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Intestinal epithelial serum amyloid A modulates bacterial growth in vitro and pro-inflammatory responses in mouse experimental colitis.

Eckhardt ER, Witta J, Zhong J, Arsenescu R, Arsenescu V, Wang Y, Ghoshal S, de Beer MC, de Beer FC, de Villiers WJ - BMC Gastroenterol (2010)

Bottom Line: Overexpression of SAA1/2 in cultured epithelial cell lines reduced the viability of co-cultured E. coli.SAA1/2 expression was increased in colon samples obtained from Crohn's Disease patients compared to controls.Altered expression of SAA in intestinal biopsies from Crohn's Disease patients suggests that SAA is involved in the disease process..

View Article: PubMed Central - HTML - PubMed

Affiliation: Graduate Center for Nutritional Sciences, University of Kentucky, Lexington, KY, USA. erik.eckhardt@uky.edu

ABSTRACT

Background: Serum Amyloid A (SAA) is a major acute phase protein of unknown function. SAA is mostly expressed in the liver, but also in other tissues including the intestinal epithelium. SAA reportedly has anti-bacterial effects, and because inflammatory bowel diseases (IBD) result from a breakdown in homeostatic interactions between intestinal epithelia and bacteria, we hypothesized that SAA is protective during experimental colitis.

Methods: Intestinal SAA expression was measured in mouse and human samples. Dextran sodium sulfate (DSS) colitis was induced in SAA 1/2 double knockout (DKO) mice and in wildtype controls. Anti-bacterial effects of SAA1/2 were tested in intestinal epithelial cell lines transduced with adenoviral vectors encoding the CE/J SAA isoform or control vectors prior to exposure to live Escherichia coli.

Results: Significant levels of SAA1/SAA2 RNA and SAA protein were detected by in situ hybridization and immunohistochemistry in mouse colonic epithelium. SAA3 expression was weaker, but similarly distributed. SAA1/2 RNA was present in the ileum and colon of conventional mice and in the colon of germfree mice. Expression of SAA3 was strongly regulated by bacterial lipopolysaccharides in cultured epithelial cell lines, whereas SAA1/2 expression was constitutive and not LPS inducible. Overexpression of SAA1/2 in cultured epithelial cell lines reduced the viability of co-cultured E. coli. This might partially explain the observed increase in susceptibility of DKO mice to DSS colitis. SAA1/2 expression was increased in colon samples obtained from Crohn's Disease patients compared to controls.

Conclusions: Intestinal epithelial SAA displays bactericidal properties in vitro and could play a protective role in experimental mouse colitis. Altered expression of SAA in intestinal biopsies from Crohn's Disease patients suggests that SAA is involved in the disease process..

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Intestinal SAA expression and secretion. SAA1/2 RNA, represented by the black signal in A and E, or the white signal in the counter-stained image B, was readily detectable in cross-sections of colonic epithelia (A, B), and was mainly located at the villous tips (E). SAA3 message showed similar distribution, but was much weaker (black signal in C, white in counter-stained image D). SAA immunostaining (green signal) showed a more diffuse pattern than SAA RNA, with SAA protein expressed along the crypt-villus axis (F), perhaps reflecting secretion of SAA by intestinal epithelial cells into the apical and basolateral milieu. A,C and E represent bright-field images, B and D dark-field images.
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Figure 1: Intestinal SAA expression and secretion. SAA1/2 RNA, represented by the black signal in A and E, or the white signal in the counter-stained image B, was readily detectable in cross-sections of colonic epithelia (A, B), and was mainly located at the villous tips (E). SAA3 message showed similar distribution, but was much weaker (black signal in C, white in counter-stained image D). SAA immunostaining (green signal) showed a more diffuse pattern than SAA RNA, with SAA protein expressed along the crypt-villus axis (F), perhaps reflecting secretion of SAA by intestinal epithelial cells into the apical and basolateral milieu. A,C and E represent bright-field images, B and D dark-field images.

Mentions: Sections of mouse colon were incubated with an RNA probe recognizing both Saa1 and -2 to determine whether SAA is expressed in mouse intestinal epithelium. Strong staining was observed at the level of the epithelium (Figure 1A,B), with most of the signal present at the tip of the villi (Figure 1E). Expression of the SAA3 isoform was weaker, but overlapped with the expression of SAA1/2 (Figure 1C,D). Immunostaining confirmed the presence of SAA in the mouse intestine, though the pattern was more diffuse (Figure 1F), perhaps consistent with release of SAA into apical and basolateral sides of the epithelium. To test whether SAA was indeed apically secreted into the lumen, extracts of fecal excrements from wildtype and SAA-knockout mice were analyzed by immunoblotting after SDS-polyacrylamide electrophoresis. As shown in Figure 2B, immunoreactivity was indeed observed in fecal extracts, consistent with secretion of SAA into the gut. Thus, SAA is expressed in the intestinal epithelium, and is secreted into the gut lumen.


Intestinal epithelial serum amyloid A modulates bacterial growth in vitro and pro-inflammatory responses in mouse experimental colitis.

Eckhardt ER, Witta J, Zhong J, Arsenescu R, Arsenescu V, Wang Y, Ghoshal S, de Beer MC, de Beer FC, de Villiers WJ - BMC Gastroenterol (2010)

Intestinal SAA expression and secretion. SAA1/2 RNA, represented by the black signal in A and E, or the white signal in the counter-stained image B, was readily detectable in cross-sections of colonic epithelia (A, B), and was mainly located at the villous tips (E). SAA3 message showed similar distribution, but was much weaker (black signal in C, white in counter-stained image D). SAA immunostaining (green signal) showed a more diffuse pattern than SAA RNA, with SAA protein expressed along the crypt-villus axis (F), perhaps reflecting secretion of SAA by intestinal epithelial cells into the apical and basolateral milieu. A,C and E represent bright-field images, B and D dark-field images.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2992040&req=5

Figure 1: Intestinal SAA expression and secretion. SAA1/2 RNA, represented by the black signal in A and E, or the white signal in the counter-stained image B, was readily detectable in cross-sections of colonic epithelia (A, B), and was mainly located at the villous tips (E). SAA3 message showed similar distribution, but was much weaker (black signal in C, white in counter-stained image D). SAA immunostaining (green signal) showed a more diffuse pattern than SAA RNA, with SAA protein expressed along the crypt-villus axis (F), perhaps reflecting secretion of SAA by intestinal epithelial cells into the apical and basolateral milieu. A,C and E represent bright-field images, B and D dark-field images.
Mentions: Sections of mouse colon were incubated with an RNA probe recognizing both Saa1 and -2 to determine whether SAA is expressed in mouse intestinal epithelium. Strong staining was observed at the level of the epithelium (Figure 1A,B), with most of the signal present at the tip of the villi (Figure 1E). Expression of the SAA3 isoform was weaker, but overlapped with the expression of SAA1/2 (Figure 1C,D). Immunostaining confirmed the presence of SAA in the mouse intestine, though the pattern was more diffuse (Figure 1F), perhaps consistent with release of SAA into apical and basolateral sides of the epithelium. To test whether SAA was indeed apically secreted into the lumen, extracts of fecal excrements from wildtype and SAA-knockout mice were analyzed by immunoblotting after SDS-polyacrylamide electrophoresis. As shown in Figure 2B, immunoreactivity was indeed observed in fecal extracts, consistent with secretion of SAA into the gut. Thus, SAA is expressed in the intestinal epithelium, and is secreted into the gut lumen.

Bottom Line: Overexpression of SAA1/2 in cultured epithelial cell lines reduced the viability of co-cultured E. coli.SAA1/2 expression was increased in colon samples obtained from Crohn's Disease patients compared to controls.Altered expression of SAA in intestinal biopsies from Crohn's Disease patients suggests that SAA is involved in the disease process..

View Article: PubMed Central - HTML - PubMed

Affiliation: Graduate Center for Nutritional Sciences, University of Kentucky, Lexington, KY, USA. erik.eckhardt@uky.edu

ABSTRACT

Background: Serum Amyloid A (SAA) is a major acute phase protein of unknown function. SAA is mostly expressed in the liver, but also in other tissues including the intestinal epithelium. SAA reportedly has anti-bacterial effects, and because inflammatory bowel diseases (IBD) result from a breakdown in homeostatic interactions between intestinal epithelia and bacteria, we hypothesized that SAA is protective during experimental colitis.

Methods: Intestinal SAA expression was measured in mouse and human samples. Dextran sodium sulfate (DSS) colitis was induced in SAA 1/2 double knockout (DKO) mice and in wildtype controls. Anti-bacterial effects of SAA1/2 were tested in intestinal epithelial cell lines transduced with adenoviral vectors encoding the CE/J SAA isoform or control vectors prior to exposure to live Escherichia coli.

Results: Significant levels of SAA1/SAA2 RNA and SAA protein were detected by in situ hybridization and immunohistochemistry in mouse colonic epithelium. SAA3 expression was weaker, but similarly distributed. SAA1/2 RNA was present in the ileum and colon of conventional mice and in the colon of germfree mice. Expression of SAA3 was strongly regulated by bacterial lipopolysaccharides in cultured epithelial cell lines, whereas SAA1/2 expression was constitutive and not LPS inducible. Overexpression of SAA1/2 in cultured epithelial cell lines reduced the viability of co-cultured E. coli. This might partially explain the observed increase in susceptibility of DKO mice to DSS colitis. SAA1/2 expression was increased in colon samples obtained from Crohn's Disease patients compared to controls.

Conclusions: Intestinal epithelial SAA displays bactericidal properties in vitro and could play a protective role in experimental mouse colitis. Altered expression of SAA in intestinal biopsies from Crohn's Disease patients suggests that SAA is involved in the disease process..

Show MeSH
Related in: MedlinePlus