Lessons for TB Treatment from the Zebrafish

Lalita Ramakrishnan

Francisco RocaDavid Tobin

Muse DavisHilary Clay

Hannah VolkmanESCMID eLibrary

© by author

Global burden of tuberculosis

Estimated TB incidence rates, 2016 (WHO - Global TB report 2017)

ESCMID eLibrary

© by author

Multidrug resistance is widespread

Percentage of new TB cases with MDR/RR-TB (WHO - Global TB report 2017)

ESCMID eLibrary

© by author

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

ESCMID eLibrary

© by author

Reviewed in Cambier, Falkow, Ramakrishnan, Cell 2015

TB is a complex disease

ESCMID eLibrary

© by author

Rediscovering Mycobacterium marinum, a close genetic relative of Mycobacterium tuberculosis

Photo by Paul Edelstein

ESCMID eLibrary

© by author

Mycobacterium marinum infection of the hand

Ramakrishnan , NEJM, 1997

ESCMID eLibrary

© by author

Zebrafish TB Resembles Human TB

Human Zebrafish

ESCMID eLibrary

© by author

TB granulomas form in the zebrafish larvaZebrafish larvae form tuberculous granulomas

M. marinum

ESCMID eLibrary

© by author

A high throughput platform for infection studies

ESCMID eLibrary

© by author

Genetic screen reveals host susceptibility loci

wildtype

mutant

Tobin et al., Cell 2010

ESCMID eLibrary

© by author

A Genetic Screen Reveals Hypersusceptible Mutants

wildtype fh112

LTA4H Mutants Exhibit Bacterial Cording

WT LTA4H mutant

ESCMID eLibrary

© by author

TB granulomas form in the zebrafish larvaThe cording revealed that the granulomas had become necrotic

M. marinum

ESCMID eLibrary

© by author

Leukotriene A4 hydrolase is a rheostat

ESCMID eLibrary

© by author

LTA4H deficiency and excess both

increase TB susceptibility in zebrafish and humans

Tobin et al., Cell 2010

Tobin et al., Cell 2012

ESCMID eLibrary

© by author

LTA4H Genotype

LTA

4H

Pro

tein

C/C C/T T/T

A human LTA4H promoter polymorphism

regulates gene expression

ESCMID eLibrary

© by author

TB meningitis is the most severe form of disease

ESCMID eLibrary

© by author

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)

ESCMID eLibrary

© by author

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)

ESCMID eLibrary

© by author

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

ESCMID eLibrary

© by author

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

ESCMID eLibrary

© by author

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

© by author

Guy Thwaites Mary-Claire King

Tom Hawn

Jeremy FarrarDeanna HaggeSarah DunstanNguyen D. BangSaroswati KhadgeNuxia ZouTran T.H. ChauThuong Nguyen Thuy Thuong

ESCMID eLibrary

© by author

What makes LTA4H extremes susceptible?

ESCMID eLibrary

© by author

Low and high LTA4H activity both cause infected

macrophage necrosis by dysregulating TNF

ESCMID eLibrary

© by author

TNF excess triggers microbicidal reactive oxygen species in the mitochondrion which are bactericidal

ESCMID eLibrary

© by author

Mitochondrial ROS promotefirst increased bactericidal activity, then necrosis

Roca and Ramakrishnan, Cell 2013

ESCMID eLibrary

© by author

Mitochondrial ROS promote necrosis through themitochondrial matrix protein Cyclophilin D

Roca and Ramakrishnan, Cell 2013

ESCMID eLibrary

© by author

Surprise! Lysosomal components are also requiredfor this necrosis pathway

ESCMID eLibrary

© by author

TNF-induced mitROS induce lysosomal ceramide production

ESCMID eLibrary

© by author

Lysosomal cathepsin D activates BAX

ESCMID eLibrary

© by author

• 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

ESCMID eLibrary

© by author

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.

ESCMID eLibrary

© by author

BAX targets the ER to cause Ca2+ translocation into themitochondrion

ESCMID eLibrary

© by author

BAX causes mitochondrial Ca2+ overload in infected macrophages

ESCMID eLibrary

© by author

Mitochondrial calcium overload requires upstream components also

ESCMID eLibrary

© by author

BAX activates Ryanodine receptors in the ER to cause mitochondrial calcium overload

ESCMID eLibrary

© by author

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

ESCMID eLibrary

© by author

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

ESCMID eLibrary

© by author

LTA4H/TNF-high pathway suggests downstream drugs

ESCMID eLibrary

© by author

Reduction of cellular calcium with Ca2+ channel blocking drugs inhibits necrosis

ESCMID eLibrary

© by author

• 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

ESCMID eLibrary

© by author

Verapamil targets M. tuberculosis by inhibiting macrophage-induced antibiotic tolerance

Adams et al., Cell 2011Adams, Szumowski, Ramakrishnan, JID 2014

ESCMID eLibrary

© by author

Verapamil targets M. tuberculosis by inhibiting macrophage-induced antibiotic tolerance

Verapamil also targets the host by inhibiting a pathogenic macrophage necrosis pathway

ESCMID eLibrary

© by author

Photo: Maya Ramakrishnan

ESCMID eLibrary

© by author

Boiling River, Yellowstone National Park

Photo: Neeta Satam

ESCMID eLibrary

© by author

Boiling River, Yellowstone National Park

Photo: Neeta Satam

Tricyclic antidepressantsDantroleneNifedipineDiltiazemFlunarizineVerapamil

Ibuprofen

ESCMID eLibrary

© by author