osteoprotegerin and commensal bacteria
TRANSCRIPT
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