osteoprotegerin plays an unrecognized role in the etiopathogenesis of inflammatory bowel diseases
TRANSCRIPT
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Recent developments in understanding the role of osteoprotegerin in colonic intestinal epithelium at Sylvester lab
Raghunath Ramanarasimhaiah
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Which evolved first? Gut or bone!
http://answers.mheducation.com/sciences/life-science/biology/animal-kingdom
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Expression of OPG in different cell types
van Agtmaal EL, et al. The shear stress-induced transcription factor KLF2 affects dynamics and angiopoietin-2 content of Weibel-Palade bodies. PLoS One. 2012;7(6):e38399.
Chollet ME, et al. Evidence of a colocalisation of osteoprotegerin (OPG) with von Willebrand factor (VWF) in platelets and megakaryocytes alpha granules. Studies from normal and grey platelets. Br J Haematol. 2010 Mar;148(5):805-7.
Sylvester lab
MG63 osteoblastic cells
OPG DAPI Merge
Caco 2 Intestinal epithelial cells
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Expression of OPG in different tissues
Carda C, et al. Osteoprotegerin (OPG) and RANKL expression and distribution in developing human craniomandibular joint. Tissue Cell. 2005 Jun;37(3):247-55.
Ueland T, et al. Dysregulated osteoprotegerin/RANK ligand/RANK axis in clinical and experimental heart failure. Circulation. 2005 May 17;111(19):2461-8.
Sylvester lab
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Questions
• Does treatment with a detergent increase detectable concentration of osteoprotegerin (OPG) in spent Caco 2 media?
• Does exposure to commensal bacteria alter OPG expression in Caco 2 cells?
• Does OPG bind to bacteria? If yes, can it be released from the bacteria?
• Expression of OPG in appendix?
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Question
• Does treatment with a detergent increase detectable concentration of OPG in spent Caco 2 media?
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Experimental procedureSpin down
Collect supernatant
ELISAIL1β
Media only
Add 1% Tween 20
Incubate on ice for 30 M
12 well culture plate supporting Caco 2
Over night370C
Spin down
Collect supernatant
Add 1% Tween 20
Incubate on ice for 30 M
ELISA
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Detergent treatment of Caco 2 supernatant does not increase the detectable OPG by ELISA
3000
6000
9000
12000
15000
1% Tween 20
IL1β
- - ++
+ -
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Cell culture inserts
• Seed and grow Caco 2 cells as a monolayer• After achieving confluence, allow 21 days for
polarization• Check TEER (transepithelial electrical
resistance)
Apical
Cell monolayer
Baso-lateral
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Questions
• Does exposure to commensal bacteria (ATCC 25922) alter OPG expression in Caco 2 cells?
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Experimental procedure
Live E.coli
Spindown Collect supernatant
ELISAApical
Baso lateral
Over night370C
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OPG expression appears suppressed on exposure to commensal bacteria
A B A B A B A B A B A B106 105 104 102 EC Sup Media
2000
4000
6000
8000
10000
No. of EC/insert A: ApicalB: BasolateralEC: Escherichia coliEC Sup: Filtered spent medium of EC
pg/m
L O
PG
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Questions
• Does OPG bind to bacteria? If yes, can it be released from the bacteria?
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Experimental procedure
Spindown Collect supernatant
ELISAOn ice 30 M
0.05% Tween 20
Spindown Collect supernatant
ELISAOn ice 30 MELISA
Spindown
Live E.coli
Killed E.coli
Over night370C
TEER
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Live Escherichia coli
+
Live E.coli 106 104 102 EC sup EC med Con med
2000
4000
6000
- +- +- +- +- +-
EC sup: E.coli spent mediaEC med: Fresh LB brothCon med: Caco 2 media
Tween 20 (0.05%)
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Killed Escherichia coli
Tween 20 (0.05%)
Killed E.coli 106 104 102 EC sup EC med Con med
2000
4000
6000
+- +- +- +- +- +-
EC sup: E.coli spent mediaEC med: Fresh LB brothCon med: Caco 2 media
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Baso-lateral compartment
L K + - + - + -E.coli
106 104 102 EC sup EC med Con med
2000
4000
6000
L K L K
L: LiveK: KilledEC sup: E.coli spent mediaEC med: Fresh LB brothCon med: Caco 2 media
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Conclusions
• ELISA detects the true concentration of OPG in Caco 2 supernatants and is not altered by detergent (0.05% Tween 20)
• Osteoprotegerin (OPG) binds to commensal bacteria and the bound OPG can be released by detergent treatment
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Next
• Perform a Western blot on E.coli co-cultured with Caco 2 cells
• Charge the baso-lateral compartment of the inserts with E.coli and assess the OPG expression by Caco 2 cells in both apical and baso-lateral compartments
• Expression of OPG in appendix of subjects infested with Enterobius vermicularis
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Does intestinal epithelial cell and bacteria influence immune-competent cell profile?
Monocyte/macrophage cell line (mature/ immature)
Caco 2 cells
Live/killed intestinal commensal bacteria
Live/killed intestinal pathogenic bacteria
Activation markers and cytokines
OPG and cytokine profile
No bacteria
No Caco 2
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OPG-/- Caco 2 cells
• Zinc-finger nucleases?• Alterations in the phenotype?• Can the cells still transform into functional
intestinal epithelial cells?• Perform the above mentioned experiments to
investigate the role of OPG!
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Does intestinal epithelial cell and bacteria influence immune-competent cell profile?
Monocyte/macrophage cell line (mature/ immature)
OPG-/- Caco 2 cells
Live/killed intestinal commensal bacteria
Live/killed intestinal pathogenic bacteria
Activation markers and cytokines
Cytokine profile
No bacteria
No Caco 2