Derek M. McKay

Activation of neuronal P2X7 receptor-pannexin-1 mediates death of enteric neurons during colitis

Inflammatory bowel diseases (IBDs) are chronic relapsing and remitting conditions associated with long-term gut dysfunction resulting from alterations to the enteric nervous system and a loss of enteric neurons. The mechanisms underlying inflammation-induced enteric neuron death are unknown. Here using in vivo models of experimental colitis we report that inflammation causes enteric neuron death by activating a neuronal signaling complex composed of P2X7 receptors (P2X7Rs), pannexin-1 (Panx1) channels, the Asc adaptor protein and caspases. Inhibition of P2X7R, Panx1, Asc or caspase activity prevented inflammation-induced neuron cell death. Preservation of enteric neurons by inhibiting Panx1 in vivo prevented the onset of inflammation-induced colonic motor dysfunction. Panx1 expression was reduced in Crohns disease but not ulcerative colitis. We conclude that activation of neuronal Panx1 underlies neuron death and the subsequent development of abnormal gut motility in IBD. Targeting Panx1 represents a new neuroprotective strategy to ameliorate the progression of IBD-associated dysmotility.

Corticotropin releasing hormone (CRH) regulates macromolecular permeability via mast cells in normal human colonic biopsies in vitro

Objective: Persistent stress and life events affect the course of ulcerative colitis and irritable bowel syndrome by largely unknown mechanisms. Corticotropin-releasing hormone (CRH) has been implicated as an important mediator of stress-induced abnormalities in intestinal mucosal function in animal models, but to date no studies in human colon have been reported. The aim was to examine the effects of CRH on mucosal barrier function in the human colon and to elucidate the mechanisms involved in CRH-induced hyper-permeability. Design: Biopsies from 39 volunteers were assessed for macromolecular permeability (horseradish peroxidise (HRP), 51Cr-EDTA), and electrophysiology after CRH challenge in Ussing chambers. The biopsies were examined by electron and confocal microscopy for HRP and CRH receptor localisation, respectively. Moreover, CRH receptor mRNA and protein expression were examined in the human mast cell line, HMC-1. Results: Mucosal permeability to HRP was increased by CRH (2.8±0.5 pmol/cm2/h) compared to vehicle exposure (1.5±0.4 pmol/cm2/h), p = 0.032, whereas permeability to 51Cr-EDTA and transmucosal electrical resistance were unchanged. The increased permeability to HRP was abolished by α-helical CRH (9-41) (1.3±0.6 pmol/cm2/h) and the mast cell stabiliser, lodoxamide (1.6±0.6 pmol/cm2/h). Electron microscopy showed transcellular passage of HRP through colonocytes. CRH receptor subtypes R1 and R2 were detected in the HMC-1 cell line and in lamina propria mast cells in human colon. Conclusions: Our results suggest that CRH mediates transcellular uptake of HRP in human colonic mucosa via CRH receptor subtypes R1 and R2 on subepithelial mast cells. CRH-induced macromolecular uptake in human colon mucosa may have implications for stress-related intestinal disorders.

Bacterial DNA evokes epithelial IL-8 production by a MAPK-dependent, NF-κB-independent pathway

Recognition of bacterial products by the innate immune system is dependent on pattern‐recognition receptors: toll‐like receptor 9 (TLR‐9) in the case of bacterial DNA. We hypothesized that bacterial DNA can directly affect enteric epithelial cells. RT‐PCR revealed constitutive TLR‐9 mRNA expression in three human colonic epithelial cell lines (T84, HT‐29, Caco‐2) and THP‐1 monocytes. Epithelial cells, in six‐well culture plates or on filter supports, were exposed to E. coli DNA (1–50 µg/ml), synthetic CpG‐rich oligonucleotides, or calf thymus DNA for 6–48 h. Exposure to E. coli DNA resulted in an increase in IL‐8 mRNA, and a time‐and dose‐dependent increase in IL‐8 secretion. Also, CpG oligonucleotides induced epithelial IL‐8 production, whereas calf thymus DNA did not. Exposure to E. coli DNA resulted in phosphorylation of ERK 1/2 MAPK and inhibitors of ERK activity (PD98059, UO126) significantly reduced the evoked IL‐8 production. In contrast, inhibitors of NFκB activity (PDTC, SN50) did not block E. coli DNA‐induced IL‐8 production. Electrophoretic mobility shift assays revealed that E. coli DNA stimulated epithelial AP‐1 but not NFκB activation. The barrier (i.e., transepithelial resistance) and ion transport parameters of epithelial monolayers (assessed in Ussing chambers) were unaltered following E. coli DNA exposure. Thus model gut epithelia express TLR‐9 mRNA and, while maintaining their barrier function, can respond to E. coli DNA by increased IL‐8 production.

Enhanced translocation of bacteria across metabolically stressed epithelia is reduced by butyrate

Background: The gut microflora in some patients with Crohns disease can be reduced in numbers of butyrate‐producing bacteria and this could result in metabolic stress in the colonocytes. Thus, we hypothesized that the short‐chain fatty acid, butyrate, is important in the maintenance and regulation of the barrier function of the colonic epithelium. Methods: Confluent monolayers of the human colon‐derived T84 or HT‐29 epithelial cell lines were exposed to dinitrophenol (DNP (0.1 mM), uncouples oxidative phosphorylation) + Escherichia coli (strain HB101, 106 cfu) ± butyrate (3–50 mM). Transepithelial resistance (TER), and bacterial internalization and translocation were assessed over a 24‐hour period. Epithelial ultrastructure was assessed by transmission electron microscopy. Results: Epithelia under metabolic stress display decreased TER and increased numbers of pseudopodia that is consistent with increased internalization and translocation of the E. coli. Butyrate (but not acetate) significantly reduced the bacterial translocation across DNP‐treated epithelia but did not ameliorate the drop in TER in the DNP+E. coli exposed monolayers. Inhibition of bacterial transcytosis across metabolically stressed epithelia was associated with reduced I‐&kgr;B phosphorylation and hence NF‐&kgr;B activation. Conclusions: Reduced butyrate‐producing bacteria could result in increased epithelial permeability particularly in the context of concomitant exposure to another stimulus that reduces mitochondria function. We speculate that prebiotics, the substrate for butyrate synthesis, is a valuable prophylaxis in the regulation of epithelial permeability and could be of benefit in preventing relapses in IBD. (Inflamm Bowel Dis 2010)

Cholinergic regulation of epithelial ion transport in the mammalian intestine

Acetylcholine (ACh) is critical in controlling epithelial ion transport and hence water movements for gut hydration. Here we review the mechanism of cholinergic control of epithelial ion transport across the mammalian intestine. The cholinergic nervous system affects basal ion flux and can evoke increased active ion transport events. Most studies rely on measuring increases in short‐circuit current (ISC = active ion transport) evoked by adding ACh or cholinomimetics to intestinal tissue mounted in Ussing chambers. Despite subtle species and gut regional differences, most data indicate that, under normal circumstances, the effect of ACh on intestinal ion transport is mainly an increase in Cl‐ secretion due to interaction with epithelial M3 muscarinic ACh receptors (mAChRs) and, to a lesser extent, neuronal M1 mAChRs; however, AChR pharmacology has been plagued by a lack of good receptor subtype‐selective compounds. Mice lacking M3 mAChRs display intact cholinergically‐mediated intestinal ion transport, suggesting a possible compensatory mechanism. Inflamed tissues often display perturbations in the enteric cholinergic system and reduced intestinal ion transport responses to cholinomimetics. The mechanism(s) underlying this hyporesponsiveness are not fully defined. Inflammation‐evoked loss of mAChR‐mediated control of epithelial ion transport in the mouse reveals a role for neuronal nicotinic AChRs, representing a hitherto unappreciated braking system to limit ACh‐evoked Cl‐ secretion. We suggest that: i) pharmacological analyses should be supported by the use of more selective compounds and supplemented with molecular biology techniques targeting specific ACh receptors and signalling molecules, and ii) assessment of ion transport in normal tissue must be complemented with investigations of tissues from patients or animals with intestinal disease to reveal control mechanisms that may go undetected by focusing on healthy tissue only.

The pro-inflammatory cytokine, interleukin-6, enhances the polarization of alternatively activated macrophages.

Macrophages are important innate immune cells that are associated with two distinct phenotypes: a pro-inflammatory (or classically activated) subset with prototypic macrophage functions such as inflammatory cytokine production and bactericidal activity, and an anti-inflammatory (or alternatively activated (AAM)) subset linked with wound healing and tissue repair processes. In this study, we examined the effect of interlukein-6 on human and murine macrophage polarization. The results indicate that despite being commonly associated with pro-inflammatory functions and being implicated in the pathogenesis/pathophysiology of numerous inflammatory diseases, interleukin-6 can enhance the polarization of AAMs, based on increased expression of hallmark markers: arginase-1, Ym1 and CD206; this effect required the AAM differentiating cytokines, IL-4 and IL-13. Co-treatment of AAMs with IL-6 resulted in spontaneous release of IL-10, suppressed LPS-induced nitric oxide production and inhibited cytokine production by activated CD4+ T cells – immunoregulatory features not observed in the ‘parent’ IL-4+IL-13-induced AAM. The effect of IL-6 required signal transducer and activator of transcription (STAT)-3, was partially dependent on up-regulation of the IL4Rα chain, and was independent of autocrine IL-10. In the presence of IFNγ, IL-6 promoted the production of IL-1β and TNFα suggesting that this cytokine can enhance the phenotype to which a macrophage has committed. This finding may explain the pleiotrophic nature of IL-6, where it is associated with the perpetuation and enhancement of disease in inflammatory situations, but is also necessary for resolution of inflammation and adequate wound healing to occur in others. Thus, the potential benefit of IL-6 in promoting an AAM, with its’ anti-inflammatory and wound healing ability, may need to be considered in immunotherapies aimed at in vivo modulation or inhibition of IL-6.

Extracts of the Rat Tapeworm, Hymenolepis diminuta, Suppress Macrophage Activation In Vitro and Alleviate Chemically Induced Colitis in Mice

ABSTRACT Analysis of parasite-host interactions can reveal the intricacies of immunity and identify ways to modulate immunopathological reactions. We assessed the ability of a phosphate-buffered saline-soluble extract of adult Hymenolepis diminuta to suppress macrophage (human THP-1 cell line, murine peritoneal macrophages) activity in vitro and the impact of treating mice with this extract on colitis induced by dinitrobenzene sulfonic acid (DNBS). A high-molecular-mass fraction of adult H. diminuta (HdHMW) or excretory/secretory products reduced macrophage activation: lipopolysaccharide (LPS)-induced interleukin-1β (IL-1β), IL-6, and tumor necrosis factor alpha (TNF-α) and poly(I:C)-induced TNF-α and IL-6 were suppressed by HdHMW. The active component in the HdHMW extract was minimally sensitive to boiling and trypsin digestion, whereas the use of sodium metaperiodate, as a general deglycosylation strategy, indicated that the immunosuppressive effect of HdHMW was at least partially dependent on a glycan: treating the HdHMW with neuraminidase and α-mannosidase failed to inhibit its blockade of LPS-induced TNF-α production by THP-1 macrophages. Mice treated with DNBS developed colitis, as typified by wasting, shortening of the colon, macroscopic and microscopic tissue damage, and an inflammatory infiltrate. Mice cotreated with HdHMW (three intraperitoneal injections) displayed significantly less inflammatory disease, and this was accompanied by reduced TNF-α production and increased IL-10 and IL-4 production by mitogen-stimulated spleen cells. However, cotreatment of mice with neutralizing anti-IL-10 antibodies had only a minor impact on the anticolitic effect of the HdHMW. We speculate that purification of the immunosuppressive factor(s) from H. diminuta has the potential to lead to the development of novel immunomodulatory drugs to treat inflammatory disease.

Parasitic helminths : a pharmacopeia of anti-inflammatory molecules

SUMMARY Infection with parasitic helminths takes a heavy toll on the health and well-being of humans and their domestic livestock, concomitantly resulting in major economic losses. Analyses have consistently revealed bioactive molecules in extracts of helminths or in their excretory/secretory products that modulate the immune response of the host. It is our view that parasitic helminths are an untapped source of immunomodulatory substances that, in pure form, could become new drugs (or models for drug design) to treat disease. Here, we illustrate the range of immunomodulatory molecules in selected parasitic trematodes, cestodes and nematodes, their impact on the immune cells in the host and how the host may recognize these molecules. There are many examples of the partial characterization of helminth-derived immunomodulatory molecules, but these have not yet translated into new drugs, reflecting the difficulty of isolating and fully characterizing proteins, glycoproteins and lipid-based molecules from small amounts of parasite material. However, this should not deter the investigator, since analytical techniques are now being used to accrue considerable structural information on parasite-derived molecules, even when only minute quantities of tissue are available. With the introduction of methodologies to purify and structurally-characterize molecules from small amounts of tissue and the application of high throughput immunological assays, one would predict that an assessment of parasitic helminths will yield a variety of novel drug candidates in the coming years.

The therapeutic helminth

By definition, parasites harm their hosts. Yet substantial evidence from animal models of human disease support the hypothesis that infection with helminths can suppress the development of other maladies. Here, the view is presented that assessment of the immunophysiological response to helminths could identify that infection with specific parasites would be therapeutically useful (although many helminths could not fulfil this role) and lead to precise knowledge of the immune events following infection, to identify ways to intervene in disease processes (in the absence of infection per se) that can be used to treat, and eventually cure, inflammatory and autoimmune disease.

Salvinorin A inhibits colonic transit and neurogenic ion transport in mice by activating κ-opioid and cannabinoid receptors

Abstract  The major active ingredient of the plant Salvia divinorum, salvinorin A (SA) has been used to treat gastrointestinal (GI) symptoms. As the action of SA on the regulation of colonic function is unknown, our aim was to examine the effects of SA on mouse colonic motility and secretion in vitro and in vivo. The effects of SA on GI motility were studied using isolated preparations of colon, which were compared with preparations from stomach and ileum. Colonic epithelial ion transport was evaluated using Ussing chambers. Additionally, we studied GI motility in vivo by measuring colonic propulsion, gastric emptying, and upper GI transit. Salvinorin A inhibited contractions of the mouse colon, stomach, and ileum in vitro, prolonged colonic propulsion and slowed upper GI transit in vivo. Salvinorin A had no effect on gastric emptying in vivo. Salvinorin A reduced veratridine‐, but not forskolin‐induced epithelial ion transport. The effects of SA on colonic motility in vitro were mediated by κ‐opioid receptors (KORs) and cannabinoid (CB) receptors, as they were inhibited by the antagonists nor‐binaltorphimine (KOR), AM 251 (CB1 receptor) and AM 630 (CB2 receptor). However, in the colon in vivo, the effects were largely mediated by KORs. The effects of SA on veratridine‐mediated epithelial ion transport were inhibited by nor‐binaltorphimine and AM 630. Salvinorin A slows colonic motility in vitro and in vivo and influences neurogenic ion transport. Due to its specific regional action, SA or its derivatives may be useful drugs in the treatment of lower GI disorders associated with increased GI transit and diarrhoea.