Patent Application
20230149355
The disclose relates to tryptophan catabolites and methods of use thereof. In particular, the disclosure relates to tryptophan catabolites and uses thereof in methods of treating gastrointestinal disorders, suppressing appetite, and promoting weight loss in a subject.
Transient receptor potential (TRP) channels act as molecular sensors of multiple stimuli, including changes in pH, chemicals, temperature, and osmolarity. The TRP family of ion channels is divided into six subfamilies, classified as canonical (TRPC), vanilloid (TRPV), ankyrin (TRPA), melastatin (TRPM), polycystin (TRPP), and mucolipin (TRPML). Transient receptor potential ankyrin 1 (TRPA1) is an excitatory calcium-permeable non-selective cation channel expressed in multiple cell types in the central and peripheral nervous system. This includes a subpopulation of nociceptive primary sensory neurons from dorsal root ganglia and trigeminal ganglia that innervate epidermis as well as visceral organs like the lung, heart, and lower gastrointestinal (GI) tract. In addition to nociceptive sensory neurons, TRPA1 is expressed in a subpopulation of vagal sensory neurons that innervate almost all the visceral organs. In addition to nerve cells, TRPA1 is also expressed in astrocytes, oligodendrocytes and Schwann cells. TRPA1 is a common target for chemically-diverse pronociceptive agonists generated in multiple pathophysiological pain conditions. Thereby, pain therapy that reduces TRPA1 agonism can be expected to be superior compared to many other drugs targeting single nociceptive signaling pathways.
In addition to pain, TRPA1 is also known as a gate keeper for inflammation. Primary afferent sensory neurons that innervate target organs release inflammatory neuropeptides in the local area of tissue damage. TRPA1 channels are required for neuronal excitation, the release of inflammatory neuropeptides, and subsequent pain hypersensitivity. TRPA1 is also activated by the release of inflammatory agents from non-neuronal cells in the area of tissue injury or disease.
TRPA1 is also widely expressed outside the nervous system where its function is less understood. In humans, TRPA1 is expressed in high levels in the gastrointestinal tract, urinary bladder and lymphoid tissues including epithelial cells (such as enteroendocrine cells in the intestinal epithelium, urothelium lining the lower urinary tract, endothelium (such as endothelium of rat cerebral and cerebellar arteries) as well as immune cells (such as macrophages and CD4+ T-cells).
Tryptophan is an essential amino acid in humans and other animals, and is also catabolized by bacteria and animals into diverse derivatives. Bacterial products of tryptophan catabolism are diverse with distinct properties, but many are still undefined. Humans and other animals can also metabolize tryptophan into diverse bioactive products including the neurotransmitter serotonin (5-HT). Although high levels of tryptophan are found in the gut, it as well as its microbial and host catabolites can be found throughout the body. Some microbial tryptophan derivatives are better understood than others. For some tryptophan catabolites there are known effects on host biology without known mechanisms of action. Moreover, new microbial tryptophan catabolites continue to be discovered, and many have no defined function. Importantly, there is no existing information linking bacterial tryptophan catabolites and TRP channels in any cell type in humans or any other animals.
In some aspects, provided herein are methods of treating or preventing a gastrointestinal disorder in a subject. In some embodiments, provided herein is a method of treating or preventing a gastrointestinal disorder in a subject, comprising providing to the subject a composition comprising a tryptophan catabolite. Any suitable tryptophan catabolite may be used. In some embodiments, the tryptophan catabolite is indole, 3-methylindole (Skatole), indole-3-carboxaldehdye (IAld), Indoleacetic acid (IAA), Indoleacrylic acid (IA), indole-3-ethanol (IE), Indole-3-lactic acid (ILA), 3-indolepropionic acid (IPA), or tryptamine.
The methods described herein may be used for treatment of any suitable gastrointestinal disorder. In some embodiments, the gastrointestinal disorder is a gastrointestinal motility disorder. In some embodiments, the gastrointestinal disorder is intestinal pseudo-obstruction, small bowel bacterial overgrowth, small intestinal bacterial overgrowth, constipation, outlet obstruction type constipation, diarrhea, or tropical sprue. In some embodiments, the gastrointestinal disorder is diarrhea associated with diarrhea-predominant irritable bowel syndrome (IBS-D) or constipation associated with constipation-predominant irritable bowel syndrome (IBS-C). In some embodiments, the gastrointestinal disorder is irritable bowel syndrome (IBS). In some embodiments, the IBS is constipation predominant IBS (IBS-C), diarrhea predominant IBS (IBS-D), or post-infections IBS (PI-IBS). In some embodiments, the gastrointestinal disorder is colitis. In some embodiments, the gastrointestinal disorder is Crohn's disease. In some embodiments, the composition comprises indole or indole-3-carboxaldehdye.
In some aspects, provided herein are methods of inducing weight loss and/or suppressing appetite in a subject. In some embodiments, provided herein is a method of inducing weight loss in a subject, comprising administering to the subject a composition comprising a tryptophan catabolite. In some embodiments, provided herein is a method of suppressing appetite in a subject, comprising administering to the subject a composition comprising a tryptophan catabolite. Any suitable tryptophan catabolite may be used. In some embodiments, the tryptophan catabolite is indole, 3-methylindole (Skatole), indole-3-carboxaldehdye (IAld), Indoleacetic acid (IAA), Indoleacrylic acid (IA), indole-3-ethanol (IE), Indole-3-lactic acid (ILA), 3-indolepropionic acid (IPA), or tryptamine.
In some aspects, provided herein are methods of cleansing the colon of a subject. In some embodiments, provided herein is a method of cleansing the colon of a subject, comprising administering to the subject a composition comprising a tryptophan catabolite. Any suitable tryptophan catabolite may be used. In some embodiments, the tryptophan catabolite is indole, 3-methylindole (Skatole), indole-3-carboxaldehdye (IAld), Indoleacetic acid (IAA), Indoleacrylic acid (IA), indole-3-ethanol (IE), Indole-3-lactic acid (ILA), 3-indolepropionic acid (IPA), or tryptamine.
For any of the methods described herein, the subject may be human.
In some aspects, provided herein are compositions comprising a tryptophan catabolite. The tryptophan catabolite may be indole, 3-methylindole (Skatole), indole-3-carboxaldehdye (IAld), Indoleacetic acid (IAA), Indoleacrylic acid (IA), indole-3-ethanol (IE), Indole-3-lactic acid (ILA), 3-indolepropionic acid (IPA), or tryptamine. The compositions find use in a variety of methods, including treating or preventing a gastrointestinal disorder, inducing weight loss, suppressing appetite, and/or cleansing the colon of a subject. The subject may be human. The gastrointestinal disorder may be a gastrointestinal motility disorder. For example, provided herein are compositions for use in a method of treating or preventing a gastrointestinal motility disorder such as intestinal pseudo-obstruction, small bowel bacterial overgrowth, small intestinal bacterial overgrowth, constipation, outlet obstruction type constipation, diarrhea, or tropical sprue. As another example, provided herein are compositions for use in a method of treating or preventing a gastrointestinal disorder such as diarrhea associated with diarrhea-predominant irritable bowel syndrome (IBS-D) or constipation associated with constipation-predominant irritable bowel syndrome (IBS-C). Other suitable gastrointestinal disorders include, for example, is irritable bowel syndrome (IBS). For example, the IBS may be constipation-predominant IBS (IBS-C), diarrhea predominant IBS (IBS-D), or post-infections IBS (PI-IBS). In some embodiments, the gastrointestinal disorder is colitis. In some embodiments, the gastrointestinal disorder is Crohn's disease.
Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.
The use of the terms “a” and “an” and “the” and “at least one” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.
The use of the term “at least one” followed by a list of one or more items (for example, “at least one of A and B”) is to be construed to mean one item selected from the listed items (A or B) or any combination of two or more of the listed items (A and B), unless otherwise indicated herein or clearly contradicted by context.
As used herein, the term “about” is used to provide flexibility to a numerical range endpoint by providing that a given value may be “slightly above” or “slightly below” the endpoint without affecting the desired result. In some embodiments, “about” may refer to variations of in some embodiments ±20%, in some embodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, in some embodiments 0.5%, and in some embodiments ±0.10% from the specified amount.
As used herein, the terms “comprise”, “include”, and linguistic variations thereof denote the presence of recited feature(s), element(s), method step(s), etc. without the exclusion of the presence of additional feature(s), element(s), method step(s), etc.
Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise-indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if a concentration range is stated as 1% to 50%, it is intended that values such as 2% to 40%, 10% to 30%, or 1% to 3%, etc., are expressly enumerated in this specification. These are only examples of what is specifically intended, and all possible combinations of numerical values between and including the lowest value and the highest value enumerated are to be considered to be expressly stated in this disclosure.
The term “amino acid” refers to natural amino acids, unnatural amino acids, and amino acid analogs, all in their D and L stereoisomers, unless otherwise indicated, if their structures allow such stereoisomeric forms.
Natural amino acids include alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine (Cys or C), glutamine (Gln or Q), glutamic acid (Glu or E), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), Lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y) and valine (Val or V).
As used herein, the terms “co-administration” and variations thereof refer to the administration of at least two agent(s) or therapies to a subject. In some embodiments, the co-administration of two or more agents or therapies is concurrent. In other embodiments, a first agent/therapy is administered prior to a second agent/therapy. Those of skill in the art understand that the formulations and/or routes of administration of the various agents or therapies used may vary. The appropriate dosage for co-administration can be readily determined by one skilled in the art. In some embodiments, when agents or therapies are co-administered, the respective agents or therapies are administered at lower dosages than appropriate for their administration alone. Accordingly, co-administration may be especially desirable in embodiments where the co-administration of two or more agents results in sensitization of a subject to beneficial effects of one of the agents via co-administration of the other agent.
The term “carrier” as used herein refers to any pharmaceutically acceptable solvent of agents that will allow a therapeutic composition to be administered to the subject. A “carrier” as used herein, therefore, refers to such solvent as, but not limited to, water, saline, physiological saline, oil-water emulsions, gels, or any other solvent or combination of solvents and compounds known to one of skill in the art that is pharmaceutically and physiologically acceptable to the recipient human or animal. The term “pharmaceutically acceptable” as used herein refers to a compound or composition that will not impair the physiology of the recipient human or animal to the extent that the viability of the recipient is compromised. For example, “pharmaceutically acceptable” may refer to a compound or composition that does not substantially produce adverse reactions, e.g., toxic, allergic, or immunological reactions, when administered to a subject.
The term “intestine” or “intestines” as used interchangeably herein refer to the long-continuous tube running from the stomach to the anus. The term “intestine” includes the small intestine, the large intestine, and the rectum.
The term “gastrointestinal tract” or “GI tract” as used herein refers to the tract from the mouth to the anus. The gastrointestinal tract includes the mouth, esophagus, stomach, and intestines. The “gastrointestinal tract” may also be referred to herein as the “gut”.
The term “motility” when used in reference to the GI tract refers to the contraction of muscles that mix and propel contents in the GI tract. The term “gastrointestinal motility disorder” refers to any number of conditions in which motility in the GI tract is abnormal, which may cause one or more undesirable symptoms in an afflicted subject.
As used herein, the terms “prevent,” “prevention,” and preventing” may refer to reducing the likelihood of a particular condition or disease state (e.g., a gastrointestinal disorder) from occurring in a subject not presently experiencing or afflicted with the condition or disease state. The terms do not necessarily indicate complete or absolute prevention. For example “preventing a gastrointestinal disorder” refers to reducing the likelihood of the gastrointestinal disorder occurring in a subject not presently experiencing or diagnosed with the disorder. The term may also refer to delaying the onset of a particular condition or disease state (e.g., a gastrointestinal disorder) in a subject not presently experiencing or afflicted with the condition or disease state. In order to “prevent” a condition, a composition or method need only reduce the likelihood and/or delay the onset of the condition, not completely block any possibility thereof “Prevention,” encompasses any administration or application of a therapeutic or technique to reduce the likelihood or delay the onset of a disease developing (e.g., in a mammal, including a human).
As used herein, the terms “subject” and “patient” are used interchangeably herein and refer to both human and nonhuman animals. The term “nonhuman animals” includes all vertebrates, e.g., mammals and non-mammals, such as nonhuman primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, and the like. In some embodiments, the subject is a human. In some embodiments, the subject is a human. In particular embodiments, the subject may be male. In other embodiments, the subject may be female. In some embodiments, the subject is suffering from or at risk of developing a gastrointestinal disorder. In some embodiments, the subject is overweight or obese.
As used herein, “treat”, “treating”, “treatment”, and variations thereof refer to the clinical intervention made in response to a disease, disorder or physiological condition manifested by a patient or to which a patient may be susceptible. The aim of treatment includes the alleviation or prevention of symptoms, slowing or stopping the progression or worsening of a disease, disorder, or condition and/or the remission of the disease, disorder or condition. In some embodiments, treating a gastrointestinal disorder refers to the management and care of the subject for combating and reducing one or more symptoms of the disorder. Treating a gastrointestinal disorder may reduce, inhibit, ameliorate and/or improve the onset of the symptoms or complications, alleviating the symptoms or complications of the disorder, or eliminating the disorder.
In some aspects, provided herein are compositions. In some embodiments, provided herein are compositions comprising a TRPA1 agonist. In some embodiments, the TRPA1 agonist is a tryptophan catabolite. The tryptophan catabolite may be any suitable compound that is generated during the catabolism of tryptophan. In some embodiments, the tryptophan catabolite is indole, 3-methylindole (Skatole), indole-3-carboxaldehdye (IAld), Indoleacetic acid (IAA), Indoleacrylic acid (IA), indole-3-ethanol (IE), Indole-3-lactic acid (ILA), 3-indolepropionic acid (IPA), or tryptamine. In some embodiments, the compositions comprise a plurality of tryptophan derivatives. Any suitable combination of tryptophan derivatives may be used. The formula and structure of common tryptophan catabolites are highlighted in Table 1. In some embodiments, the composition may comprise pharmaceutically acceptable salts, solvates, hydrates, prodrugs, or derivatives of a tryptophan catabolite.
In some embodiments, the tryptophan catabolite is made synthetically. In some embodiments, the tryptophan catabolite is isolated from a naturally occurring source. For example, the tryptophan catabolite may be produced by one or more bacteria, in which case the tryptophan catabolite may be referred to herein as a “bacterial tryptophan catabolite”.
In some embodiments, the compositions described herein may be used in methods of modulating GI tract motility. In some embodiments, the compositions described herein may be used in methods of modulating intestinal motility. For example, the compositions may be provided to a subject to modulate motility in any suitable area of the intestine, including the small intestine or the large intestine. Disruption of normal intestinal motility occurs in a variety of GI motility disorders, therefore modulating intestinal motility may be beneficial for treating or preventing a GI motility disorder.
In some embodiments, the compositions described herein may be used in methods of treating or preventing one or more gastrointestinal disorders. The gastrointestinal disorder may be a functional gastrointestinal disorder, wherein the GI tract looks normal when examined by does not move properly. In some embodiments, the gastrointestinal disorder is a structural gastrointestinal disorder, where the GI tract looks abnormal when examined and does not work properly. In some embodiments, the gastrointestinal disorder is a gastrointestinal motility disorder. In some embodiments, the gastrointestinal motility disorder is intestinal pseudo-obstruction, small bowel bacterial overgrowth, small intestinal bacterial overgrowth, constipation, outlet obstruction type constipation (i.e. pelvic floor dyssynergia), or diarrhea. In some embodiments, the gastrointestinal disorder is diarrhea is associated with diarrhea-predominant irritable bowel syndrome (IBS-D). In some embodiments, the gastrointestinal disorder is constipation associated with constipation-predominant irritable bowel syndrome (IBS-C).
In some embodiments, the gastrointestinal disorder is irritable bowel syndrome (IBS). The irritable bowel syndrome may be constipation predominant (IBS-C) or diarrhea predominant (IBS-D). The irritable bowel syndrome may be post-infectious IBS (PI-IBS). In some embodiments, the gastrointestinal disorder is colitis (e.g. infectious colitis, ulcerative colitis). In some embodiments, the gastrointestinal disorder is Crohn's disease. In some embodiments, the gastrointestinal disorder is tropical sprue. Tropical sprue is a malabsorption disease commonly found in tropical regions, marked by abnormal flattening of the villi and inflammation of the lining of the small intestine. Tropical sprue typically starts with an acute attack of diarrhea, fever, and malaise and may ultimately lead to a chronic phase of diarrhea, steatorrhea, weight loss, anorexia, malaise, and nutritional deficiencies. Tropical sprue may be caused by persistent bacterial, viral, or parasitic infections. Tropical sprue may also be caused by folic acid deficiencies, disrupted intestinal motility, and persistent small intestinal bacterial overgrowth. which may be affiliated with small intestinal bacterial overgrowth and/or irritable bowel syndrome.
In some embodiments, compositions comprising a tryptophan catabolite as described herein find use in methods of cleansing the colon. For example, in some aspects the compositions described herein may be provided to the subject for use in a method of cleansing the colon of the subject, such as in preparation for a colonoscopy.
In some embodiments, the compositions herein find use in methods of reducing visceral pain in a subject. For example, the visceral pain may be associated with one or more gastrointestinal disorders, such as constipation. Treating one or more gastrointestinal disorders (e.g. constipation) may result in treatment of the constipation along with associated symptoms, including visceral pain.
In addition to modulating gut motility and treating/preventing gastrointestinal disorders, the compositions described herein find use methods of modulating communication between the gastrointestinal tract and the nervous system in the subject (e.g. modulating gut-brain communication). The vagus nerve is the primary sensory pathway by which visceral information is transmitted to the CNS. The vagus nerve may play a role in communicating gut microbial information to the brain. In particular, the results provided herein demonstrate that, in addition to spinal sensory nerves, EEC-vagal signaling is an important pathway for transmitting specific gut microbial signals to the CNS. The vagal ganglia project directly onto the hindbrain, and that vagal-hindbrain pathway has key roles in appetite and metabolic regulation. Accordingly, compositions comprising a tryptophan catabolite as described herein may be used in methods of regulating appetite in a subject. For example, compositions comprising a tryptophan catabolite may be used in a method of suppressing appetite in a subject. Inhibiting appetite may lead to diminished food intake and/or weight loss in the subject. As another example, compositions comprising a tryptophan catabolite may be used in methods of promoting weight loss in a subject.
The composition may further comprise one or more pharmaceutically acceptable carriers. Suitable carriers depend on the intended route of administration to the subject. Contemplated routes of administration include those oral, rectal, nasal, topical (including transdermal, buccal and sublingual), vaginal, parenteral (including subcutaneous, intramuscular, intravenous and intradermal) and pulmonary administration. In some embodiments, the composition or compositions are conveniently presented in unit dosage form and are prepared by any method known in the art of pharmacy. Such methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association (e.g., mixing) the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
Formulations of the present disclosure suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets, wherein each preferably contains a predetermined amount of the one or more therapeutic agents as a powder or granules; as a solution or suspension in an aqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. In other embodiments, the composition is presented as a bolus, electuary, or paste, etc. In some embodiments, compositions suitable for oral administration may include such further agents as sweeteners, thickeners and flavoring agents. Still other formulations optionally include food additives (suitable sweeteners, flavorings, colorings, etc.), phytonutrients (e.g., flax seed oil), minerals (e.g., Ca, Fe, K, etc.), vitamins, and other acceptable compositions (e.g., conjugated linoelic acid), extenders, preservatives, and stabilizers, etc.
Various delivery systems are known and can be used to administer compositions described herein, e.g., encapsulation in liposomes, microparticles, microcapsules, receptor-mediated endocytosis, and the like. In specific embodiments, it may be desirable to administer the compositions of the disclosure locally to the area in need of treatment (e.g. directly to the intestine); this may be achieved by, for example, and not by way of limitation, local infusion during surgery, injection, or by means of a catheter.
Any suitable amount of the tryptophan catabolite may be provided to the subject. In general, suitable amounts are empirically determined and vary with the pathology being treated, the subject being treated and the efficacy and toxicity of the catabolite. It is understood that therapeutically effective amounts vary based upon factors including the age, gender, and weight of the subject, among others. It also is intended that the compositions and methods of this disclosure may be co-administered with other suitable compositions and therapies.
In general, suitable doses of the tryptophan catabolite may range from about 1 ng catabolite/kg body weight to about 1 g/kg. For example, a suitable dose may be from about 1 ng/kg to about 1 g/kg, about 100 ng/kg to about 900 mg/kg, about 200 ng/kg to about 800 mg/kg, about 300 ng/kg to about 700 mg/kg, about 400 ng/kg to about 600 mg/kg, about 500 ng/kg to about 500 mg/kg, about 600 ng/kg to about 400 mg/kg, about 700 ng/kg to about 300 mg/kg, about 800 ng/kg to about 200 mg/kg, about 900 ng/kg to about 100 mg/kg, about 1 μg/kg to about 50 mg/kg, about 10 μg/kg to about 10 mg/kg, about 100 μg/kg to about 1 mg/kg, about 200 μg/kg to about 900 μg/kg, about 300 μg/kg to about 800 μg/kg, about 400 μg/kg to about 700 μg/kg, or about 500 μg/kg to about 600 μg/kg.
The composition may be provided to the subject at any desired frequency. For example, the composition may be provided to the subject more than once per day (e.g. twice per day, three times per day, four times per day, and the like), once per day, once every other day, once a week, and the like. The one composition may be provided to the subject for any desired duration. For example, the composition may be administered to the subject for at least one week, at least two weeks, at least three weeks, at least one month, at least two months, at least three months, at least six months, at least one year, at least two years, at least three years, at least four years, at least five years, at least ten years, at least twenty years, or for the lifetime of the subject.
The composition may further comprise one or more additional suitable agents, such as a suitable agent for treatment or preventing of a gastrointestinal disorder or a suitable agent for appetite suppression and/or weight loss. Alternatively or in addition, the compositions may be co-administered to the subject along a separate composition comprising the additional agent. Co-administration may be simultaneously or sequentially, in any order.
Overview: The intestinal epithelium senses nutritional and microbial stimuli using epithelial sensory enteroendocrine cells (EECs). EECs can communicate nutritional information to the nervous system, but similar mechanisms for microbial information are unknown. In vivo real-time measurements of EEC and nervous system activity in zebrafish demonstrate that the bacteria Edwardsiella tarda specifically activates EECs through the receptor transient receptor potential ankyrin A1 (Trpa1) and increases intestinal motility in an EEC-dependent manner. Microbial, pharmacological, or optogenetic activation of Trpa1+ EECs directly stimulates vagal sensory ganglia and activates cholinergic enteric neurons through 5-HT. Herein, a subset of indole derivatives of tryptophan catabolism produced by E. tarda and other gut microbes were investigated. It was shown that indole derivatives potently activate zebrafish EEC Trpa1 signaling and also directly stimulate human and mouse Trpa1 and intestinal 5-HT secretion. These results establish a molecular pathway by which EECs regulate enteric and vagal neuronal pathways in response to specific microbial signals.
Background: The intestine harbors complex microbial communities that shape intestinal physiology, modulate systemic metabolism, and regulate brain function. These effects on host biology are often evoked by distinct microbial stimuli including microbe-associated molecular patterns (MAMPs) and microbial metabolites derived from digested carbohydrates, proteins, lipids, and bile acid (Brown and Hazen, 2015, Liu et al., 2020, Coleman and Haller, 2017). The intestinal epithelium is the primary interface that mediates this host-microbe communication (Kaiko and Stappenbeck, 2014). The mechanisms by which the intestinal epithelium senses distinct microbial stimuli and transmits that information to the rest of the body remains incompletely understood.
The intestinal epithelium has evolved specialized enteroendocrine cells (EECs) that exhibit conserved sensory functions in insects, fishes, and mammals (Guo et al., 2019, Ye et al., 2019, Furness et al., 2013). Distributed along the entire digestive tract, EECs are activated by diverse luminal stimuli to secrete hormones or neuronal transmitters in a calcium dependent manner (Furness et al., 2013). Recent studies have revealed that EECs form synaptic connections with sensory neurons (Kaelberer et al., 2018, Bellono et al., 2017, Bohorquez et al., 2015). The connection between EECs and neurons forms a direct route for the intestinal epithelium to transmit nutrient sensory information to the brain (Kaelberer et al., 2018). EECs are classically known for their ability to sense nutrients (Symonds et al., 2015) but whether they can be directly stimulated by microbes or microbially derived products is unclear. Short chain fatty acids and branched chain fatty acids from microbial carbohydrate and amino acid catabolism activate EECs via G-protein coupled receptors (Bellono et al., 2017, Lu et al., 2018).
With the growing understanding of gut microbiota and their metabolites, identifying the EEC receptors that recognize distinct microbial stimuli as well as the downstream pathways by which EECs transmit microbial stimuli to regulate local and systemic host physiology, has emerged as an important goal.
The vertebrate intestine is innervated by the intrinsic enteric nervous system (ENS) and extrinsic neurons from autonomic nerves, including sensory nerve fibers from the nodose vagal ganglia and dorsal root ganglia in the spinal cord (Furness et al., 1999). Both vagal and spinal sensory nerve fibers transmit visceral stimuli to the central nervous system and modulate a broad spectrum of brain functions (Brookes et al., 2013). Stimulating EECs with the microbial metabolite isovalerate activates spinal sensory nerves through 5-hydroxytryptamine (5-HT) secretion (Bellono et al., 2017). Whether and how gut microbial stimuli modulate ENS or vagal sensory activity through EECs is still unknown.
To identify stimuli that activate EECs in live animals, a new transgenic zebrafish line that permits recording of EEC activity by expressing the calcium modulated photoactivatable ratiometric integrator (CaMPARI) protein in EECs under control of the neurod1 promoter was developed (
To test the validity of this EEC-CaMPARI system, larvae were stimulated with different nutrients known to activate zebrafish EECs (Ye et al., 2019). Exposure to only water as a vehicle control revealed an expected low basal red:green CaMPARI ratio (
The EEC-CaMPARI system was next used to investigate whether EECs acutely respond to live bacterial stimulation in vivo. Tg(neurod1:CaMPARI) zebrafish were exposed to individual bacterial strains for 20 mins in zebrafish housing water (GZM), followed by photoconversion and imaging of CaMPARI fluorescence. For these experiments, a panel of 11 bacterial strains including 3 model species (P. aeruginosa, E. coli, B. subtilis), 7 commensal strains isolated from the zebrafish intestine (Rawls et al., 2006, Stephens et al., 2016), and the pathogen E. tarda FL6-60 (also called E. piscicida (Abayneh et al., 2013, Bujan et al., 2018) were used;
Edwardsiella tarda FL6-60
Edwardsiella tarda LSE40; pmkb:mCherry
Edwardsiella tarda 23685
Edwardsiella tarda 15974
Aeromonas veronii ZOR0002
Shewanella sp. ZOR0012
Enterobacter sp. ZOR0014
Vibrio sp. ZWU0020
Chryseobacterium sp. ZOR0023
Exiguobacterium acetylicum ZWU0009
Bacillus subtilis 168
Pseudomonas aeruginosa PAK
Plesiomonas sp. ZOR0011
Escherichia coli MG1655
EECs express a variety of sensory receptors that can be activated by different environmental stimuli. To investigate the mechanisms by which EECs perceive E. tarda stimulation, EECs were isolated from zebrafish larvae and RNA-seq analysis was performed. Transcript levels in FACS-sorted EECs (cldn15la:EGFP+; neurod1:TagRFP+) were compared to all other intestinal epithelial cells (IECs) (cldn15la:EGFP+; neurod1:TagRFP−) (
Trpa1 is a nociception receptor that is known to mediate pain sensation in nociceptive neurons (Lapointe and Altier, 2011). A broad spectrum of chemical irritants, including many compounds that are derived from food spices, activate Trpa1 (Nilius et al., 2011). In addition to chemical irritants, certain bacterial products, including lipopolysaccharide (LPS) and hydrogen sulfide (H2S), stimulated nociceptive neurons in a Trpa1-dependent manner (Meseguer et al., 2014). The zebrafish genome encodes two trpa1 paralogs, trpa1a and trpa1b (Prober et al., 2008). The data presented herein establish that trpa1b, but not trpa1a, is expressed by a subset of zebrafish EECs and is required for EEC activation by Trpa1 agonist AITC (
To determine how E. tarda-induced Trpa1 signaling in EECs affects the host, trpa1b+/+ and trpa1b−/− zebrafish larvae were exposed to an E. tarda strain expressing mCherry fluorescent protein. High-dose (107 CFU/mL) E. tarda exposure for 3 days decreased survival rate and caused gross pathology (
In addition to EECs, Trpa1 is also expressed in mesenchymal cells within the intestine (
To understand the mechanisms by which EEC Trpa1 regulates gut microbial homeostasis, an opto-pharmacological approach that permits temporal control of EEC Trpa1 activation through UV light exposure was used (
To further test if signaling from Trpa1+ EECs is sufficient to activate intestinal motility, \ a new optogenetic system was used in which a mCherry tagged Channelrhodopsin (ChR2-mCherry) is expressed in EECs from the neurod1 promoter (
Finally, it was tested whether microbial activation of Trpa1 signaling in EECs also increased intestinal motility. Using microgavage (Cocchiaro and Rawls, 2013), delivery of live E. tarda into the intestinal lumen was found to significantly promote intestinal peristalsis and motility compared to PBS-gavaged controls (
To test the role of the ENS in Trpa1-activated intestinal motility, zebrafish that lack an ENS due to mutation of the receptor tyrosine kinase gene ret (Taraviras et al., 1999) were used. Immunofluorescence demonstrated that ret−/− zebrafish lack all identifiable enteric nerves (marked by NBT transgenes,
The ENS is a complex network composed of many different neuronal subtypes. Among these subtypes, cholinergic neurons secrete the excitatory neurotransmitter acetylcholine to stimulate other enteric neurons or smooth muscle (Pan and Gershon, 2000, Qu et al., 2008) and are essential for normal intestinal motility (Johnson et al., 2018). One of the key enzymes for the synthesis of acetylcholine in the ENS is choline acetyltransferase (Chat) (Furness et al., 2014). Using TgBAC(chata:Gal4); Tg(UAS:NTR-mCherry) transgenic zebrafish, cholinergic enteric neurons in the zebrafish intestine were observed (
To investigate whether activation of Trpa1+ EECs stimulates chata+ enteric neurons, TgBAC(chata:Gal4); Tg(UAS:Gcamp6s) zebrafish were used, which permit recording of in vivo calcium activity in chata+ neurons (
Previous mouse studies demonstrated that Trpa1 mRNA is highly enriched in 5-HT-secreting EC cells in the small intestine of mammals (Nozawa et al., 2009). Immunofluorescence staining indicated that, similar to mammals, 5-HT expression in the zebrafish intestinal epithelium is also highly enriched in Trpa1+ EECs (
The intestine is innervated by both intrinsic ENS and extrinsic sensory nerves from the brain and spinal cord (Brookes et al., 2013). In mammals, afferent neuronal cell bodies of the vagus nerve reside in the nodose ganglia and travel from the intestine to the brainstem to convey visceral information to the CNS. However, in zebrafish, it is unknown if the vagal sensory system innervates the intestine. The zebrafish vagal sensory ganglia can be labelled using TgBAC(neurod1: EGFP) or immunofluorescence staining of the neuronal marker acetylated α Tubulin (Ac-uTub) (
To further visualize the vagal sensory network in zebrafish, Tg(isl1:EGFP) zebrafish were used in which EGFP is expressed in vagal sensory ganglia and overlaps with neurod1 (
Next, it was investigated whether this vagal network is activated in response to enteric microbial stimulation with E. tarda. Tg(neurod1:Gcamp6f); Tg(neurod1:TagRFP) zebrafish larvae were gavaged with either PBS or live E. tarda bacteria. 30 min after enteric stimulation with Trpa1 agonist AITC or E. tarda, but not after PBS vehicle stimulation, Gcamp6f fluorescence intensity significantly increased in a subset of vagal sensory neurons (
Tryptophan Catabolites Secreted from E. tarda Activate the EEC Trpa1 Gut-Brain Pathway
In order to identify the molecular mechanism by which E. tarda activates Trpa1 in EECs, the effects of live and killed E. tarda cells and cell-free supernatant (CFS) from E. tarda cultures on EEC calcium activity were examined (
It was next tested if E. tarda tryptophan catabolites are sufficient to activate EECs. Indole and IAld, but not other tested tryptophan catabolites, strongly activated zebrafish EECs in a trpa1b-dependent manner (
Next, it was investigated whether indole and IAld can mimic live E. tarda bacterial stimulation and activate a similar gut-brain pathway through EEC Trpa1 signaling. Results indicated that enteric delivery of indole or IAld by microgavage increased Gcamp6f fluorescence in a subset of vagal sensory neurons (
Microbially Derived Tryptophan Catabolites Interact with the Host Through Trpa1
Trpa1 is a primary nociceptor involved in pain sensation and neuroinflammation. Trpa1 can be activated by several environmental chemical irritants and inflammatory mediators (Bautista et al., 2006), however, it was not known if and how Trpa1 might be activated by microbes. Tryptophan is an essential amino acid that is released in the intestinal lumen by dietary protein digestion or microbial synthesis. Gut microbes can catabolize tryptophan to produce a variety of metabolites, among which indole was the first discovered and often the most abundant (Smith, 1897). These tryptophan-derived metabolites secreted by gut bacteria can act as interspecies and interkingdom signaling molecules. Some microbially-derived tryptophan catabolites including indole and IAld may regulate host immune homeostasis and intestinal barrier function through ligand binding to the transcription factors, Ahr and Pxr (Venkatesh et al., 2014, Zelante et al., 2013). Another microbial tryptophan catabolite, tryptamine, activates epithelial 5-HT4R and increases anion-dependent fluid secretion in the proximal mouse colon (Bhattarai et al., 2018). Though several tryptophan metabolites including IAld can act as Ahr agonists (Zelante et al., 2013), conflicting effects of indole on AhR activation have been reported (Heath-Pagliuso et al., 1998, Hubbard et al., 2015, Jin et al., 2014). The results presented herein indicate that E. tarda or Trpa1+ EEC-induced intestinal motility is not mediated via AhR (
Trpa1+ EECs are abundant in the small intestine but not in the colon of human and rodents (Yang et al., 2019, Nozawa et al., 2009). The data presented herein demonstrate that microbially derived tryptophan metabolites are restricted to the colon and largely absent in small intestine under normal physiological conditions (
Nerve fibers do not penetrate the gut epithelium therefore, sensation is believed to be a transepithelial phenomenon as the host senses gut contents through the relay of information from EECs to the ENS (Gershon, 2004). Using an in vitro preparation of mucosa-submucosa, mechanical or electrical stimulation of mucosa was shown to activate submucosal neuronal ganglia, an effect blocked by a 5-HTiR antagonist (Pan and Gershon, 2000). Consistent with these previous findings, zebrafish data suggest a model that 5-HT released from Trpa1+ EECs stimulates intrinsic primary afferent neurons (IPANs) which then activate secondary neurons to promote intestinal motility through the local enteric EEC-ENS circuitry. 90% of 5-HT in the intestine is produced by EC cells, and therefore, EC cell 5-HT secretion was thought to be important in regulating intestinal motility (Gershon, 2013). This hypothesis, however, was challenged by recent findings that depletion of EC 5-HT production in Tph1−/− mice had only minor effects on gastric emptying, intestinal transit, and colonic motility (Li et al., 2011). Therefore, the physiological role of EC 5-HT production and secretion remains unclear. The data presented herein suggests that EEC 5-HT production may be necessary for intestinal motility changes in response to environmental chemical or microbial stimuli, but not for intestinal motility under normal physiological conditions. Mice raised germ-free displayed lower 5-HT content in the colon, however no significant difference of 5-HT production was observed in the small intestine compared to colonized mice (Yano et al., 2015). Whether gut microbiota regulate small intestinal 5-HT secretion and signaling remains unknown. The data shown herein suggest a model in which specific microbial communities or constituent species stimulate 5-HT secretion from Trpa1+ EECs to modulate small intestinal motility by producing tryptophan catabolites. This may provide a new mechanism by which gut microbiota can regulate 5-HT signaling in the small intestine. Indole, IAld and other tryptophan catabolites are produced by a wide range of gut bacteria, so results herein should be applicable to commensal and pathogenic bacteria and their host interactions.
The vagus nerve is the primary sensory pathway by which visceral information is transmitted to the CNS. Recent evidence suggests that the vagus nerve may play a role in communicating gut microbial information to the brain (Fulling et al., 2019, Breit et al., 2018, Bonaz et al., 2018). For example, the beneficial effects of Bifidobacterium longum and Lactobacillus rhamnosus in neurogenesis and behavior were abolished following vagotomy (Bercik et al., 2011, Bravo et al., 2011). However, direct evidence for whether and how vagal sensory neurons perceive and respond to gut bacteria has been lacking. The results herein demonstrate that bacterial tryptophan catabolites activate vagal sensory ganglia through EEC Trpa1 signaling. Previous findings have shown that EC cells transmit microbial metabolite and chemical irritant stimuli to pelvic fibers from the spinal cord dorsal root ganglion (Bellono et al., 2017). Findings here demonstrate that, in addition to spinal sensory nerves, EEC-vagal signaling is an important pathway for transmitting specific gut microbial signals to the CNS. The vagal ganglia project directly onto the hindbrain, and that vagal-hindbrain pathway has key roles in appetite and metabolic regulation (Grill and Hayes, 2009, Han et al., 2018, Travagli et al., 2006, Berthoud et al., 2006). The present findings raise the possibility that certain tryptophan catabolites, including indole, may directly impact these processes as well as emotional behavior and cognitive function (Jaglin et al., 2018). If so, this pathway could be manipulated to treat gut microbiota-associated neurological disorders.
All zebrafish experiments conformed to the US Public Health Service Policy on Humane Care and Use of Laboratory Animals, using protocol numbers A115-16-05 and A096-19-04 approved by the Institutional Animal Care and Use Committee of Duke University. For experiments involving conventionally raised zebrafish larvae, adults were bred naturally in system water and fertilized eggs were transferred to 100 mm petri dishes containing ˜25 mL of egg water at approximately 6 hours post-fertilization. The resulting larvae were raised under a 14 h light/10 h dark cycle in an air incubator at 28° C. at a density of 2 larvae/mL water. All the experiments performed in this study ended at 6 dpf unless specifically indicated. The strains used in this study are listed in Table 2. All lines were maintained on a mixed Ekkwill (EKW) background.
All bacterial strains in this study were cultured at 30° C. in Trypticase soy broth (TSB) or Gnotobiotic zebrafish medium (GZM) (Pham et al., 2008). Tryptic Soy Agar (TSA) plate was used for streaking bacterial from glycerol stock or performing colony forming unit (CFU) experiments. The antibiotic carbenicillin was used to select E. tarda LSE that express mCherry at the working concentration of 100 μg/mL.
The Gateway Tol2 cloning approach was used to generate the neurod1:CaMPARI and neurod1:cre plasmids (Kawakami, 2007, Kwan et al., 2007). The 5 kb pDONR-neurod1 P5E promoter was previously reported (McGraw et al., 2012). The pME-cre plasmid was reported previously (Cronan et al., 2016). The pcDNA3-CaMPARI plasmid was reported previously (Fosque et al., 2015) and obtained from Addgene. The CaMPARI gene was cloned into pDONR-221 plasmid using BP clonase (Invitrogen, 11789-020) to generate PME-CaMPARI. pDONR-neurod1 P5E and PME-CaMPARI were cloned into pDestTol2pA2 using LR Clonase (ThermoFisher, 11791). Similarly, pDONR-neurod1 P5E and pME-cre were cloned into pDestTol2CG2 containing a cmlc2:EGFP marker. The final plasmid was sequenced and injected into the wild-type EKW zebrafish strain and the F2 generation of alleles Tg(neurod1:CaMPARI)rdu78 and Tg(neurod1:cre; cmlcl2: EGFP)rdu79 were used for this study.
To make transgenic lines, that permit specific EEC ablation, Tg(neurod1:cre) and TgBAC(gata5:loxp-mCherry-stop-loxp-DTA) new transgenic system were used. This system consists of two new transgene alleles—one expressing Cre recombinase from the neurod1 promoter (in EECs, CNS, and islets) and a second expressing the diphtheria toxin (DTA) in gata5+ cells (in EECs, other IECs, heart, and perhaps other cell types) only in the presence of Cre (
Tg(tph1b:mCherry-NTR)pd275 zebrafish were generated using I-SceI transgenesis in an Ekkwill (EK) background. Golden Gate Cloning with BsaI-HF restriction enzyme (NEB) and T4 DNA ligase (NEB) was used to generate the tph1b:mCherry-NTR plasmid by cloning the 5 kb tph1b promoter sequence (tph1bP GG F: GGTCTCGATCGGtctaaggtgaatctgtcacattc (SEQ ID NO: 23); tph1bP GG R: GGTCTCGGCTACggatggatgctcttgttttatag (SEQ ID NO: 24)), mCherry (mC GG F: GGTCTCGTAGCC gccgccaccatggtgag (SEQ ID NO: 25); mC GG2 R: GGTCTCGGTACCcttgtacagctcgtccatgccgcc (SEQ ID NO: 26)), a P2A polycistronic sequence and triple mutant variant nitroreductase (Mathias et al., 2014) (mutNTR GG F: GGTCTCGGTACCtacttgtacaagggaagcggagc (SEQ ID NO: 27); mutNTR GG2 R: GGTCTCCCATGC caggatcggtcgtgctcga (SEQ ID NO: 28)), into a pENT7 vector backbone with a poly-A tail and I-SceI sites (pENT7 mCN GG F: GGTCTCGCATGGacacctccccctgaacctg (SEQ ID NO: 29); pENT7 mCN GG R: GGTCTCCCGATC gtcaaaggtttggggtccgc (SEQ ID NO: 30)). 500 pL of 25 ng/μL plasmid, 333 U/mL I-SceI (NEB), 1×I-SceI buffer, 0.05% Phenol Red (Sigma-Aldrich) solution was injected into EK 1-cell zebrafish embryos. F0 founders were discovered by screening for fluorescence in outcrossed F1 embryos.
To isolate zebrafish EECs and other IECs, two transgenic zebrafish lines were crossed, one that specifically expresses enhanced green fluorescent protein (EGFP) in all intestinal epithelial cells (TgBAC(cldn15la:EGFP)) (Alvers et al., 2014) and a second that expresses red fluorescent protein (RFP) in EECs, pancreatic islets, and the central nervous system (CNS) (Tg(neurod1:TagRFP)) (McGraw et al., 2012). The FACS-isolated EECs were identified by cldn15la:EGFP+; neurod1:TagRFP+; and the other IECs were identified by cldn15la: EGFP+; neurod1:TagRFP−. Conventionalized (CV) and germ-free (GF) TgBAC(cldn15la: EGFP); Tg(neurod1:TagRFP) ZM000 fed zebrafish larvae were derived and reared using a published protocol (Pham et al., 2008) for Flow Activated Cell Sorting (FACS) to isolate zebrafish EECs and other IECs. The protocol for FACS was adopted from a previous publication (Espenschied et al., 2019). Replicate pools of 50-100 double transgenic TgBAC(cldn15la:EGFP); Tg(neurod1:TagRFP) zebrafish larvae were euthanized with Tricaine and washed with deyolking buffer (55 mM NaCl, 1.8 mM KCl and 1.25 mM NaHCO3) before they were transferred to dissociation buffer [HBSS supplemented with 5% heat-inactivated fetal bovine serum (HI-FBS, Sigma, F2442) and 10 mM HEPES (Gibco, 15630-080)]. Larvae were dissociated using a combination of enzymatic disruption using Liberase (Roche, 05 401 119 001, 5 μg/mL final), DNaseI (Sigma, D4513, 2 μg/mL final), Hyaluronidase (Sigma, H3506, 6 U/mL final) and Collagenase XI (Sigma, C7657, 12.5 U/mL final) and mechanical disruption using a gentleMACS dissociator (Miltenyi Biotec, 130-093-235). 400 μL of ice-cold 120 mM EDTA (in 1×PBS) was added to each sample at the end of the dissociation process to stop the enzymatic digestion. Following addition of 10 mL Buffer 2 [HBSS supplemented with 5% HI-FBS, 10 mM HEPES and 2 mM EDTA], samples were filtered through 30 μm cell strainers (Miltenyi Biotec, 130-098-458). Samples were then centrifuged at 1800 rcf for 15 minutes at room temperature. The supernatant was decanted, and cell pellets were resuspended in 500 μL Buffer 2. FACS was performed with a MoFlo XDP cell sorter (Beckman Coulter) at the Duke Cancer Institute Flow Cytometry Shared Resource. Single-color control samples were used for compensation and gating. Viable EECs or IECs were identified as 7-AAD negative.
Samples from three independent experimental replicates were performed. 250-580 EECs (n=3 for each CV and GF group) and 100 IECs (n=3 for each CV and GF group) from each experiment were used for library generation and RNA sequencing. Total RNA was extracted from cell pellets using the Argencourt RNAdvance Cell V2 kit (Beckman) following the manufacturer's instructions. RNA amplification prior to library preparation had to be performed. The Clontech SMART-Seq v4 Ultra Low Input RNA Kit (Takara) was used to generate full-length cDNA. mRNA transcripts were converted into cDNA through Clontech's oligo(dT)-priming method. Full length cDNA was then converted into an Illumina sequencing library using the Kapa Hyper Prep kit (Roche). In brief, cDNA was sheared using a Covaris instrument to produce fragments of about 300 bp in length. Illumina sequencing adapters were then ligated to both ends of the 300 bp fragments prior to final library amplification. Each library was uniquely indexed allowing for multiple samples to be pooled and sequenced on two lanes of an Illumina HiSeq 4000 flow cell. Each HiSeq 4000 lane could generate >330M 50 bp single end reads per lane. This pooling strategy generated enough sequencing depth (˜55M reads per sample) for estimating differential expression. Sample preparation and sequencing was performed at the GCB Sequencing and Genomic Technologies Shared Resource.
Zebrafish RNA-seq reads were mapped to the danRer10 genome using HISAT2(Galaxy Version 2.0.5.1) using default settings. Normalized counts and pairwise differentiation analysis were carried out via DESeq2 (Love et al., 2014) with the web based-galaxy platform: https://usegalaxv.org/. For the purpose of this study, only the CV EEC (n=3) and CV IEC (n=3) comparison and analysis are shown. The default significance threshold of FDR <5% was used for comparison. Hierarchical clustering of replicates and a gene expression heat map of RNA-seq data were generated using the online expression heatmap tool: http://heatmapper.ca/expression/. The human and mouse RNA-seq raw counts data were obtained from the NCBI GEO repository: human, GSE114853; mouse, GSE114913 (Roberts et al., 2019). Pairwise differentiation analysis of human jejunum CHGA+ (n=11) and CHGA− (n=11) and mouse duodenum Neurod1+ (n=3) and Neurod1− (n=3) was performed using DESeq2. The mouse and zebrafish ortholog Gene ID conversion was downloaded from Ensemble. The genes that were significantly enriched (PFDR<0.05) in the human and mouse EEC data sets were used to query the zebrafish EEC RNA seq dataset and data were plotted using Graphpad Prism7. RNA-seq data generated in this study can be accessed under Gene Expression Omnibus accession GSE151711.
CaMPARI undergoes permanent green-to-red photoconversion (PC) under 405 nm light when calcium is present. This permanent conversion records the calcium activity for all areas illuminated by PC-light. Red fluorescence intensity correlates with calcium activity during photoconversion (Fosque et al., 2015). In the Tg(neurod1:CaMPARI) zebrafish line, the CaMPARI (calcium-modulated photoactivatable ratiometric integrator) transgene is expressed under control of the −5 kb promoter cloned from the zebrafish neurod1 locus. CaMPARI mRNA is transcribed and the CaMPARI protein is expressed in cells that are able to activate the neurod1 promoter. There are multiple cell types in the zebrafish body that are sufficient to activate the neurod1 promoter, including all EECs in the intestine (Ye et al., 2019). CaMPARI protein is a calcium indicator protein that binds calcium and converts from green fluorescence to red fluorescence in the presence of UV light. This protein is engineered and described in detail in a previous publication (Fosque et al., 2015). This transgenic model was used to measure the level of intracellular calcium in EECs. Similar to neurons, it is well known that when extracellular stimulants act on various receptors on EECs, this leads to an increase of intracellular calcium either due to calcium influx through calcium channels in the plasma membrane or release of calcium stored in the ER. Through either of these pathways, increased intracellular calcium then directly triggers EECs to release hormone/neurotransmitter vesicles. To record in vivo EEC activity using the CaMPARI platform, conventionally raised Tg(neurod1:CaMPARI) zebrafish larvae were sorted at 3 dpf and maintained in Gnotobiotic Zebrafish Media (GZM) (Pham et al., 2008) with 1 larvae/mL density. At 6 dpf, for each experimental group, ˜20 larvae were transferred into 50 mL conical tubes in 2 mL GZM medium. The larvae were adjusted to the new environment for 30 mins before stimuli were added to each conical tube. For nutrient stimulation, since linoleate, oleate and laurate are not soluble in water, a bovine serum albumin (BSA) conjugated fatty acid solution was generated as described previously (Ye et al., 2019). 2 mL linoleate, oleate, laurate, butyrate or glucose was added to the testing tube containing ˜20 zebrafish larvae in 3 mL GZM. The final stimulant concentrations were: linoleate (1.66 mM), oleate (1.66 mM), laurate (1.66 mM), butyrate (2 mM) and glucose (500 mM). Zebrafish larvae were stimulated for 2 mins (fatty acids) or 5 mins (glucose) before the UV pulse. For bacterial stimulation, single colonies of the different bacterial strains were cultured aerobically in tryptic soy broth (TSB) at 30° C. overnight (rotating 50-60 rpm, Thermo Fisher Tissue Culture Rotator CEL-GRO #1640Q)(see strains listed in Table 2). O/N TSB cultured bacteria were harvested, washed with GZM and resuspended in 2 mL GZM. 2 mL bacteria were then added to a test tube containing ˜20 zebrafish larvae in 3 mL GZM. The final concentration of the bacterial is ˜108 CFU/ml. Zebrafish were then stimulated for 20 mins before treated with a UV pulse. A customized LED light source (400 nm-405 nm, Hongke Lighting CO. LTD) was used to deliver a UV light pulse (100 W power, DC32-34 V and 3500 mA) for 30 seconds. Following the UV pulse, zebrafish larvae were transferred to 6-well plates. To block spontaneous intestinal motility and facilitate in vivo imaging, zebrafish larvae were incubated in 20 μM 4-DAMP (mAChR blocker), 10 μM atropine (mAChR blocker) and 20 μM clozapine (5-HTR blocker) for 30 mins. Zebrafish larvae were then anesthetized with Tricaine (1.64 mg/ml) and mounted in 1% low melting agarose and imaged using a 780 Zeiss upright confocal microscope in the Duke Light Microscope Core Facility. Z-stack confocal images were taken of the mid-intestinal region in individual zebrafish. The laser intensity and gain were set to be consistent across different experimental groups. The resulting images were then processed and analyzed using FIJI software (Schindelin et al., 2012). To quantify the number of activated EECs, the color threshold was set for the CaMPARI red channel. EECs surpassing the color threshold were counted as activated EECs. The CaMPARI green channel was used to quantify the total number of EECs in each sample. The ratio of activated EECs to the total EEC number was calculated as the percentage of activated EECs. As reported in
To record in vivo EEC activity using the Tg(neurod1:Gcamp6f) system, a published protocol with slight modification (Ye et al., 2019) was used. In brief, unanesthetized zebrafish larvae were gently mounted in 3% methylcellulose. Excess water was removed and zebrafish larvae were gently positioned with right side up. Zebrafish were then moved onto an upright Leica M205 FA fluorescence stereomicroscope equipped with a Leica DFC 365FX camera. The zebrafish larvae were allowed to recover for 2 mins before 100 μL of test agent was pipetted directly in front of the mouth region. Images were then recorded every 10 seconds. The data shown in
Whole mount immunofluorescence staining was performed as previously described (Ye et al., 2019). In brief, ice cold 2.5% formalin was used to fix zebrafish larvae overnight at 4° C. The samples were then washed with PT solution (PBS+0.75% Triton-100). The skin and remaining yolk were then removed using forceps under a dissecting microscope. The deyolked samples were then permeabilized with methanol for more than 2 hrs at −20° C. Samples were then blocked with 4% BSA at room temperature for more than 1 hr. The primary antibody was diluted in PT solution and incubated at 4° C. for more than 24 hrs. Following primary antibody incubation, the samples were washed with PT solution and incubated overnight with secondary antibody with Hoechst 33342 for DNA staining. Imaging was performed with Zeiss 780 inverted confocal and Zeiss 710 inverted confocal microscopes with 40× oil lens. The secondary antibodies in this study were from Alexa Fluor Invitrogen were used at a dilution of 1:250.
To quantify vagal activity by pERK staining, published protocol with slight modification (Randlett et al., 2015) was used. Zebrafish larvae were quickly collected by funneling through a 0.75 mm cell strainer and dropped into a 5 mL petri dish containing ice cold fix buffer (2.5% formalin+0.25% Triton 100). Larvae were fixed overnight at 4° C., then washed 3 times in PT (PBS+0.3% Triton 100), treated with 150 mM Tris-HCl (PH=9) for 15 mins at 70° C., washed with PT and digested with 0.05% trypsin-EDTA on ice for 45 mins. Following digestion, samples were then washed with PT and transferred into block solution [PT+1% bovine serum albumin (BSA, Fisher)+2% normal goat serum (NGS, Sigma)+1% dimethyl sulfoxide (DMSO)]. The primary antibodies [pERK (Cell signaling); tERK (Cell signaling); GFP (Aves Lab)] were diluted in block solution (1:150 for pERK; 1:150 for tERK and 1:500 for GFP) and samples were incubated in 100 μl of primary antibody overnight at 4° C. Following primary antibody incubation, samples were then washed with PT and incubated with secondary antibody overnight at 4° C. Samples were then washed with PBS, mounted in 1% LMA and imaged using a Zeiss 780 upright confocal microscope.
Zebrafish E. tarda Colonization
For E. tarda colonization experiments, fertilized zebrafish eggs were collected, sorted and transferred into a cell culture flask containing 80 mL GZM at 0 dpf. At 3 dpf, dead embryos and 60 mL GZM were removed and replaced with 50 mL fresh GZM in each flask. To facilitate consistent commensal gut bacterial colonization, an additional 10 mL of filtered system water (5 μm filter, SLSV025LS, Millipore) were added to each flask. Overnight E. tarda mCherry culture was harvested, washed three times with GZM. 150 μL of GZM-washed E. tarda mCherry culture were inoculated into each flask. The E. tarda concentration is ˜106 CFU/ml. Daily water changes (60 ml) was performed and 200 μL autoclaved solution of ZM000 food (ZM Ltd.) was added from 3 dpf to 6 dpf as previously described (Pham et al., 2008). At 6 dpf, zebrafish larvae were subjected to fluorescence imaging analysis or CFU quantification. For fluorescence imaging analysis, zebrafish larvae were anesthetized with Tricaine (1.64 mg/ml), mounted in 3% methylcellulose and imaged with a Leica M205 FA upright fluorescence stereomicroscope equipped with a Leica DFC 365FX camera. For CFU quantification, digestive tracts were dissected and transferred into 1 mL sterile PBS which was then mechanically disassociated using a Tissue-Tearor (BioSpec Products, 985370). 100 μL of serially diluted solution was then spread on a Tryptic soy agar (TSA) plate with Carbenicillin (100 μg/ml) and cultured overnight at 30° C. under aerobic conditions. The mCherry+ colonies were quantified from each plate and E. tarda colony forming units (CFUs) per fish were calculated.
For delivering bacterial or chemicals specifically to the intestine, the established microgavage technique (Cocchiaro and Rawls, 2013) was adopted. Zebrafish were anesthetized with 1 mg/mL α-Bungarotoxin (α-BTX) and the gavage procedure was performed as previously described using microinjection station (Cocchiaro and Rawls, 2013). For bacteria gavage experiments, 1 ml of overnight bacterial culture was harvested, pelleted, washed with PBS and resuspended in 100 ul PBS.˜8 nl was then delivered into the zebrafish intestine using microgavage. The gavaged zebrafish was then transferred into egg water and mounted in 1% LMA for imaging. For chemical gavage experiments, ˜8 nl of AITC (100 mM), indole (1 mM) and IAld (1 mM) was gavaged into the intestine.
For Trpa1 inhibition, Trpa1 antagonist HC030031 (280 μM) was treated 2 hours before and during the 30 mins of E. tarda stimulation. For AhR inhibition, two AhR inhibitors, CH030031 and folic acid, were selected based on previous publications (Puyskens et al., 2020, Kim et al., 2020). CH030031 is a well-established specific AhR inhibitor (Choi et al., 2012). Whereas folic acid is shown to act as a competitive AhR antagonist at the concentration as low as 10 ng/ml (Kim et al., 2020). For the E. tarda treatment experiment, DMSO, CH030031 (1 μM) or folic acid (10 μM) was added into zebrafish water at 3 dpf zebrafish at the same time as E. tarda administration. The AhR inhibitors were replenished during daily water changes, and zebrafish were analyzed at 6 dpf. For the Optovin-UV experiment, overnight Optovin treated zebrafish were treated for 2 hours with DMSO, CH030031 (10 μM) or folic acid (10 μM). As demonstrated by previous study, 2-hour 10 μM and 3-day 0.5 μM CH030031 treatment is sufficient to inhibit larvae zebrafish AhR signaling (Puyskens et al., 2020, Sun et al., 2019b, Yue et al., 2017). The concentration of FA was chosen based on zebrafish tolerance and a previous study shown the treatment of early zebrafish embryos with 0.05 μM FA inhibits AhR signaling (Yue et al., 2017).
For EEC Trpa1 activation using the Optovin platform, zebrafish larvae were treated with 10 μM Optovin overnight. Following Optovin treatment, unanesthetized zebrafish were mounted in 1% LMA and imaged under a 780 upright Zeiss confocal microscope using 20× water objective lenses. For all the experiments, the mid-intestine region was imaged (
Photoactivation of channelrhodopsin (ChR2) in EECs was performed in Tg(neurod1:Gal4, cmlc2; EGFP); Tg(UAS:ChR2-mCherry) transgenic zebrafish. In this model, ChR2 expression in EECs is mosaic. At 6 dpf, unanesthetized zebrafish larvae were mounted in 1% LMA. Photoactivation and imaging were performed with a Zeiss 780 upright confocal using 20× water objective lenses. Individual ChR2+ EECs were selected as ROI (
To determine whether Optovin-UV or ChR2 was sufficient to activate EECs, Tg(neurod1:Gcamp6f) zebrafish were used. To facilitate EEC calcium imaging under the confocal microscope, zebrafish larvae were incubated in 20 μM 4-DAMP, 10 μM atropine and 20 μM clozapine for 30 mins before mounting in 1% LMA to reduce spontaneous motility. The Gcamp6f signal was recorded with 488 nm laser intensity less than 0.5. The zebrafish intestinal motility is quantified through recorded image series of zebrafish intestine using the method similar as previously described (Ganz et al., 2018). Intestinal p velocity and v velocity were used to estimate intestinal motility in zebrafish as previously described using the PIV-Lab MATLAB app (Ganz et al., 2018). A positive value of the μ velocity indicates an anterograde intestinal movement and a negative value of the μ velocity indicates a retrograde intestinal movement. The time-course p velocity number is plotted as heatmaps. When calculating the mean velocity, only the mean velocity magnitude was calculated, which therefore doesn't account for the movement direction. The MTrackJ FIJI plugin was used to quantify the mean velocity magnitude (Meijering et al., 2012).
To assess whether Trpa1+ EEC activation induced intestinal motility change is due to the indirect communication through vagal afferent and efferent system, zebrafish CNS with the intestine was anatomically disconnected by decapitating. Optovin-treated unanesthetized zebrafish were mounted and placed on the 780 Zeiss upright confocal station as described above. The zebrafish head was then removed with a razor blade. The same imaging and 405 nm activation of the mid-intestinal region was performed as described above.
Enteric cholinergic neuron and vagal ganglion calcium imaging TgBAC(chata:Gal4); Tg(UAS:Gcamp6s); Tg(NBT:DsRed) or TgBAC(chata:Gal4); Tg(UAS:Gcamp6s); Tg(UAS:NTR-mCherry) zebrafish were used to record in vivo calcium activity in enteric cholinergic neurons. The NBT promoter labels all ENS neurons while the Chata promoter labels only cholinergic enteric neurons. DsRed or mCherry fluorescence was used as reference for cholinergic neuron Gcamp quantification. Zebrafish larvae were incubated in 20 μM 4-DAMP for 30 mins before mounting in 1% LMA to reduce spontaneous motility and facilitate in vivo imaging using a Zeiss 780 upright confocal microscope with 20× water lenses. Serial images were taken at 5 s/frame. To record cholinergic neuron calcium activation, zebrafish was pretreated with Optovin and 40 nm light was applied at the frequency of 1 pulse/5s to the intestinal epithelium ROI. The Gcamp6s to DsRed fluorescence in cholinergic neurons was calculated for recorded.
Tg(neurod1:Gcamp6f); Tg(neurod1:TagRFP) zebrafish were used to record vagal sensory ganglia calcium activity in vivo. Zebrafish were anesthetized with 1 mg/mL α-Bungarotoxin (α-BTX) and gavaged with chemical compounds or bacteria as described (Naumann et al., 2016). Zebrafish larvae were mounted in 1% LMA and imaged under a Zeiss 780 upright confocal microscope. Z-stack images of the entire vagal ganglia were collected as serial images at 10 mins/frame and processed in FIJI. Individual vagal sensory neurons were identified and the Gcamp6f to TagRFP fluorescence ratios of individual vagal sensory neurons were calculated.
Quantitative real-time PCR was performed as described previously (Murdoch et al., 2019). In brief, 20 zebrafish larvae digestive tracts were dissected and pooled into 1 mL TRIzol (ThermoFisher, 15596026). mRNA was then isolated with isopropanol precipitation and washed with 70% ethanol. 500 ng mRNA was used for cDNA synthesis using the iScript kit (Bio-Rad, 1708891). Quantitative PCR was performed in triplicate 25 μL reactions using 2×SYBR Green SuperMix (PerfeCTa, Hi Rox, Quanta Biosciences, 95055) run on an ABI Step One Plus qPCR instrument using gene specific primers (Supplementary file 1). Data were analyzed with the ΔΔCt method. 18S was used as a housekeeping gene to normalize gene expression.
HEK-293T cells were cultured in DMEM (Thermofisher Scientific, Waltham, Mass.) and supplemented with 10% fetal bovine serum (FBS) (Thermofisher Scientific), penicillin (100 units/mL) and streptomycin (0.1 mg/mL). Cells were plated on 100 mm tissue culture plates coated with poly-D-lysine (Sigma Aldrich, Saint Louis, Mo.) and grown to ˜60% confluence. The cells were transiently transfected for 16-24 hours with either human or mouse orthologs of TRPA1 using Fugene 6 transfection reagents and Opti-MEM (Thermofisher Scientific) according to the manufacturer's protocol. Subsequently, cells were trypsinized, re-suspended and re-plated onto poly-D-lysine coated 96-well plates (Krystal black walled plates, Genesee Scientific) at 5×105 cells/mL (100 μL/well) and allowed to grow for another 16-20 hrs prior to the experiments. Cells were maintained as monolayers in a 5% CO2 incubator at 37° C.
Measurements of changes in intracellular Ca2+ concentrations ([Ca2+]i) were performed as described previously (Caceres et al., 2017). In brief, cells in 96-well plates were loaded with Calcium 6, a no-wash fluorescent indicator, for 1.5 hrs (Molecular Devices, San Jose, Calif.) and then transferred to a FlexStation III benchtop scanning fluorometer chamber (Molecular Devices). Fluorescence measurements in the FlexStation were performed at 37° C. (Ex:485 nm, Em: 525 nm at every 1.8 s). After recording baseline fluorescence, agonists (indole, IAld, cinnamaldehyde) were added and fluorescence was monitored for a total of 60 s. To determine the effects of TRPA1 inhibition on agonist response, TRPA1 transfected HEK-293 cells were pretreated with various concentrations of A967079 (Medchem101, Plymouth Meeting, Pa.), a specific antagonist of TRPA1, and then exposed to either 100 μM indole or IAld. The change in fluorescence was measured as Fmax-F0, where Fmax is the maximum fluorescence and F0 is the baseline fluorescence measured in each well. The EC50 and IC50 values and associated 95% confidence intervals for agonist (Indole and IAld) stimulation of Ca2+ influx and A967079 inhibition of agonist-induced Ca2+ influx, respectively, were determined by non-linear regression analysis with a 4-parameter logistic equation (Graphpad Prism, San Diego, Calif.). Indole and IAld concentration-response data was normalized to 1 mM cinnamaldehyde for EC50's calculations and A967079 concentration-response data was normalized to 100 μM indole or IAld for IC50's calculations.
The chemical profiling of Trp-Indole derivatives was performed using 1 L culture of E. tarda. The strain was inoculated in 3 mL of TSB medium and cultivated for 1 day on a rotary shaker at 180 rpm at 30° C. under aerobic conditions. After 1 day, 1 mL of E. tarda liquid culture was inoculated in 1 L of TSB medium in a 4-L Pyrex flask. The E. tarda culture was incubated at 30° C. for 24 hr under aerobic conditions. For time-course screening, 10 mL from the E. tarda TSB culture was collected at 0, 6, 18, and 24 hours. Each 10 mL sample of E. tarda culture was extracted with 15 mL of ethyl acetate (EtOAc). The EtOAc layer was separated from the aqueous layer and residual water was removed by addition of anhydrous sodium sulfate. Each EtOAc fraction was dried under reduced pressure, then resuspended in 500 μL of 50% MeOH/50% H2O and 50 μL of each sample were analyzed using an Agilent Technologies 6130 quadrupole mass spectrometer coupled with an Agilent Technologies 1200-series HPLC (Agilent Technologies, Waldbron, Germany). The chemical screening was performed with a Kinetex® EVO C18 column (100×4.6 mm, 5 μm) using the gradient solvent system (10% ACN/90% H2O to 100% ACN over 20 min at a flow rate of 0.7 mL/min).
For HPLC-MS analysis of E. tarda in GZM medium, the remaining 1 L culture of E. tarda in TSB culture was centrifuged at 7,000 rpm for 30 min. Pellets were transferred to 1 L of GZM medium in a 4-L Pyrex flask and cultivated on a rotary shaker at 30° C. for 24 hr. For time-course screening, 10 mL from the E. tarda GZM culture was collected at 0, 1, 6, and 24 hours. Sample preparation and HPLC-MS analysis of E. tarda culture GZM medium were performed using same procedures as described above for TSB. Trp-Indole derivatives of E. tarda culture broths were identified by comparing the retention time and extracted ion chromatogram with authentic standards. Extracted ions were selected for Indole (m/z 117, Sigma-Aldrich), IAld (m/z 145, Sigma-Aldrich), IAAld (m/z 159, Ambeed), IEt (m/z 161, Sigma-Aldrich), IAM (m/z 174, Sigma-Aldrich), IAA (m/z 175, Sigma-Aldrich), and IpyA (m/z 203, Sigma-Aldrich).
For HPLC-MS analysis of Trp-indole derivatives from 15 different bacterial strains in TSB medium, each of the strains (Acinetobacter sp. ZOR0008, Aeromonas veronii ZOR0002, Bacillus subtilis 168, Chryseobacterium sp. ZOR0023, Edwardsiella tarda 15974, Edwardsiella tarda 23685, Edwardsiella tarda LSE40, Edwardsiella tarda FL6-60, Enterobacter sp. ZOR0014, Escherichia coli MG1655, Exiguobacterium acetylicum sp. ZWU0009, Plesiomonas sp. ZOR0011, Pseudomonas aeruginosa PAK, Shewanella sp. ZOR0012, and Vibrio sp. ZWU0020) were inoculated in 3 mL of TSB medium and cultivated for 1 day on a rotary shaker at 180 rpm at 30° C. under aerobic conditions. After 1 day, 1 mL of each liquid culture was inoculated in 100 mL of TSB medium in 500 mL Pyrex flasks and cultivated on a rotary shaker at 30° C. overnight. A 10 mL sample was taken from each culture and extracted and analyzed via HPLC-MS as explained above. CFU was calculated for each bacterial liquid culture and the HPLC-MS data was normalized to the CFU.
For HPLC-MS analysis of Trp-indole derivatives from 15 different bacterial strains in GZM medium, the remaining 100 mL culture of each strain was centrifuged at 4500 rpm for 20 min. Pellets were transferred to 100 mL of GZM medium in 500 mL Pyrex flasks and cultivated on a rotary shaker at 30° C. overnight. Sample preparation and HPLC-MS analysis of each GZM culture were performed using the same procedure as described above.
For HPLC-MS analysis of Trp-indole derivatives from murine small intestine and large intestine, three 10-week old female and three 10-week old male conventionally-reared specific pathogen-free C57BL/6J mice were ordered from Jackson Lab. The mice were not fast in advance and euthanized with 5% isoflurane. The 2/5-4/5 portion of the small intestinal region and the colon caudal to cecum was collected from each mouse and transferred to a 50 mL conical tube that was placed on dry-ice. 80% methanol was then added according to the tissue weight (50 μL/mg tissue). The intestine was then homogenized with a Tissue-Tearor (BioSpec Products, 985370). Following homogenizing, the tryptophan metabolites were extracted and analyzed with HPLC-MS as explained above. The relative metabolite abundance was normalized to tissue weight. These mouse experiments conformed to the US Public Health Service Policy on Humane Care and Use of Laboratory Animals, using protocol number A170-17-07 approved by the Institutional Animal Care and Use Committee of Duke University.
Measurement of Serotonin Release from Mouse and Human Small Intestine
These experiments using C57BL/6J mice were approved by the Flinders University Animal Welfare Committee (number 965-19) and human ileum tissue was collected from resected small and large intestine from patients that gave written informed consent under the approval of the Southern Adelaide Clinical Human Research Ethics Committee (number 50.07) as previous (Sun et al., 2019a). Mice were euthanized at 8 to 12 weeks by isoflurane overdose followed by cervical dislocation. The duodenum was removed and placed in Krebs solution oxygenated with 95% O2, 5% CO2. A midline incision was made along the duodenum to create a flat sheet, the section was pinned with the mucosal side up in an organ bath lined with Sylgard and containing oxygenated Krebs solution. Serotonin release was measured using amperometry. A carbon-fibre electrode (5-μm diameter, ProCFE; Dagan Corporation, Minneapolis, Minn.), was lowered above the mucosa and 400 mV potential was applied to the electrode causing oxidation of serotonin (Zelkas et al., 2015). 10 mM Indole and/or 50 μM HC030031 were applied to tissue by constantly perfusing the bath. The change in amplitude due to serotonin oxidation was recorded using an EPC-10 amplifier and Pulse software (HEKA Electronic, Lambrecht/Pfalz, Germany), and samples at 10 kHz and low-pass filtered at 1 kHz. Data was assessed as peak current during each treatment. Data was analyzed comparing all groups using one-way ANOVA with Tukey's post-hoc test. For the mouse experiments, 6 independent experiments were performed in 6 mouse duodenal samples. For the human experiments, 4 independent experiments were performed in 3 human samples.
The appropriate sample size for each experiment was suggested by preliminary experiments evaluating variance and effects. Using significance level of 0.05 and power of 90%, a biological replicate sample number 8 was suggested for EEC CaMPARI analysis. For each experiment, wildtype or indicated transgenic zebrafish embryos were randomly allocated to test groups prior to treatment. Individual data points, mean and standard deviation are plotted in each figure. The raw data points in each figure are represented as solid dots. Data were analyzed using GraphPad Prism 7 software. For experiments comparing just two differentially treated populations, a Student's t-test with equal variance assumptions was used. For experiments measuring a single variable with multiple treatment groups, a single factor ANOVA with post hoc means testing (Tukey) was utilized. Statistical evaluation for each figure was marked * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001 or ns (no significant difference, P>0.05).
This application claims priority to U.S. Provisional Patent Application No. 63/014,816, filed Apr. 23, 2020, the entire contents of which are incorporated herein by reference for all purposes.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2021/028602 | 4/22/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63014186 | Apr 2020 | US |
