Impairment of adenosine A3 receptor activity disrupts neutrophil migratory capacity and impacts innate immune function in vivo.

Adenosine possesses potent anti-inflammatory properties which are partly mediated by G(i) -coupled adenosine A3 receptors (A3Rs). A3R agonists have shown clinical benefit in a number of inflammatory conditions although some studies in A3R-deficient mice suggest a pro-inflammatory role. We hypothesised that, in addition to cell signalling effects, A3R compounds might inhibit neutrophil chemotaxis by disrupting the purinergic feedback loop controlling leukocyte migration. Human neutrophil activation triggered rapid upregulation of surface A3R expression which was disrupted by pre-treatment with either agonist (Cl-IB-MECA) or antagonist (MRS1220). Both compounds reduced migration velocity and neutrophil transmigration capacity without impacting the response to chemokines per se. Similar effects were observed in murine neutrophils, while cells from A3R-deficient mice displayed a constitutively impaired migratory phenotype indicating compound-induced desensitisation and genetic ablation had the same functional outcome. In a dextran sodium sulphate-induced colitis model, A3R-deficient mice exhibited reduced colon pathology and decreased tissue myeloperoxidase levels at day 8 - consistent with reduced neutrophil recruitment. However, A3R-deficient mice were unable to resolve the dextran sodium sulphate-induced inflammation and had elevated numbers of tissue-associated bacteria by day 21. Our data indicate that A3Rs play a role in neutrophil migration and disrupting this function has the potential to adversely affect innate immune responses.

Glial cell-derived neurotrophic factor enhances synaptic communication and 5-hydroxytryptamine 3a receptor expression in enteric neurons.

BACKGROUND & AIMS: Glial cell-derived neurotrophic factor (GDNF) is essential for the development of the enteric nervous system during embryogenesis. We have observed the presence of Gdnf transcripts in the gastrointestinal tract of adult mice, and its early up-regulation after inflammation. We therefore investigated the effects of GDNF on enteric neuronal function in vitro. METHODS: Primary neuronal cultures were established from isolated myenteric plexi, and characterized by immunostaining and Ca(2+) imaging. Gene expression of several ion channels was analyzed by quantitative polymerase chain reaction (PCR) and the electrophysiologic properties of the neurons were studied by patch clamp. RESULTS: GDNF enhanced synaptogenesis and intercellular communication in primary myenteric neuronal cultures. Expression profiling revealed that GDNF exposure results in an up-regulation of Htr3a expression in the cultures and a similar increase was observed in inflamed colonic tissue where Gdnf expression was also increased. The increased Htr3a expression was accompanied by a functional increase in the response of neurons to acute challenge with 5-hydroxytryptamine (5-HT). GDNF treatment also caused inhibition of delayed rectifying voltage-gated potassium (Kv) currents, which correlated with the up-regulation of Htr3a and 5-HT-induced responses. Furthermore, pharmacologic blockade of Kv channels mimicked the effect of GDNF by increasing Htr3a expression as well as enhancing 5-HT-induced responses in the cultured myenteric neurons. CONCLUSIONS: GDNF promotes synaptic communication in cultured myenteric neurons. It also up-regulates 5-HT(3a)-receptor expression via modulation of Kv channel activity. Up-regulation of Gdnf after gastrointestinal inflammation might play an important role in the pathophysiology of gastrointestinal diseases.

GPR39 is coupled to TMEM16A in intestinal fibroblast-like cells.

GPR39 is a GPCR implicated as a regulator of gastrointestinal motility, although the mechanism remains elusive. Here, we report that GPR39 is expressed by a specific cell population cultured from mouse small intestine muscle layers, which was subsequently identified as fibroblast-like cells (FLCs) that have recently been shown to modulate gut motility. Application of the GPR39 agonist, Zn(2+), induced large currents and membrane depolarization in FLCs cultured from wild-type mice, but not Gpr39(-/-) mice. This Zn(2+)-induced current could be suppressed by application of a TMEM16A antagonist, CaCC(inh)-A01, or by silencing Tmem16a expression. These data suggest that GPR39 might modulate gut motility via regulating TMEM16A function in FLCs.

Host genetics and environmental factors regulate ecological succession of the mouse colon tissue-associated microbiota.

BACKGROUND: The integration of host genetics, environmental triggers and the microbiota is a recognised factor in the pathogenesis of barrier function diseases such as IBD. In order to determine how these factors interact to regulate the host immune response and ecological succession of the colon tissue-associated microbiota, we investigated the temporal interaction between the microbiota and the host following disruption of the colonic epithelial barrier. METHODOLOGY/PRINCIPAL FINDINGS: Oral administration of DSS was applied as a mechanistic model of environmental damage of the colon and the resulting inflammation characterized for various parameters over time in WT and Nod2 KO mice. RESULTS: In WT mice, DSS damage exposed the host to the commensal flora and led to a migration of the tissue-associated bacteria from the epithelium to mucosal and submucosal layers correlating with changes in proinflammatory cytokine profiles and a progressive transition from acute to chronic inflammation of the colon. Tissue-associated bacteria levels peaked at day 21 post-DSS and declined thereafter, correlating with recruitment of innate immune cells and development of the adaptive immune response. Histological parameters, immune cell infiltration and cytokine biomarkers of inflammation were indistinguishable between Nod2 and WT littermates following DSS, however, Nod2 KO mice demonstrated significantly higher tissue-associated bacterial levels in the colon. DSS damage and Nod2 genotype independently regulated the community structure of the colon microbiota. CONCLUSIONS/SIGNIFICANCE: The results of these experiments demonstrate the integration of environmental and genetic factors in the ecological succession of the commensal flora in mammalian tissue. The association of Nod2 genotype (and other host polymorphisms) and environmental factors likely combine to influence the ecological succession of the tissue-associated microflora accounting in part for their association with the pathogenesis of inflammatory bowel diseases.

Increased prokineticin 2 expression in gut inflammation: role in visceral pain and intestinal ion transport.

BACKGROUND: Prokineticin 2 (PROK2) is an inflammatory cytokine-like molecule expressed predominantly by macrophages and neutrophils infiltrating sites of tissue damage. Given the established role of prokineticin signaling on gastrointestinal function, we have explored Prok2 gene expression in inflammatory conditions of the gastrointestinal tract and assessed the possible consequences on gut physiology. METHODS: Prokineticin expression was examined in normal and colitic tissues using qPCR and immunohistochemistry. Functional responses to PROK2 were studied using calcium imaging and a novel antagonist, Compound 3, used to determine the role of PROK2 and prokineticin receptors in inflammatory visceral pain and ion transport. KEY RESULTS: Prok2 gene expression was up-regulated in biopsy samples from ulcerative colitis patients, and similar elevations were observed in rodent models of inflammatory colitis. Prokineticin receptor 1 (PKR1) was localized to the enteric neurons and extrinsic sensory neurons, whereas Pkr2 expression was restricted to sensory ganglia. In rats, PROK2-increased intracellular calcium levels in cultured enteric and dorsal root ganglia neurons, which was blocked by Compound 3. Moreover, PROK2 acting at prokineticin receptors stimulated intrinsic neuronally mediated ion transport in rat ileal mucosa. In vivo, Compound 3 reversed intracolonic mustard oil-induced referred allodynia and TNBS-induced visceral hypersensitivity, but not non-inflammatory, stress-induced visceral pain. CONCLUSIONS & INFERENCES: Elevated Prok2 levels, as a consequence of gastrointestinal tract inflammation, induce visceral pain via prokineticin receptors. This observation, together with the finding that PROK2 can modulate intestinal ion transport, raises the possibility that inhibitors of PROK2 signaling may have clinical utility in gastrointestinal disorders, such as irritable bowel syndrome and inflammatory bowel disease.

Spleen versus pancreas: strict control of organ interrelationship revealed by analyses of Bapx1-/- mice.

During early stages of pancreatic development, the mesenchyme that contributes to the spleen overlies the dorsal pancreatic endoderm. Here, we show that interactions between splenic mesenchyme and pancreas proceed via a highly orchestrated morphogenetic program. Disruption of morphogenesis, as occurs in the Bapx1(Nkx3.2)(-/-) embryo, results in transformation of these tissues into well-organized, ectopic gut-like structures. Bapx1 plays a crucial organizing role effecting position and separation of the spleen and pancreas to prevent this metaplastic transformation. Similar transformations occur in organ cultures employing wild-type pancreatic endoderm and spleen mesenchyme, revealing the developmental plasticity of the pancreas and that precise spatial and temporal control of tissue interactions are required for development of both organs.

Bapx1 regulates patterning in the middle ear: altered regulatory role in the transition from the proximal jaw during vertebrate evolution.

The middle ear apparatus is composed of three endochondrial ossicles (the stapes, incus and malleus) and two membranous bones, the tympanic ring and the gonium, which act as structural components to anchor the ossicles to the skull. Except for the stapes, these skeletal elements are unique to mammals and are derived from the first and second branchial arches. We show that, in combination with goosecoid (Gsc), the Bapx1 gene defines the structural components of the murine middle ear. During embryogenesis, Bapx1 is expressed in a discrete domain within the mandibular component of the first branchial arch and later in the primordia of middle ear-associated bones, the gonium and tympanic ring. Consistent with the expression pattern of Bapx1, mouse embryos deficient for Bapx1 lack a gonium and display hypoplasia of the anterior end of the tympanic ring. At E10.5, expression of Bapx1 partially overlaps that of Gsc and although Gsc is required for development of the entire tympanic ring, the role of Bapx1 is restricted to the specification of the gonium and the anterior tympanic ring. Thus, simple overlapping expression of these two genes appears to account for the patterning of the elements that compose the structural components of the middle ear and suggests that they act in concert. In addition, Bapx1 is expressed both within and surrounding the incus and the malleus. Examination of the malleus shows that the width, but not the length, of this ossicle is decreased in the mutant mice. In non-mammalian jawed vertebrates, the bones homologous to the mammalian middle ear ossicles compose the proximal jaw bones that form the jaw articulation (primary jaw joint). In fish, Bapx1 is responsible for the formation of the joint between the quadrate and articular (homologues of the malleus and incus, respectively) enabling an evolutionary comparison of the role of a regulatory gene in the transition of the proximal jawbones to middle ear ossicles. Contrary to expectations, murine Bapx1 does not affect the articulation of the malleus and incus. We show that this change in role of Bapx1 following the transition to the mammalian ossicle configuration is not due to a change in expression pattern but results from an inability to regulate Gdf5 and Gdf6, two genes predicted to be essential in joint formation.

The splanchnic mesodermal plate directs spleen and pancreatic laterality, and is regulated by Bapx1/Nkx3.2.

The mechanism by which left-right (LR) information is interpreted by organ primordia during asymmetric morphogenesis is largely unknown. We show that spleen and pancreatic laterality is dependent on a specialised, columnar mesodermal-derived cell layer referred to here as the splanchnic mesodermal plate (SMP). At early embryonic stages, the SMP is bilateral, surrounding the midline-located stomach and dorsal pancreatic bud. Under control of the LR asymmetry pathway, the left SMP is maintained and grows laterally. Mice carrying the dominant hemimelia (Dh) mutation lack the SMP. Significantly, the mice are asplenic and the pancreas remains positioned along the embryonic midline. In the absence of Fgf10 expression, the spleno-pancreatic mesenchyme and surrounding SMP grow laterally but contain no endodermal component, showing that leftward growth is autonomous and independent of endoderm. In the Bapx1(-/-) mutants, the SMP is defective. Normally, the SMP is a source for both Fgf9 and Fgf10; however, in the Bapx1 mutant, Fgf10 expression is downregulated and the dorsal pancreas remains at the midline. We conclude that the SMP is an organiser responsible for the leftward growth of the spleno-pancreatic region and that Bapx1 regulates SMP functions required for pancreatic laterality.