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TNFAIP3 maintains intestinal barrier function and supports epithelial cell tight junctions.

Kolodziej LE, Lodolce JP, Chang JE, Schneider JR, Grimm WA, Bartulis SJ, Zhu X, Messer JS, Murphy SF, Reddy N, Turner JR, Boone DL - PLoS ONE (2011)

Bottom Line: In cultured human intestinal epithelial cell lines, TNFAIP3 expression regulated both TNF-induced and myosin light chain kinase-regulated tight junction dynamics but did not affect myosin light chain kinase activity.We also found that TNFAIP3 deubiquitinates polyubiquitinated occludin.These in vivo and in vitro studies support the role of TNFAIP3 in promoting intestinal epithelial barrier integrity and demonstrate its novel ability to maintain intestinal homeostasis through tight junction protein regulation.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of Chicago, Chicago, Illinois, United States of America.

ABSTRACT
Tight junctions between intestinal epithelial cells mediate the permeability of the intestinal barrier, and loss of intestinal barrier function mediated by TNF signaling is associated with the inflammatory pathophysiology observed in Crohn's disease and celiac disease. Thus, factors that modulate intestinal epithelial cell response to TNF may be critical for the maintenance of barrier function. TNF alpha-induced protein 3 (TNFAIP3) is a cytosolic protein that acts in a negative feedback loop to regulate cell signaling induced by Toll-like receptor ligands and TNF, suggesting that TNFAIP3 may play a role in regulating the intestinal barrier. To investigate the specific role of TNFAIP3 in intestinal barrier function we assessed barrier permeability in TNFAIP3(-/-) mice and LPS-treated villin-TNFAIP3 transgenic mice. TNFAIP3(-/-) mice had greater intestinal permeability compared to wild-type littermates, while villin-TNFAIP3 transgenic mice were protected from increases in permeability seen within LPS-treated wild-type littermates, indicating that barrier permeability is controlled by TNFAIP3. In cultured human intestinal epithelial cell lines, TNFAIP3 expression regulated both TNF-induced and myosin light chain kinase-regulated tight junction dynamics but did not affect myosin light chain kinase activity. Immunohistochemistry of mouse intestine revealed that TNFAIP3 expression inhibits LPS-induced loss of the tight junction protein occludin from the apical border of the intestinal epithelium. We also found that TNFAIP3 deubiquitinates polyubiquitinated occludin. These in vivo and in vitro studies support the role of TNFAIP3 in promoting intestinal epithelial barrier integrity and demonstrate its novel ability to maintain intestinal homeostasis through tight junction protein regulation.

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TNFAIP3 regulates TNF-induced tight junction dynamics in intestinal epithelial cells.Transepithelial electrical resistance (TER) was measured serially in cells grown in semi-permeable supports untreated (open symbols) or treated with TNF (10 ng/ml; closed symbols) for the indicated times. Values represent percentage of initial resistance measurements (% Init Ω). Pools of cells were stably transduced with (A) GFP alone (GFP control; triangles) or GFP and TNFAIP3 (TNFAIP3 +++; squares); or (B) scrambled shRNA (control shRNA; triangles) or TNFAIP3 shRNA (circles). (**p<0.01, ***p<0.001; TNF-treated GFP controls vs. untreated GFP controls (A) or TNF-treated control shRNA/TNF-treated TNFAIP3 shRNA vs. their respective untreated lines (B)).
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pone-0026352-g005: TNFAIP3 regulates TNF-induced tight junction dynamics in intestinal epithelial cells.Transepithelial electrical resistance (TER) was measured serially in cells grown in semi-permeable supports untreated (open symbols) or treated with TNF (10 ng/ml; closed symbols) for the indicated times. Values represent percentage of initial resistance measurements (% Init Ω). Pools of cells were stably transduced with (A) GFP alone (GFP control; triangles) or GFP and TNFAIP3 (TNFAIP3 +++; squares); or (B) scrambled shRNA (control shRNA; triangles) or TNFAIP3 shRNA (circles). (**p<0.01, ***p<0.001; TNF-treated GFP controls vs. untreated GFP controls (A) or TNF-treated control shRNA/TNF-treated TNFAIP3 shRNA vs. their respective untreated lines (B)).

Mentions: To test whether TNFAIP3 plays a direct role in the regulation of IEC tight junction integrity, we infected the IEC line HCT116 with lentivirus containing the TNFAIP3 cDNA in order to generate epithelial cell lines that stably and constitutively overexpress TNFAIP3 (Figure S3). IECs grown in semi-permeable supports were treated with TNF in the basolateral compartment and barrier function was assessed by changes in transepithelial electrical resistance (TER) over time. TNF induced a TER drop of ∼15% (p<0.001) within 1 hour in cells expressing the control GFP vector, but this TER drop was blocked in cells overexpressing TNFAIP3 (Figure 5A). Using annexin V and propidium iodide staining we did not observe any differences in the number of dead or apoptotic cells over a similar time course, consistent with the inability of TNF alone to induce death in most cell types (Figure S4) [44]. Thus, the heterologous expression of TNFAIP3 in IECs can prevent TNF-induced decreases in barrier function in these cells.


TNFAIP3 maintains intestinal barrier function and supports epithelial cell tight junctions.

Kolodziej LE, Lodolce JP, Chang JE, Schneider JR, Grimm WA, Bartulis SJ, Zhu X, Messer JS, Murphy SF, Reddy N, Turner JR, Boone DL - PLoS ONE (2011)

TNFAIP3 regulates TNF-induced tight junction dynamics in intestinal epithelial cells.Transepithelial electrical resistance (TER) was measured serially in cells grown in semi-permeable supports untreated (open symbols) or treated with TNF (10 ng/ml; closed symbols) for the indicated times. Values represent percentage of initial resistance measurements (% Init Ω). Pools of cells were stably transduced with (A) GFP alone (GFP control; triangles) or GFP and TNFAIP3 (TNFAIP3 +++; squares); or (B) scrambled shRNA (control shRNA; triangles) or TNFAIP3 shRNA (circles). (**p<0.01, ***p<0.001; TNF-treated GFP controls vs. untreated GFP controls (A) or TNF-treated control shRNA/TNF-treated TNFAIP3 shRNA vs. their respective untreated lines (B)).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3198775&req=5

pone-0026352-g005: TNFAIP3 regulates TNF-induced tight junction dynamics in intestinal epithelial cells.Transepithelial electrical resistance (TER) was measured serially in cells grown in semi-permeable supports untreated (open symbols) or treated with TNF (10 ng/ml; closed symbols) for the indicated times. Values represent percentage of initial resistance measurements (% Init Ω). Pools of cells were stably transduced with (A) GFP alone (GFP control; triangles) or GFP and TNFAIP3 (TNFAIP3 +++; squares); or (B) scrambled shRNA (control shRNA; triangles) or TNFAIP3 shRNA (circles). (**p<0.01, ***p<0.001; TNF-treated GFP controls vs. untreated GFP controls (A) or TNF-treated control shRNA/TNF-treated TNFAIP3 shRNA vs. their respective untreated lines (B)).
Mentions: To test whether TNFAIP3 plays a direct role in the regulation of IEC tight junction integrity, we infected the IEC line HCT116 with lentivirus containing the TNFAIP3 cDNA in order to generate epithelial cell lines that stably and constitutively overexpress TNFAIP3 (Figure S3). IECs grown in semi-permeable supports were treated with TNF in the basolateral compartment and barrier function was assessed by changes in transepithelial electrical resistance (TER) over time. TNF induced a TER drop of ∼15% (p<0.001) within 1 hour in cells expressing the control GFP vector, but this TER drop was blocked in cells overexpressing TNFAIP3 (Figure 5A). Using annexin V and propidium iodide staining we did not observe any differences in the number of dead or apoptotic cells over a similar time course, consistent with the inability of TNF alone to induce death in most cell types (Figure S4) [44]. Thus, the heterologous expression of TNFAIP3 in IECs can prevent TNF-induced decreases in barrier function in these cells.

Bottom Line: In cultured human intestinal epithelial cell lines, TNFAIP3 expression regulated both TNF-induced and myosin light chain kinase-regulated tight junction dynamics but did not affect myosin light chain kinase activity.We also found that TNFAIP3 deubiquitinates polyubiquitinated occludin.These in vivo and in vitro studies support the role of TNFAIP3 in promoting intestinal epithelial barrier integrity and demonstrate its novel ability to maintain intestinal homeostasis through tight junction protein regulation.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of Chicago, Chicago, Illinois, United States of America.

ABSTRACT
Tight junctions between intestinal epithelial cells mediate the permeability of the intestinal barrier, and loss of intestinal barrier function mediated by TNF signaling is associated with the inflammatory pathophysiology observed in Crohn's disease and celiac disease. Thus, factors that modulate intestinal epithelial cell response to TNF may be critical for the maintenance of barrier function. TNF alpha-induced protein 3 (TNFAIP3) is a cytosolic protein that acts in a negative feedback loop to regulate cell signaling induced by Toll-like receptor ligands and TNF, suggesting that TNFAIP3 may play a role in regulating the intestinal barrier. To investigate the specific role of TNFAIP3 in intestinal barrier function we assessed barrier permeability in TNFAIP3(-/-) mice and LPS-treated villin-TNFAIP3 transgenic mice. TNFAIP3(-/-) mice had greater intestinal permeability compared to wild-type littermates, while villin-TNFAIP3 transgenic mice were protected from increases in permeability seen within LPS-treated wild-type littermates, indicating that barrier permeability is controlled by TNFAIP3. In cultured human intestinal epithelial cell lines, TNFAIP3 expression regulated both TNF-induced and myosin light chain kinase-regulated tight junction dynamics but did not affect myosin light chain kinase activity. Immunohistochemistry of mouse intestine revealed that TNFAIP3 expression inhibits LPS-induced loss of the tight junction protein occludin from the apical border of the intestinal epithelium. We also found that TNFAIP3 deubiquitinates polyubiquitinated occludin. These in vivo and in vitro studies support the role of TNFAIP3 in promoting intestinal epithelial barrier integrity and demonstrate its novel ability to maintain intestinal homeostasis through tight junction protein regulation.

Show MeSH
Related in: MedlinePlus