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Guanylate cyclase C deficiency causes severe inflammation in a murine model of spontaneous colitis.

Harmel-Laws E, Mann EA, Cohen MB, Steinbrecher KA - PLoS ONE (2013)

Bottom Line: Spontaneous colitis in GC-C(-/-)IL-10(-/-) animals was significantly more severe relative to GC-C(+/+)IL-10(-/-) mice.F4/80 and myeloperoxidase positive cells as well as proinflammatory gene expression were elevated in GC-C(-/-)IL-10(-/-) mucosa relative to control animals.These data as well as the apparent intestinal inflammation in human GC-C mutant kindred underscore the importance of GC-C in regulating the response to injury and inflammation within the gut.

View Article: PubMed Central - PubMed

Affiliation: Division of Gastroenterology, Hepatology, and Nutrition, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, United States of America.

ABSTRACT

Background: Guanylate Cyclase C (GC-C; Gucy2c) is a transmembrane receptor expressed in intestinal epithelial cells. Activation of GC-C by its secreted ligand guanylin stimulates intestinal fluid secretion. Familial mutations in GC-C cause chronic diarrheal disease or constipation and are associated with intestinal inflammation and infection. Here, we investigated the impact of GC-C activity on mucosal immune responses.

Methods: We utilized intraperitoneal injection of lipopolysaccharide to elicit a systemic cytokine challenge and then measured pro-inflammatory gene expression in colonic mucosa. GC-C(+/+) and GC-C(-/-) mice were bred with interleukin (IL)-10 deficient animals and colonic inflammation were assessed. Immune cell influx and cytokine/chemokine expression was measured in the colon of wildtype, IL-10(-/-), GC-C(+/+)IL-10(-/-) and GC-C(-/-)IL-10(-/-) mice. GC-C and guanylin production were examined in the colon of these animals and in a cytokine-treated colon epithelial cell line.

Results: Relative to GC-C(+/+) animals, intraperitoneal lipopolysaccharide injection into GC-C(-/-) mice increased proinflammatory gene expression in both whole colon tissue and in partially purified colonocyte isolations. Spontaneous colitis in GC-C(-/-)IL-10(-/-) animals was significantly more severe relative to GC-C(+/+)IL-10(-/-) mice. Unlike GC-C(+/+)IL-10(-/-) controls, colon pathology in GC-C(-/-)IL-10(-/-) animals was apparent at an early age and was characterized by severely altered mucosal architecture, crypt abscesses, and hyperplastic subepithelial lesions. F4/80 and myeloperoxidase positive cells as well as proinflammatory gene expression were elevated in GC-C(-/-)IL-10(-/-) mucosa relative to control animals. Guanylin was diminished early in colitis in vivo and tumor necrosis factor α suppressed guanylin mRNA and protein in intestinal goblet cell-like HT29-18-N2 cells.

Conclusions: The GC-C signaling pathway blunts colonic mucosal inflammation that is initiated by systemic cytokine burst or loss of mucosal immune cell immunosuppression. These data as well as the apparent intestinal inflammation in human GC-C mutant kindred underscore the importance of GC-C in regulating the response to injury and inflammation within the gut.

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GC-C−/−IL-10−/− mice have severe colitis and pre-cancerous lesions at 8–10 weeks of age.(A) While mice lacking IL-10 alone had moderate inflammatory disease at 8–10 weeks of age, GCC−/−IL-10−/− were severely affected and had clear indication of progression toward adenocarcinoma (inset). (B) Disease scores confirm that there is significantly more pathology in GC-C/IL-10 double knockout mice. n = 8–14 mice per group; *p<0.03.
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pone-0079180-g002: GC-C−/−IL-10−/− mice have severe colitis and pre-cancerous lesions at 8–10 weeks of age.(A) While mice lacking IL-10 alone had moderate inflammatory disease at 8–10 weeks of age, GCC−/−IL-10−/− were severely affected and had clear indication of progression toward adenocarcinoma (inset). (B) Disease scores confirm that there is significantly more pathology in GC-C/IL-10 double knockout mice. n = 8–14 mice per group; *p<0.03.

Mentions: We bred mice deficient in GC-C with IL-10−/− animals and developed GC-C−/−IL-10−/− and control GC-C+/+IL-10−/− mice that were >10 generations into the C57BL/6J genetic background. As expected, we found no indication of inflammation in wildtype or GC-C−/− colon. IL-10−/− mice had colitis that was mild by 6 weeks of age in CCHMC specific pathogen free vivarium. However, we found that loss of GC-C results in the accelerated appearance of colitis in IL-10−/− animals. Compound knockout GC-C−/−IL-10−/− mice presented with obvious signs of gastrointestinal disease, including diarrhea and rectal prolapse, by 6 weeks of age. Histological analysis indicated that GC-C−/−IL-10−/− animals had severe epithelial hyperplasia and apoptosis, frequent crypt abscesses, and significant mixed inflammatory infiltrate (Figure 1A). Crypt-surface architecture was massively disrupted in these mice and transmural inflammation was common. Histological disease scoring confirmed that mice lacking both GC-C and IL-10 develop more severe disease by six weeks of age (Figure 1B). We also analyzed 8–10 week old mice and found, as expected, that GC-C−/−IL-10−/− mice continued to have more significant disease as compared to IL-10−/− mice (Figure 2A). Notably, in addition to profound transmural inflammation and disruption of normal epithelial crypt-surface placement, many of the more severely affected GC-C−/−IL-10−/− mice had developed clear indications of inflammation-associated epithelial transformation and progression toward adenocarcinoma with frequent gland penetration into the intestinal musculature (Figure 2A, inset). IL-10−/− mice of this age did not display similar precancerous lesions. Disease scoring clearly indicated that GC-C−/− mice that lack IL-10 have significantly more intestinal inflammation as compared to animals lacking only IL-10 (Figure 2B).


Guanylate cyclase C deficiency causes severe inflammation in a murine model of spontaneous colitis.

Harmel-Laws E, Mann EA, Cohen MB, Steinbrecher KA - PLoS ONE (2013)

GC-C−/−IL-10−/− mice have severe colitis and pre-cancerous lesions at 8–10 weeks of age.(A) While mice lacking IL-10 alone had moderate inflammatory disease at 8–10 weeks of age, GCC−/−IL-10−/− were severely affected and had clear indication of progression toward adenocarcinoma (inset). (B) Disease scores confirm that there is significantly more pathology in GC-C/IL-10 double knockout mice. n = 8–14 mice per group; *p<0.03.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3823613&req=5

pone-0079180-g002: GC-C−/−IL-10−/− mice have severe colitis and pre-cancerous lesions at 8–10 weeks of age.(A) While mice lacking IL-10 alone had moderate inflammatory disease at 8–10 weeks of age, GCC−/−IL-10−/− were severely affected and had clear indication of progression toward adenocarcinoma (inset). (B) Disease scores confirm that there is significantly more pathology in GC-C/IL-10 double knockout mice. n = 8–14 mice per group; *p<0.03.
Mentions: We bred mice deficient in GC-C with IL-10−/− animals and developed GC-C−/−IL-10−/− and control GC-C+/+IL-10−/− mice that were >10 generations into the C57BL/6J genetic background. As expected, we found no indication of inflammation in wildtype or GC-C−/− colon. IL-10−/− mice had colitis that was mild by 6 weeks of age in CCHMC specific pathogen free vivarium. However, we found that loss of GC-C results in the accelerated appearance of colitis in IL-10−/− animals. Compound knockout GC-C−/−IL-10−/− mice presented with obvious signs of gastrointestinal disease, including diarrhea and rectal prolapse, by 6 weeks of age. Histological analysis indicated that GC-C−/−IL-10−/− animals had severe epithelial hyperplasia and apoptosis, frequent crypt abscesses, and significant mixed inflammatory infiltrate (Figure 1A). Crypt-surface architecture was massively disrupted in these mice and transmural inflammation was common. Histological disease scoring confirmed that mice lacking both GC-C and IL-10 develop more severe disease by six weeks of age (Figure 1B). We also analyzed 8–10 week old mice and found, as expected, that GC-C−/−IL-10−/− mice continued to have more significant disease as compared to IL-10−/− mice (Figure 2A). Notably, in addition to profound transmural inflammation and disruption of normal epithelial crypt-surface placement, many of the more severely affected GC-C−/−IL-10−/− mice had developed clear indications of inflammation-associated epithelial transformation and progression toward adenocarcinoma with frequent gland penetration into the intestinal musculature (Figure 2A, inset). IL-10−/− mice of this age did not display similar precancerous lesions. Disease scoring clearly indicated that GC-C−/− mice that lack IL-10 have significantly more intestinal inflammation as compared to animals lacking only IL-10 (Figure 2B).

Bottom Line: Spontaneous colitis in GC-C(-/-)IL-10(-/-) animals was significantly more severe relative to GC-C(+/+)IL-10(-/-) mice.F4/80 and myeloperoxidase positive cells as well as proinflammatory gene expression were elevated in GC-C(-/-)IL-10(-/-) mucosa relative to control animals.These data as well as the apparent intestinal inflammation in human GC-C mutant kindred underscore the importance of GC-C in regulating the response to injury and inflammation within the gut.

View Article: PubMed Central - PubMed

Affiliation: Division of Gastroenterology, Hepatology, and Nutrition, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, United States of America.

ABSTRACT

Background: Guanylate Cyclase C (GC-C; Gucy2c) is a transmembrane receptor expressed in intestinal epithelial cells. Activation of GC-C by its secreted ligand guanylin stimulates intestinal fluid secretion. Familial mutations in GC-C cause chronic diarrheal disease or constipation and are associated with intestinal inflammation and infection. Here, we investigated the impact of GC-C activity on mucosal immune responses.

Methods: We utilized intraperitoneal injection of lipopolysaccharide to elicit a systemic cytokine challenge and then measured pro-inflammatory gene expression in colonic mucosa. GC-C(+/+) and GC-C(-/-) mice were bred with interleukin (IL)-10 deficient animals and colonic inflammation were assessed. Immune cell influx and cytokine/chemokine expression was measured in the colon of wildtype, IL-10(-/-), GC-C(+/+)IL-10(-/-) and GC-C(-/-)IL-10(-/-) mice. GC-C and guanylin production were examined in the colon of these animals and in a cytokine-treated colon epithelial cell line.

Results: Relative to GC-C(+/+) animals, intraperitoneal lipopolysaccharide injection into GC-C(-/-) mice increased proinflammatory gene expression in both whole colon tissue and in partially purified colonocyte isolations. Spontaneous colitis in GC-C(-/-)IL-10(-/-) animals was significantly more severe relative to GC-C(+/+)IL-10(-/-) mice. Unlike GC-C(+/+)IL-10(-/-) controls, colon pathology in GC-C(-/-)IL-10(-/-) animals was apparent at an early age and was characterized by severely altered mucosal architecture, crypt abscesses, and hyperplastic subepithelial lesions. F4/80 and myeloperoxidase positive cells as well as proinflammatory gene expression were elevated in GC-C(-/-)IL-10(-/-) mucosa relative to control animals. Guanylin was diminished early in colitis in vivo and tumor necrosis factor α suppressed guanylin mRNA and protein in intestinal goblet cell-like HT29-18-N2 cells.

Conclusions: The GC-C signaling pathway blunts colonic mucosal inflammation that is initiated by systemic cytokine burst or loss of mucosal immune cell immunosuppression. These data as well as the apparent intestinal inflammation in human GC-C mutant kindred underscore the importance of GC-C in regulating the response to injury and inflammation within the gut.

Show MeSH
Related in: MedlinePlus