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091012 Johanna Eberhard Mycobacterium tuberculosis evades macrophage defenses by inhibiting plasma membrane repair Maziar Divangahi, Minjian Chen, Huixian Gan, Danielle Desjardins, Tyler T Hickman, David M Lee, Sarah Fortune, Samuel M Behar & Heinz G Remold June 2009 Plasma membrane repair Based on exocytosis of endomembranes • Derived from Golgi vesicles or lysosomes • Calcium dependant • Calcium sensor: • Synaptotagmin-7 (Syt-7) • Neuronal Calcium sensor-1(NCS-1) Introduction Mycobacterium tuberculosis Is phagocytosed by pulmonary macrophages Virulent: Avirulent: Introduction blocks phagosome maturation, leads to macrophage death by necrosis induces apoptosis of macrophages Mtb infection Prostaglandin E2 (PGE2 ) Introduction Lipoxin A4 (LXA4) Aim of study Analyse mechanisms by which virulent Mtb induces necrosis or inhibits apoptosis – Does Mtb induce plasma membrane disruptions? – Which inner membranes are involved in the repair? Introduction Virulent Mtb causes persistent microdisruptions -FDX influxFlow cytometry/ human macrophages • Less FDX influx in avirulent infected Mφ than in virulent infected Mφ Results Recruitment of lysosomal membranes -Translocation of LAMP-1Flow cytometry/ human macrophages After 12 h of infection: • Translocation of LAMP-1 stronger in H37Ra infected Mφ than in virulent infected Mφ Results Recruitment of lysosomal membranes -Translocation of LAMP-1 and Syt-7Flow cytometry/ human macrophages • Higher Syt-7 expression after infection with H37Ra not with H37Rv Results Recruitment of Golgi derived membranes -Translocation of mannosidase II, not of BiPFlow cytometry/ human macrophages • Higher translocation of Mannosidase II after avirulent infection • No BiP translocation, no involvement of ER derived membranes Results Summary I virulent Mtb causes persistent membrane disruptions Membranes both from the lysosomal and Golgi compartments are involved in macrophage plasma membrane repair What is the function of calcium sensors in the recruitment of these membranes? Results Calcium-sensors in plasma membrane repair -Silencing of Syt-7 expressionImmunoblot/ human macrophages Flow cytometry/ human macrophages • impaired LAMP-1 translocation but enhanced Mannosidase II, Posphatidylserine and annexin-1 expression Results Inhibition of Golgi membrane recruitment - blocking with Brefeldin AFlow cytometry/ human macrophages • Inhibition of mannosidase II, phosphatidylserine and annexin-1 translocation, but not of LAMP-1 Results Calcium sensors in plasma membrane repair -Silencing of NCS-1 expressionImmunoblot/ human Mφ Flow cytometry/ human Mφ • Inhibition of the translocation of Golgi membranes, Phosphatetidylserine and annexin-1 Results Inhibition of plasma membrane repair -FDX influx and Necrosis (7-AminoActinomycinD)Flow cytometry/ human Mφ • Higher FDX influx after Syt-7 or NCS-1 silencing or treatment with BFA • Promotion of Mφ necrosis via Syt-7 or NCS-1 silencing Results Summary II Recruitment of lysosomal and Golgi derived vesicels is critical in the repair of plasma membrane damage after Mtb infection and is recuired to prevent necrosis Is the upregulation of cAMP sufficient to trigger membrane repair? Results Effect of cAMP upregulation on membrane repair -Acivation of adenylate cyclase with forskolinFlow cytometry/ human Mφ • Greater translocation of LAMP-1 and Syt-7 • no effect on translocation of Golgi membranes Results Effect of PGE2 on membrane repair Flow cytometry/ human Mφ • Reconstitution of plasma membrane repair after addition of PGE2 • No increase of Golgi membrane recruitment Results Kinases involved in PGE2 activated membrane repair - Inhibition of PKA and PI(3)K Flow cytometry/ human Mφ PKA inhibitor PI(3)K inhibitor • PGE2 requires PI(3)K activation Results Effect of PGE2 on membrane repair - Infection of Mφ from Wt and Ptges -/- mice Flow cytometry/ Ra infected mouse Mφ • No recruitment of lysosomal membranes in Mφ deficient in Prostaglandin E synthase • No effect on Golgi membrane recruitment Results Summary III • recruitment of lysosomal membranes is PGE2 dependent • recruitment of Golgi-derived membranes is independent of PGE2 How do PGE2 and LXA4 affect the outcome of Mtb infection? Results Influence of LXA4 and PGE on the death modality of Mφ in Mtb infecion Cell death detection ELISA / mouse Mφ • More necrosis and less apoptosis in Ptges deficient Mφ • More apoptosis and less necrosis in Alox5 deficient Mφ Results Influence of LXA4 and PGE2 in control of Mtb growth Colony count / mouse Mφ • enhanced Mtb growth in Ptges deficient Mφ • Lower growth of Mtb in Alox5 deficient Mφ Results Innate control of infection in vivo -virulent infection of ko and wt mice Pulmonary lavage /cell death detection ELISA/ mouse APCs • More apoposis in the cells of Alox5 deficient mice Results Innate control of infection in vivo - adoptive transfer of Mφ into Rag1 deficient miceColony count/ transferred mouse Mφ • Bacterial burden higher in Rag-1 deficient mice with Ptges deficient Mφ • lower with Alox5 deficient Mφ Results Bacterial burden in spleen and lung Colony count/ transferred mouse Mφ • Durable effect in spleen and lung Results Summary IV • Transfer of Alox5 deficient Mφ restricts virulent Mtb replication in vivo • The effect is determined by the Mφ genotype and independent of adaptive immunity • The balance of PGE2 and LXA4 production affects the outcome of infection Is PGE2 involved in Syt-7 synthesis? Results Induction of Syt-7 transcription by exogenous PGE2 Real –time PCR /uninfected mouse Mφ • PGE2 induces Syt-7 expression in uninfected mouse Mφ Results No Induction of LAMP-1 transcription Real –time PCR /uninfected mouse Mφ • No effect of exogeneous PGE2 on LAMP-1 transcription Results Induction of Syt-7 transcription in virulent Mtb infection Real –time PCR/ uninfected and infected Mφ • Synergistical effect of exogenous PGE2 and virulent Mtb Results Induction of Syt-7 transcription in virulent Mtb infection Real –time PCR/ Rv infected Mφ • Higher Syt -7 expression in Alox 5 deficient Mφ than in wt or Ptges deficient Mφ Results In vivo induction of Syt-7 transcription Real –time PCR/ lungs of wt mice 7d after aerosol infection • Greater abundance of Syt-7 transcripts in avirulent infected mice than in wt or virulent infected mice Results Summary V • Virulent Mtb evades innate immunity by suppressing the production of PGE2 What is the direct link among Syt-7 function , the death modality of Mtb infected Mφ and the outcome of infection? Results Translocation of LAMP-1 Flow cytometry/ uninfected and Rv infected mouse Mφ • Translocation of Lamp-1 in Alox-5 deficient Mφ • No translocaion in wt or Ptges deficient Mφ Results Visualization of LAMP-1 translocation Confocal microscopy/ Mφ un-or infected with GFP labeled virulent Mtb • Little amounts of LAMP-1 on the surface of Ptges deficient or wt Mφ • extensive recruitment of LAMP-1 to the surface of Alox5 deficient Mφ Red: luminal domain of LAMP-1 Results Influence of Syt-7 on the death modality Cell death detection ELISA / Alox5 deficient Mφ infected with Rv Mtb with and without silencing of Syt-7 • Without silencing: More apoptosis than necrosis in Alox5 Mφ • After silencing: more necrosis than apoptosis => Syt-7:critical for preventing necrosis Results Influence of Syt-7 on bacterial growth Colony count/ Rv infected mouse Mφ • Limited bacerial growth in Alox5 deficient Mφ compared to WT • After silencing: impaired bacterial restriction => Direct involvement of Syt-7 in the innate control of Mtb infection Results Summary • Virulent Mtb perturbs the repair of plasma membrane microdisruptions • Lysosomal and Golgi-derived vesicles are involved in plasma membrane repair • Two distinct calcium sencor proteins regulate lysosomal and Glogi dependent plasma membrane repair • Lysosome-dependent membrane repair was promoted by PGE2 (regulates Syt-7) • In absence of PGE2/Syt-7 Macrophages undergo necrosis and are unable to control Mtb growth Summary Conclusion Inhibiting membrane repair by blocking PGE2 production represents a critical mechanism that allows virulent bacteria to replicate, to induce necrosis and escape from the host macrophage and infect other cells Conclusion Thank you for your attention! The end… Tuberculosis: unsealing the apoptotic envelope Steven A Porcelli & William R Jacobs Jr nature immunology volume 9 number 10 october 2008 Avirulent mycobacteria, including mutant forms of MTb that have lost their ability to cause disease, stimulate the macrophage to undergo apoptosis, which results in a 'cellular corpse' with an impermeable envelope that prevents bacteria from escaping. This process leads to containment and killing of the bacteria and is also associated with rapid priming of antigen-specific T cell responses. In contrast, virulent mycobacteria such as wild-type strains of MTb cause macrophage death by a process that proceeds to necrosis, which produces a permeable cell membrane that enables bacteria to escape and spread. eicosanoids • comprise Prostaglandins and related compounds • Mostly produced from arachidonic acid (a 20-carbon polyunsaturated fatty acid) • considered "local hormones" • effects on target cells close to their site of formation • Are rapidly degraded, so they are not transported to distal sites within the body • participate in intercellular signaling and intracellular signal cascades • various roles in inflammation, immune system modulation, control of reproductive processes and tissue growth cyclopentane ring Total amount of proteins