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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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Guanylin production is nearly absent during colitis associated with IL-10 deficiency.(A) GC-C mRNA is not affected by intestinal inflammation, as measured by realtime RT-PCR. n = 5–6 per group (B) Guanylin gene expression is highly reduced in GC-C−/−IL-10−/− colon. n = 5 per group; *p<0.005 (C) Western blotting indicates that guanylin protein expression is suppressed by colonic inflammation in both IL-10−/− and GC-C−/−IL-10−/− mice. On this representative blot, each lane represents a sample from an individual mouse. β-tubulin is shown to demonstrate equal loading within each lane.
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pone-0079180-g006: Guanylin production is nearly absent during colitis associated with IL-10 deficiency.(A) GC-C mRNA is not affected by intestinal inflammation, as measured by realtime RT-PCR. n = 5–6 per group (B) Guanylin gene expression is highly reduced in GC-C−/−IL-10−/− colon. n = 5 per group; *p<0.005 (C) Western blotting indicates that guanylin protein expression is suppressed by colonic inflammation in both IL-10−/− and GC-C−/−IL-10−/− mice. On this representative blot, each lane represents a sample from an individual mouse. β-tubulin is shown to demonstrate equal loading within each lane.

Mentions: Recent studies indicate that familial polymorphisms in the GC-C gene may be important for susceptibility to intestinal disorders including intestinal inflammation [8], [10]. Our data in GC-C−/−IL-10−/− mice indicates that loss of GC-C signaling influences the timing and severity of immune cell-mediated colitis. We next investigated whether the converse was also true, that is, if intestinal inflammation influenced the expression of GC-C or its primary colonic ligand, guanylin. Relative to wildtype mice, realtime RT-PCR indicated that GC-C expression was not changed in adult IL-10−/− mice with active colitis (Figure 6A). Guanylin is produced in goblet cells of the intestine and goblet cell depletion often accompanies the latter, more severe stages of intestinal inflammation. IL-10−/− mice which had moderate colitis and only slight goblet cell loss had significantly diminished guanylin mRNA levels (Figure 6B). As expected, guanylin expression was greatly reduced in severely affected GC-C−/−IL-10−/− animals that had significant goblet cell depletion. Diminished guanylin production was even more evident in the analysis of colonic protein extracts using western blotting which demonstrated substantial loss of guanylin protein in both IL-10−/− as well as GC-C−/−IL-10−/− colon (Figure 6C). We speculate that the progressive loss of guanylin during inflammation leads to diminishing activation of GC-C and that this may be linked to disease severity in spontaneous models of murine colitis.


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)

Guanylin production is nearly absent during colitis associated with IL-10 deficiency.(A) GC-C mRNA is not affected by intestinal inflammation, as measured by realtime RT-PCR. n = 5–6 per group (B) Guanylin gene expression is highly reduced in GC-C−/−IL-10−/− colon. n = 5 per group; *p<0.005 (C) Western blotting indicates that guanylin protein expression is suppressed by colonic inflammation in both IL-10−/− and GC-C−/−IL-10−/− mice. On this representative blot, each lane represents a sample from an individual mouse. β-tubulin is shown to demonstrate equal loading within each lane.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3823613&req=5

pone-0079180-g006: Guanylin production is nearly absent during colitis associated with IL-10 deficiency.(A) GC-C mRNA is not affected by intestinal inflammation, as measured by realtime RT-PCR. n = 5–6 per group (B) Guanylin gene expression is highly reduced in GC-C−/−IL-10−/− colon. n = 5 per group; *p<0.005 (C) Western blotting indicates that guanylin protein expression is suppressed by colonic inflammation in both IL-10−/− and GC-C−/−IL-10−/− mice. On this representative blot, each lane represents a sample from an individual mouse. β-tubulin is shown to demonstrate equal loading within each lane.
Mentions: Recent studies indicate that familial polymorphisms in the GC-C gene may be important for susceptibility to intestinal disorders including intestinal inflammation [8], [10]. Our data in GC-C−/−IL-10−/− mice indicates that loss of GC-C signaling influences the timing and severity of immune cell-mediated colitis. We next investigated whether the converse was also true, that is, if intestinal inflammation influenced the expression of GC-C or its primary colonic ligand, guanylin. Relative to wildtype mice, realtime RT-PCR indicated that GC-C expression was not changed in adult IL-10−/− mice with active colitis (Figure 6A). Guanylin is produced in goblet cells of the intestine and goblet cell depletion often accompanies the latter, more severe stages of intestinal inflammation. IL-10−/− mice which had moderate colitis and only slight goblet cell loss had significantly diminished guanylin mRNA levels (Figure 6B). As expected, guanylin expression was greatly reduced in severely affected GC-C−/−IL-10−/− animals that had significant goblet cell depletion. Diminished guanylin production was even more evident in the analysis of colonic protein extracts using western blotting which demonstrated substantial loss of guanylin protein in both IL-10−/− as well as GC-C−/−IL-10−/− colon (Figure 6C). We speculate that the progressive loss of guanylin during inflammation leads to diminishing activation of GC-C and that this may be linked to disease severity in spontaneous models of murine colitis.

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