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Targeted colonic claudin-2 expression renders resistance to epithelial injury, induces immune suppression, and protects from colitis.

Ahmad R, Chaturvedi R, Olivares-Villagómez D, Habib T, Asim M, Shivesh P, Polk DB, Wilson KT, Washington MK, Van Kaer L, Dhawan P, Singh AB - Mucosal Immunol (2014)

Bottom Line: Importantly, claudin-2 expression increased colonocyte proliferation and provided protection against colitis-induced colonocyte death in a PI-3Kinase/Bcl-2-dependent manner.Importantly, these immunosuppressive changes were associated with increased synthesis of the immunoregulatory cytokine TGF-β by colonic epithelial cells in Cl-2TG mice compared with WT littermates.Taken together, our findings reveal a critical albeit complex role of claudin-2 in intestinal homeostasis by regulating epithelial permeability, inflammation and proliferation and suggest novel therapeutic opportunities.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, Vanderbilt University School of Medicine, Nashville, Tennessee, USA.

ABSTRACT
Expression of claudin-2, a tight junction protein, is highly upregulated during inflammatory bowel disease (IBD) and, due to its association with epithelial permeability, has been postulated to promote inflammation. Notably, claudin-2 has also been implicated in the regulation of intestinal epithelial proliferation. However, precise role of claudin-2 in regulating colonic homeostasis remains unclear. Here, we demonstrate, using Villin-Claudin-2 transgenic mice, that increased colonic claudin-2 expression augments mucosal permeability as well as colon and crypt length. Most notably, despite leaky colon, Cl-2TG mice were significantly protected against experimental colitis. Importantly, claudin-2 expression increased colonocyte proliferation and provided protection against colitis-induced colonocyte death in a PI-3Kinase/Bcl-2-dependent manner. However, Cl-2TG mice also demonstrated marked suppression of colitis-induced increases in immune activation and associated signaling, suggesting immune tolerance. Accordingly, colons from naive Cl-2TG mice harbored significantly increased numbers of regulatory (CD4(+)Foxp3(+)) T cells than WT littermates. Furthermore, macrophages isolated from Cl-2TG mouse colon exhibited immune anergy. Importantly, these immunosuppressive changes were associated with increased synthesis of the immunoregulatory cytokine TGF-β by colonic epithelial cells in Cl-2TG mice compared with WT littermates. Taken together, our findings reveal a critical albeit complex role of claudin-2 in intestinal homeostasis by regulating epithelial permeability, inflammation and proliferation and suggest novel therapeutic opportunities.

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Cl-2TG mice are protected from DSS-induced acute colitisTo induce colitis, mice received DSS (4% w/v) in the drinking water (10 days). (A). Weight loss during the course of DSS administration; (B). Disease activity index (DAI) changes among DSS-treated groups; C(i). Colon length (cm) in control and DSS-treated mice, C(ii). Representative colon images, and C(iii). Colon weight/cm; (D). Cumulative injury scores. Control mice did not show inflammation and associated injury; (E). Representative H&E staining of the colonic tissues from control and DSS-treated mice; (F). Representative photomicrographs demonstrating immunostaining to determine colonic myeloperoxidase (MPO) activity from control and DSS-treated mice. Values are presented as mean ± sem. *p<0.05, **p<0.01, ***p<0.001. Scale bars=500 or 50 μm.
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Figure 2: Cl-2TG mice are protected from DSS-induced acute colitisTo induce colitis, mice received DSS (4% w/v) in the drinking water (10 days). (A). Weight loss during the course of DSS administration; (B). Disease activity index (DAI) changes among DSS-treated groups; C(i). Colon length (cm) in control and DSS-treated mice, C(ii). Representative colon images, and C(iii). Colon weight/cm; (D). Cumulative injury scores. Control mice did not show inflammation and associated injury; (E). Representative H&E staining of the colonic tissues from control and DSS-treated mice; (F). Representative photomicrographs demonstrating immunostaining to determine colonic myeloperoxidase (MPO) activity from control and DSS-treated mice. Values are presented as mean ± sem. *p<0.05, **p<0.01, ***p<0.001. Scale bars=500 or 50 μm.

Mentions: Despite increased mucosal permeability, Cl-2TG mice did not have signs of mucosal inflammation. To examine whether Cl-2TG mice are more susceptible to colitis, Cl-2TG and WT mice were subjected to chemically-induced colitis by providing drinking water containing DSS. As expected, DSS-treated WT mice demonstrated significant body weight loss (p<0.01; Figure-2A). However, the DSS-induced weight loss was inhibited in Cl-2TG mice. The cumulative clinical disease index further supported protection of Cl-2TG mice against DSS colitis (Figure-2B). Consistent with these findings, colon length in DSS-treated WT mice was significantly decreased (p<0.001) compared with control mice {Figure-2C(i–iii)}, whereas this parameter was not significantly different between DSS-treated and control Cl-2TG mice. Independent evaluation by a gastrointestinal pathologist confirmed a significantly lower injury score (p<0.001) in DSS-treated Cl-2TG versus WT mice (Figure-2D). Importantly, depth of inflammation, crypt damage and % involved by crypt damage were all significantly different between DSS-treated WT and Cl-2TG mice (Figure S-2). Histological analysis further demonstrated marked preservation of the crypt architecture and decreased inflammation in DSS-treated Cl-2TG mice, as compared with WT mice where crypt architecture was largely disrupted and the severe loss of epithelia was accompanied by massive inflammation (Figure-2E). A marked decrease in myeloperoxidase (MPO)-activity in DSS-treated Cl-2TG mice further suggested decreased neutrophil infiltration in the colon (Figure-2F). Because our animals were generated on a B6D2F1/J background, and because colitis is influenced by the genetic background of the animals employed, we performed similar experiments with claudin-2 transgenic animals that were backcrossed more than 10 times with C57BL/6 mice, which demonstrated similar differences as those observed for the mixed background animals {Figure S-3(A–E)}. Taken together, these data showed that Cl-2TG mice were, contrary to expectation, protected against experimentally induced colitis.


Targeted colonic claudin-2 expression renders resistance to epithelial injury, induces immune suppression, and protects from colitis.

Ahmad R, Chaturvedi R, Olivares-Villagómez D, Habib T, Asim M, Shivesh P, Polk DB, Wilson KT, Washington MK, Van Kaer L, Dhawan P, Singh AB - Mucosal Immunol (2014)

Cl-2TG mice are protected from DSS-induced acute colitisTo induce colitis, mice received DSS (4% w/v) in the drinking water (10 days). (A). Weight loss during the course of DSS administration; (B). Disease activity index (DAI) changes among DSS-treated groups; C(i). Colon length (cm) in control and DSS-treated mice, C(ii). Representative colon images, and C(iii). Colon weight/cm; (D). Cumulative injury scores. Control mice did not show inflammation and associated injury; (E). Representative H&E staining of the colonic tissues from control and DSS-treated mice; (F). Representative photomicrographs demonstrating immunostaining to determine colonic myeloperoxidase (MPO) activity from control and DSS-treated mice. Values are presented as mean ± sem. *p<0.05, **p<0.01, ***p<0.001. Scale bars=500 or 50 μm.
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Figure 2: Cl-2TG mice are protected from DSS-induced acute colitisTo induce colitis, mice received DSS (4% w/v) in the drinking water (10 days). (A). Weight loss during the course of DSS administration; (B). Disease activity index (DAI) changes among DSS-treated groups; C(i). Colon length (cm) in control and DSS-treated mice, C(ii). Representative colon images, and C(iii). Colon weight/cm; (D). Cumulative injury scores. Control mice did not show inflammation and associated injury; (E). Representative H&E staining of the colonic tissues from control and DSS-treated mice; (F). Representative photomicrographs demonstrating immunostaining to determine colonic myeloperoxidase (MPO) activity from control and DSS-treated mice. Values are presented as mean ± sem. *p<0.05, **p<0.01, ***p<0.001. Scale bars=500 or 50 μm.
Mentions: Despite increased mucosal permeability, Cl-2TG mice did not have signs of mucosal inflammation. To examine whether Cl-2TG mice are more susceptible to colitis, Cl-2TG and WT mice were subjected to chemically-induced colitis by providing drinking water containing DSS. As expected, DSS-treated WT mice demonstrated significant body weight loss (p<0.01; Figure-2A). However, the DSS-induced weight loss was inhibited in Cl-2TG mice. The cumulative clinical disease index further supported protection of Cl-2TG mice against DSS colitis (Figure-2B). Consistent with these findings, colon length in DSS-treated WT mice was significantly decreased (p<0.001) compared with control mice {Figure-2C(i–iii)}, whereas this parameter was not significantly different between DSS-treated and control Cl-2TG mice. Independent evaluation by a gastrointestinal pathologist confirmed a significantly lower injury score (p<0.001) in DSS-treated Cl-2TG versus WT mice (Figure-2D). Importantly, depth of inflammation, crypt damage and % involved by crypt damage were all significantly different between DSS-treated WT and Cl-2TG mice (Figure S-2). Histological analysis further demonstrated marked preservation of the crypt architecture and decreased inflammation in DSS-treated Cl-2TG mice, as compared with WT mice where crypt architecture was largely disrupted and the severe loss of epithelia was accompanied by massive inflammation (Figure-2E). A marked decrease in myeloperoxidase (MPO)-activity in DSS-treated Cl-2TG mice further suggested decreased neutrophil infiltration in the colon (Figure-2F). Because our animals were generated on a B6D2F1/J background, and because colitis is influenced by the genetic background of the animals employed, we performed similar experiments with claudin-2 transgenic animals that were backcrossed more than 10 times with C57BL/6 mice, which demonstrated similar differences as those observed for the mixed background animals {Figure S-3(A–E)}. Taken together, these data showed that Cl-2TG mice were, contrary to expectation, protected against experimentally induced colitis.

Bottom Line: Importantly, claudin-2 expression increased colonocyte proliferation and provided protection against colitis-induced colonocyte death in a PI-3Kinase/Bcl-2-dependent manner.Importantly, these immunosuppressive changes were associated with increased synthesis of the immunoregulatory cytokine TGF-β by colonic epithelial cells in Cl-2TG mice compared with WT littermates.Taken together, our findings reveal a critical albeit complex role of claudin-2 in intestinal homeostasis by regulating epithelial permeability, inflammation and proliferation and suggest novel therapeutic opportunities.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, Vanderbilt University School of Medicine, Nashville, Tennessee, USA.

ABSTRACT
Expression of claudin-2, a tight junction protein, is highly upregulated during inflammatory bowel disease (IBD) and, due to its association with epithelial permeability, has been postulated to promote inflammation. Notably, claudin-2 has also been implicated in the regulation of intestinal epithelial proliferation. However, precise role of claudin-2 in regulating colonic homeostasis remains unclear. Here, we demonstrate, using Villin-Claudin-2 transgenic mice, that increased colonic claudin-2 expression augments mucosal permeability as well as colon and crypt length. Most notably, despite leaky colon, Cl-2TG mice were significantly protected against experimental colitis. Importantly, claudin-2 expression increased colonocyte proliferation and provided protection against colitis-induced colonocyte death in a PI-3Kinase/Bcl-2-dependent manner. However, Cl-2TG mice also demonstrated marked suppression of colitis-induced increases in immune activation and associated signaling, suggesting immune tolerance. Accordingly, colons from naive Cl-2TG mice harbored significantly increased numbers of regulatory (CD4(+)Foxp3(+)) T cells than WT littermates. Furthermore, macrophages isolated from Cl-2TG mouse colon exhibited immune anergy. Importantly, these immunosuppressive changes were associated with increased synthesis of the immunoregulatory cytokine TGF-β by colonic epithelial cells in Cl-2TG mice compared with WT littermates. Taken together, our findings reveal a critical albeit complex role of claudin-2 in intestinal homeostasis by regulating epithelial permeability, inflammation and proliferation and suggest novel therapeutic opportunities.

Show MeSH
Related in: MedlinePlus