Intestinal inflammation caused by magnesium deficiency alters basal and oxidative stress-induced intestinal function

The aim of this study was to determine the effect of magnesium deficiency on small intestinal morphology and function. Rats were assigned to 4 groups and placed on magnesium sufficient or deficient diet for 1 or 3 weeks. Infiltration of neutrophils and mucosal injury were assessed in stained sections of small intestine. Magnesium deficiency alone induced a significant increase in neutrophil infiltration and increased vascular ICAM-1 expression, in the absence of changes in mucosal injury or expression of proinflammatory mediators. Magnesium deficiency was associated with hyposecretory epithelial cell responses and vascular macromolecular leak in the small intestine and lung, which was attributed partly to reduced expression of NOS-3. To determine the effect of hypomagnesmia on the intestinal responses to a known oxidative stress, groups of rats were randomized to either sham operation or superior mesenteric artery occlusion for 10 (non-injurious) or 30 (injurious) minutes followed by a 1- or 4-hour reperfusion period. In response to mesenteric ischemia/reperfusion, deficient rats showed exaggerated PMN influx, but similar mucosal injury. Intestinal ischemia in sufficient animals induced vascular macromolecular leak in the small intestine and lung at 4 hours of reperfusion, with levels similar to those observed in untreated deficient rats. Acute magnesium repletion of deficient rats 24 h before surgery attenuated the exaggerated inflammation in deficient rats. These data show that magnesium deficiency induced a subclinical inflammation in the small intestine in the absence of mucosal injury, but with significant functional changes in local and remote organs and increased sensitivity to oxidative stress. The opinions contained herein are those of the authors and are not to be construed as official policy or reflecting the views of the Department of Defense     

Impact of vitamin E or selenium deficiency on nematode-induced alterations in murine intestinal function

The effects of deficiencies in the antioxidant nutrients, vitamin E and selenium, on the host response to gastrointestinal nematode infection are unknown. The aim of the study was to determine the effect of antioxidant deficiencies on nematode-induced alterations in intestinal function in mice. BALB/c mice were fed control diets or diets deficient in selenium or vitamin E and the response to a secondary challenge inoculation with Heligmosomoides polygyrus was determined. Egg and worm counts were assessed to determine host resistance. Sections of jejunum were mounted in Ussing chambers to measure changes in permeability, absorption, and secretion, or suspended in organ baths to determine smooth muscle contraction. Both selenium and vitamin E deficient diets reduced resistance to helminth infection. Vitamin E, but not selenium, deficiency prevented nematode-induced decreases in glucose absorption and hyper-contractility of smooth muscle. Thus, vitamin E status is an important factor in the physiological response to intestinal nematode infection and may contribute to antioxidant-dependent protective mechanisms in the small intestine.     

Immune regulation of protease-activated receptor-1 expression in murine small intestine during Nippostrongylus brasiliensis infection

Infection with gastrointestinal nematodes exerts profound effects on both immune and physiological responses of the host. Helminth infection induces a hypercontractility of intestinal smooth muscle that is dependent on the Th2 cytokines, IL-4 and IL-13, and may contribute to worm expulsion. Protease-activated receptors (PARs) are expressed throughout the gut, and activation of PAR-1 was observed in asthma, a Th2-driven pathology. In the current study we investigated the physiologic and immunologic regulation of PAR-1 in the murine small intestine, specifically 1) the effect of PAR-1 agonists on small intestinal smooth muscle contractility, 2) the effects of Nippostrongylus brasiliensis infection on PAR-1 responses, 3) the roles of IL-13 and IL-4 in N. brasiliensis infection-induced alterations in PAR-1 responses, and 4) the STAT6 dependence of these responses. We demonstrate that PAR-1 activation induces contraction of murine intestinal smooth muscle that is enhanced during helminth infection. This hypercontractility is associated with an elevated expression of PAR-1 mRNA and protein. N. brasiliensis-induced changes in PAR-1 function and expression were seen in IL-4-deficient mice, but not in IL-13- or STAT6-deficient mice, indicating the dependence of IL-13 on the STAT6 signaling pathway independent of IL-4.     

Functional importance of regional differences in localized gene expression of receptors for IL-13 in murine gut

IL-13 induces a STAT6-dependent hypercontractility of intestinal smooth muscle that is mediated by binding to the IL-13Ralpha1 component of the type 2 IL-4R that is linked to STAT6. IL-13 also binds to the IL-13Ralpha2 that is not linked to STAT6 and functions to limit the effects of IL-13 in vivo. In this study we assessed the contributions of regional and cellular differences in the distribution of the IL-13R components to the physiological regulation of smooth muscle function in wild-type mice and mice deficient in STAT6 or IL-13Ralpha2. The expression of IL-13 and IL-13Ralpha2 was higher in colon than in small intestine. Laser capture microdissection of specific cell types revealed that the expression of IL-13Ralpha2 was higher in the smooth muscle layer compared with levels in the epithelial cells of the mucosa. In contrast, there was a uniform distribution of IL-13alpha1 in smooth muscle, epithelia, and myenteric neurons. The significant hypercontractility of smooth muscle in mice deficient in IL-13Ralpha2, but not in STAT6, shows the physiological importance of IL-13 binding to IL-13Ralpha2. The pronounced differences in the expression of IL-13Ralpha2 suggest that the gut has developed sophisticated mechanisms for controlling the physiological and pathophysiological activities of IL-13.     

Mice deficient in the CXCR2 ligand, CXCL1 (KC/GRO-alpha), exhibit increased susceptibility to dextran sodium sulfate (DSS)-induced colitis

The role of TLRs and MyD88 in the maintenance of gut integrity in response to dextran sodium sulfate (DSS)-induced colitis was demonstrated recently and led to the conclusion that the innate immune response to luminal commensal flora provides necessary signals that facilitate epithelial repair and permits a return to homeostasis after colonic injury. In this report, we demonstrate that a deficit in a single neutrophil chemokine, CXCL1/KC, also results in a greatly exaggerated response to DSS. Mice with a targeted mutation in the gene that encodes this chemokine responded to 2.5% DSS in their drinking water with significant weight loss, bloody stools, and a complete loss of gut integrity in the proximal and distal colon, accompanied by a predominantly mononuclear infiltrate, with few detectable neutrophils. In contrast, CXCL1/KC(- /-) and wild-type C57BL/6J mice provided water showed no signs of inflammation and, at this concentration of DSS, wild-type mice showed only minimal histopathology, but significantly more infiltrating neutrophils. This finding implies that neutrophil infiltration induced by CXCL1/KC is an essential component of the intestinal response to inflammatory stimuli as well as the ability of the intestine to restore mucosal barrier integrity.     

Atomic resolution structures and solution behavior of enzyme-substrate complexes of Enterobacter cloacae PB2 pentaerythritol tetranitrate reductase. Multiple conformational states and implications for the mechanism of nitroaromatic explosive degradation

The structure of pentaerythritol tetranitrate (PETN) reductase in complex with the nitroaromatic substrate picric acid determined previously at 1.55 A resolution indicated additional electron density between the indole ring of residue Trp-102 and the nitro group at C-6 of picrate. The data suggested the presence of an unusual bond between substrate and the tryptophan side chain. Herein, we have extended the resolution of the PETN reductase-picric acid complex to 0.9 A. This high-resolution analysis indicates that the active site is partially occupied with picric acid and that the anomalous density seen in the original study is attributed to the population of multiple conformational states of Trp-102 and not a formal covalent bond between the indole ring of Trp-102 and picric acid. The significance of any interaction between Trp-102 and nitroaromatic substrates was probed further in solution and crystal complexes with wild-type and mutant (W102Y and W102F) enzymes. Unlike with wild-type enzyme, in the crystalline form picric acid was bound at full occupancy in the mutant enzymes, and there was no evidence for multiple conformations of active site residues. Solution studies indicate tighter binding of picric acid in the active sites of the W102Y and W102F enzymes. Mutation of Trp-102 does not impair significantly enzyme reduction by NADPH, but the kinetics of decay of the hydride-Meisenheimer complex are accelerated in the mutant enzymes. The data reveal that decay of the hydride-Meisenheimer complex is enzyme catalyzed and that the final distribution of reaction products for the mutant enzymes is substantially different from wild-type enzyme. Implications for the mechanism of high explosive degradation by PETN reductase are discussed.     

Crystal structure of bacterial morphinone reductase and properties of the C191A mutant enzyme

The crystal structure of the NADH-dependent bacterial flavoenzyme morphinone reductase (MR) has been determined at 2.2-A resolution in complex with the oxidizing substrate codeinone. The structure reveals a dimeric enzyme comprising two 8-fold beta/alpha barrel domains, each bound to FMN, and a subunit folding topology and mode of flavin-binding similar to that found in Old Yellow Enzyme (OYE) and pentaerythritol tetranitrate (PETN) reductase. The subunit interface of MR is formed by interactions from an N-terminal beta strand and helices 2 and 8 of the barrel domain and is different to that seen in OYE. The active site structures of MR, OYE, and PETN reductase are highly conserved reflecting the ability of these enzymes to catalyze "generic" reactions such as the reduction of 2-cyclohexenone. A region of polypeptide presumed to define the reducing coenzyme specificity is identified by comparison of the MR structure (NADH-dependent) with that of PETN reductase (NADPH-dependent). The active site acid identified in OYE (Tyr-196) and conserved in PETN reductase (Tyr-186) is replaced by Cys-191 in MR. Mutagenesis studies have established that Cys-191 does not act as a crucial acid in the mechanism of reduction of the olefinic bond found in 2-cyclohexenone and codeinone.     

Complement inhibitor, complement receptor 1-related gene/protein y-Ig attenuates intestinal damage after the onset of mesenteric ischemia/reperfusion injury in mice.

Complement receptor 1-related gene/protein y (Crry) is a murine membrane protein that regulates the activity of both classical and alternative complement pathways. We used a recombinant soluble form of Crry fused to the hinge, CH2, and CH3 domains of mouse IgG1 (Crry-Ig) to determine whether inhibition of complement activation prevents and/or reverses mesenteric ischemia/reperfusion-induced injury in mice. Mice were subjected to 30 min of ischemia, followed by 2 h of reperfusion. Crry-Ig was administered either 5 min before or 30 min after initiation of the reperfusion phase. Pretreatment with Crry-Ig reduced local intestinal mucosal injury and decreased generation of leukotriene B(4) (LTB(4)). When given 30 min after the beginning of the reperfusion phase, Crry-Ig resulted in a decrease in ischemia/reperfusion-induced intestinal mucosal injury comparable to that occurring when it was given 5 min before initiation of the reperfusion phase. The beneficial effect of Crry-Ig administered 30 min after the initiation of reperfusion coincided with a decrease in PGE(2) generation despite the fact that it did not prevent local infiltration of neutrophils and did not have a significant effect on LTB(4) production. These data suggest that complement inhibition protects animals from reperfusion-induced intestinal damage even if administered as late as 30 min into reperfusion and that the mechanism of protection is independent of neutrophil infiltration or LTB(4) inhibition.     

Critical role of IL-25 in nematode infection-induced alterations in intestinal function

IL-25 (IL-17E) is a member of the IL-17 cytokine family. IL-25-deficient mice exhibit impaired Th2 immunity against nematode infection, implicating IL-25 as a key component in mucosal immunity. The sources of IL-25 and mechanisms responsible for the induction of Th2 immunity by IL-25 in the gastrointestinal tract remain poorly understood. There is also little information on the regulation of IL-25 during inflammation or its role in gut function. In the current study, we investigated the regulation of IL-25 during Nippostrongylus brasiliensis infection and the contribution of IL-25 to the infection-induced alterations in intestinal function. We found that epithelial cells, but not immune cells, are the major source of IL-25 in the small intestine. N. brasiliensis infection-induced upregulation of IL-25 depends upon IL-13 activation of STAT6. IL-25(-/-) mice had diminished intestinal smooth muscle and epithelial responses to N. brasiliensis infection that were associated with an impaired Th2 protective immunity. Exogenous IL-25 induced characteristic changes similar to those after nematode infection but was unable to restore the impaired host immunity against N. brasiliensis infection in IL-13(-/-) mice. These data show that IL-25 plays a critical role in nematode infection-induced alterations in intestinal function that are important for host protective immunity, and IL-13 is the major downstream Th2 cytokine responsible for the IL-25 effects.