Transcript Role of autophagy in the pathogenicity of Crohn*s disease

Role of autophagy in the
pathogenicity of Crohn’s disease
Presented by JIANPING LI
Overview
 Background of autophagy
What is know about the role of autophagy in the
pathogenicity of Chron’s disease
Paper 1
Paper 2
What is still poorly understood about the role of
autophagy in the pathogenicity of Chron’s disease
Hypothesis and specific aim
Autophagy
 termed by Christian de
Duve in 1963
 Greek word, auto- “ self”
and phagein “ to eat”
 A suicide process?
(Suicide Pie by Dr. Hypercube, Sep 10th , 2010)
Autophagy
 Conservative catabolic
pathway
Bulk degrade cellular
contents
Highly regulated by
(Shvets et al, 2008 Autophagy 4: 621-628)
autophagy related proteins
(Atgs)
Basal autophagy maintain
cellular homeostasis
Defective autophagy: disease
Autophagy
LC3 I
LC3 II
(Adopted from Maiuri et al., Nature Reviews Molecular Cell Biology 8, 741-752)
Monitor autophagy and autophagic flux
 LC3 II: membrane protein in
autophagosome
 Number of LC 3 II correlated
linearly with autophagosome
 Accumulation of LC 3 II:
blockage of the fusion of
autophagosome and
lysosome, e.g. Bafilomycin
A1, LAMP2-RNAi
(Iovanna et al., 2010, DOI:
10.3998/panc.2010.13)
Overview
 Background of autophagy
What is know about the role of autophagy in the
pathogenicity of Chron’s disease
Paper 1
Paper 2
What is still poorly understood about the role of
autophagy in the pathogenicity of Chron’s disease
Hypothesis and specific aim
What is know about the role of autophagy in the
pathogenicity of Chron’s disease
 Genetic association linked defective ATG16L and
IGRM to the susceptibility to Crohn’s disease (Hampe et
al., 2007, Nat. Genet. 39, 207–211)
Autophagy related gene, Nod2 was identified as
‘risk’ allele for Crohn’s disease (Kanneganti et al., 2007, Immunity
27, 549–559)
Sirolimus (rapamycin) treatment improved
symptoms of refractory Crohn’s disease in clinic
(Massey et al., 2008, Gut 57(9):1294-6).
A lot of animal model with defective function of
Atgs pointed out autophagy might involve in the
pathogenicity of Crohn’s disease
What is know about the role of autophagy in the
pathogenicity of Chron’s disease
(Adopted from Herbert W Virgin & Beth Levine, 2009, Nature Immunology 10, 461 – 470)
Overview
 Background of autophagy
What is know about the role of autophagy in the
pathogenicity of Chron’s disease
Paper 1
Paper 2
What is still poorly understood about the role of
autophagy in the pathogenicity of Chron’s disease
Hypothesis and specific aim
Paper 1
Background and hypothesis of Paper 1
 Abnormal colonization of adherent-invasive
Escherichia coli (AIEC) in ileal lesions of CD
patients
AIECs multiply in gut epithelium and macrophagy
after invasion.
 Defective autophagy decrease the ability of AIEC
replication in the gut epithelium
Hypothesis: impaired autophagy allows
intramacrophagic mutiplication of AIEC and
promotes inflammatory cytokine response.
Fig 1. determine autophagy induced in response
to AIEC infectionin human THP-1 macrophages
 Conclusion: autophagy was induced in response to
infection of different AIEC strains
Fig 1(Cont’d). determine autophagy induced in
response to AIEC infectionin human THP-1
macrophages
 Conclusion: AIEC bacteria were targeted by
autophagy for degradation
Fig. 2 determine whether AIEC persist within
autophagosomes at late time point post infection
Fig. 2 (Cont’d) determine whether AIEC persist within
autophagosomes at late time point post infection
Conclusion: LF82
targeted early by
autophagy was rapidly
degraded whereas
bacteria escaped from
autophogosoms persist
in phagolysosomal
structures within
macrophagy
Fig. 3 determine impact of impaired expression of
ATG16L, IRGM or NOD2 on persistence of AIEC
within macrophagy
Fig. 3 (Cont’d) determine impact of impaired
expression of ATG16L, IRGM or NOD2 on
persistence of AIEC within macrophagy
 Conclusion:
impaired autophagy
by disrupting
ATG16L, IRGM
favors survival of
AIEC
Fig 4. determine impact of IRGM overexpresssion
on AIEC intramacrophagic persistence
Conclusion:
 Overexpressed IRGM increased autophagy flux
 The intramacrophagic AIEC decrease by the enhanced
autophagic activiy
Fig 4. (cont’d) determine impact of IRGM
overexpresssion on AIEC intramacrophagic
persistence
 Conclusion:
Overexpressed
IRGM induced
cell death in
dose-dependent
manner
Fig 5. determine AIEC intramacrophagic persistence
in macrophages from NOD2 -/- mice
Fig 5(cont’d) . determine AIEC intramacrophagic
persistence in macrophages from NOD2 -/- mice
 Conclusion:
Persistence of AIEC
in NOD2 -/macrophages was
enhanced as
comparing to in the
wildtype
macrophages.
Fig 6. Investigated pro-inflammatory cytokine
response of macrophages displaying autophagy
deficiency upon AIEC infection
Conclusion: AIEC induced secretion of high level of
TNFα and IL 6
Fig 6 (cont’d) . Investigated pro-inflammatory
cytokine response of macrophages displaying
autophagy deficiency upon AIEC infection
Conclusion: impaired
autophagy resulted in
significantly increase of
level of proinflammatory cytokine
TNFα upon AIEC
infection
Fig 7 examine effect of forced activated
autophagy on AIEC bacterial replication
Fig 7 (cont’d) examine effect of forced activated
autophagy on AIEC bacterial replication
Conclusion: forced induced autophagy at early time post infection
restrained AIEC replication and slowed down pro-inflammatory response
induced by the bacteria
Summaries from paper 1
 Autophagy was rapidly induced upon AIEC infection
within macrophages
Autophagy restrain AIEC multiplication within
macrophages and slow down pro-inflammatory
cytokine responses induced by the bacteria
Mutation of autophagy related genes, ATG16L, IRGM,
NOD2, ‘risk’ susceptible alleles in Crohn’s disease,
favors persistence of AIEC and stimulates proinflammatory cytokines response within macrophages
Overview
 Background of autophagy
What is know about the role of autophagy in the
pathogenicity of Chron’s disease
Paper 1
Paper 2
What is still poorly understood about the role of
autophagy in the pathogenicity of Chron’s disease
Hypothesis and specific aim
Paper 2
Background and hypothesis of paper 2
ubiquitin-mediated targeting in autophagy pathway:
clear intracellular bacteria
Ubiquitin ligase responsible for modifying surface
of intracellular bacteria with ubiquitin
Parkin: ubiquitin ligase, participating in mitophay,
associated with susceptibility to infection of
intracellular bacteria
Hypothesis: parkin mediates resistence to
intracellular pathogen via autophagy pathway.
Fig 1. determine whether parkin involves ubiquintin
mediated targeting M. Tuberculosis to autophagy
 Conclusion: ligase
activity of parkin
is essential to colocalization of
ubiquitin with M.
tuberculosis
during infection
Fig 1. extended data Quantification of parkin colocalization and effect of LRSAM1 knockdown in
BMDMs
 Conclusion: ligase activity of parkin is essential to colocalization of ubiquitin with M. tuberculosis during
infection
Fig 2. Parkin mediates K63-ubiquitin colocalization of TB and recruitment of ubiqutinautophagy receptors
 Conclusion:
Parkin mediating
K63-linked
polyubiquitin
around TB and
facilitates
recruitment of
multiple ubiquitinadaptors in the
autophagy
pathway
Fig 2. extended data to confirmed K63-ubiquitin
co-localization of TB
 Conclusion: most of co-localization of TB was
mediated by K63-ubiqitin instead of K48-ubiquitin
Fig 3. extended data address whether bacterial or
host proteins become unbiquited
 Conclusion: parkin facilitates the linkage of K63-linked
ubiquitin chains surrounding TB containing
phagosomes
Fig 3. to determine whether parkin mediates autophaic
targeting of TB and restrains bacterial replication
 Conclusion: parkinmediated ubiquination
leads to the
autophagic targeting
of TB and inhibits
bacterial replication
Fig 4. to determine whether parkin was required in
vivo during TB of mice
 Conclusion: parkin
is essential in vivo
for controlling
intracellular bacteria
Fig 2. extended data to confirm the role of parkin
in TB immunity in mice by analyzing human TB
patients
 Parkin was highly expressed in granuloma lesions of
human TB patiens, which agree with the results
suggested in mice.
Fig 5. to address whether parkin is conserved in
immunity
 Conclusion: parkin
play an
evolutionarily
conserved role in
innate immunity
Summaries from paper 2
 Parkin facilitates co-localization with TB is
required for TB autophagy
Parkin was able to inhibited TB replication,
suggesting a conserved mechanism fighting
intracellular bacteria infection
Overview
 Background of autophagy
What is know about the role of autophagy in the
pathogenicity of Chron’s disease
Paper 1
Paper 2
What is still poorly understood about the role of
autophagy in the pathogenicity of Chron’s disease
Hypothesis and specific aim
What is still poorly understood about the role of
autophagy in the pathogenicity of Chron’s disease
 No direct evidence point out that autophagy
defects contribute to human crohn’s disease
How autophagy regulate pro-inflammatory
cytokine response in crohn’s disease patients is
poorly understood?
How gut commensal subvert autophagy pathway in
crohn’s disease patients is obscured.
Overview
 Background of autophagy
What is know about the role of autophagy in the
pathogenicity of Chron’s disease
Paper 1
Paper 2
What is still poorly understood about the role of
autophagy in the pathogenicity of Chron’s disease
Hypothesis and specific aim
Hypothesis and specific aim
Hypothesis: dysfunctional interaction of
autophagy and adherent-invasive Escherichia coli
resulted from defective PARK2 gene promotes the
pathogenesis of Crohn’s disease
Specific aim: to determine role of parkin fight for
gut commensal intracellular bacteria AIEC in
crohn’s disease model; to address whether parkin
serve as cytokine modulator in macrophages upon
AIEC infection
Thank you