Effects of BDL and GW4064 on serum bilirubin concentrations and FXR-regulated genes. ( A ) Total bilirubin was measured in serum from WT and FXR-KO mice ( n ϭ 5–9) subjected to sham operation and vehicle (Veh) treatment, BDL and vehicle treatment, or BDL and GW4064 (GW) treatment. The presence of different lowercase letters indicates statistical significance ( P Ͻ 0.01) within each strain. ( B – H ) Total RNA was prepared from ileum of WT and FXR-KO mice ( n ϭ 5–9) subjected to sham operation and vehicle (Veh) treatment, BDL and vehicle treatment, or BDL and GW4064 (GW) treatment. Expression of the indicated genes was measured by RTQ-PCR by using cyclophilin as the internal control. Data represent the mean Ϯ SEM and are plotted as fold change relative to mRNA levels in sham-operated mice treated with vehicle. The presence of different lowercase letters indicates statistical significance ( P Ͻ 0.05) within each strain. 

Contexts in source publication

Context 1

... the ileum and cecum. In WT mice, BDL resulted in an increase in bacterial number in the mesenteric lymph node complex, and GW4064 treatment completely inhibited this effect. The effect was particularly pronounced for aerobic bacteria (Fig. 3 E ). FXR-KO mice had Ϸ 10-fold higher levels of aerobic bacteria in mesenteric lymph nodes as com- pared with WT animals (Fig. 3 E ; note differences in scales for WT and FXR-KO mice), and BDL caused a trend toward increased bacterial counts (Fig. 3 E and F ). This trend was not reversed by GW4064 administration (Fig. 3 E and F ). The increase in in vivo bacterial translocation suggested that BDL causes a disruption of the intestinal barrier function that is inhibited by FXR. To examine barrier integrity, immunostaining was done for occludin, a component of the epithelial tight junctions. In the villus epithelium of WT mice, occludin staining was continuous except where interrupted by the normal presence of mucus-secreting goblet cells (Fig. 4 A ). In contrast, many enterocytes in the BDL mice expressed little or no occludin (Fig. 4 A ). Consistent with these data, transmission electron microscopy revealed large ruptures in the mucosal surface of BDL WT mice, with bacteria penetrating deep into the epithelium (Fig. 4 C and D ). Hematoxylin and eosin staining revealed that the breakdown in barrier function was accompanied by dilated lymphatics and a pronounced interstitial edema, as previously described (Fig. 4 B ) (3, 25, 26, 34). In many villi, the severity of the edema caused separation of the villus epithelium from the underlying lamina propria (Fig. 4 B ). Bacteria were seen within edematous areas by electron microscopy (Fig. 4 D ). Consistent with the edema resulting from an inflammatory response, there was a marked increase in the number of neutrophils infiltrating the villi of BDL mice (Fig. 4 G ). Neutrophils are known to contribute to epithelial permeability in inflammatory diseases of the intestine (35). GW4064 prevented the pathological effects caused by BDL. Ilea from BDL mice administered GW4064 showed a more continuous pattern of occludin immunostaining (Fig. 4 A ), little or no edema (Fig. 4 B ), and a reduction in the number of neutrophils to the level seen in sham-operated mice (Fig. 4 G ). Virtually no mucosal damage was detected in electron microscopy studies done by using tissue from GW4064-treated mice (data not shown). Thus, activation of FXR prevents deterioration of the epithelial barrier and the accompanying inflamma- tion caused by biliary obstruction. No changes were seen in ileal rates of cell proliferation (assessed by immunostaining with Ki67 antibody) or apoptosis (assessed by TUNEL assays) in the different treatment groups (data not shown). Thus, FXR blocks the changes caused by BDL in WT mice without affecting cell proliferation or programmed cell death. In marked contrast to the continuous occludin immunostaining seen in sham-operated WT mice, occludin staining was discontinuous and weak in FXR-KO mice, indicating a deterioration in the epithelial barrier even without BDL (Fig. 4 A ). Consistent with this result and the higher incidence of bacterial translocation in FXR-KO mice (Fig. 3 E ), bacteria and edema were present in the junctions between epithelial cells in FXR-KO animals (Fig. 4 E ). Bacteria were also present in the dilated lymphatic vessels (Fig. 4 F ). These data demonstrate that the lack of FXR leads to mucosal injury and bacterial translocation even in the absence of additional insult. BDL did not exacerbate the edema (data not shown) or further increase the elevated neu- trophil numbers (Fig. 4 G ) in the FXR-KO mice. Moreover, GW4064 administration had no effect on any of these parame- ters in FXR-KO mice (Fig. 4 G and data not shown). Biliary obstruction causes bacterial overgrowth and translocation in the small intestine that can be reversed by administration of bile acids (3, 4, 25–30). It has been proposed that the detergent properties of bile acids are responsible for their enteroprotective actions. Although bile acids were not assessed for direct antibacterial actions in this study, our data suggest a more sophis- ticated mechanism wherein activation of the bile acid receptor FXR protects against bacterial proliferation and its detrimental effects in the distal small intestine. The products of several genes regulated by FXR in ileum, including Ang1 , iNos , and Il18 , have established antimicrobial actions. It is not possible to tell from our data whether these genes are regulated by FXR in enterocytes or other cell types, including resident immune cells. The expression profile of Ang1 as well as Fgf15 , Shp , Car12 , and Ibabp correlate well with FXR-mediated enteroprotection in the BDL model (Fig. 2), suggesting that the protective actions of FXR are likely to involve multiple downstream genes. Expression of iNos and Il18 is more complex in that their expression is increased by BDL in both WT and FXR-KO mice (Fig. 2), which likely reflects their regulation by proinflammatory signaling cascades that are activated by BDL. Nevertheless, iNOS and IL18 may contribute to FXR-induced enteroprotection under physiologic or other pathophysiologic conditions. Notably, elevated concentrations of iNOS and IL18 are associated with mucosal damage and inflammatory bowel disease (21, 36). Regulation of these genes by bile acids, which are released from the gallbladder into the small intestine during feeding, may ensure an adequate level of enteroprotection during periods of increased microbial expo- sure while preventing the overproduction of proteins that can cause inflammation and intestinal disease. In summary, our results demonstrate that FXR plays a crucial role in protecting the distal small intestine against bacterial overgrowth and the resulting disruption of the epithelial barrier. These findings raise the intriguing possibility that potent, synthetic FXR agonists may have therapeutic utility in patients with obstructed or reduced bile flow, including those on total par- enteral nutrition, who are susceptible to bacterial overgrowth and translocation (37, 38). Transcriptional Profiling Analysis. Total RNA was extracted from tissues by using RNA STAT-60 (Tel-Test, Friendswood, TX), and cDNA was prepared. Preparation of in vitro transcription products, oligonucleotide array hybridization, and scanning were performed by using Affymetrix high-density oligonucleotide array mouse genome 430A 2.0 chips according to Affymetrix protocols. To minimize discrepancies due to vari- ables, the raw expression data were scaled by using Affymetrix MICROARRAY SUITE 5.0 software, and pairwise comparisons were performed. The trimmed mean signal of all probe sets was adjusted to a user-specified target signal value (200) for each array for global scaling. No specific exclusion criteria were applied. Additional analysis was performed by using the freeware program BULLFROG 7 ...

Context 2

... in the regulatory regions of target genes. FXR is much studied in liver, where it regulates a program of genes involved in maintaining bile acid homeostasis (8). FXR is also expressed in the intestine, where it regulates several genes, including the ileal bile acid binding protein and fibroblast growth factor 15 (9–12). However, little is known about the overarching function of FXR in the gut. In this report we have examined the role of FXR in the ileum. We demonstrate that it plays a crucial role in preventing bacterial overgrowth and maintaining the integrity of the intestinal epithelium. Real-time quantitative PCR (RTQ-PCR) was used to measure FXR mRNA concentrations in duodenum, jejunum, ileum, colon, and liver. FXR was expressed in each of these tissues, with highest mRNA levels in ileum (Fig. 1 A ). A previous study showed that FXR is expressed in the villus epithelium in late-stage mouse embryos (13). Consistent with this finding, in situ hybridization analysis with ileum sections revealed that FXR is expressed in the villus epithelium in adult mouse ileum, with highest expression in the intervillus regions (Fig. 1 B ). Little or no FXR mRNA was detected in the crypts of Lieberk ̈hn, lamina propria, and tunica muscularis. To gain insight into the function of FXR in the small intestine, transcriptional profiling experiments were done by using mice lacking the gene encoding sterol 27-hydroxylase (CYP27), a key enzyme for bile acid synthesis. The CYP27-knockout (KO) mice produce only low levels of bile acids and thus are essentially devoid of endogenous FXR agonists (14). RNA was prepared from ileal mucosa of CYP27-KO mice administered either the potent, synthetic FXR agonist GW4064 (15) or vehicle for 14 h. Microarray analysis yielded a list of 15 genes whose expression was changed Ն 2-fold by GW4064 administration (Table 1). Among these were the established FXR target genes small heterodimer partner ( Shp ), fibroblast growth factor 15 ( Fgf15 ), and ileal bile acid binding protein ( Ibabp ), which encode proteins involved in bile acid homeostasis (9–12, 16, 17). Interestingly, several of the other genes in Table 1 have established roles in mucosal defense in the intestine. NO, the product of inducible NO synthase (iNOS), has direct antimicrobial effects and regulates many different aspects of the innate immune response, including mucus secretion, vascular tone, and epithelial barrier function (18, 19). The proinflammatory cytokine IL18 stimulates resistance to an array of pathogens, including intracellular and extracellular bacteria and mycobacteria, and appears to have a protective role during the early, acute phase of mucosal immune response (20, 21). Angiogenin (ANG1) and RNase A family member 4 (RNASE4) are closely related proteins that arise by differential splicing from a common gene locus (22). Although the function of RNASE4 is not established, ANG1 is part of the acute phase response to infection and has potent antibacterial and antimycotic actions (23). Carbonic anhydrase 12 (CAR12) is a member of a family of proteins involved in the maintenance of pH and ion balance (24). The regulation of all of these genes by FXR was confirmed by RTQ-PCR by using WT, CYP27-KO, and FXR-KO mice administered either GW4064 or vehicle (Fig. 5, which is published as supporting information on the PNAS web site). Notably, FXR had different effects on different genes. For example, activation of FXR had little or no effect on Ibabp or iNos expression in WT mice, but its elimination caused a marked decrease in the basal expression of these genes (Fig. 5). In contrast, FXR activation induced Il18 but did not contribute to its basal expression (Fig. 5). Based on these data, the possibility that Il18 induction by FXR is pharmacological rather than physiological cannot be excluded. Decreased bile secretion in rodents by either ligation of the common bile duct or induction of cirrhosis causes changes in the small intestine, including bacterial overgrowth and translocation across the mucosal barrier (3, 4, 25–30). Oral administration of bile acids inhibits these effects (3, 4). The genes regulated by FXR in ileum suggested that it might contribute to the enteroprotective actions of bile acids. To test this hypothesis, groups of WT and FXR-KO mice (31) were administered either GW4064 or vehicle for 2 days and then subjected to bile duct ligation (BDL) or sham operation. After 5 days, during which GW4064 or vehicle treatment was continued, the mice were killed and their intestines were analyzed for FXR target gene expression and bacterial content. As expected, BDL caused jaundice manifested by increased total serum bilirubin concentrations in both WT and FXR-KO mice (Fig. 2 A ). GW4064 administration had no effect on total serum bilirubin concentrations in BDL mice. BDL also had marked effects on genes regulated by FXR in ileum. In WT mice, expression of Fgf15 , Shp , Ang1 , Car12 , and Ibabp was reduced by BDL (Fig. 2). GW4064 administration increased expression of these genes in BDL mice to levels above those seen in sham- operated animals. None of these genes was induced by GW4064 in FXR-KO mice. In contrast, the iNos and Il18 genes showed a more complex expression pattern (Fig. 2 D and G ). Although both genes are induced by FXR (Fig. 5), there was a trend toward increased expression in BDL mice that occurred in an FXR- independent fashion (Fig. 2 D and G ). Because both iNos and Il18 are induced by proinflammatory stimuli, including lipopoly- saccharide and various cytokines (32, 33), this likely reflects regulation of these genes by additional signaling pathways that are activated by BDL. The effect of BDL and GW4064 treatment on the bacterial content of ileum and cecum was assessed. As expected, BDL increased the number of aerobic and anaerobic bacteria in the ileum and cecum of WT mice (Fig. 3 A – D ). The increase was particularly pronounced for aerobic bacteria (Fig. 3 A and C ). Notably, bacterial overgrowth in the lumen of both tissues was completely blocked by administration of GW4064 to BDL mice (Fig. 3 A – D ). To determine whether FXR is required for the antibacterial actions of GW4064, the same set of experiments was performed in FXR-KO mice. Two important differences were seen between WT and FXR-KO mice. First, GW4064 did not decrease aerobic or anaerobic bacteria counts in the ileum or cecum of FXR-KO mice (Fig. 3 A – D ). The reason for the marked variability in the bacterial counts in BDL FXR-KO mice is not known. Nevertheless, these data demonstrate that the antibacterial actions of GW4064 require FXR. Second, sham-operated FXR-KO mice had significantly higher aerobic bacteria counts than sham- operated, WT mice (Fig. 3 A and C ). These data support a role for FXR in suppressing bacterial proliferation under normal physiologic conditions. Because FXR-KO mice have reduced hepatic bile salt export pump (ABCB11) expression (31), de- creased bile acid concentrations in the intestinal lumen of these animals may contribute to the bacterial overgrowth. In comple- mentary in vitro experiments, GW4064 had no bacteriostatic effects in aerobic cultures of ileal contents even at 10 ␮ M concentrations (data not shown), demonstrating that GW4064 itself does not have direct antibacterial activity. As a measure of bacterial translocation across the mucosal barrier, bacteria were also quantified in the mesenteric lymph node complex for each of the treatment groups (Fig. 3 E and F ). The pattern for the bacterial counts was similar to that seen for bacterial overgrowth in the ileum and cecum. In WT mice, BDL resulted in an increase in bacterial number in the mesenteric lymph node complex, and GW4064 treatment completely inhibited this effect. The effect was particularly pronounced for aerobic bacteria (Fig. 3 E ). FXR-KO mice had Ϸ 10-fold higher levels of aerobic bacteria in mesenteric lymph nodes as com- pared with WT animals (Fig. 3 E ; note differences in scales for WT and FXR-KO mice), and BDL caused a trend toward increased bacterial counts (Fig. 3 E and F ). This trend was not reversed by GW4064 administration (Fig. 3 E and F ). The increase in in vivo bacterial translocation suggested that BDL causes a disruption of the intestinal barrier function that is inhibited by FXR. To examine barrier integrity, immunostaining was done for occludin, a component of the epithelial tight junctions. In the villus epithelium of WT mice, occludin staining was continuous except where interrupted by the normal presence of mucus-secreting goblet cells (Fig. 4 A ). In contrast, many enterocytes in the BDL mice expressed little or no occludin (Fig. 4 A ). Consistent with these data, transmission electron microscopy revealed large ruptures in the mucosal surface of BDL WT mice, with bacteria penetrating deep into the epithelium (Fig. 4 C and D ). Hematoxylin and eosin staining revealed that the breakdown in barrier function was accompanied by dilated lymphatics and a pronounced interstitial edema, as previously described (Fig. 4 B ) (3, 25, 26, 34). In many villi, the severity of the edema caused separation of the villus epithelium from the underlying lamina propria (Fig. 4 B ). Bacteria were seen within edematous areas by electron microscopy (Fig. 4 D ). Consistent with the edema resulting from an inflammatory response, there was a marked increase in the number of neutrophils infiltrating the villi of BDL mice (Fig. 4 G ). Neutrophils are known to contribute to epithelial permeability in inflammatory diseases of the intestine (35). GW4064 prevented the pathological effects caused by BDL. Ilea from BDL mice administered GW4064 showed a more continuous pattern of occludin immunostaining (Fig. 4 A ), little or no edema (Fig. 4 B ), and a reduction in the number of neutrophils to the level seen in sham-operated mice (Fig. 4 G ). Virtually no mucosal damage was detected in electron microscopy studies done by using tissue ...

Context 3

... barrier, bacteria were also quantified in the mesenteric lymph node complex for each of the treatment groups (Fig. 3 E and F ). The pattern for the bacterial counts was similar to that seen for bacterial overgrowth in the ileum and cecum. In WT mice, BDL resulted in an increase in bacterial number in the mesenteric lymph node complex, and GW4064 treatment completely inhibited this effect. The effect was particularly pronounced for aerobic bacteria (Fig. 3 E ). FXR-KO mice had Ϸ 10-fold higher levels of aerobic bacteria in mesenteric lymph nodes as com- pared with WT animals (Fig. 3 E ; note differences in scales for WT and FXR-KO mice), and BDL caused a trend toward increased bacterial counts (Fig. 3 E and F ). This trend was not reversed by GW4064 administration (Fig. 3 E and F ). The increase in in vivo bacterial translocation suggested that BDL causes a disruption of the intestinal barrier function that is inhibited by FXR. To examine barrier integrity, immunostaining was done for occludin, a component of the epithelial tight junctions. In the villus epithelium of WT mice, occludin staining was continuous except where interrupted by the normal presence of mucus-secreting goblet cells (Fig. 4 A ). In contrast, many enterocytes in the BDL mice expressed little or no occludin (Fig. 4 A ). Consistent with these data, transmission electron microscopy revealed large ruptures in the mucosal surface of BDL WT mice, with bacteria penetrating deep into the epithelium (Fig. 4 C and D ). Hematoxylin and eosin staining revealed that the breakdown in barrier function was accompanied by dilated lymphatics and a pronounced interstitial edema, as previously described (Fig. 4 B ) (3, 25, 26, 34). In many villi, the severity of the edema caused separation of the villus epithelium from the underlying lamina propria (Fig. 4 B ). Bacteria were seen within edematous areas by electron microscopy (Fig. 4 D ). Consistent with the edema resulting from an inflammatory response, there was a marked increase in the number of neutrophils infiltrating the villi of BDL mice (Fig. 4 G ). Neutrophils are known to contribute to epithelial permeability in inflammatory diseases of the intestine (35). GW4064 prevented the pathological effects caused by BDL. Ilea from BDL mice administered GW4064 showed a more continuous pattern of occludin immunostaining (Fig. 4 A ), little or no edema (Fig. 4 B ), and a reduction in the number of neutrophils to the level seen in sham-operated mice (Fig. 4 G ). Virtually no mucosal damage was detected in electron microscopy studies done by using tissue from GW4064-treated mice (data not shown). Thus, activation of FXR prevents deterioration of the epithelial barrier and the accompanying inflamma- tion caused by biliary obstruction. No changes were seen in ileal rates of cell proliferation (assessed by immunostaining with Ki67 antibody) or apoptosis (assessed by TUNEL assays) in the different treatment groups (data not shown). Thus, FXR blocks the changes caused by BDL in WT mice without affecting cell proliferation or programmed cell death. In marked contrast to the continuous occludin immunostaining seen in sham-operated WT mice, occludin staining was discontinuous and weak in FXR-KO mice, indicating a deterioration in the epithelial barrier even without BDL (Fig. 4 A ). Consistent with this result and the higher incidence of bacterial translocation in FXR-KO mice (Fig. 3 E ), bacteria and edema were present in the junctions between epithelial cells in FXR-KO animals (Fig. 4 E ). Bacteria were also present in the dilated lymphatic vessels (Fig. 4 F ). These data demonstrate that the lack of FXR leads to mucosal injury and bacterial translocation even in the absence of additional insult. BDL did not exacerbate the edema (data not shown) or further increase the elevated neu- trophil numbers (Fig. 4 G ) in the FXR-KO mice. Moreover, GW4064 administration had no effect on any of these parame- ters in FXR-KO mice (Fig. 4 G and data not shown). Biliary obstruction causes bacterial overgrowth and translocation in the small intestine that can be reversed by administration of bile acids (3, 4, 25–30). It has been proposed that the detergent properties of bile acids are responsible for their enteroprotective actions. Although bile acids were not assessed for direct antibacterial actions in this study, our data suggest a more sophis- ticated mechanism wherein activation of the bile acid receptor FXR protects against bacterial proliferation and its detrimental effects in the distal small intestine. The products of several genes regulated by FXR in ileum, including Ang1 , iNos , and Il18 , have established antimicrobial actions. It is not possible to tell from our data whether these genes are regulated by FXR in enterocytes or other cell types, including resident immune cells. The expression profile of Ang1 as well as Fgf15 , Shp , Car12 , and Ibabp correlate well with FXR-mediated enteroprotection in the BDL model (Fig. 2), suggesting that the protective actions of FXR are likely to involve multiple downstream genes. Expression of iNos and Il18 is more complex in that their expression is increased by BDL in both WT and FXR-KO mice (Fig. 2), which likely reflects their regulation by proinflammatory signaling cascades that are activated by BDL. Nevertheless, iNOS and IL18 may contribute to FXR-induced enteroprotection under physiologic or other pathophysiologic conditions. Notably, elevated concentrations of iNOS and IL18 are associated with mucosal damage and inflammatory bowel disease (21, 36). Regulation of these genes by bile acids, which are released from the gallbladder into the small intestine during feeding, may ensure an adequate level of enteroprotection during periods of increased microbial expo- sure while preventing the overproduction of proteins that can cause inflammation and intestinal disease. In summary, our results demonstrate that FXR plays a crucial role in protecting the distal small intestine against bacterial overgrowth and the resulting disruption of the epithelial barrier. These findings raise the intriguing possibility that potent, synthetic FXR agonists may have therapeutic utility in patients with obstructed or reduced bile flow, including those on total par- enteral nutrition, who are susceptible to bacterial overgrowth and translocation (37, 38). Transcriptional Profiling Analysis. Total RNA was extracted from tissues by using RNA STAT-60 (Tel-Test, Friendswood, TX), and cDNA was prepared. Preparation of in vitro transcription products, oligonucleotide array hybridization, and scanning were performed by using Affymetrix high-density oligonucleotide array mouse genome 430A 2.0 chips according to Affymetrix protocols. To minimize discrepancies due to vari- ables, the raw expression data were scaled by using Affymetrix MICROARRAY SUITE 5.0 software, and pairwise comparisons were performed. The trimmed mean signal of all probe sets was adjusted to a user-specified target signal value (200) for each array for global scaling. No specific exclusion criteria were applied. Additional analysis was performed by using the freeware program BULLFROG 7 ...

Context 4

... receptor family of ligand-activated transcription factors that is activated by bile acids including cholic acid and chenodeoxycholic acid (8). FXR modulates gene expression by binding as a heterodimer with the retinoid X receptors to DNA response elements in the regulatory regions of target genes. FXR is much studied in liver, where it regulates a program of genes involved in maintaining bile acid homeostasis (8). FXR is also expressed in the intestine, where it regulates several genes, including the ileal bile acid binding protein and fibroblast growth factor 15 (9–12). However, little is known about the overarching function of FXR in the gut. In this report we have examined the role of FXR in the ileum. We demonstrate that it plays a crucial role in preventing bacterial overgrowth and maintaining the integrity of the intestinal epithelium. Real-time quantitative PCR (RTQ-PCR) was used to measure FXR mRNA concentrations in duodenum, jejunum, ileum, colon, and liver. FXR was expressed in each of these tissues, with highest mRNA levels in ileum (Fig. 1 A ). A previous study showed that FXR is expressed in the villus epithelium in late-stage mouse embryos (13). Consistent with this finding, in situ hybridization analysis with ileum sections revealed that FXR is expressed in the villus epithelium in adult mouse ileum, with highest expression in the intervillus regions (Fig. 1 B ). Little or no FXR mRNA was detected in the crypts of Lieberk ̈hn, lamina propria, and tunica muscularis. To gain insight into the function of FXR in the small intestine, transcriptional profiling experiments were done by using mice lacking the gene encoding sterol 27-hydroxylase (CYP27), a key enzyme for bile acid synthesis. The CYP27-knockout (KO) mice produce only low levels of bile acids and thus are essentially devoid of endogenous FXR agonists (14). RNA was prepared from ileal mucosa of CYP27-KO mice administered either the potent, synthetic FXR agonist GW4064 (15) or vehicle for 14 h. Microarray analysis yielded a list of 15 genes whose expression was changed Ն 2-fold by GW4064 administration (Table 1). Among these were the established FXR target genes small heterodimer partner ( Shp ), fibroblast growth factor 15 ( Fgf15 ), and ileal bile acid binding protein ( Ibabp ), which encode proteins involved in bile acid homeostasis (9–12, 16, 17). Interestingly, several of the other genes in Table 1 have established roles in mucosal defense in the intestine. NO, the product of inducible NO synthase (iNOS), has direct antimicrobial effects and regulates many different aspects of the innate immune response, including mucus secretion, vascular tone, and epithelial barrier function (18, 19). The proinflammatory cytokine IL18 stimulates resistance to an array of pathogens, including intracellular and extracellular bacteria and mycobacteria, and appears to have a protective role during the early, acute phase of mucosal immune response (20, 21). Angiogenin (ANG1) and RNase A family member 4 (RNASE4) are closely related proteins that arise by differential splicing from a common gene locus (22). Although the function of RNASE4 is not established, ANG1 is part of the acute phase response to infection and has potent antibacterial and antimycotic actions (23). Carbonic anhydrase 12 (CAR12) is a member of a family of proteins involved in the maintenance of pH and ion balance (24). The regulation of all of these genes by FXR was confirmed by RTQ-PCR by using WT, CYP27-KO, and FXR-KO mice administered either GW4064 or vehicle (Fig. 5, which is published as supporting information on the PNAS web site). Notably, FXR had different effects on different genes. For example, activation of FXR had little or no effect on Ibabp or iNos expression in WT mice, but its elimination caused a marked decrease in the basal expression of these genes (Fig. 5). In contrast, FXR activation induced Il18 but did not contribute to its basal expression (Fig. 5). Based on these data, the possibility that Il18 induction by FXR is pharmacological rather than physiological cannot be excluded. Decreased bile secretion in rodents by either ligation of the common bile duct or induction of cirrhosis causes changes in the small intestine, including bacterial overgrowth and translocation across the mucosal barrier (3, 4, 25–30). Oral administration of bile acids inhibits these effects (3, 4). The genes regulated by FXR in ileum suggested that it might contribute to the enteroprotective actions of bile acids. To test this hypothesis, groups of WT and FXR-KO mice (31) were administered either GW4064 or vehicle for 2 days and then subjected to bile duct ligation (BDL) or sham operation. After 5 days, during which GW4064 or vehicle treatment was continued, the mice were killed and their intestines were analyzed for FXR target gene expression and bacterial content. As expected, BDL caused jaundice manifested by increased total serum bilirubin concentrations in both WT and FXR-KO mice (Fig. 2 A ). GW4064 administration had no effect on total serum bilirubin concentrations in BDL mice. BDL also had marked effects on genes regulated by FXR in ileum. In WT mice, expression of Fgf15 , Shp , Ang1 , Car12 , and Ibabp was reduced by BDL (Fig. 2). GW4064 administration increased expression of these genes in BDL mice to levels above those seen in sham- operated animals. None of these genes was induced by GW4064 in FXR-KO mice. In contrast, the iNos and Il18 genes showed a more complex expression pattern (Fig. 2 D and G ). Although both genes are induced by FXR (Fig. 5), there was a trend toward increased expression in BDL mice that occurred in an FXR- independent fashion (Fig. 2 D and G ). Because both iNos and Il18 are induced by proinflammatory stimuli, including lipopoly- saccharide and various cytokines (32, 33), this likely reflects regulation of these genes by additional signaling pathways that are activated by BDL. The effect of BDL and GW4064 treatment on the bacterial content of ileum and cecum was assessed. As expected, BDL increased the number of aerobic and anaerobic bacteria in the ileum and cecum of WT mice (Fig. 3 A – D ). The increase was particularly pronounced for aerobic bacteria (Fig. 3 A and C ). Notably, bacterial overgrowth in the lumen of both tissues was completely blocked by administration of GW4064 to BDL mice (Fig. 3 A – D ). To determine whether FXR is required for the antibacterial actions of GW4064, the same set of experiments was performed in FXR-KO mice. Two important differences were seen between WT and FXR-KO mice. First, GW4064 did not decrease aerobic or anaerobic bacteria counts in the ileum or cecum of FXR-KO mice (Fig. 3 A – D ). The reason for the marked variability in the bacterial counts in BDL FXR-KO mice is not known. Nevertheless, these data demonstrate that the antibacterial actions of GW4064 require FXR. Second, sham-operated FXR-KO mice had significantly higher aerobic bacteria counts than sham- operated, WT mice (Fig. 3 A and C ). These data support a role for FXR in suppressing bacterial proliferation under normal physiologic conditions. Because FXR-KO mice have reduced hepatic bile salt export pump (ABCB11) expression (31), de- creased bile acid concentrations in the intestinal lumen of these animals may contribute to the bacterial overgrowth. In comple- mentary in vitro experiments, GW4064 had no bacteriostatic effects in aerobic cultures of ileal contents even at 10 ␮ M concentrations (data not shown), demonstrating that GW4064 itself does not have direct antibacterial activity. As a measure of bacterial translocation across the mucosal barrier, bacteria were also quantified in the mesenteric lymph node complex for each of the treatment groups (Fig. 3 E and F ). The pattern for the bacterial counts was similar to that seen for bacterial overgrowth in the ileum and cecum. In WT mice, BDL resulted in an increase in bacterial number in the mesenteric lymph node complex, and GW4064 treatment completely inhibited this effect. The effect was particularly pronounced for aerobic bacteria (Fig. 3 E ). FXR-KO mice had Ϸ 10-fold higher levels of aerobic bacteria in mesenteric lymph nodes as com- pared with WT animals (Fig. 3 E ; note differences in scales for WT and FXR-KO mice), and BDL caused a trend toward increased bacterial counts (Fig. 3 E and F ). This trend was not reversed by GW4064 administration (Fig. 3 E and F ). The increase in in vivo bacterial translocation suggested that BDL causes a disruption of the intestinal barrier function that is inhibited by FXR. To examine barrier integrity, immunostaining was done for occludin, a component of the epithelial tight junctions. In the villus epithelium of WT mice, occludin staining was continuous except where interrupted by the normal presence of mucus-secreting goblet cells (Fig. 4 A ). In contrast, many enterocytes in the BDL mice expressed little or no occludin (Fig. 4 A ). Consistent with these data, transmission electron microscopy revealed large ruptures in the mucosal surface of BDL WT mice, with bacteria penetrating deep into the epithelium (Fig. 4 C and D ). Hematoxylin and eosin staining revealed that the breakdown in barrier function was accompanied by dilated lymphatics and a pronounced interstitial edema, as previously described (Fig. 4 B ) (3, 25, 26, 34). In many villi, the severity of the edema caused separation of the villus epithelium from the underlying lamina propria (Fig. 4 B ). Bacteria were seen within edematous areas by electron microscopy (Fig. 4 D ). Consistent with the edema resulting from an inflammatory response, there was a marked increase in the number of neutrophils infiltrating the villi of BDL mice (Fig. 4 G ). Neutrophils are known to contribute to epithelial permeability in inflammatory diseases of the intestine (35). GW4064 prevented the pathological effects caused by BDL. Ilea from BDL mice administered GW4064 showed a more continuous pattern of occludin ...

Context 5

... binding protein and fibroblast growth factor 15 (9–12). However, little is known about the overarching function of FXR in the gut. In this report we have examined the role of FXR in the ileum. We demonstrate that it plays a crucial role in preventing bacterial overgrowth and maintaining the integrity of the intestinal epithelium. Real-time quantitative PCR (RTQ-PCR) was used to measure FXR mRNA concentrations in duodenum, jejunum, ileum, colon, and liver. FXR was expressed in each of these tissues, with highest mRNA levels in ileum (Fig. 1 A ). A previous study showed that FXR is expressed in the villus epithelium in late-stage mouse embryos (13). Consistent with this finding, in situ hybridization analysis with ileum sections revealed that FXR is expressed in the villus epithelium in adult mouse ileum, with highest expression in the intervillus regions (Fig. 1 B ). Little or no FXR mRNA was detected in the crypts of Lieberk ̈hn, lamina propria, and tunica muscularis. To gain insight into the function of FXR in the small intestine, transcriptional profiling experiments were done by using mice lacking the gene encoding sterol 27-hydroxylase (CYP27), a key enzyme for bile acid synthesis. The CYP27-knockout (KO) mice produce only low levels of bile acids and thus are essentially devoid of endogenous FXR agonists (14). RNA was prepared from ileal mucosa of CYP27-KO mice administered either the potent, synthetic FXR agonist GW4064 (15) or vehicle for 14 h. Microarray analysis yielded a list of 15 genes whose expression was changed Ն 2-fold by GW4064 administration (Table 1). Among these were the established FXR target genes small heterodimer partner ( Shp ), fibroblast growth factor 15 ( Fgf15 ), and ileal bile acid binding protein ( Ibabp ), which encode proteins involved in bile acid homeostasis (9–12, 16, 17). Interestingly, several of the other genes in Table 1 have established roles in mucosal defense in the intestine. NO, the product of inducible NO synthase (iNOS), has direct antimicrobial effects and regulates many different aspects of the innate immune response, including mucus secretion, vascular tone, and epithelial barrier function (18, 19). The proinflammatory cytokine IL18 stimulates resistance to an array of pathogens, including intracellular and extracellular bacteria and mycobacteria, and appears to have a protective role during the early, acute phase of mucosal immune response (20, 21). Angiogenin (ANG1) and RNase A family member 4 (RNASE4) are closely related proteins that arise by differential splicing from a common gene locus (22). Although the function of RNASE4 is not established, ANG1 is part of the acute phase response to infection and has potent antibacterial and antimycotic actions (23). Carbonic anhydrase 12 (CAR12) is a member of a family of proteins involved in the maintenance of pH and ion balance (24). The regulation of all of these genes by FXR was confirmed by RTQ-PCR by using WT, CYP27-KO, and FXR-KO mice administered either GW4064 or vehicle (Fig. 5, which is published as supporting information on the PNAS web site). Notably, FXR had different effects on different genes. For example, activation of FXR had little or no effect on Ibabp or iNos expression in WT mice, but its elimination caused a marked decrease in the basal expression of these genes (Fig. 5). In contrast, FXR activation induced Il18 but did not contribute to its basal expression (Fig. 5). Based on these data, the possibility that Il18 induction by FXR is pharmacological rather than physiological cannot be excluded. Decreased bile secretion in rodents by either ligation of the common bile duct or induction of cirrhosis causes changes in the small intestine, including bacterial overgrowth and translocation across the mucosal barrier (3, 4, 25–30). Oral administration of bile acids inhibits these effects (3, 4). The genes regulated by FXR in ileum suggested that it might contribute to the enteroprotective actions of bile acids. To test this hypothesis, groups of WT and FXR-KO mice (31) were administered either GW4064 or vehicle for 2 days and then subjected to bile duct ligation (BDL) or sham operation. After 5 days, during which GW4064 or vehicle treatment was continued, the mice were killed and their intestines were analyzed for FXR target gene expression and bacterial content. As expected, BDL caused jaundice manifested by increased total serum bilirubin concentrations in both WT and FXR-KO mice (Fig. 2 A ). GW4064 administration had no effect on total serum bilirubin concentrations in BDL mice. BDL also had marked effects on genes regulated by FXR in ileum. In WT mice, expression of Fgf15 , Shp , Ang1 , Car12 , and Ibabp was reduced by BDL (Fig. 2). GW4064 administration increased expression of these genes in BDL mice to levels above those seen in sham- operated animals. None of these genes was induced by GW4064 in FXR-KO mice. In contrast, the iNos and Il18 genes showed a more complex expression pattern (Fig. 2 D and G ). Although both genes are induced by FXR (Fig. 5), there was a trend toward increased expression in BDL mice that occurred in an FXR- independent fashion (Fig. 2 D and G ). Because both iNos and Il18 are induced by proinflammatory stimuli, including lipopoly- saccharide and various cytokines (32, 33), this likely reflects regulation of these genes by additional signaling pathways that are activated by BDL. The effect of BDL and GW4064 treatment on the bacterial content of ileum and cecum was assessed. As expected, BDL increased the number of aerobic and anaerobic bacteria in the ileum and cecum of WT mice (Fig. 3 A – D ). The increase was particularly pronounced for aerobic bacteria (Fig. 3 A and C ). Notably, bacterial overgrowth in the lumen of both tissues was completely blocked by administration of GW4064 to BDL mice (Fig. 3 A – D ). To determine whether FXR is required for the antibacterial actions of GW4064, the same set of experiments was performed in FXR-KO mice. Two important differences were seen between WT and FXR-KO mice. First, GW4064 did not decrease aerobic or anaerobic bacteria counts in the ileum or cecum of FXR-KO mice (Fig. 3 A – D ). The reason for the marked variability in the bacterial counts in BDL FXR-KO mice is not known. Nevertheless, these data demonstrate that the antibacterial actions of GW4064 require FXR. Second, sham-operated FXR-KO mice had significantly higher aerobic bacteria counts than sham- operated, WT mice (Fig. 3 A and C ). These data support a role for FXR in suppressing bacterial proliferation under normal physiologic conditions. Because FXR-KO mice have reduced hepatic bile salt export pump (ABCB11) expression (31), de- creased bile acid concentrations in the intestinal lumen of these animals may contribute to the bacterial overgrowth. In comple- mentary in vitro experiments, GW4064 had no bacteriostatic effects in aerobic cultures of ileal contents even at 10 ␮ M concentrations (data not shown), demonstrating that GW4064 itself does not have direct antibacterial activity. As a measure of bacterial translocation across the mucosal barrier, bacteria were also quantified in the mesenteric lymph node complex for each of the treatment groups (Fig. 3 E and F ). The pattern for the bacterial counts was similar to that seen for bacterial overgrowth in the ileum and cecum. In WT mice, BDL resulted in an increase in bacterial number in the mesenteric lymph node complex, and GW4064 treatment completely inhibited this effect. The effect was particularly pronounced for aerobic bacteria (Fig. 3 E ). FXR-KO mice had Ϸ 10-fold higher levels of aerobic bacteria in mesenteric lymph nodes as com- pared with WT animals (Fig. 3 E ; note differences in scales for WT and FXR-KO mice), and BDL caused a trend toward increased bacterial counts (Fig. 3 E and F ). This trend was not reversed by GW4064 administration (Fig. 3 E and F ). The increase in in vivo bacterial translocation suggested that BDL causes a disruption of the intestinal barrier function that is inhibited by FXR. To examine barrier integrity, immunostaining was done for occludin, a component of the epithelial tight junctions. In the villus epithelium of WT mice, occludin staining was continuous except where interrupted by the normal presence of mucus-secreting goblet cells (Fig. 4 A ). In contrast, many enterocytes in the BDL mice expressed little or no occludin (Fig. 4 A ). Consistent with these data, transmission electron microscopy revealed large ruptures in the mucosal surface of BDL WT mice, with bacteria penetrating deep into the epithelium (Fig. 4 C and D ). Hematoxylin and eosin staining revealed that the breakdown in barrier function was accompanied by dilated lymphatics and a pronounced interstitial edema, as previously described (Fig. 4 B ) (3, 25, 26, 34). In many villi, the severity of the edema caused separation of the villus epithelium from the underlying lamina propria (Fig. 4 B ). Bacteria were seen within edematous areas by electron microscopy (Fig. 4 D ). Consistent with the edema resulting from an inflammatory response, there was a marked increase in the number of neutrophils infiltrating the villi of BDL mice (Fig. 4 G ). Neutrophils are known to contribute to epithelial permeability in inflammatory diseases of the intestine (35). GW4064 prevented the pathological effects caused by BDL. Ilea from BDL mice administered GW4064 showed a more continuous pattern of occludin immunostaining (Fig. 4 A ), little or no edema (Fig. 4 B ), and a reduction in the number of neutrophils to the level seen in sham-operated mice (Fig. 4 G ). Virtually no mucosal damage was detected in electron microscopy studies done by using tissue from GW4064-treated mice (data not shown). Thus, activation of FXR prevents deterioration of the epithelial barrier and the accompanying inflamma- tion caused by biliary obstruction. No changes were seen in ileal rates of cell proliferation (assessed by immunostaining ...

Context 6

... of FXR in the ileum. We demonstrate that it plays a crucial role in preventing bacterial overgrowth and maintaining the integrity of the intestinal epithelium. Real-time quantitative PCR (RTQ-PCR) was used to measure FXR mRNA concentrations in duodenum, jejunum, ileum, colon, and liver. FXR was expressed in each of these tissues, with highest mRNA levels in ileum (Fig. 1 A ). A previous study showed that FXR is expressed in the villus epithelium in late-stage mouse embryos (13). Consistent with this finding, in situ hybridization analysis with ileum sections revealed that FXR is expressed in the villus epithelium in adult mouse ileum, with highest expression in the intervillus regions (Fig. 1 B ). Little or no FXR mRNA was detected in the crypts of Lieberk ̈hn, lamina propria, and tunica muscularis. To gain insight into the function of FXR in the small intestine, transcriptional profiling experiments were done by using mice lacking the gene encoding sterol 27-hydroxylase (CYP27), a key enzyme for bile acid synthesis. The CYP27-knockout (KO) mice produce only low levels of bile acids and thus are essentially devoid of endogenous FXR agonists (14). RNA was prepared from ileal mucosa of CYP27-KO mice administered either the potent, synthetic FXR agonist GW4064 (15) or vehicle for 14 h. Microarray analysis yielded a list of 15 genes whose expression was changed Ն 2-fold by GW4064 administration (Table 1). Among these were the established FXR target genes small heterodimer partner ( Shp ), fibroblast growth factor 15 ( Fgf15 ), and ileal bile acid binding protein ( Ibabp ), which encode proteins involved in bile acid homeostasis (9–12, 16, 17). Interestingly, several of the other genes in Table 1 have established roles in mucosal defense in the intestine. NO, the product of inducible NO synthase (iNOS), has direct antimicrobial effects and regulates many different aspects of the innate immune response, including mucus secretion, vascular tone, and epithelial barrier function (18, 19). The proinflammatory cytokine IL18 stimulates resistance to an array of pathogens, including intracellular and extracellular bacteria and mycobacteria, and appears to have a protective role during the early, acute phase of mucosal immune response (20, 21). Angiogenin (ANG1) and RNase A family member 4 (RNASE4) are closely related proteins that arise by differential splicing from a common gene locus (22). Although the function of RNASE4 is not established, ANG1 is part of the acute phase response to infection and has potent antibacterial and antimycotic actions (23). Carbonic anhydrase 12 (CAR12) is a member of a family of proteins involved in the maintenance of pH and ion balance (24). The regulation of all of these genes by FXR was confirmed by RTQ-PCR by using WT, CYP27-KO, and FXR-KO mice administered either GW4064 or vehicle (Fig. 5, which is published as supporting information on the PNAS web site). Notably, FXR had different effects on different genes. For example, activation of FXR had little or no effect on Ibabp or iNos expression in WT mice, but its elimination caused a marked decrease in the basal expression of these genes (Fig. 5). In contrast, FXR activation induced Il18 but did not contribute to its basal expression (Fig. 5). Based on these data, the possibility that Il18 induction by FXR is pharmacological rather than physiological cannot be excluded. Decreased bile secretion in rodents by either ligation of the common bile duct or induction of cirrhosis causes changes in the small intestine, including bacterial overgrowth and translocation across the mucosal barrier (3, 4, 25–30). Oral administration of bile acids inhibits these effects (3, 4). The genes regulated by FXR in ileum suggested that it might contribute to the enteroprotective actions of bile acids. To test this hypothesis, groups of WT and FXR-KO mice (31) were administered either GW4064 or vehicle for 2 days and then subjected to bile duct ligation (BDL) or sham operation. After 5 days, during which GW4064 or vehicle treatment was continued, the mice were killed and their intestines were analyzed for FXR target gene expression and bacterial content. As expected, BDL caused jaundice manifested by increased total serum bilirubin concentrations in both WT and FXR-KO mice (Fig. 2 A ). GW4064 administration had no effect on total serum bilirubin concentrations in BDL mice. BDL also had marked effects on genes regulated by FXR in ileum. In WT mice, expression of Fgf15 , Shp , Ang1 , Car12 , and Ibabp was reduced by BDL (Fig. 2). GW4064 administration increased expression of these genes in BDL mice to levels above those seen in sham- operated animals. None of these genes was induced by GW4064 in FXR-KO mice. In contrast, the iNos and Il18 genes showed a more complex expression pattern (Fig. 2 D and G ). Although both genes are induced by FXR (Fig. 5), there was a trend toward increased expression in BDL mice that occurred in an FXR- independent fashion (Fig. 2 D and G ). Because both iNos and Il18 are induced by proinflammatory stimuli, including lipopoly- saccharide and various cytokines (32, 33), this likely reflects regulation of these genes by additional signaling pathways that are activated by BDL. The effect of BDL and GW4064 treatment on the bacterial content of ileum and cecum was assessed. As expected, BDL increased the number of aerobic and anaerobic bacteria in the ileum and cecum of WT mice (Fig. 3 A – D ). The increase was particularly pronounced for aerobic bacteria (Fig. 3 A and C ). Notably, bacterial overgrowth in the lumen of both tissues was completely blocked by administration of GW4064 to BDL mice (Fig. 3 A – D ). To determine whether FXR is required for the antibacterial actions of GW4064, the same set of experiments was performed in FXR-KO mice. Two important differences were seen between WT and FXR-KO mice. First, GW4064 did not decrease aerobic or anaerobic bacteria counts in the ileum or cecum of FXR-KO mice (Fig. 3 A – D ). The reason for the marked variability in the bacterial counts in BDL FXR-KO mice is not known. Nevertheless, these data demonstrate that the antibacterial actions of GW4064 require FXR. Second, sham-operated FXR-KO mice had significantly higher aerobic bacteria counts than sham- operated, WT mice (Fig. 3 A and C ). These data support a role for FXR in suppressing bacterial proliferation under normal physiologic conditions. Because FXR-KO mice have reduced hepatic bile salt export pump (ABCB11) expression (31), de- creased bile acid concentrations in the intestinal lumen of these animals may contribute to the bacterial overgrowth. In comple- mentary in vitro experiments, GW4064 had no bacteriostatic effects in aerobic cultures of ileal contents even at 10 ␮ M concentrations (data not shown), demonstrating that GW4064 itself does not have direct antibacterial activity. As a measure of bacterial translocation across the mucosal barrier, bacteria were also quantified in the mesenteric lymph node complex for each of the treatment groups (Fig. 3 E and F ). The pattern for the bacterial counts was similar to that seen for bacterial overgrowth in the ileum and cecum. In WT mice, BDL resulted in an increase in bacterial number in the mesenteric lymph node complex, and GW4064 treatment completely inhibited this effect. The effect was particularly pronounced for aerobic bacteria (Fig. 3 E ). FXR-KO mice had Ϸ 10-fold higher levels of aerobic bacteria in mesenteric lymph nodes as com- pared with WT animals (Fig. 3 E ; note differences in scales for WT and FXR-KO mice), and BDL caused a trend toward increased bacterial counts (Fig. 3 E and F ). This trend was not reversed by GW4064 administration (Fig. 3 E and F ). The increase in in vivo bacterial translocation suggested that BDL causes a disruption of the intestinal barrier function that is inhibited by FXR. To examine barrier integrity, immunostaining was done for occludin, a component of the epithelial tight junctions. In the villus epithelium of WT mice, occludin staining was continuous except where interrupted by the normal presence of mucus-secreting goblet cells (Fig. 4 A ). In contrast, many enterocytes in the BDL mice expressed little or no occludin (Fig. 4 A ). Consistent with these data, transmission electron microscopy revealed large ruptures in the mucosal surface of BDL WT mice, with bacteria penetrating deep into the epithelium (Fig. 4 C and D ). Hematoxylin and eosin staining revealed that the breakdown in barrier function was accompanied by dilated lymphatics and a pronounced interstitial edema, as previously described (Fig. 4 B ) (3, 25, 26, 34). In many villi, the severity of the edema caused separation of the villus epithelium from the underlying lamina propria (Fig. 4 B ). Bacteria were seen within edematous areas by electron microscopy (Fig. 4 D ). Consistent with the edema resulting from an inflammatory response, there was a marked increase in the number of neutrophils infiltrating the villi of BDL mice (Fig. 4 G ). Neutrophils are known to contribute to epithelial permeability in inflammatory diseases of the intestine (35). GW4064 prevented the pathological effects caused by BDL. Ilea from BDL mice administered GW4064 showed a more continuous pattern of occludin immunostaining (Fig. 4 A ), little or no edema (Fig. 4 B ), and a reduction in the number of neutrophils to the level seen in sham-operated mice (Fig. 4 G ). Virtually no mucosal damage was detected in electron microscopy studies done by using tissue from GW4064-treated mice (data not shown). Thus, activation of FXR prevents deterioration of the epithelial barrier and the accompanying inflamma- tion caused by biliary obstruction. No changes were seen in ileal rates of cell proliferation (assessed by immunostaining with Ki67 antibody) or apoptosis (assessed by TUNEL assays) in the different treatment groups (data not shown). Thus, FXR blocks the changes caused by BDL in WT mice without ...