osteoprotegerin plays an unrecognized role in the etiopathogenesis of inflammatory bowel diseases
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Recent developments in understanding the role of osteoprotegerin in colonic intestinal epithelium at Sylvester lab
Raghunath Ramanarasimhaiah
Which evolved first? Gut or bone!
http://answers.mheducation.com/sciences/life-science/biology/animal-kingdom
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
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
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?
Question
• Does treatment with a detergent increase detectable concentration of OPG in spent Caco 2 media?
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
Detergent treatment of Caco 2 supernatant does not increase the detectable OPG by ELISA
3000
6000
9000
12000
15000
1% Tween 20
IL1β
- - ++
+ -
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
Questions
• Does exposure to commensal bacteria (ATCC 25922) alter OPG expression in Caco 2 cells?
Experimental procedure
Live E.coli
Spindown Collect supernatant
ELISAApical
Baso lateral
Over night370C
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
Questions
• Does OPG bind to bacteria? If yes, can it be released from the bacteria?
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
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%)
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
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
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
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
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
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!
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
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