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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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Intestinal epithelial cell-specific expression of TNFAIP3 protects against LPS-induced loss of barrier function.Explanted intestinal loops from WT (triangles) or villin-TNFAIP3 transgenic mice (squares) were assayed for barrier function beginning 45 minutes after injection of LPS (0.1 mg/mouse, ∼5 mg/kg i.p.; filled symbols) or in untreated animals (open symbols). The flux of FITC-dextran over time out of the loops was measured in relative fluorescent units (RFU) per cm of intestinal tissue. Increased FITC-dextran flux indicates decreased barrier function. (*p<0.05, ***p<0.001; WT untreated vs. WT LPS treated).
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pone-0026352-g003: Intestinal epithelial cell-specific expression of TNFAIP3 protects against LPS-induced loss of barrier function.Explanted intestinal loops from WT (triangles) or villin-TNFAIP3 transgenic mice (squares) were assayed for barrier function beginning 45 minutes after injection of LPS (0.1 mg/mouse, ∼5 mg/kg i.p.; filled symbols) or in untreated animals (open symbols). The flux of FITC-dextran over time out of the loops was measured in relative fluorescent units (RFU) per cm of intestinal tissue. Increased FITC-dextran flux indicates decreased barrier function. (*p<0.05, ***p<0.001; WT untreated vs. WT LPS treated).

Mentions: To assess the direct role of IEC expression of TNFAIP3 in barrier function we generated villin-TNFAIP3 mice that constitutively overexpress a TNFAIP3 transgene in the IECs of the small intestine, cecum and colon (Figures S1, S2). Villin-TNFAIP3 mice are healthy and display normal growth and reproduction up to 6 months of age. We stimulated inflammation in WT and villin-TNFAIP3 mice by injecting LPS into the peritoneal cavity 45 minutes prior to euthanasia. LPS induces a systemic inflammatory response affecting a variety of cell types including IECs, and its short-term effects are mediated largely by the induction of TNF release from innate immune cells, as well as enterocytes in vitro [40]–[41]. LPS induces endogenous TNF release which, when overproduced by intestinal epithelial cells, causes an IBD pathology [42]. The LPS challenge induced greater transepithelial flux of FITC-dextran across the ileum in explanted loops from WT mice compared to villin-TNFAIP3 mice, indicating that epithelial-specific overexpression of TNFAIP3 promotes resistance to inflammatory disruption of the intestinal barrier (Figure 3). Occludin is removed from apical sites of the epithelium and internalized in LPS-treated mice, and recent studies implicate occludin relocation by endocytosis in TNF-induced barrier permeability [14], [43]. Similar to these previously published reports, we observed occludin loss from the apical surface and an overall diminution of occludin intensity in IEC following administration of LPS to WT mice; however, occludin loss was markedly reduced in the intestine of LPS-treated villin-TNFAIP3 mice (Figure 4). This prevention of occludin redistribution suggests a mechanism by which TNFAIP3 affects the structure or integrity of tight junctions in IECs. Together these results demonstrate that TNFAIP3 is required for the maintenance of intestinal barrier function and that TNFAIP3 plays an important role in modulating epithelial permeability, in part by controlling occludin localization or stability in IEC.


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)

Intestinal epithelial cell-specific expression of TNFAIP3 protects against LPS-induced loss of barrier function.Explanted intestinal loops from WT (triangles) or villin-TNFAIP3 transgenic mice (squares) were assayed for barrier function beginning 45 minutes after injection of LPS (0.1 mg/mouse, ∼5 mg/kg i.p.; filled symbols) or in untreated animals (open symbols). The flux of FITC-dextran over time out of the loops was measured in relative fluorescent units (RFU) per cm of intestinal tissue. Increased FITC-dextran flux indicates decreased barrier function. (*p<0.05, ***p<0.001; WT untreated vs. WT LPS treated).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3198775&req=5

pone-0026352-g003: Intestinal epithelial cell-specific expression of TNFAIP3 protects against LPS-induced loss of barrier function.Explanted intestinal loops from WT (triangles) or villin-TNFAIP3 transgenic mice (squares) were assayed for barrier function beginning 45 minutes after injection of LPS (0.1 mg/mouse, ∼5 mg/kg i.p.; filled symbols) or in untreated animals (open symbols). The flux of FITC-dextran over time out of the loops was measured in relative fluorescent units (RFU) per cm of intestinal tissue. Increased FITC-dextran flux indicates decreased barrier function. (*p<0.05, ***p<0.001; WT untreated vs. WT LPS treated).
Mentions: To assess the direct role of IEC expression of TNFAIP3 in barrier function we generated villin-TNFAIP3 mice that constitutively overexpress a TNFAIP3 transgene in the IECs of the small intestine, cecum and colon (Figures S1, S2). Villin-TNFAIP3 mice are healthy and display normal growth and reproduction up to 6 months of age. We stimulated inflammation in WT and villin-TNFAIP3 mice by injecting LPS into the peritoneal cavity 45 minutes prior to euthanasia. LPS induces a systemic inflammatory response affecting a variety of cell types including IECs, and its short-term effects are mediated largely by the induction of TNF release from innate immune cells, as well as enterocytes in vitro [40]–[41]. LPS induces endogenous TNF release which, when overproduced by intestinal epithelial cells, causes an IBD pathology [42]. The LPS challenge induced greater transepithelial flux of FITC-dextran across the ileum in explanted loops from WT mice compared to villin-TNFAIP3 mice, indicating that epithelial-specific overexpression of TNFAIP3 promotes resistance to inflammatory disruption of the intestinal barrier (Figure 3). Occludin is removed from apical sites of the epithelium and internalized in LPS-treated mice, and recent studies implicate occludin relocation by endocytosis in TNF-induced barrier permeability [14], [43]. Similar to these previously published reports, we observed occludin loss from the apical surface and an overall diminution of occludin intensity in IEC following administration of LPS to WT mice; however, occludin loss was markedly reduced in the intestine of LPS-treated villin-TNFAIP3 mice (Figure 4). This prevention of occludin redistribution suggests a mechanism by which TNFAIP3 affects the structure or integrity of tight junctions in IECs. Together these results demonstrate that TNFAIP3 is required for the maintenance of intestinal barrier function and that TNFAIP3 plays an important role in modulating epithelial permeability, in part by controlling occludin localization or stability in IEC.

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