stimulated macrophage,and observation the effects of intra-phagosome and extracellular ESAT6 stimuli on macrophagesFirst,we constructed the ESAT6 nanocapsules with acidic degradable organic molecules.The morphology of ESAT6 nanocapsules was characterized by Scanning Electron Microscopy and Atomic Force Microscopy.The subcellular localization of ESAT6 nanocapsules after phagocytosis of macrophages was observed by Laser Confocal Microscopy to determine whether ESAT6 nanocapsules were successfully located in phagocytic bodies.Subsequently,we observed the effects of different concentrations of ESAT6 on THP-1 macrophage survival?apoptosis,death?,proinflammatory activation?IL1,IL6,IL8,IL10 and TNF? levels?and macrophage polarization state glucose metabolism?glucose consumption,lactic acid production?.2.The study of the transcription level of the overall response characteristics from macrophages treated with ESAT6 via intra-phagosome and extracellular stimuli modes.We investigated the effect of ESAT6 on the transcription level of THP-1 macrophages in the two stimuli modes by GO analysis,IPA signaling pathway and molecular network analysis after high-throughput RNA sequencing.3.To analyze the similarities and differences in macrophage transcriptional response to intra-phagosome and extracellular ESAT6 stimuli modes.We first get the differentially expressed genes of macrophages after treated with ESAT6 via intra-phagosome and extracellular stimuli modes.Then the functional characteristics of these genes were obtained by GO analysis.4.To explore the possible mechanism that ESAT6 regulates the proinflammatory activation of THP-1 macrophages.Based on the results of sequencing data,the mechanism of differential expression genes CMPK2 and MALAT1 in ESAT6-induced macrophage proinflammatory activation was explored by using siRNA approach.5.To observe the value of CMPK2,MALAT1 and inflammatory cytokine pathway molecules in the identification of different tuberculosis infection status.We collected the peripheral blood samples of patients with different tuberculosis infection status,to observe the difference of expression of inflammatory signal molecules in PBMC of patients with different tuberculosis infection status,and to study their value in differential diagnosis of tuberculosis infection.Results1.We successfully established the intra-phagosome ESAT6 stimulus mode.First,we successfully synthesized ESAT6 nanocapsules,characterized by Atomic Force Microscopy and Scanning Electron Microscopy,the diameter of nanocapsules was in the range of 200-500 nm.It was confirmed by Laser Confocal Microscopy that the nanocapsules were co-located with the lysosomes after macrophage phagocytosis.These results demonstrated that the purpose of delivering ESAT6 antigen into the phagosome of macrophages can be achieved by nanocapsules approach.Second,we observed that the effect of ESAT6 on THP-1 macrophage survival,proinflammatory activation and glucose metabolism was significantly different between intraphagosome and extra-cellular ESAT6 stimuli modes.Both stimulation modes can induce THP-1 macrophage apoptosis,but the extracellular mode tends to promote the late apoptosis and necrosis of THP-1 cells.Under the intra-phagosome ESAT6 stimulus at 24 h,the level of necrosis was not obvious in THP-1 macrophages.In the early stage of ESAT6 stimulation?4h?,extracellular ESAT6 stimulation promotes the secretion of proinflammatory cytokines in macrophages;whereas intra-phagosome ESAT6 stimulus tends to inhibit the proinflammatory effect of macrophages.In terms of glucose metabolism,ESAT6 significantly increased the uptake and lactate production of macrophages from extracellular stimuli.The levels of carbohydrate intake and lactic acid production were significantly decreased in intra-phagosome ESAT6 stimulus group.2.The overall level of transcriptional response characteristics in macrophages treated with ESAT6 via intra-phagosome and extracellular ESAT6 stimuli modes.RNA sequencing data analysis revealed a great number of molecular and signal pathways that haven't been investigated in tuberculosis research.In the extracellular treatment of ESAT6 4 hours "Acute Phase Response Signaling" was significantly enriched,and "cAMP Signaling" was significantly enriched in the 24-hour group,suggesting that the proinflammatory effect of extracellular ESAT6.While in macrophages stimulated with ESAT6 vs intra-phagosome mode at 4 h or 24 h,the most significant enrichment of the five upstream regulatory molecules are closely related to the inflammatory response.Their downstream signal molecules in the sequencing data were down-regulated,suggesting that the proinflammatory response of THP-1 macrophages was inhibited by intra-phagosome ESAT6 stimulation.3.The proinflammatory response in macrophages treated with ESAT6 via Extracellular and intra-phagosome stimuli were different.We focused on the datasets?ESAT6₄h_up + nESAT6₄h_down?,which includes genes up-regulated after extracellular stimuli at 4 hours,while down-regulated after intra-phagosome stimulation.GO analysis of the data set,revealed the most significant enrichment of the five Biological Process terms "cellular response to interleukin-1","cellular response to tumor necrosis factor","monocyte chemotaxis","cellular response to interferon-gamma" and "Chemokine-mediated signaling pathway" are closely related to macrophage inflammatory response and macrophage activation.It is further suggested that in the early stage of ESAT6 stimulation?4h?,extracellular ESAT6 stimulation leads to macrophage proinflammatory effects,whereas ESAT6 stimulation in phagocytic vesicles tends to inhibit the proinflammatory effect of macrophages.4.ESAT6 regulates the possible mechanism of THP-1 macrophage proinflammatory activationFirst,the extracellular ESAT6 treatment of THP-1 macrophages,both the RNA sequencing and qPCR data showed that CMPK2 levels were significantly increased.After interfering with CMPK2,the secretion of IL1? in THP-1 macrophages was not affected,but the macrophage glucose consumption was decreased and phagocytosis was inhibited.ESAT6 treatment of THP-1 macrophages,increased the intracellular HIF1? mRNA and protein expression levels.When interfering the expression of HIF1? mRNA,production of lactate,generation of ROS and capability of phagocytosis were reduced in THP-1 macrophages compared to the control siRNA groups,respectively.Bioinformatics predicted that HIF1? can bind to the CMPK2 promoter region and activate the expression of CMPK2.These above data suggested that ESAT6 may activate CMPK2 expression through HIF1? transcription and promote the proinflammatory effect of THP-1.Second,both the RNA sequencing and qPCR data showed that after ESAT6 extracellular stimulation,MALAT1 expression levels in THP-1 macrophages were significantly downregulated.Based on the stimulation of ESAT6,after the siRNA interference of MALAT1,levels of proinflammatory cytokines IL1?,IL8 and TNF? increased significantly in THP-1 macrophages.This suggested that ESAT6 could inhibit the inhibitory effect of MALAT1 and promote the proinflammatory function of THP-1.5.The value of CMPK2,MALAT1 and inflammatory cytokine pathway molecules in the identification of different tuberculosis infectious population.We observed the expression levels of CMPK2,MALAT1 and multiple cytokines and cytokine receptors in patients with active tuberculosis infection,latent tuberculosis infection,and other non-tuberculosis other lung infections.And we found that MALAT1 level can be used in identification of active tuberculosis infection and non-tuberculosis lung infections.At the same time,IL10 + IL12 combination showed the highest power in identification between active tuberculosis infection and latent tuberculosis infection status.ConclusionIn this study we revealed that the nanocapsule approach can be used to study the effect of pathogen antigen in phagocytic vesicle on macrophages.Extracellular ESAT6 stimulation promotes macrophage proinflammatory effects,whereas stimulation of ESAT6 in phagocytic vesicles inhibits macrophage proinflammatory responses.ESAT6 may promote the macrophage inflammatory activation by up-regulating the expression of CMPK2 and down-regulating MALAT1 expression.The level of MALAT1 and inflammatory signaling molecules can be used to identify different tuberculosis infections status.SignificanceThis study provides a new approach to study the effects of virulence factors from intracellularly infected bacteria on host macrophages via intra-phagosome stimulus mode.And the insight that the different effect of ESAT6 on macrophages inflammatory responses due to different stimuli modes,will extend our understanding of the ESAT6 function in tuberculosis infection,and the development of new tuberculosis infection control measures according to the different effects of ESAT6.