Download Mycobacterium tuberculosis evades macrophage defenses by

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