Functional Dyspepsia Microbiome Study

Recurrent abdominal pain has long been acknowledged to be one of the most common chronic pain entities in children. In the US, ~13-17% of school-aged children and adolescents report abdominal pain that occurs at least weekly and 21% of these report pain severe enough to interfere with daily activities. (1) For most of these children (~90%), no specific organic disease is found. However, these children experience decreases in quality of life that are comparable to children with identifiable organic diseases such as inflammatory bowel disease and gastro-esophageal reflux. (2) In addition, children with no identifiable organic disease do not appear to get better on their own and often continue to have problems with abdominal pain and associated symptoms into adulthood. (3,4)

Over 90% of children with chronic abdominal pain will fit the diagnostic criteria for a functional gastrointestinal disorder (FGID). (5) As defined by Rome criteria, the FGIDs are a set of diagnoses with specific symptom profiles in the absence of structural or biochemical abnormalities to explain symptoms. FGIDs related to chronic or recurrent abdominal pain include irritable bowel syndrome (IBS), functional dyspepsia (FD), functional abdominal pain (FAP), and abdominal migraine. (6) FGIDs are probably best understood utilizing a biopsychosocial model which states that abdominal pain occurs as a result of varying contributions from, and interactions between, biological factors, psychological factors, and social factors. The biological factors most implicated in the generation of abdominal pain include motility disturbances, visceral hypersensitivity, and inflammation. Stress/anxiety is the most implicated psychologic factor. Anxiety may interact with both motility and inflammation. For example, the investigators have demonstrated an association between anxiety and mucosal mast cells in children with post-prandial distress syndrome, a subcategory of FD. (7) The scaffolding for the interaction between the various factors has been termed the brain-gut axis consisting of both neural and humoral pathways.

There is increasing evidence that the investigators need to expand this paradigm to include interaction of the biopsychosocial model with the intestinal flora, i.e. a brain-gut-microbiota axis (B-G-M axis). It is becoming increasingly clear that host anxiety and stress can effect the composition of the microbiome. (8) Likewise, the microbiome can influence neural development, brain chemistry, and a wide range of behavioral phenomena including emotional behavior, pain perception, and how the stress system responds. (9) Both immune and neural pathways are involved intimately in visceral pain perception and intestinal microbiota can modulate this communication. (8) The general scaffolding of the brain-gut-microbiota axis includes the CNS, neuroendocrine and neuroimmune systems, autonomic nervous system, and the enteric nervous system. (10) Microbiota communicate with the brain-gut axis through different mechanisms including direct interaction with mucosal cells, via immune cells, and via contact with neural endings. (11)

The B-G-M axis has been studied primarily under four conditions: 1. germ free intestines, 2. infection or bacterial introduction, 3. antibiotic exposure, and 4. probiotic treatment. Germ free mice exhibit anxiety-like behavior accompanied by decreased N-methyl-D-aspartate receptor subunit mRNA expression in the amygdala and increased BDNF expression and decreased serotonin receptor 1A expression in the dentate granule layer of the hippocampus. (12) The brain is aware of the introduction of pathogenic microbes into the GI tract and this results in brain stem nuclei becoming activated and in some instances, associated with the development of anxiety-like behavior. (8) This occurs within hours after introduction of pathogens at subclinical thresholds. (8) Even minute doses of microbes that do not trigger an immune response are capable of influencing neurotransmission in the paraventricular hypothalamus, the central nucleus of the amygdale, and the bed nucleus of the stria terminalis, three regions involved in the processing of emotions related to anxiety and mood. (13) Citerobacter rodentium (CR) is a bacteria that has been evaluated in a number of studies involving mice. CR-infected mice demonstrated anxious-like behavior at 7-8 hours. (14) CR has been demonstrated to induce anxiety-like behavior associated with no change in plasma levels of IFN-γ, TNF-α, or IL-12 but with evidence of increased vagal transmission without mucosal inflammation. (15) Additional work has also demonstrated that Helicobacter pylori infected mice exhibit anxiety-like behavior corrected by treatment with a probiotic, Bifidobacterium. Oral antibiotics have also been shown to induce anxiety-like behavior in mice. (8) This behavior change is transient and behavior normalizes once normal flora has been restored. This effect is not seen with systemic antibiotic administration. Changes in behavior are accompanied by an increase in brain-derived neurotrophic factor (BDNF) in the hippocampus and amygdala. (8) Lastly, clinical evidence is mounting to support a role for probiotics in reducing anxiety and the stress response as well as improving mood in patients with IBS and those with chronic fatigue. (14) Probiotics have been shown to reduce anxiety in rats and to have beneficial psychological effects with a decrease in serum cortisol in humans. (14) Lactobacillus reuteri has been shown to decrease anxiety and the stress-induced increase in corticosterone in mice. (A) In addition, there are a number of other studies demonstrating the effects of probiotics on anxiety in humans. (8,9,13)

The DCX-domain family of proteins has been demonstrated to be involved in signal transduction and cytoskeletal regulation. The investigators have demonstrated that DCDC3a (DCLK1/DCAMKL1) is an intestinal epithelial stem cell marker expressed in the intestinal mesenchymal stem cell niche which plays a critical role during intestinal tissue damage and repair. DCDC2 is altered in the mouse model by Citrobacter rodentium infection and TNBS induced colitis. DCDC2 knockout mice exhibit an increased anxiety phenotype. (16)

BDNF has been found to promote neuronal survival and differentiation and to guide axon extension both in vitro and in vivo. (17) Evidence suggests that BDNF may act as a stress-responsive intercellular messenger modifying HPA axis activity. (17) Short-term acute stress induces a significant increase in BDNF mRNA and protein in both younger and older groups but changes in the younger group are substantially greater. (18) BDNF has interactions with both inflammation and pain. BDNF knock out mice show a weaker visceral response to colorectal distention. (19) A human study compared 40 adults with IBS to 21 healthy controls. (20) Biopsies from patients with IBS revealed significant upregulation of BDNF as compared to controls. (20)

The transient receptor potential vanilloid type-1 (TRPV1) is expressed throughout the gastrointestinal tract in myenteric ganglia, muscular layers, and mucosa and TRPV1 expression has been reported on mast cells. (21) TRPV1 is expressed by intestinal sensory nerves and activated by capsaicin, heat, acid, and inflammation. (21) TRPV1 receptor has been implicated as a mechanosensor involved in integrating painful stimuli and in the generation of neurogenic inflammation and hyperalgesia. (22) In rodent models of inflammation, TPRV1 is upregulated with subsequent visceral hyperalgesia to mechanical and chemical stimuli which can be attenuated by TRPV1 antagonism. (22,23) In the rat model, it appears that mast cells are required for upregulation TRPV1 under both infectious and stress conditions. (24) In humans with IBS, there is a significant increase in rectosigmoid TRPV1-immunoreactive fibers and TRPV1 and mast cells are related to abdominal pain scores. (21) Likewise, IBS in quiescent inflammatory bowel disease is associated with an increase in rectosigmoid colon TRPV1-immunoreactive fibers as compared to asymptomatic quiescent IBD or healthy controls and again these fibers correlate with the abdominal pain score. (25) There is evidence of a significant role for TRPV1 in visceral sensitivity and TRPV1 appears to interact with inflammation in general, mast cells in particular, and to be influenced by intestinal infection and stress.

The major initiator of the body's physiological stress response is the release of corticotropin releasing hormone (CRH). CRH, produced within the hypothalamus (as well as by immune cells, including human lymphocytes and mast cells), is the principal regulator of the basal and stress-induced pituitary-adrenal axis which, in turn, activates glucocorticoid (e.g. cortisol) and adrenal androgen secretion. Though results have been variable, the majority of studies support an enhanced HPA axis responsiveness in at least a subset of adults with IBS. (26-30) CRH receptors (CRH-R) are widely expressed within the gastrointestinal tract and immune cells, where CRH-R activation has multiple effects which may be relevant in the etiology of FGIDs. These effects include alteration of autonomic balance, visceral sensitivity, motility disturbances, and inflammation. The most important mechanism by which CRH may generate pain is through interaction with inflammatory cells, directly and via epithelial cells and enteric nerves. CRH mediates visceral hypersensitivity by activating mucosal mast cells with subsequent mediator sensitization of afferent sensory enteric nerves. (31,32) Gastric mast cells have been shown to be increased in adults with dyspepsia and may contain both pre-formed and newly synthesized cytokines (including IL-4, IL-5, IL-6, and TNF-α, among others). (33-34) The investigators have demonstrated delayed gastric emptying and increased gastric dysrhythmia in children/adolescents with FD with elevated antral mast cell density. (35) The investigators have also demonstrated this dysrhythmia to be associated with increased post-prandial pain. (36) Adult humans have demonstrated selective luminal release of tryptase and histamine from jejunal mast cells under cold stress; the magnitude of release was shown to be similar to that induced by antigen exposure in food allergic patients. (37) This is a rapid response with peak histamine and tryptase concentrations occurring between 15 and 30 minutes. Mast cells may also recruit eosinophils as a secondary effector cell. Eosinophils are increased in the duodenal mucosa of 71% of children with FD and they have also been found to be increased in adults with FD. (38,39) These eosinophils are highly activated and the investigators have demonstrated clinical improvement with treatment directed at mucosal eosinophils. (40,41) The investigators have previously found a high correlation between anxiety scores and mucosal eosinophil density, providing preliminary support for the role of CRH in downstream eosinophil recruitment and activation. (42) Stress also has been shown to shift the relative proportion and trafficking of T helper lymphocytes towards a Th2 or "allergic" phenotype. This shift is driven by catecholamines and central CRH as well as CRH from peripheral nerves and inflammatory cells. The Th2 phenotype is associated with release of IL 4,IL 10, and IL 13, which stimulate growth and activation of mast cells and eosinophils. (43) CRH also has been associated with increased expression of TNF-α, MCP1, and IL 8, which have, in turn, been associated with hyperalgesia. (44-46) As described above, CRH can initiate an inflammatory cascade which may lead to pain directly or indirectly by causing dysmotility and visceral hypersensitivity.

The purpose of the current study is to describe the microbiome in children with FD and to explore relationships between the microbiome and postprandial distress syndrome, anxiety scores, and mucosal biomarkers or anxiety.

SPECIFIC AIMS

1. To determine the frequencies and relative proportions for specific bacteria or bacteria phyla in children with FD in both duodenal mucosal specimens and stool samples.

2. To determine if the frequencies or proportions of specific bacteria or bacteria phyla differ between children with and without PDS.

3. To determine bi-variate correlations between bacteria/phyla frequency, bacteria/ phyla proportion, anxiety scores, and mucosal biomarkers, respectively.