Advances in Inflammatory Bowel DiseasesHollywood FL, 2013

Pro-inflammatory cytokines alter tight junction self-assembly and barrier function through

claudin heterotypic incompatibility.

Christopher T. Capaldo, Ph.D.Emory University

DISCLOSURES

• Nothing to disclose

• Barrier defects are linked to IBD.

• Epithelial Tight Junctions provide the paracellular barrier.

• Barrier properties depend on transmembrane claudins.

• Pro-inflammatory cytokines, such as IFN-g and TNF-a, disrupt Tight Junctions and claudin function.

• Tight Junctions are dynamic structures.(Sasaki PNAS, 2003. Shen J Cell Bio.2008)

• Fluorescence Recovery After Photobleaching (FRAP)

• Are these dynamics disrupted in the presence of pro inflammatory cytokines?

IFN-g/TNF- a increase claudin-4 dynamics

(48hrs)

Fluorescence Recovery After Photobleaching (FRAP)

• Increased recovery• Little or no planar TJ

diffusion.

IFN-g/TNF- a increases claudin-4 partitioning into the mobile Junction sub-pool.

• Cytokine alter Mf/If partitioning.

• Cytokines do not alter t1/2 of recovery.

• Does this reflect a change in TJ structure?

cont

rol

Freeze fracture EM

Susanne Krug, Fromm Lab

IFN-g/TNF-a treatment does not alter gross TJ structure.

IFN

-g/T

NF

-a

(200nm)

IFN

-g/T

NF

-a

1 20

20

40

60

80

100

120

140

Ohm

s*cm

2control IFN-g

TNF-a

n=6

If not TJ structure, then is TJ composition changed?

Claudin-4 excludes claudin-2 from the TJ.

• Claudin 2 and 4 are heterotypically incompatible across cells, but not within the same cell.

(Daugherty 2007, Piontek, 2011)

• Is this responsible for cytokine-induced changes in mobility?

• Does this play a role in cytokine –induced barrier loss?

Claudin-4 excludes claudin-2 from the TJ.

Claudin 4 rescues cytokine-induced changes in mobility and barrier function

***

TJ Immobile stands

Lateral pool

TJ mobile pool

Summary and working model

IFN-g/TNF-a Claudin 4

Claudin 2• IFN-g/TNF- a increase

claudin 2 levels (1).• Claudin 2 competes

with claudin 4 at the junction.

• Resulting in increased mobile pool claudin 4 and lower barrier function over time.

(1. Mankertz, 2009)

Nusrat labAsma NusratRoland HilgarthMattie FeasleMike KwonAttila FarkasJeremy Benedik

Koval LabParkos LabShroyer Lab

Fromm LabSusanne Krug

Funded by a CCFA Career Development Award