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Role of autophagy in the pathogenicity of Crohn’s disease Presented by JIANPING LI.

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Presentation on theme: "Role of autophagy in the pathogenicity of Crohn’s disease Presented by JIANPING LI."— Presentation transcript:

1 Role of autophagy in the pathogenicity of Crohn’s disease Presented by JIANPING LI

2 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

3 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 10 th, 2010)

4 Autophagy  Conservative catabolic pathway  Bulk degrade cellular contents  Highly regulated by autophagy related proteins (Atgs)  Basal autophagy maintain cellular homeostasis  Defective autophagy: disease (Shvets et al, 2008 Autophagy 4: )

5 Autophagy (Adopted from Maiuri et al., Nature Reviews Molecular Cell Biology 8, ) LC3 ILC3 II

6 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: /panc )

7 Overview  B ackground of autophagy WW hat is know about the role of autophagy in the pathogenicity of Chron’s disease PP aper 1 PP aper 2 WW hat is still poorly understood about the role of autophagy in the pathogenicity of Chron’s disease HH ypothesis and specific aim

8 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

9 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)

10 Overview  B ackground of autophagy WW hat is know about the role of autophagy in the pathogenicity of Chron’s disease PP aper 1 PP aper 2 WW hat is still poorly understood about the role of autophagy in the pathogenicity of Chron’s disease HH ypothesis and specific aim

11 Paper 1

12  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. Background and hypothesis of Paper 1

13 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

14 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

15 Fig. 2 determine whether AIEC persist within autophagosomes at late time point post infection

16 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

17 Fig. 3 determine impact of impaired expression of ATG16L, IRGM or NOD2 on persistence of AIEC within macrophagy

18 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

19 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

20 Fig 4. (cont’d) determine impact of IRGM overexpresssion on AIEC intramacrophagic persistence  Conclusion: Overexpressed IRGM induced cell death in dose-dependent manner

21 Fig 5. determine AIEC intramacrophagic persistence in macrophages from NOD2 -/- mice

22 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.

23 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

24 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 pro- inflammatory cytokine TNFα upon AIEC infection

25 Fig 7 examine effect of forced activated autophagy on AIEC bacterial replication

26 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

27 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 pro- inflammatory cytokines response within macrophages

28 Overview  B ackground of autophagy WW hat is know about the role of autophagy in the pathogenicity of Chron’s disease PP aper 1 PP aper 2 WW hat is still poorly understood about the role of autophagy in the pathogenicity of Chron’s disease HH ypothesis and specific aim

29 Paper 2

30 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.

31 Fig 1. determine whether parkin involves ubiquintin mediated targeting M. Tuberculosis to autophagy  Conclusion: ligase activity of parkin is essential to co- localization of ubiquitin with M. tuberculosis during infection

32 Fig 1. extended data Quantification of parkin co- localization and effect of LRSAM1 knockdown in BMDMs  Conclusion: ligase activity of parkin is essential to co- localization of ubiquitin with M. tuberculosis during infection

33 Fig 2. Parkin mediates K63-ubiquitin co- localization of TB and recruitment of ubiqutin- autophagy receptors  Conclusion: Parkin mediating K63-linked polyubiquitin around TB and facilitates recruitment of multiple ubiquitin- adaptors in the autophagy pathway

34 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

35 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

36 Fig 3. to determine whether parkin mediates autophaic targeting of TB and restrains bacterial replication  Conclusion: parkin- mediated ubiquination leads to the autophagic targeting of TB and inhibits bacterial replication

37 Fig 4. to determine whether parkin was required in vivo during TB of mice  Conclusion: parkin is essential in vivo for controlling intracellular bacteria

38 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.

39 Fig 5. to address whether parkin is conserved in immunity  Conclusion: parkin play an evolutionarily conserved role in innate immunity

40 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

41 Overview  B ackground of autophagy WW hat is know about the role of autophagy in the pathogenicity of Chron’s disease PP aper 1 PP aper 2 WW hat is still poorly understood about the role of autophagy in the pathogenicity of Chron’s disease HH ypothesis and specific aim

42 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.

43 Overview  B ackground of autophagy WW hat is know about the role of autophagy in the pathogenicity of Chron’s disease PP aper 1 PP aper 2 WW hat is still poorly understood about the role of autophagy in the pathogenicity of Chron’s disease HH ypothesis and specific aim

44 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

45 Thank you


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