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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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SAA expression in mouse colon and ileum and in response to LPS. RNA was isolated from the colon and ileum of germfree or conventionally raised mice, and cDNA was amplified with oligonucleotides recognizing a common region of SAA1/2 (A). In conventional mice, SAA1/2 was detected in colon and ileum, whereas in germfree mice, SAA1/2 was only detectable in the colon. In (B), CMT93 cells were incubated with the indicated amounts of LPS for 16 h, and SAA1/2 and SAA3 expression were determined with realtime PCR. Whereas SAA1/2 message remained rather constant, SAA3 expression markedly increased with increased LPS exposure. Each group contained data on 4 wells. Shown are average ± S.E.M. The asterisk indicates statistically significant differences (P < 0.05) compared with un-induced cells (ANOVA).
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Figure 3: SAA expression in mouse colon and ileum and in response to LPS. RNA was isolated from the colon and ileum of germfree or conventionally raised mice, and cDNA was amplified with oligonucleotides recognizing a common region of SAA1/2 (A). In conventional mice, SAA1/2 was detected in colon and ileum, whereas in germfree mice, SAA1/2 was only detectable in the colon. In (B), CMT93 cells were incubated with the indicated amounts of LPS for 16 h, and SAA1/2 and SAA3 expression were determined with realtime PCR. Whereas SAA1/2 message remained rather constant, SAA3 expression markedly increased with increased LPS exposure. Each group contained data on 4 wells. Shown are average ± S.E.M. The asterisk indicates statistically significant differences (P < 0.05) compared with un-induced cells (ANOVA).

Mentions: To confirm intestinal expression of SAA, RNA was extracted from intestinal segments, and cDNA was analyzed by PCR with primers recognizing a common fragment of mRNA encoded by Saa1 and 2. As shown in Figure 3A, strong signal was detected in the colon, with weaker signals in the ileum. This finding could suggest a link between bacterial load and SAA expression. To test this possibility, intestinal tissue was obtained from germfree mice and analyzed for expression of SAA1/2. Surprisingly, whereas expression was nearly absent from the ileum, significant message was still detectable in the colons of germfree mice, suggesting that colonic expression is constitutive and may be independent of the presence of bacterial factors. To further test regulation of SAA expression in intestinal epithelial cells as a function of bacterial load, we used CMT93 cells, a mouse rectal epithelial line. Whereas SAA1/2 expression did not increase with increasing exposure to LPS and was already high without added LPS, SAA3 expression appeared to be strongly inducible (Figure 3B).


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)

SAA expression in mouse colon and ileum and in response to LPS. RNA was isolated from the colon and ileum of germfree or conventionally raised mice, and cDNA was amplified with oligonucleotides recognizing a common region of SAA1/2 (A). In conventional mice, SAA1/2 was detected in colon and ileum, whereas in germfree mice, SAA1/2 was only detectable in the colon. In (B), CMT93 cells were incubated with the indicated amounts of LPS for 16 h, and SAA1/2 and SAA3 expression were determined with realtime PCR. Whereas SAA1/2 message remained rather constant, SAA3 expression markedly increased with increased LPS exposure. Each group contained data on 4 wells. Shown are average ± S.E.M. The asterisk indicates statistically significant differences (P < 0.05) compared with un-induced cells (ANOVA).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: SAA expression in mouse colon and ileum and in response to LPS. RNA was isolated from the colon and ileum of germfree or conventionally raised mice, and cDNA was amplified with oligonucleotides recognizing a common region of SAA1/2 (A). In conventional mice, SAA1/2 was detected in colon and ileum, whereas in germfree mice, SAA1/2 was only detectable in the colon. In (B), CMT93 cells were incubated with the indicated amounts of LPS for 16 h, and SAA1/2 and SAA3 expression were determined with realtime PCR. Whereas SAA1/2 message remained rather constant, SAA3 expression markedly increased with increased LPS exposure. Each group contained data on 4 wells. Shown are average ± S.E.M. The asterisk indicates statistically significant differences (P < 0.05) compared with un-induced cells (ANOVA).
Mentions: To confirm intestinal expression of SAA, RNA was extracted from intestinal segments, and cDNA was analyzed by PCR with primers recognizing a common fragment of mRNA encoded by Saa1 and 2. As shown in Figure 3A, strong signal was detected in the colon, with weaker signals in the ileum. This finding could suggest a link between bacterial load and SAA expression. To test this possibility, intestinal tissue was obtained from germfree mice and analyzed for expression of SAA1/2. Surprisingly, whereas expression was nearly absent from the ileum, significant message was still detectable in the colons of germfree mice, suggesting that colonic expression is constitutive and may be independent of the presence of bacterial factors. To further test regulation of SAA expression in intestinal epithelial cells as a function of bacterial load, we used CMT93 cells, a mouse rectal epithelial line. Whereas SAA1/2 expression did not increase with increasing exposure to LPS and was already high without added LPS, SAA3 expression appeared to be strongly inducible (Figure 3B).

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