Lessons for TB Treatment from the Zebrafish
Lalita Ramakrishnan
Francisco RocaDavid Tobin
Muse DavisHilary Clay
Hannah VolkmanESCMID eLibrary
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Global burden of tuberculosis
Estimated TB incidence rates, 2016 (WHO - Global TB report 2017)
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Multidrug resistance is widespread
Percentage of new TB cases with MDR/RR-TB (WHO - Global TB report 2017)
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The Lubeck Diaster - 1929
251 infants accidentally given three doses of virulent M. tuberculosis as newborns
72 died of TB
173 showed some clinical or radiological features of TB but survived after differing levels of disease
Genetic susceptibilities influence TB outcomes
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Reviewed in Cambier, Falkow, Ramakrishnan, Cell 2015
TB is a complex disease
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Rediscovering Mycobacterium marinum, a close genetic relative of Mycobacterium tuberculosis
Photo by Paul Edelstein
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TB granulomas form in the zebrafish larvaZebrafish larvae form tuberculous granulomas
M. marinum
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Genetic screen reveals host susceptibility loci
wildtype
mutant
Tobin et al., Cell 2010
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A Genetic Screen Reveals Hypersusceptible Mutants
wildtype fh112
LTA4H Mutants Exhibit Bacterial Cording
WT LTA4H mutant
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TB granulomas form in the zebrafish larvaThe cording revealed that the granulomas had become necrotic
M. marinum
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LTA4H deficiency and excess both
increase TB susceptibility in zebrafish and humans
Tobin et al., Cell 2010
Tobin et al., Cell 2012
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LTA4H Genotype
LTA
4H
Pro
tein
C/C C/T T/T
A human LTA4H promoter polymorphism
regulates gene expression
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100 200 300 4000.4
0.6
0.8
1.0
Days after enrollment
Su
rviv
al
LTA4H genotype influences inflammation and
survival in TB meningitis
Overall survival
(Thwaites et al., NEJM, 2004)
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LTA4H genotype influences inflammation and
survival in TB meningitis
LTA4H Genotype-Adjusted SurvivalSurvival from TBM by genotype
0 100 200 300 4000.6
0.7
0.8
0.9
1.0
C/C
T/T
C/T
Days after enrollment
Su
rviv
al
P=0.02
HIGH
HET
HIGH
LOW
100 200 300 4000.4
0.6
0.8
1.0
Days after enrollment
Su
rviv
al
Overall survival
(Thwaites et al., NEJM, 2004)
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100 200 300 4000.4
0.6
0.8
1.0
Days after enrollment
Su
rviv
al
DEXAMETHASONE
PLACEBO(Thwaites et al., NEJM, 2004)
Overall Response
LTA4H genotype influences treatment response
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0 100 200 300 4000.4
0.6
0.8
1.0
Days after enrollment
Su
rviv
al
0 100 200 300 4000.4
0.6
0.8
1.0
Days after enrollment
Su
rviv
al
100 200 300 4000.4
0.6
0.8
1.0
Days after enrollment
Su
rviv
al
DEXAMETHASONE
PLACEBO(Thwaites et al., NEJM, 2004)
Overall Response
LTA4H genotype influences treatment response
Genotype-adjusted Response
PLACEBO DEXAMETHASONE
HIGH
HET
HIGHHET
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100 200 300 4000.4
0.6
0.8
1.0
Days after enrollment
Su
rviv
al
DEXAMETHASONE
PLACEBO(Thwaites et al., NEJM, 2004)
Overall Response
LTA4H genotype influences treatment response
Genotype-adjusted Response
PLACEBO DEXAMETHASONE
0 100 200 300 4000.4
0.6
0.8
1.0
Days after enrollment
Su
rviv
al HET
HIGH
LOW
0 100 200 300 4000.4
0.6
0.8
1.0
Days after enrollment
Su
rviv
al HET
HIGH
LOW
P=0.005ESCMID eLibrary
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Guy Thwaites Mary-Claire King
Tom Hawn
Jeremy FarrarDeanna HaggeSarah DunstanNguyen D. BangSaroswati KhadgeNuxia ZouTran T.H. ChauThuong Nguyen Thuy Thuong
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Low and high LTA4H activity both cause infected
macrophage necrosis by dysregulating TNF
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TNF excess triggers microbicidal reactive oxygen species in the mitochondrion which are bactericidal
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Mitochondrial ROS promotefirst increased bactericidal activity, then necrosis
Roca and Ramakrishnan, Cell 2013
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Mitochondrial ROS promote necrosis through themitochondrial matrix protein Cyclophilin D
Roca and Ramakrishnan, Cell 2013
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Surprise! Lysosomal components are also requiredfor this necrosis pathway
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• BAX mediates apoptosis by oligomerizing at and piercing pores in the MOM, which releases pro-apoptogenic factors.
• BAX also mediates mitochondrial necrosis independent of oligomerization and in collaboration with cyclophilin D.
Lysosomal cathepsin D activates BAX
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Lysosomal cathepsin D activates BAX
However mitochondrially-tagged BAX fails to cause TNF-mediated necrosis.
• BAX mediates apoptosis by oligomerizing at and piercing pores in the MOM, which releases pro-apoptogenic factors.
• BAX also mediates mitochondrial necrosis independent of oligomerization and in collaboration with cyclophilin D.
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BAX activates Ryanodine receptors in the ER to cause mitochondrial calcium overload
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Glucocorticoids worsen outcome of LTA4H low TB meningitis
PLACEBO DEXAMETHASONE
0 100 200 300 4000.4
0.6
0.8
1.0
Days after enrollment
Su
rviv
al
HET
HIGH
LOW
0 100 200 300 4000.4
0.6
0.8
1.0
Days after enrollmentS
urv
ival HET
HIGH
LOW
VietnamThwaites et al, NEJM 2004
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Glucocorticoids worsen outcome of LTA4H low TB meningitis
DEXAMETHASONE
HET/LOW
HIGH
0 100 200 300 4000.4
0.6
0.8
1.0
Days after enrollmentS
urv
ival HET
HIGH
LOW
VietnamThwaites et al, NEJM 2004
HIGH
HET/LOW
Indonesiavan Laarhoven et al,JID 2017
VietnamThuong et al,JID 2017
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Reduction of cellular calcium with Ca2+ channel blocking drugs inhibits necrosis
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• Studying TB can uncover fundamental new biology
• This pathway may be a common mechanism of necrosis in TB regardless of LTA4H genotype:
- Evidence of high content of ceramide in necrotic TB granulomas (Kim et al., EMBO Mol Med 2010)
- Key components of pathway (RIPK3, ROS and cyclophilin D) cause necrosis of human macrophages (Zhao et al., Mucosal Immunol 2017)
• Safe and inexpensive drugs can be repurposed as host-targeting drugs for both drug sensitive and resistant TB
Conclusions
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Verapamil targets M. tuberculosis by inhibiting macrophage-induced antibiotic tolerance
Adams et al., Cell 2011Adams, Szumowski, Ramakrishnan, JID 2014
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Verapamil targets M. tuberculosis by inhibiting macrophage-induced antibiotic tolerance
Verapamil also targets the host by inhibiting a pathogenic macrophage necrosis pathway
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