Ursodeoxycholic acid and lithocholic acid exert anti-inflammatory actions in the colon.

Ward JB, Lajczak NK, Kelly OB, O'Dwyer AM, Giddam AK, Ní Gabhann J, Franco P, Tambuwala MM, Jefferies CA, Keely S, Roda A, Keely SJ. Ursodeoxycholic acid and lithocholic acid exert anti-inflammatory actions in the colon. Am J Physiol Gastrointest Liver Physiol 312: G550-G558, 2017. First published March 30, 2017; doi:10.1152/ajpgi.00256.2016.-Inflammatory bowel diseases (IBD) comprise a group of common and debilitating chronic intestinal disorders for which currently available therapies are often unsatisfactory. The naturally occurring secondary bile acid, ursodeoxycholic acid (UDCA), has well-established anti-inflammatory and cytoprotective actions and may therefore be effective in treating IBD. We aimed to investigate regulation of colonic inflammatory responses by UDCA and to determine the potential impact of bacterial metabolism on its therapeutic actions. The anti-inflammatory efficacy of UDCA, a nonmetabolizable analog, 6α-methyl-UDCA (6-MUDCA), and its primary colonic metabolite lithocholic acid (LCA) was assessed in the murine dextran sodium sulfate (DSS) model of mucosal injury. The effects of bile acids on cytokine (TNF-α, IL-6, Il-1ß, and IFN-γ) release from cultured colonic epithelial cells and mouse colonic tissue in vivo were investigated. Luminal bile acids were measured by gas chromatography-mass spectrometry. UDCA attenuated release of proinflammatory cytokines from colonic epithelial cells in vitro and was protective against the development of colonic inflammation in vivo. In contrast, although 6-MUDCA mimicked the effects of UDCA on epithelial cytokine release in vitro, it was ineffective in preventing inflammation in the DSS model. In UDCA-treated mice, LCA became the most common colonic bile acid. Finally, LCA treatment more potently inhibited epithelial cytokine release and protected against DSS-induced mucosal inflammation than did UDCA. These studies identify a new role for the primary metabolite of UDCA, LCA, in preventing colonic inflammation and suggest that microbial metabolism of UDCA is necessary for the full expression of its protective actions.NEW & NOTEWORTHY On the basis of its cytoprotective and anti-inflammatory actions, the secondary bile acid ursodeoxycholic acid (UDCA) has well-established uses in both traditional and Western medicine. We identify a new role for the primary metabolite of UDCA, lithocholic acid, as a potent inhibitor of intestinal inflammatory responses, and we present data to suggest that microbial metabolism of UDCA is necessary for the full expression of its protective effects against colonic inflammation.

Ursodeoxycholic acid inhibits TNFα-induced IL-8 release from monocytes.

Monocytes are critical to the pathogenesis of inflammatory bowel disease (IBD) as they infiltrate the mucosa and release cytokines that drive the inflammatory response. Ursodeoxycholic acid (UDCA), a naturally occurring bile acid with anti-inflammatory actions, has been proposed as a potential new therapy for IBD. However, its effects on monocyte function are not yet known. Primary monocytes from healthy volunteers or cultured U937 monocytes were treated with either the proinflammatory cytokine, TNFα (5 ng/ml) or the bacterial endotoxin, lipopolysaccharide (LPS; 1 µg/ml) for 24 h, in the absence or presence of UDCA (25-100 µM). IL-8 release into the supernatant was measured by ELISA. mRNA levels were quantified by qPCR and changes in cell signaling proteins were determined by Western blotting. Toxicity was assessed by measuring lactate dehydrogenase (LDH) release. UDCA treatment significantly attenuated TNFα-, but not LPS-driven, release of IL-8 from both primary and cultured monocytes. UDCA inhibition of TNFα-driven responses was associated with reduced IL-8 mRNA expression. Both TNFα and LPS stimulated NFκB activation in monocytes, while IL-8 release in response to both cytokines was attenuated by an NFκB inhibitor, BMS-345541. Interestingly, UDCA inhibited TNFα-, but not LPS-stimulated, NFκB activation. Finally, TNFα, but not LPS, induced phosphorylation of TNF receptor associated factor (TRAF2), while UDCA cotreatment attenuated this response. We conclude that UDCA specifically inhibits TNFα-induced IL-8 release from monocytes by inhibiting TRAF2 activation. Since such actions would serve to dampen mucosal immune responses in vivo, our data support the therapeutic potential of UDCA for IBD.

Oxygen in the regulation of intestinal epithelial transport.

The transport of fluid, nutrients and electrolytes to and from the intestinal lumen is a primary function of epithelial cells. Normally, the intestine absorbs approximately 9 l of fluid and 1 kg of nutrients daily, driven by epithelial transport processes that consume large amounts of cellular energy and O2. The epithelium exists at the interface of the richly vascularised mucosa, and the anoxic luminal environment and this steep O2 gradient play a key role in determining the expression pattern of proteins involved in fluid, nutrient and electrolyte transport. However, the dynamic nature of the splanchnic circulation necessitates that the epithelium can evoke co-ordinated responses to fluctuations in O2 availability, which occur either as a part of the normal digestive process or as a consequence of several pathophysiological conditions. While it is known that hypoxia-responsive signals, such as reactive oxygen species, AMP-activated kinase, hypoxia-inducible factors, and prolyl hydroxylases are all important in regulating epithelial responses to altered O2 supply, our understanding of the molecular mechanisms involved is still limited. Here, we aim to review the current literature regarding the role that O2 plays in regulating intestinal transport processes and to highlight areas of research that still need to be addressed.

Farnesoid X receptor agonists attenuate colonic epithelial secretory function and prevent experimental diarrhoea in vivo.

OBJECTIVE: Bile acids are important regulators of intestinal physiology, and the nuclear bile acid receptor, farnesoid X receptor (FXR), is emerging as a promising therapeutic target for several intestinal disorders. Here, we investigated a role for FXR in regulating intestinal fluid and electrolyte transport and the potential for FXR agonists in treating diarrhoeal diseases. DESIGN: Electrogenic ion transport was measured as changes in short-circuit current across voltage-clamped T84 cell monolayers or mouse tissues in Ussing chambers. NHE3 activity was measured as BCECF fluorescence in Caco-2 cells. Protein expression was measured by immunoblotting and cell surface biotinylation. Antidiarrhoeal efficacy of GW4064 was assessed using two in vivo mouse models: the ovalbumin-induced diarrhoea model and cholera toxin (CTX)-induced intestinal fluid accumulation. RESULTS: GW4064 (5 µmol/L; 24 h), a specific FXR agonist, induced nuclear translocation of the receptor in T84 cells and attenuated Cl(-) secretory responses to both Ca(2+) and cAMP-dependent agonists. GW4064 also prevented agonist-induced inhibition of NHE3 in Caco-2 cells. In mice, intraperitoneal administration of GW4064 (50 mg/mL) also inhibited Ca(2+) and cAMP-dependent secretory responses across ex vivo colonic tissues and prevented ovalbumin-induced diarrhoea and CTX-induced intestinal fluid accumulation in vivo. At the molecular level, FXR activation attenuated apical Cl(-) currents by inhibiting expression of cystic fibrosis transmembrane conductance regulator channels and inhibited basolateral Na(+)/K(+)-ATPase activity without altering expression of the protein. CONCLUSIONS: These data reveal a novel antisecretory role for the FXR in colonic epithelial cells and suggest that FXR agonists have excellent potential for development as a new class of antidiarrheal drugs.

Ursodeoxycholic acid attenuates colonic epithelial secretory function.

Dihydroxy bile acids, such as chenodeoxycholic acid (CDCA), are well known to promote colonic fluid and electrolyte secretion, thereby causing diarrhoea associated with bile acid malabsorption. However, CDCA is rapidly metabolised by colonic bacteria to ursodeoxycholic acid (UDCA), the effects of which on epithelial transport are poorly characterised. Here, we investigated the role of UDCA in the regulation of colonic epithelial secretion. Cl(-) secretion was measured across voltage-clamped monolayers of T84 cells and muscle-stripped sections of mouse or human colon. Cell surface biotinylation was used to assess abundance/surface expression of transport proteins. Acute (15 min) treatment of T84 cells with bilateral UDCA attenuated Cl(-) secretory responses to the Ca(2+) and cAMP-dependent secretagogues carbachol (CCh) and forskolin (FSK) to 14.0 ± 3.8 and 40.2 ± 7.4% of controls, respectively (n = 18, P < 0.001). Investigation of the molecular targets involved revealed that UDCA acts by inhibiting Na(+)/K(+)-ATPase activity and basolateral K(+) channel currents, without altering their cell surface expression. In contrast, intraperitoneal administration of UDCA (25 mg kg(-1)) to mice enhanced agonist-induced colonic secretory responses, an effect we hypothesised to be due to bacterial metabolism of UDCA to lithocholic acid (LCA). Accordingly, LCA (50-200 µm) enhanced agonist-induced secretory responses in vitro and a metabolically stable UDCA analogue, 6α-methyl-UDCA, exerted anti-secretory actions in vitro and in vivo. In conclusion, UDCA exerts direct anti-secretory actions on colonic epithelial cells and metabolically stable derivatives of the bile acid may offer a new approach for treating intestinal diseases associated with diarrhoea.

Hydroxylase inhibition attenuates colonic epithelial secretory function and ameliorates experimental diarrhea.

Hydroxylases are oxygen-sensing enzymes that regulate cellular responses to hypoxia. Transepithelial Cl(-) secretion, the driving force for fluid secretion, is dependent on O(2) availability for generation of cellular energy. Here, we investigated the role of hydroxylases in regulating epithelial secretion and the potential for targeting these enzymes in treatment of diarrheal disorders. Ion transport was measured as short-circuit current changes across voltage-clamped monolayers of T(84) cells and mouse colon. The antidiarrheal efficacy of dimethyloxallyl glycine (DMOG) was tested in a mouse model of allergic disease. Hydroxylase inhibition with DMOG attenuated Ca(2+)- and cAMP-dependent secretory responses in voltage-clamped T(84) cells to 20.2 ± 2.6 and 38.8 ± 6.7% (n=16; P≤0.001) of those in control cells, respectively. Antisecretory actions of DMOG were time and concentration dependent, being maximal after 18 h of DMOG (1 mM) treatment. DMOG specifically inhibited Na(+)/K(+)-ATPase pump activity without altering its expression or membrane localization. In mice, DMOG inhibited agonist-induced secretory responses ex vivo and prevented allergic diarrhea in vivo. In conclusion, hydroxylases are important regulators of epithelial Cl(-) and fluid secretion and present a promising target for development of new drugs to treat transport disorders.