Gut Permeability and Colitis

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production of short-chain fatty acids, including acetate, propionate, and butyrate, which in turn causes a dosedependent suppression of proliferation of colonic cells both in vivo and in experimental models. Indeed, hydrothermally treated resistant starch enhanced apoptosis and decreased cell proliferation in colon cancer cell lines (Carcinogenesis 2006;27:1849 –1859), and large bowel and hepatic portal venous butyrate levels correlated negatively with tumor indices in azoxymethane-induced CRC in rats (Carcinogenesis 2008;28:2190 –2194). There are very few investigations on the antineoplastic effects of resistant starch in humans, and their results are inconclusive. One of the most recent showed that resistant starch supplementation for up to 4 weeks in patients with CRC reduced the proportion of mitotic cells in the upper part of the crypt in normal-appearing colorectal mucosa, and induced CDK4 and GADD45A gene expression, thus providing an explanation for the potential chemopreventive effect of this dietary agent (Gut 2009; 58:413– 420). Unfortunately, results of Burn’s study did not confirm this appealing possibility, at least in the short term (N Engl J Med 2008;359:2567–2578). In conclusion, because chemoprevention has failed to demonstrate any beneficial effect on CRC incidence in Lynch syndrome, prevention of this neoplasm should continue relying in periodic colonoscopy surveillance, the most effective approach so far for improving survival in such a setting. CARMEN LÓPEZ–RAMOS, MD ANTONI CASTELLS, MD

GUT PERMEABILITY AND COLITIS Arieta MC, Madsen K, Doyle J, et al. (Department of Medicine, University of Alberta, Walter C. Mackenzie Health Science Centre, Edmonton, Alberta, Canada). Reducing small intestinal permeability attenuates colitis in the IL10 gene-deficient mouse. Gut 2009;58: 41– 48. Gut epithelial permeability, the integrity of its physical barrier function, plays an important part in the development of inflammatory bowel disease and autoimmunity in certain animal models. Because increased gut permeability has intriguing associations with Crohn’s disease, celiac disease, and HIV/AIDS, it follows that emerging information concerning the regulation of this physical barrier could point to new therapies for testing in human diseases. One example of this research is the discovery of zonulin regulation of gut epithelial permeability. A mammalian homolog of a Vibrio cholerae toxin, zonulin is secreted by lamina propria cells and activates apical receptors on gut epithelial cells leading to phosphorylation of tight junction proteins and increased permeability owing to enlargement of the paracellular space. Recently, a novel peptide inhibitor has been developed to block

zonulin from binding to its receptor, with its effects limited to the small intestine where the receptor is located. Human trials with this drug (AT-1001, Alba Therapeutics) are underway in celiac disease, a setting where excess zonulin secretion occurs and increased gut permeability can precede clinical disease (Lancet 2000;355:1518 – 1519). This report in Gut suggests that inhibition of zonulin binding can reverse the preexisting small bowel permeability defect in a well-characterized mouse model of human Crohn’s disease, the IL-10 – deficient mouse, and furthermore can ameliorate features of the colitis, a site distant from the small bowel permeability defect (interestingly, the colon itself is unaffected directly by zonulin). These findings would further suggest that permeability defects in the small bowel contribute to the immune response at distant sites such as the colon (a paradigm already suspected in the BB rat model of autoimmune type 1 diabetes). These investigators used the IL-10 – deficient (IL10⫺/⫺) mouse as a model of colitis; histologically detectable colonic inflammation develops spontaneously at around 10 weeks after conventional animal housing (ie, after exposure to environmental bacteria). These mice typically do not show histologic evidence of small intestinal enteritis, however. This increase in permeability also was not dependent on excessive inflammatory cytokine production (interferon-␥ and tumor necrosis factor-␣) in the small intestine. IL10⫺/⫺ mice and controls were treated with the zonulin antagonist AT-1001 (added to the drinking water) or placebo for 17 weeks. Using validated measures of small intestinal and colonic permeability, they showed that immediately after weaning, the IL10⫺/⫺ mice had increased small bowel permeability compared with wildtype control mice at all weekly timepoints measured. The small bowel permeability defect was prevented by treatment with the zonulin-antagonist AT-1001. This improvement in the epithelial layer leakiness was seen using both in vivo (lactulose/mannitol fractional excretion ratio) and in vitro (mannitol flux and transepithelial electrical potential difference using mucosa mounted in Ussing chambers) methods. Importantly, there are also apparent effects of AT-1001 treatment on the course of the colitis, but the data from the studies of the colonic response raise some questions. First, we do not know whether there are baseline permeability defects in the colon similar to what exist in the small bowel of IL10⫺/⫺ mice right after weaning. This is important to know; a zonulin-independent colonic permeability defect could contribute to the spontaneous colitis despite effects on small bowel permeability. However, the increase in colonic permeability measured as increased mannitol flux and lowered electrical resistance in mucosal sections, at the 8-week time point just preceding the first signs of colonic damage (increased sucralose excretion at 10 weeks), is prevented by treatment with the zonulin antagonist. Furthermore, although treatment with the zonulin antagonist significantly pre-

August 2009

vents increases in colonic permeability (sucralose excretion) over time, it is interesting that the placebo and treatment groups have the same effect on bringing colonic permeability back to baseline by the end of the 17-week study period, suggesting that critical timepoints for zonulin effects on colonic responses may be early in the induction of the spontaneous colitis. Additionally, there may be some methodologic issues with the use of sucralose excretion in this model; at 17 weeks, the sucralose excretion scores are the same, whereas the histologic scores and inflammatory cytokine secretion are significantly different (and factors such as epithelial cell apoptosis and epithelial ulceration can increase sucralose excretion); it is not clear why the sucralose excretion is not higher in a more inflamed colon. Finally, although measures of neutrophil infiltration of colonic tissue were unchanged at the end of treatment between groups, the inflammatory cytokine secretion increases seen in placebotreated animals were prevented by zonulin antagonist treatment, similar to what was seen in the small intestine. Comment. This report is timely for 2 reasons. First, the role of increased intestinal permeability in disease pathophysiology is being expanded beyond its traditionally accepted role as being limited to gastrointestinal diseases. Increased permeability of the gut epithelial layer has long been recognized as a factor contributing to the severity of local intestinal lamina propria inflammation, like in Crohn’s disease and celiac disease. However, increased gut permeability is also a factor contributing to the development of autoimmune type I diabetes in the BB rat model where it is a necessary but not sufficient component for disease initiation (Am J Physiol 1999; 276:G951–G957). Interestingly, type I diabetic humans also have increased small bowel permeability and even increased zonulin serum levels (Diabetes 2006;55:1443– 1449); increased small bowel permeability may be an early event in autoimmune diabetes, but it is not known if a permeability defect is actually a requirement for diabetes to develop, and if so whether it would be a constitutional or a transient defect. Regardless, the result of the permeability defect is a presumed increase in antigen exposure with consequent autoimmune inflammation establishing itself with increased frequency in genetically susceptible hosts. On the other hand, gut permeability defects can result in general immune activation, as seems to be the case in HIV/AIDS. Increased microbial translocation of the gut epithelium as reflected by increased plasma lipopolysaccharide levels is found in patients with chronic HIV and AIDS. This can be reduced by successful antiretroviral therapy. In fact, small bowel permeability (measured by mannitol flux and impedence spectroscopy on endoscopic biopsies) is significantly elevated in untreated versus treatment-suppressed HIV patients and seronegative controls (Gut 2009;58:220 –227). In this case, enhanced gut permeability is likely related to epithelial barrier response

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to local gut infection and cytokine production, and the systemic effect of enhanced immune activation (with lymphocyte proliferation and retrovirus replication) is thought to play a role in HIV disease progression. The second reason this report is timely is that AT-1001 is currently being evaluated for treatment of celiac disease in humans. With the introduction of this agent into clinical trials, there is the potential for testing this drug in a number of autoimmune and inflammatory states that may rely on increased gut permeability for initiation or persistence of the disease. This report does raise an interesting question about the mechanism of increased gut permeability in inflammatory bowel disease and in this mouse model. Assuming that the IL10⫺/⫺ mice have baseline increases in small bowel permeability (but not colonic permeability) before developing colitis, and by correcting this defect the colitis can be prevented, how then does the small intestine control the colon immune response? Perhaps as in the case of the BB rat, a more permeable small bowel allows increased exposure to luminal antigen, and naïve T cells becoming antigen experienced in mesenteric lymph nodes of the small intestine then home to the lamina propria throughout the gut, including the colon where they are activated (notably, in the AT-1001–treated mice, interferon-␥ secretion was significantly reduced in the small and large bowel). Alternatively, the small bowel permeability defect may lead to a general immune activation that can lower the threshold development of colitis on a susceptible genetic background. This report aims to demonstrate that control of increased gut epithelial permeability can alter the natural history of colitis. This is most clearly seen in the data showing early precolitis improvement in small bowel and colonic permeability (before 10 weeks) and later suppression of inflammatory cytokine production in the colon of zonulin antagonist-treated mice. Of course this model of spontaneous colitis in IL10⫺/⫺ mice does not recapitulate the immune background of Crohn’s disease patients, but it is difficult to not point out that patients with Crohn’s disease and their unaffected first-degree relatives also display increased small bowel permeability and even a heightened sensitivity to nonsteroidal anti-inflammatory drug–induced increases in permeability. It would be interesting to know whether the permeability changes are associated more with the occurrence of Crohn’s colitis compared with isolated small bowel disease. Having said this, it is not known whether this permeability defect in human Crohn’s disease is zonulin dependent or not. Furthermore, once inflammation is active, it is well recognized that inflammatory cytokines can increase epithelial barrier permeability, leading to increased exposure of the lamina propria to luminal contents, suggesting that targeting cytokines as well as targeting the components of the epithelial barrier itself could both contribute to normalizing permeability defects.

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With this in mind, maintenance of the epithelial barrier either directly by stabilizing the tight junction, or indirectly by blocking inflammatory cytokines, is a novel approach to treating IBD. PETER MANNON, MD, MPH

TARGETING FARNESOID X RECEPTOR IN HEPATIC AND BILIARY INFLAMMATORY DISEASES Wang YD, Chen WD, Wang M, et al. (Department of Gene Regulation and Drug Discovery, Beckman Research Institute, City of Hope National Medical Center, Duarte, California). Farnesoid X receptor antagonizes nuclear factor kappaB in hepatic inflammatory response. Hepatology 2008;48:1632–1643. In chronic liver diseases, inflammation is a critical pathophysiologic step leading to cirrhosis and cancer. Inflammation is mediated by cytokines, chemokines, and enzymes produced by hematopoietic and epithelial cells. The expression of inflammation mediators is under the molecular control of the nuclear factor-␬B (NF-␬B). NF-␬B is a heterodimeric transcription factor composed of a p65 (RelA) and a p52 subunit that is activated by proinflammatory stimuli, such as pathogen-associated molecular patterns (PAMPs). NF-␬B has been shown to cross-talk with nuclear receptors (NRs) in the gut–liver axis to suppress inflammation. Marked liver inflammation is observed in farnesoid X receptor (FXR) ablated mice suggesting an interaction of NF-␬B with FXR in the liver (Cancer Res 2007;67:863– 867). Wang et al show that hepatocytes from FXR ablated (FXR⫺/⫺) mice display a higher proinflammatory gene induction to NF-␬B activators (12-O-tetradecanoylphorbol-13-acetate [TPA], tumor necrosis factor [TNF]-␣, and lipopolysaccharide [LPS]) than hepatocytes from wild-type (WT) mice. Conversely, they show that the pharmacologic activation of FXR lessens the induction of proinflammatory gene expression triggered by NF-␬B in hepatoblastoma cells. The results presented by Wang et al suggest that activation of FXR may counteract the ability of NF-␬B to transactivate proinflammatory genes, such as TNF-␣, cyclo-oxygenase-2, interleukin (IL)-1 and IL-6. Moreover, FXR was shown to reduce inducible nitric oxide synthase gene expression in hepatocytes; however, in the latter case, the effect of FXR may be independent of NF-␬B, as suggested by the data presented by Wang et al and previous published work (Proc Natl Acad Sci U S A 2006; 103:3920 –3925; Arterioscler Thromb Vasc Biol 2007;27: 2606 –2611). The authors further demonstrated the direct interaction of FXR with NF-␬B by reporter plasmid transfection experiments, in which NF-␬B activity was reduced in cells overexpressing FXR. In these experiments, FXR activation

by the synthetic FXR agonist, GW4064, had no significant effect on the induction or amplification of the inhibition of NF-␬B activity triggered by TPA, LPS or p65 overexpression. These results suggest that the inhibiting effect of FXR is mostly ligand independent and possibly linked to the sequestration of cytoplasmic NF-␬B. However, against this assumption, the authors document an absence of influence of FXR on NF-␬B nuclear translocation. Finally, the authors demonstrate that the ability of NF-␬B to bind to specific DNA sequences is inhibited by FXR. FXR⫺/⫺ mice challenged with LPS displayed both higher proinflammatory gene expression and liver necrosis when compared with WT mice. Conversely, infection of FXR⫺/⫺ mice with a FXR expressing adenovirus diminishes the induction of proinflammatory gene expression triggered by LPS. These results therefore indicate that the crosstalk between FXR and NF-␬B is observed in vivo. Beside proinflammatory genes, NF-␬B also controls the expression of anti-apoptotic genes. Interestingly, FXR overexpression or activation had no consequence on the induction of anti-apoptotic genes elicited by NF-␬B. The authors thus show that the cross-talk between NF-␬B and FXR is restricted to the control of proinflammatory gene expression. Comment. NRs are ligand-activated transcription factors

that are involved in development and physiology. Because NRs are central in a wide range of metabolic pathways, they are increasingly recognized has potent therapeutic targets. In the NR receptor superfamily, FXR is attractive to the hepatologist because it is both highly expressed in the liver and involved in the adaptive response to cholestasis. FXR is activated by conjugated and unconjugated bile acids (BA) with chenodeoxycholic acid (CDCA) being the most potent natural agonist. The identification of potent synthetic ligands, such as GW4064 and 6-ethyl-CDCA, has recently opened the way for effective pharmacologic FXR targeting. FXR is the master regulator of BA homeostasis and enterohepatic circulation. In the intestine, FXR activation modulates the expression of specific BA transporters by repressing the human apical sodium BA transporter (ASBT) and inducing the basolateral organic solute transporters (OST-␣ and OST-␤). Furthermore, activated FXR increases the expression of the fibroblast growth factor 15 (FGF-15), known as FGF-19 in humans. FGF-15, by binding to the type-4 FGF receptor, represses both ASBT in enterocytes and CYP7A1 in hepatocytes (Am J Physiol Gastrointest Liver Physiol 2008;295:G996 –G1003; Cell Metab 2005;2:217–225). The transcription of the CYP7A1 gene is also repressed by hepatic FXR activation through the induction of the nuclear repressor SHP (Mol Cell 2000;6: 517–526). In hepatocytes, FXR activation limits hepatic BA accumulation by negatively regulating the main basolateral BA uptake system, NTCP (Gastroenterology 2001;121:140 –