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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: However, precise role of claudin-2 in regulating colonic homeostasis remains unclear.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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Colonic macrophages in Cl-2TG mice exhibit inflammatory anergy(A). Representative images demonstrating macrophage (F4/80) infiltration in the colonic mucosa in control and DSS-treated WT and Cl-2TG mice. F4/80+ cells were co-immunostained with anti-iNOS antibody; B(i–ii). Colonic or peritoneal macrophages were isolated from naïve WT and Cl-2TG mice and were subjected to immune activation using LPS and IFN-γ for 24 hours. mRNA expression of TNF-α and IL-6 was determined using qRT-PCR; B(iii). Supernatant from in vitro activated macrophages isolated from Cl-2TG and WT-mice was subjected to ELISA analysis using antigen-specific antibody. Values are presented as mean ± sem. **p<0.01, ***p<0.001. Scale bars=500 μm.
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Figure 8: Colonic macrophages in Cl-2TG mice exhibit inflammatory anergy(A). Representative images demonstrating macrophage (F4/80) infiltration in the colonic mucosa in control and DSS-treated WT and Cl-2TG mice. F4/80+ cells were co-immunostained with anti-iNOS antibody; B(i–ii). Colonic or peritoneal macrophages were isolated from naïve WT and Cl-2TG mice and were subjected to immune activation using LPS and IFN-γ for 24 hours. mRNA expression of TNF-α and IL-6 was determined using qRT-PCR; B(iii). Supernatant from in vitro activated macrophages isolated from Cl-2TG and WT-mice was subjected to ELISA analysis using antigen-specific antibody. Values are presented as mean ± sem. **p<0.01, ***p<0.001. Scale bars=500 μm.

Mentions: Our additional analysis further showed marked decreases in infiltrating F4/80+ cells in the colon of DSS-treated Cl-2TG versus WT mice. Expression of iNOS by F4/80+ cells supported their pro-inflammatory M1-phenotype (Figure-8A). Importantly, resistance to colitis and immune tolerance/adaptation in mice harboring a leakier colon similar to Cl-2TG mice has been reported18. Therefore, we asked whether this is also the case in Cl-2TG mice. Considering the primary role of macrophages in innate immune responses, we therefore analyzed whether immune responses of colonic macrophages in Cl-2TG mice are compromised. Since claudin-2 overexpression in Cl-2TG mice was restricted to the gut epithelium, immune function of peritoneal macrophages was not expected to be affected and therefore peritoneal macrophages isolated from the same animals were used as positive control (Figure S-6). The colonic and peritoneal macrophages from naïve WT and Cl-2TG mice were then stimulated with lipopolysaccharide (LPS, 1 μg/ml) and IFN-γ (100 IU/ml). As shown in Figure-8B(i–ii), IL-6 and TNF-α mRNA expression were upregulated significantly in activated peritoneal macrophages isolated from WT and Cl-2TG mice. Interestingly, however, expression of these mRNAs was upregulated only in the colonic macrophages from WT but not Cl-2TG mice. ELISA analysis demonstrated similar upregulation of inflammatory cytokines, including IL-6, IL-1β and KC only in WT colonic macrophages [Figure-8B(iii)]. Thus, our data suggested inflammatory anergy among macrophages residing in the colonic submucosa of Cl-2TG mice.


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)

Colonic macrophages in Cl-2TG mice exhibit inflammatory anergy(A). Representative images demonstrating macrophage (F4/80) infiltration in the colonic mucosa in control and DSS-treated WT and Cl-2TG mice. F4/80+ cells were co-immunostained with anti-iNOS antibody; B(i–ii). Colonic or peritoneal macrophages were isolated from naïve WT and Cl-2TG mice and were subjected to immune activation using LPS and IFN-γ for 24 hours. mRNA expression of TNF-α and IL-6 was determined using qRT-PCR; B(iii). Supernatant from in vitro activated macrophages isolated from Cl-2TG and WT-mice was subjected to ELISA analysis using antigen-specific antibody. Values are presented as mean ± sem. **p<0.01, ***p<0.001. Scale bars=500 μm.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4221190&req=5

Figure 8: Colonic macrophages in Cl-2TG mice exhibit inflammatory anergy(A). Representative images demonstrating macrophage (F4/80) infiltration in the colonic mucosa in control and DSS-treated WT and Cl-2TG mice. F4/80+ cells were co-immunostained with anti-iNOS antibody; B(i–ii). Colonic or peritoneal macrophages were isolated from naïve WT and Cl-2TG mice and were subjected to immune activation using LPS and IFN-γ for 24 hours. mRNA expression of TNF-α and IL-6 was determined using qRT-PCR; B(iii). Supernatant from in vitro activated macrophages isolated from Cl-2TG and WT-mice was subjected to ELISA analysis using antigen-specific antibody. Values are presented as mean ± sem. **p<0.01, ***p<0.001. Scale bars=500 μm.
Mentions: Our additional analysis further showed marked decreases in infiltrating F4/80+ cells in the colon of DSS-treated Cl-2TG versus WT mice. Expression of iNOS by F4/80+ cells supported their pro-inflammatory M1-phenotype (Figure-8A). Importantly, resistance to colitis and immune tolerance/adaptation in mice harboring a leakier colon similar to Cl-2TG mice has been reported18. Therefore, we asked whether this is also the case in Cl-2TG mice. Considering the primary role of macrophages in innate immune responses, we therefore analyzed whether immune responses of colonic macrophages in Cl-2TG mice are compromised. Since claudin-2 overexpression in Cl-2TG mice was restricted to the gut epithelium, immune function of peritoneal macrophages was not expected to be affected and therefore peritoneal macrophages isolated from the same animals were used as positive control (Figure S-6). The colonic and peritoneal macrophages from naïve WT and Cl-2TG mice were then stimulated with lipopolysaccharide (LPS, 1 μg/ml) and IFN-γ (100 IU/ml). As shown in Figure-8B(i–ii), IL-6 and TNF-α mRNA expression were upregulated significantly in activated peritoneal macrophages isolated from WT and Cl-2TG mice. Interestingly, however, expression of these mRNAs was upregulated only in the colonic macrophages from WT but not Cl-2TG mice. ELISA analysis demonstrated similar upregulation of inflammatory cytokines, including IL-6, IL-1β and KC only in WT colonic macrophages [Figure-8B(iii)]. Thus, our data suggested inflammatory anergy among macrophages residing in the colonic submucosa of Cl-2TG mice.

Bottom Line: However, precise role of claudin-2 in regulating colonic homeostasis remains unclear.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