The Role of Epigenomic Regulatory Pathways in the Gut-Brain Axis and Visceral Hyperalgesia.

The gut-brain axis (GBA) is broadly accepted to describe the bidirectional circuit that links the gastrointestinal tract with the central nervous system (CNS). Interest in the GBA has grown dramatically over past two decades along with advances in our understanding of the importance of the axis in the pathophysiology of numerous common clinical disorders including mood disorders, neurodegenerative disease, diabetes mellitus, non-alcohol fatty liver disease (NAFLD) and enhanced abdominal pain (visceral hyperalgesia). Paralleling the growing interest in the GBA, there have been seminal developments in our understanding of how environmental factors such as psychological stress and other extrinsic factors alter gene expression, primarily via epigenomic regulatory mechanisms. This process has been driven by advances in next-generation multi-omics methods and bioinformatics. Recent reviews address various components of GBA, but the role of epigenomic regulatory pathways in chronic stress-associated visceral hyperalgesia in relevant regions of the GBA including the amygdala, spinal cord, primary afferent (nociceptive) neurons, and the intestinal barrier has not been addressed. Rapidly developing evidence suggests that intestinal epithelial barrier dysfunction and microbial dysbiosis play a potentially significant role in chronic stress-associated visceral hyperalgesia in nociceptive neurons innervating the lower intestine via downregulation in intestinal epithelial cell tight junction protein expression and increase in paracellular permeability. These observations support an important role for the regulatory epigenome in the development of future diagnostics and therapeutic interventions in clinical disorders affecting the GBA.

Functional Bowel Disorders: A Roadmap to Guide the Next Generation of Research.

In June 2016, the National Institutes of Health hosted a workshop on functional bowel disorders (FBDs), particularly irritable bowel syndrome, with the objective of elucidating gaps in current knowledge and recommending strategies to address these gaps. The workshop aimed to provide a roadmap to help strategically guide research efforts during the next decade. Attendees were a diverse group of internationally recognized leaders in basic and clinical FBD research. This document summarizes the results of their deliberations, including the following general conclusions and recommendations. First, the high prevalence, economic burden, and impact on quality of life associated with FBDs necessitate an urgent need for improved understanding of FBDs. Second, preclinical discoveries are at a point that they can be realistically translated into novel diagnostic tests and treatments. Third, FBDs are broadly accepted as bidirectional disorders of the brain-gut axis, differentially affecting individuals throughout life. Research must integrate each component of the brain-gut axis and the influence of biological sex, early-life stressors, and genetic and epigenetic factors in individual patients. Fourth, research priorities to improve diagnostic and management paradigms include enhancement of the provider-patient relationship, longitudinal studies to identify risk and protective factors of FBDs, identification of biomarkers and endophenotypes in symptom severity and treatment response, and incorporation of emerging "-omics" discoveries. These paradigms can be applied by well-trained clinicians who are familiar with multimodal treatments. Fifth, essential components of a successful program will include the generation of a large, validated, broadly accessible database that is rigorously phenotyped; a parallel, linkable biorepository; dedicated resources to support peer-reviewed, hypothesis-driven research; access to dedicated bioinformatics expertise; and oversight by funding agencies to review priorities, progress, and potential synergies with relevant stakeholders.

Hypothesis: Caco-2 cell rotational 3D mechanogenomic turing patterns have clinical implications to colon crypts.

Colon crypts are recognized as a mechanical and biochemical Turing patterning model. Colon epithelial Caco-2 cell monolayer demonstrated 2D Turing patterns via force analysis of apical tight junction live cell imaging which illuminated actomyosin meshwork linking the actomyosin network of individual cells. Actomyosin forces act in a mechanobiological manner that alters cell/nucleus/tissue morphology. We observed the rotational motion of the nucleus in Caco-2 cells that appears to be driven by actomyosin during the formation of a differentiated confluent epithelium. Single- to multi-cell ring/torus-shaped genomes were observed prior to complex fractal Turing patterns extending from a rotating torus centre in a spiral pattern consistent with a gene morphogen motif. These features may contribute to the well-described differentiation from stem cells at the crypt base to the luminal colon epithelium along the crypt axis. This observation may be useful to study the role of mechanogenomic processes and the underlying molecular mechanisms as determinants of cellular and tissue architecture in space and time, which is the focal point of the 4D nucleome initiative. Mathematical and bioengineer modelling of gene circuits and cell shapes may provide a powerful algorithm that will contribute to future precision medicine relevant to a number of common medical disorders.

The Role of the Endocannabinoid System in the Brain-Gut Axis.

The actions of cannabis are mediated by receptors that are part of an endogenous cannabinoid system. The endocannabinoid system (ECS) consists of the naturally occurring ligands N-arachidonoylethanolamine (anandamide) and 2-arachidonoylglycerol (2-AG), their biosynthetic and degradative enzymes, and the cannabinoid (CB) receptors CB1 and CB2. The ECS is a widely distributed transmitter system that controls gut functions peripherally and centrally. It is an important physiologic regulator of gastrointestinal motility. Polymorphisms in the gene encoding CB1 (CNR1) have been associated with some forms of irritable bowel syndrome. The ECS is involved in the control of nausea and vomiting and visceral sensation. The homeostatic role of the ECS also extends to the control of intestinal inflammation. We review the mechanisms by which the ECS links stress and visceral pain. CB1 in sensory ganglia controls visceral sensation, and transcription of CNR1 is modified through epigenetic processes under conditions of chronic stress. These processes might link stress with abdominal pain. The ECS is also involved centrally in the manifestation of stress, and endocannabinoid signaling reduces the activity of hypothalamic-pituitary-adrenal pathways via actions in specific brain regions, notably the prefrontal cortex, amygdala, and hypothalamus. Agents that modulate the ECS are in early stages of development for treatment of gastrointestinal diseases. Increasing our understanding of the ECS will greatly advance our knowledge of interactions between the brain and gut and could lead to new treatments for gastrointestinal disorders.

Epigenetic regulation of genes that modulate chronic stress-induced visceral pain in the peripheral nervous system.

BACKGROUND & AIMS: Chronic stress alters the hypothalamic-pituitary-adrenal axis, increases gut motility, and increases the perception of visceral pain. We investigated whether epigenetic mechanisms regulate chronic stress-induced visceral pain in the peripheral nervous systems of rats. METHODS: Male rats were subjected to 1 hour of water avoidance stress each day, or given daily subcutaneous injections of corticosterone, for 10 consecutive days. L4-L5 and L6-S2 dorsal root ganglia (DRG) were collected and compared between stressed and control rats (placed for 1 hour each day in a tank without water). Levels of cannabinoid receptor 1 (CNR1), DNA (cytosine-5-)-methyltransferase 1 (DNMT1), transient receptor potential vanilloid type 1 (TRPV1), and EP300 were knocked down in DRG neurons in situ with small interfering RNAs. We measured DNA methylation and histone acetylation at genes encoding the glucocorticoid receptor (NR3C1), CNR1, and TRPV1. Visceral pain was measured in response to colorectal distention. RESULTS: Chronic stress was associated with increased methylation of the Nr3c1 promoter and reduced expression of this gene in L6-S2, but not L4-L5, DRGs. Stress also was associated with up-regulation in DNMT1-associated methylation of the Cnr1 promoter and down-regulation of glucocorticoid-receptor-mediated expression of CNR1 in L6-S2, but not L4-L5, DRGs. Concurrently, chronic stress increased expression of the histone acetyltransferase EP300 and increased histone acetylation at the Trpv1 promoter and expression of the TRPV1 receptor in L6-S2 DRG neurons. Knockdown of DNMT1 and EP300 in L6-S2 DRG neurons of rats reduced DNA methylation and histone acetylation, respectively, and prevented chronic stress-induced increases in visceral pain. CONCLUSIONS: Chronic stress increases DNA methylation and histone acetylation of genes that regulate visceral pain sensation in the peripheral nervous system of rats. Blocking epigenetic regulatory pathways in specific regions of the spinal cord might be developed to treat patients with chronic abdominal pain.

Elevated circulating miR-150 and miR-342-3p in patients with irritable bowel syndrome.

BACKGROUND AND AIMS: MicroRNAs (miRNAs) are small non-coding RNAs, which regulate gene expression and are thus of interest as diagnostic markers, and as clues to etiology and targets of intervention. This pilot study examined whether circulating miRNAs are differentially expressed in patients with IBS. METHODS: miRNA microarrays (NanoString) were run on the whole blood of 43 participants. RESULTS: hsa-miR-150 and hsa-miR-342-3p were found to be significantly elevated (FDR adjusted p≤0.05, ≥1.6 fold change) in IBS patients compared to healthy controls. Neither of these miRNAs showed any relationship to race or sex. hsa-miR-150 is associated with inflammatory bowel disorders and pain, and interacts with a protein kinase (AKT2) through which it may affect inflammatory pathways. hsa-miR-342-3p is predicted to interact with mRNAs involved in pain signaling, colonic motility, and smooth muscle function. CONCLUSIONS: This preliminary study reports the association of two miRNAs, detected in whole blood, with IBS. These miRNAs link to pain and inflammatory pathways both of which are thought to be dysregulated in IBS. Larger sample sizes are needed to confirm their importance and potential as biomarkers.

Glucocorticoid receptor-mediated Nr1d1 chromatin circadian misalignment in stress-induced irritable bowel syndrome.

Stress-elevated glucocorticoids cause circadian disturbances and gut-brain axis (GBA) disorders, including irritable bowel syndrome (IBS). We hypothesized that the glucocorticoid receptor (GR/NR3C1) might cause chromatin circadian misalignment in the colon epithelium. We observed significantly decreased core circadian gene Nr1d1 in water avoidance stressed (WAS) BALB/c colon epithelium, like in IBS patients. WAS decreased GR binding at the Nr1d1 promoter E-box (enhancer box), and GR could suppress Nr1d1 via this site. Stress also altered GR binding at the E-box sites along the Ikzf3-Nr1d1 chromatin and remodeled circadian chromatin 3D structures, including Ikzf3-Nr1d1 super-enhancer, Dbp, and Npas2. Intestinal deletion of Nr3c1 specifically abolished these stress-induced transcriptional alternations relevant to IBS phenotypes in BALB/c mice. GR mediated Ikzf3-Nr1d1 chromatin disease related circadian misalignment in stress-induced IBS animal model. This animal model dataset suggests that regulatory SNPs of human IKZF3-NR1D1 transcription through conserved chromatin looping have translational potential based on the GR-mediated circadian-stress crosstalk.

Corticosterone mediates reciprocal changes in CB 1 and TRPV1 receptors in primary sensory neurons in the chronically stressed rat.

BACKGROUND & AIMS: Chronic stress is associated with visceral hyperalgesia in functional gastrointestinal disorders. We investigated whether corticosterone plays a role in chronic psychological stress-induced visceral hyperalgesia. METHODS: Male rats were subjected to 1-hour water avoidance (WA) stress or subcutaneous corticosterone injection daily for 10 consecutive days in the presence or absence of corticoid-receptor antagonist RU-486 and cannabinoid-receptor agonist WIN55,212-2. The visceromotor response to colorectal distension was measured. Receptor protein levels were measured and whole-cell patch-clamp recordings were used to assess transient receptor potential vanilloid type 1 (TRPV1) currents in L6-S2 dorsal root ganglion (DRG) neurons. Mass spectrometry was used to measure endocannabinoid anandamide content. RESULTS: Chronic WA stress was associated with visceral hyperalgesia in response to colorectal distension, increased stool output and reciprocal changes in cannabinoid receptor 1 (CB1) (decreased) and TRPV1 (increased) receptor expression and function. Treatment of WA stressed rats with RU-486 prevented these changes. Control rats treated with serial injections of corticosterone in situ showed a significant increase in serum corticosterone associated with visceral hyperalgesia, enhanced anandamide content, increased TRPV1, and decreased CB1 receptor protein levels, which were prevented by co-treatment with RU-486. Exposure of isolated control L6-S2 DRGs in vitro to corticosterone reproduced the changes in CB1 and TRPV1 receptors observed in situ, which was prevented by co-treatment with RU-486 or WIN55,212-2. CONCLUSIONS: These results support a novel role for corticosterone to modulate CB1 and TRPV1-receptor pathways in L6-S2 DRGs in the chronic WA stressed rat, which contributes to visceral hyperalgesia observed in this model.

Chronic psychological stress alters gene expression in rat colon epithelial cells promoting chromatin remodeling, barrier dysfunction and inflammation.

Chronic stress is commonly associated with enhanced abdominal pain (visceral hypersensitivity), but the cellular mechanisms underlying how chronic stress induces visceral hypersensitivity are poorly understood. In this study, we examined changes in gene expression in colon epithelial cells from a rat model using RNA-sequencing to examine stress-induced changes to the transcriptome. Following chronic stress, the most significantly up-regulated genes included Atg16l1, Coq10b, Dcaf13, Nat2, Ptbp2, Rras2, Spink4 and down-regulated genes including Abat, Cited2, Cnnm2, Dab2ip, Plekhm1, Scd2, and Tab2. The primary altered biological processes revealed by network enrichment analysis were inflammation/immune response, tissue morphogenesis and development, and nucleosome/chromatin assembly. The most significantly down-regulated process was the digestive system development/function, whereas the most significantly up-regulated processes were inflammatory response, organismal injury, and chromatin remodeling mediated by H3K9 methylation. Furthermore, a subpopulation of stressed rats demonstrated very significantly altered gene expression and transcript isoforms, enriched for the differential expression of genes involved in the inflammatory response, including upregulation of cytokine and chemokine receptor gene expression coupled with downregulation of epithelial adherens and tight junction mRNAs. In summary, these findings support that chronic stress is associated with increased levels of cytokines and chemokines, their downstream signaling pathways coupled to dysregulation of intestinal cell development and function. Epigenetic regulation of chromatin remodeling likely plays a prominent role in this process. Results also suggest that super enhancers play a primary role in chronic stress-associated intestinal barrier dysfunction.

Histone H3K9 methylation regulates chronic stress and IL-6-induced colon epithelial permeability and visceral pain.

BACKGROUND: Chronic stress is associated with activation of the HPA axis, elevation in pro-inflammatory cytokines, decrease in intestinal epithelial cell tight junction (TJ) proteins, and enhanced visceral pain. It is unknown whether epigenetic regulatory pathways play a role in chronic stress-induced intestinal barrier dysfunction and visceral hyperalgesia. METHODS: Young adult male rats were subjected to water avoidance stress ± H3K9 methylation inhibitors or siRNAs. Visceral pain response was assessed. Differentiated Caco-2/BBE cells and human colonoids were treated with cortisol or IL-6 ± antagonists. Expression of TJ, IL-6, and H3K9 methylation status at gene promoters was measured. Transepithelial electrical resistance and FITC-dextran permeability were evaluated. KEY RESULTS: Chronic stress induced IL-6 up-regulation prior to a decrease in TJ proteins in the rat colon. The IL-6 level inversely correlated with occludin expression. Treatment with IL-6 decreased occludin and induced visceral hyperalgesia. Chronic stress and IL-6 increased H3K9 methylation and decreased transcriptional GR binding to the occludin gene promoter, leading to down-regulation of protein expression and increase in paracellular permeability. Intrarectal administration of a H3K9 methylation antagonist prevented chronic stress-induced visceral hyperalgesia in the rat. In a human colonoid model, cortisol decreased occludin expression, which was prevented by the GR antagonist RU486, and IL-6 increased H3K9 methylation and decreased TJ protein levels, which were prevented by inhibitors of H3K9 methylation. CONCLUSIONS & INFERENCES: Our findings support a novel role for methylation of the repressive histone H3K9 to regulate chronic stress, pro-inflammatory cytokine-mediated reduction in colon TJ protein levels, and increase in paracellular permeability and visceral hyperalgesia.

Neurogenic chronic intestinal pseudo-obstruction: antineuronal antibody-mediated activation of autophagy via Fas.

BACKGROUND & AIMS: Activation of autoimmune pathways has been implicated as a contributing mechanism to the pathophysiology in some patients with chronic intestinal pseudoobstruction (CIP). In this study we tested the hypothesis that sera from a subpopulation of patients with CIP contain autoantibodies that activate autophagy via a Fas-dependent pathway in cultured human neuroblastoma SH-Sy5Y cells. METHODS: Twenty-five patients with established neurogenic CIP (20 women, 5 men; age range, 21-57 y) were investigated and circulating antineuronal antibodies to enteric neurons were found in 6 (24%) patients. The ability of antineuronal antibodies to induce autophagy was assessed using immunohistochemical, Western immunoblot, and molecular techniques. The presence of autophagosomes was monitored using a specific immunohistochemical marker, anti-microtubule-associated light chain immunoreactivity, and colocalization with mitochondrial- and Fas-activated death domain immunofluorescence using appropriate antibodies in cells exposed to sera from matched healthy controls and patients with neurogenic CIP. RESULTS: Exposure of SH-Sy5Y cells to sera from patients with CIP containing antineuronal antibodies revealed increased binding of autoimmune immunoglobulin (IgG class) to the surface of SH-Sy5Y cells and increased formation of autophagosomes showing colocalization with mitochondria and Fas-activated death domain compared with control sera. Pretreatment of sera with either protein L agarose beads or a soluble Fas receptor (extracellular domain) chimera prevented the stimulation of autophagy. CONCLUSIONS: We provide novel evidence that antineuronal antibodies may contribute to neuronal dysfunction observed in a subset of patients with neurogenic CIP via autoantibody-mediated activation of autophagy involving the Fas receptor complex.

Chronic stress and intestinal permeability: Lubiprostone regulates glucocorticoid receptor-mediated changes in colon epithelial tight junction proteins, barrier function, and visceral pain in the rodent and human.

BACKGROUND: Chronic psychological stress is associated with increased intestinal epithelial permeability and visceral hyperalgesia. Lubiprostone, an agonist for chloride channel-2, promotes secretion and accelerates restoration of injury-induced epithelial barrier dysfunction. The mechanisms underlying how lubiprostone regulates colon epithelial barrier function and visceral hyperalgesia in chronic stress remain unknown. METHODS: Male rats were subjected to water avoidance stress for 10 consecutive days. Lubiprostone was administered daily during the stress phase. Visceromotor response to colorectal distension was measured. Human colon crypts and cell lines were treated with cortisol and lubiprostone. The transepithelial electrical resistance and FITC-dextran permeability were assayed. Chromatin immunoprecipitation was conducted to assess glucocorticoid receptor binding at tight junction gene promoters. KEY RESULTS: Lubiprostone significantly decreased chronic stress-induced visceral hyperalgesia in the rat (P < 0.05; n = 6). WA stress decreased occludin and claudin-1 and increased claudin-2 in rat colon crypts, which was prevented by lubiprostone. Cortisol treatment induced similar alterations of tight junction protein expression in Caco-2/BBE cells (P < 0.05) and significantly changed paracellular permeability in monolayers (P < 0.01). These changes were blocked by lubiprostone. Glucocorticoid receptor and its binding at occludin promoter region were decreased in cortisol-treated cells and human colon crypts, which was largely reversed by lubiprostone. In rat colonic cells, glucocorticoid receptor and its co-chaperone proteins were down-regulated after corticosterone treatment and lubiprostone reversed these changes. CONCLUSIONS & INFERENCES: Lubiprostone preferentially prevents chronic stress-induced alterations of intestinal epithelial tight junctions, barrier function, and visceral hyperalgesia that was associated with modulation of glucocorticoid receptor expression and function.

The TRPV1 receptor is associated with preferential stress in large dorsal root ganglion neurons in early diabetic sensory neuropathy.

Chronic diabetic neuropathy is associated with peripheral demyelination and degeneration of nerve fibers. The mechanism(s) underlying neuronal injury in diabetic sensory neuropathy remain poorly understood. Recently, we reported increased expression and function of transient receptor potential vanilloid 1 (TRPV1) in large dorsal root ganglion (DRG) neurons in diabetic sensory neuropathy. In this study, we examined the effects of TRPV1 activation on cell injury pathways in this subpopulation of neurons in the streptozotocin-induced diabetic rat model. Large DRG neurons from diabetic (6-8 weeks) rats displayed increased oxidative stress and activation of cell injury markers compared with healthy controls. Capsaicin (CAP) treatment induced decreased labeling of MitoTracker Red and increased cytosolic cytochrome c and activation of caspase 3 in large neurons isolated from diabetic rats. CAP treatment also induced oxidative stress in large diabetic DRG neurons, which was blocked by pre-treatment with caspase or calpain inhibitor. In addition, both mu-calpain expression and calpain activity were significantly increased in DRG neurons from diabetic rats after CAP treatment. Treatment with capsazepine, a competitive TRPV1 antagonist, markedly reduced these abnormalities in vitro and prevented activation of cell injury in large DRG neurons in diabetic rats in vivo. These results suggest that activation of the TRPV1 receptor activates pathways associated with caspase-dependent and calpain-dependent stress in large DRG neurons in STZ-diabetic rats. Activation of the TRPV1 receptor may contribute to preferential neuronal stress in large DRG neurons relatively early in diabetic sensory neuropathy.

Type 2 diabetes with neuropathy: autoantibody stimulation of autophagy via Fas.

We reported previously that sera from patients with type 2 diabetes and neuropathy induce autophagy in human neuroblastoma (SH-SY5Y) cells. Here we report that enriched immunoglobulin fractions from a subpopulation of these patients induce autophagy and colocalization with Fas-activated death domain (FADD), a component of the Fas-activated death domain receptor signaling pathway. These effects were replicated by treatment of SY5Y cells with Fas ligand, tumor necrosis factor alpha and an agonist anti-Fas antibody. Preincubation of these sera with a soluble Fas receptor chimera (extracellular domain) markedly decreased the stimulation of autophagy. The results suggest that sera from subset of individuals with type 2 diabetes and neuropathy contain autoantibodies that activate the Fas cascade.

The role of serotonin in irritable bowel syndrome: implications for management.

Irritable bowel syndrome (IBS) is a poorly understood, common, chronic condition characterized by -abdominal discomfort associated with altered bowel habits in the absence of structural or biochemical abnormalities. Despite the significant economic and personal burden associated with IBS, treatment options remain limited. Serotonin is recognized as a key neurotransmitter in intestinal secretory, sensory, and motor function. Although the pathophysiology of IBS is incompletely understood, there is evidence that abnormalities in brain-gut signaling and serotonin metabolism play a role. This article reviews the evidence that serotonin, one of the better-understood neurotransmitters with respect to its role in human central and intestinal physiology, plays a role in IBS. Serotonin signaling is discussed, with a focus on receptor subtypes and the therapeutic agents that target these receptors. Evidence that IBS is associated with perturbations in serotonin metabolism at various steps in the signaling pathway is also addressed, along with the limitations on alteration in serotonin metabolism as the sole explanation for the constellation of symptoms observed in patients with IBS.

Chronic stress-associated visceral hyperalgesia correlates with severity of intestinal barrier dysfunction.

In humans, chronic psychological stress is associated with increased intestinal paracellular permeability and visceral hyperalgesia, which is recapitulated in the chronic intermittent water avoidance stress (WAS) rat model. However, it is unknown whether enhanced visceral pain and permeability are intrinsically linked and correlate. Treatment of rats with lubiprostone during WAS significantly reduced WAS-induced changes in intestinal epithelial paracellular permeability and visceral hyperalgesia in a subpopulation of rats. Lubiprostone also prevented WAS-induced decreases in the epithelial tight junction protein, occludin (Ocln). To address the question of whether the magnitude of visceral pain correlates with the extent of altered intestinal permeability, we measured both end points in the same animal because of well-described individual differences in pain response. Our studies demonstrate that visceral pain and increased colon permeability positively correlate (0.6008, P = 0.0084). Finally, exposure of the distal colon in control animals to Ocln siRNA in vivo revealed that knockdown of Ocln protein inversely correlated with increased paracellular permeability and enhanced visceral pain similar to the levels observed in WAS-responsive rats. These data support that Ocln plays a potentially significant role in the development of stress-induced increased colon permeability. We believe this is the first demonstration that the level of chronic stress-associated visceral hyperalgesia directly correlates with the magnitude of altered colon epithelial paracellular permeability.