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