Trace Aminergic Regulation of Gastrointestinal Inflammation: A Novel Therapeutic Strategy for Ulcerative Colitis
Doctoral dissertation presented by
Katlynn Bugda Gwilt, M.S.
M.S. in Pharmacology, Northeastern University, Boston MA B.S. in Biology, Syracuse University, Syracuse NY
to
Bouvé Graduate School of Health Sciences in partial fulfillment of the requirements for the degree of
Doctor of Philosophy in Pharmacology
NORTHEASTERN UNIVERSITY BOSTON, MASSACHUSETTS
April 2019 Northeastern University Office of Graduate Student Services, BCHS
NUID: 001610563
Name: Katlynn Bugda Gwilt
Major: Pharmacology Department: Pharmaceutical Sciences
Dissertation Title: Trace Aminergic Regulation of Gastrointestinal Inflammation: A Novel Therapeutic Strategy for Ulcerative Colitis
Approval for Dissertation Requirements for Doctor of Philosophy
Dissertation Committee Chair ______Date ______Dr. Gregory Miller
Dissertation Committee Members
______Date ______Dr. David Janero
______Date ______Dr. Diomedes Logothetis
______Date ______Dr. Ralph Loring
______Date ______Dr. Leigh Plant
______Date ______Dr. Susan Westmoreland
Dean of the Bouvé College Graduate School of Health Sciences
______Date ______Dr. Susan Parish
i PREFACE Ulcerative colitis is a chronic inflammatory condition, with a rising incidence as countries become more “westernized.” Symptoms of ulcerative colitis include chronic loose stools, severe abdominal cramping, and nausea. Treatment options for ulcerative colitis remain limited to attenuating the inflammation common to the disease, or in severe cases involve surgery to remove necrotic tissue. Despite our understanding of the inflammation that plagues the disease, we have a limited understanding on the cause of disease. The current hypothesis in the pathogenesis of ulcerative colitis consists of a multifactorial etiology, involving a dynamic interplay of a genetic predisposition to disease, a pervasive role of stress, a dysregulated microbiome, and dietary correlates of flares of inflammation. Though research is focusing on these individual causes of disease there is no clear understanding how these factors come together to cause disease. Emerging studies seek to understand the „-omics‟ of inflammatory bowel diseases, including Ulcerative colitis. In 2017, a metabolomic study in the fecal content of human patients demonstrated that patients with ulcerative colitis had significantly elevated tyramine, a biogenic trace amine. Tyramine is a known ligand for the G-Protein coupled receptor, Trace Amine Associated Receptor 1 (TAAR1). TAAR1 is also responsive to common biogenic amines such as norepinephrine (NE), serotonin (5HT) and dopamine (DA) in high concentrations. While the dysregulated monoamines in disease are somewhat understood, the presence of TAAR1 and actions of tyramine have not been studied in the lower intestine. Furthermore, the observed actions of tyramine on the intestine have yet to be linked to the TAAR1 receptor mechanism. Given that tyramine is elevated in ulcerative colitis, and that sources of tyramine include diet and the gut microbiome, as well as the understood elevation of monoamines such as 5HT, DA and NE in disease, I hypothesize that TAAR1 may integrate these aberrant signals in a genetically susceptible individual (Figure 1). Here, in this dissertation, I demonstrate the presence and actions of TAAR1 in intestinal epithelial cells, macrophages, and an in vivo model of colitis, and reveal a novel therapeutic target for the treatment of UC.
Figure 1. Is TAAR1 a novel integrator of known UC disease correlates of dysregulated microbiome, stress, and dietary components in a genetically susceptible individual?
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DEDICATED TO
To my mother, Melissa Gwilt To my grandfather, George Chandler
In loving memory of Carrie Anne Chandler, 1967 – 1993 “The lofty willow always weeps and marks the spot where Carrie sleeps.”
iii Acknowledgements
I extend the deepest thanks to the members of my dissertation committee, Dr. David Janero, Dr. Diomedes Logothetis, Dr. Ralph Loring, Dr. Leigh Plant, and Dr. Susan Westmoreland for their support, encouragement and mentorship that guided me through the completion of this project. Each member of my committee very thoughtfully contributed at each step of the process with scientific advice and thought provoking and insightful discussions.
To my mentor, Dr. Gregory Miller, I cannot convey with words my gratitude for your dedication and commitment to not only me, but each and every student who crossed the threshold of your office door. With your guidance I have grown as a person, as a scientist, and as a mentor in ways I could not have imagined. You are a fantastic advisor and an outstanding role model. I hope that as I move forward in my career that I am able to emulate your compassionate nature, enthusiasm for science, and collaborative and friendly spirit in all of my work. Thank you for making this time so fun.
To Dr. Anand Sridhar, thank you for encouraging me to spread my wings and fly.
To Dr. Emanuela Gussoni, my first scientific mentor, I carry with me your dedication and scientific rigor in all of my experiments. The lessons you have taught me have stayed with me, and have been passed on to the students I have worked with. Thank you for remaining a mentor and a friend.
To my family, thank you for lifelong lessons in work ethic and perseverance that stuck with me as I completed this work. To my parents, thank you for their wise counsel and loving guidance throughout my life, and support as I pursued my dreams. To Dr. Tanya Kelly and Mrs. Francesca Bartholomew; thank you both for always being there in any way you possibly could. I couldn‟t have made it through without such supportive friends.
Thank you to all of the members of the Miller lab, past and present: Lisa Fleischer, Rachel Hoffing, Neva Olliffe, Alexandra Schueler, Sophia Mantell, and Vasileos Kreouzis. I extend my deepest gratitude to Rachel Hoffing, my colleague and friend who helped make my transition to the Miller lab seamless, and Neva Olliffe for her technical assistance and enthusiasm for science.
Thank you to the many treasured members of the Department of Pharmaceutical Sciences for their support and dedication to student success: Sarom Lay, Nancy Waggner, Dr. Barbara Waszczak, Dr. Ban An Khaw and Dean Jack Reynolds.
Thank you to Dr. Sahar El Aidy for inviting me to a fantastic two-week stay at her lab, rejuvenating my love for this project and giving me the unique opportunity to present my work and share my passion with her colleagues.
Finally, I owe a very special thank you to my best friend and loving husband Eliot for his unfailing love and understanding during my pursuit of my PhD. Without his support, technical expertise when computers crashed, reminders that I told him that “I have to go feed the cells”, rides to and from the lab at all hours of the day or night, and patience as I spent countless evenings working, my pursuit of my PhD would have been much more challenging. He reminded me to take time for myself at times when I thought it may be impossible to do so and encouraged me to continue on in the face of adversity and challenge
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List of Figures
Figure 1. Is TAAR1 a novel integrator of known UC disease correlates of dysregulated microbiome, stress, and dietary components in a genetically susceptible individual? ii Figure 2. General colon anatomy. 3 Figure 3. Tight junctions and macrophages in the GIT. 4 Figure 4. Tight junction protein regulation by PKA/PKC. 7 Figure 5. Westernization and dietary correlates of disease. 9 Figure 6. Phylogenetic abundance of microorganisms in a healthy microbiome. 11 Figure 7. Phylogenetic microbial diversity in healthy and ulcerative colitis microbiome compared to probiotic diversity. 12 Figure 8. Tyrosine decarboxylase from the small intestine of humans produces TYR ex vivo by action of tyrosine decarboxylase. 18 Figure 9. Peripheral localization of TAAR1. 26 Figure 10. Antibody validation of commercially available antibodies with immunohistochemistry (IHC). 34 Figure 11. Thermo Fisher antibody specificity. 35 Figure 12. Multiple Sequence Alignment of published antigenic determinants of commercially available antibodies with human TAAR1 (hTAAR1) and mouse TAAR (mTAAR) family members. 36 Figure 13. Multiple Sequence Alignment of protein sequence of D274 antibody with human TAAR1 (hTAAR1) and mouse TAAR (mTAAR) family members. 38 Figure 14. Immunofluorescence of D274 on HEK cells, HEK cells transfected with mTAAR1 or hTAAR1, and C57BL/6 and TAAR1-/- BMDM. 39 Figure 15. TAAR1 mRNA and protein are expressed in HT-29 cells. 55 Figure 16. TAAR1 upregulation in HT-29 cells by full agonist TYR, but not partial agonist RO5263397. 56 Figure 17. Expression and regulation of gene expression in CACO-2 cells by TYR. 57 Figure 18. TYR alters expression of tight junction proteins in HT-29 cells. 58 Figure 19. TYR can increase inter-epithelial cellular distance and mediate permeability in HT-29 cells. 59 Figure 20. PKC inhibitors block TYR mediated upregulation of ZO-1 mRNA whereas PKA inhibitors block TYR mediated upregulation of TAAR1. 60 Figure 21. Effects of RO5263397 on TJP mRNA expression, inter-epithelial cellular distance and permeability in HT-29 cells. 61 Figure 22. Bacteria culture supernatants from common probiotics and cheeses elicit a cAMP/CRE response in HEK-TAAR1 cells. 62 Figure 23. TAAR1 is expressed in mouse BMDM and is upregulated by TYR. 77 Figure 24. TYR-dependent upregulation of IL-6, IL-1β, TNF-α and iNOS. 78 Figure 25. Upregulation of TAAR1 and inflammatory cytokine gene expression is attenuated by the TAAR1-antagonist EPPTB. 79 Figure 26. TAAR1 protein expression in mouse BMDM and phenotypic alterations in response to 100nM tyramine. 80
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Figure 27. Proposed mechanism for TAAR1 mediated inflammatory response in the gastrointestinal mucosa by tyramine. 81 Figure 28. TAAR1 is expressed in the colon and small intestine of C57BL/6 mice. 94 Figure 29. TAAR1-/- colon are phenotypically different from C57BL/6 colon. 95 Figure 30. TAAR1-/- mice are protected from adverse effects of DSS induced colitis. 96 Figure 31. TAAR1-/- mice have significantly shorter lesions compared to C57BL/6 mice. 97 Figure 32. Expression of TAAR1 in the colon of healthy and diseased human colon tissue. 98 Figure 33. Score sheet for the assessment of mice with intestinal pathology in DSS- Induced Colitis. 106 Figure 34. TAAR1 co-localization with occludin in DSS-induced colitis lesion in C57BL/6 colitis lesions. 113 Figure 35. MTT Proliferation Assay in HT-29 Cells. 115 Figure 36. Transwell chemotaxis assay. 116
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List of Tables
Table 1. TAAR1 Ligand potency at mTAAR1 and hTAAR1 and presence in the gut. 23 Table 2: Commercially available antibodies and reactivity to mTAAR family members. 37 Table 3. Bioinformatics analysis determined commercially available antibody cross- reactivity to human TAAR family members. 40 Table 4. Human Primers for qRT-PCR. 67 Table 5. Antibodies and Dilutions for Chapter 2 experiments. 68 Table 6. Primers used for qRT-PCR analysis. 77 Table 7. Antibodies for experiments. 103 Table 8. Mouse Primers for PCR. 104
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List of Abbreviations
UC Ulcerative colitis TAAR1 Trace amine associated receptor -1 TYR Tyramine TAAR1 -/- Trace amine associater receptor -1 knock out (mouse) GIT Gastrointestinal tract GIT Gastrointestinal iECs Intestinal epithelial cells ZO Zonula occluden JAM Junction adhesion molecule IBD Inflammatory bowel disease CD Crohn's disease TJ Tight junction TJP Tight junction protein cAMP/PKA cyclic adenosine monophosphate / protein kinase a Ca2+/PKC calcium ion / protein kinase c SCFA Short chain fatty acid 5-ASA 5-aminosalicylate FDA Food and drug administration FODMAP Fermentable Oligosaccharides, Disaccharides, Monosaccharides and Polyols FMT fecal microbiota transplant LC/MS Liquid chromatography–mass spectrometry PEA β-phenylethylamine MAO-i Monoamine oxidase inhibitor TyrP Tyrosine transporter TyrDC Tyrosine decarboxylase PBMC Peripheral blood mononuclear cell EC50 Effective concentration 50% Ki Inhibitory constant T1AM 3-Iodothyronamine EPPTB N-(3-Ethoxy-phenyl)-4-pyrrolidin-1-yl-3-trifluoromethyl-benzamide PMN Polymorphonuclear Ccell BLAST Basic Local Alignment Search Tool NLM National library of medicine NCBI National Center for Biotechnology Information D274 Miller lab custom anti-TAAR1 antibody mTAAR mouse Trace amine associated receptor hTAAR human Trace amine associated receptor HEK Human embryonic kidney cell line PCR Polymerase chain reaction DAPI 4′,6-diamidino-2-phenylindole (blue fluorescent DNA stain) IHC Immunohistochemistry PBS Phosphate buffered saline viii
FBS Fetal bovine serum TBS Tris buffered saline LPS Lipopolysaccharide BSA Bovine serum albumin RT Room temperature (~23°C) dH2O distilled water BMDM Bone marrow derived macrophages GPCR G protein-coupled receptor CREB cAMP response element binding protein NFAT Nuclear factor of activated T-cells GEO Gene expression omnibus GAPDH Glyceraldehyde 3-phosphate dehydrogenase PKCi Protein kinase c inhibitor FITC Fluorescein isothiocyanate Cre-Luc cAMP response element binding protein- luciferase ATCC American Type Culture Collection G418 Geneticin RT-PCR Reverse transcription-polymerase chain reaction cDNA Complementary DNA qRT-PCR Quantitative reverse transcription-polymerase chain reaction BCA Bicinchoninic acid assay ECL Enhanced chemiluminescence FIJI FIJI is just image J SEM Standard error of the mean DSS Dextran sulfate sodium iNOS Inducible nitric oxide synthase fMLP N-Formylmethionyl-leucyl-phenylalanine SPF Specific pathogen free
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Table of Contents
Approval for Dissertation Requirements for Doctor of Philosophy i Preface ii Dedication iii Acknowledgements iv
List of Figures v-vi
List of Tables vii List of Abbreviations viii-ix Table of Contents x-xii
Statement of the problem 1
Review of the Literature 2
Gastrointestinal biology 2
Colonic mucosa 4
Inflammatory Bowel Diseases: Ulcerative Colitis 5
Human incidence/symptomology 5
Breakdown of epithelial barrier in UC 5
Macrophages in UC 7
Emerging knowledge in UC 8
Current treatment options for UC 12
Biogenic Amines in the GIT 16
Concentrations of Biogenic Amines in the GIT 16
Serotonin 16
Dopamine and Norepinepherine 16
Trace amine levels in the GIT 17
Trace amine presence in trigger foods 18
Trace amine production by microorganisms 20
Trace amine associate receptor 1 22
History of receptor 22
Ligands for the receptor 23
Agonists 23
Antagonists 24
Partial Agonists 24
Intracellular signaling 25
Anatomical Localization of TAAR1 25
Chapter One: Design and validation of custom TAAR1 antibody 28
BACKGROUND 29
RESULTS 31 Specificity of commercially available antibodies to mouse and human
31
TAAR1
Specificity of TAAR1 antibodies determined bioinformatically. 32
D274: A novel TAAR1 antibody. 33
FIGURES AND TABLES 34
DISCUSSION 41
MATERIALS AND METHODS 44
x
Cell culture TAAR1-Transfected HEK Cells 44
Immunohistochemistry 44
Immunostaining 45
Protein Sequence Analysis 45
Custom Antibody 46 Chapter Two: Expression, activation, and modulation of TAAR1 in intestinal
47 epithelial cells (IECs)
BACKGROUND 48
RESULTS 50
TAAR1 mRNA and protein is expressed in HT-29 cells. 50 TAAR1 expression is upregulated by full agonist treatment in HT-29 cells,
50 but not partial agonist treatment. ZO-1 gene expression in CACO-2 cells is mediated by TYR exposure, but
51
TAAR1 expression is not. Tight junction protein gene expression and protein levels are modified by
52
TYR in HT-29 cells Increased inter-epithelial cellular distance and increase in permeability in
52 response to TYR in HT-29 cells Inhibition of PKA and PKC pathways blocks the effects of TYR in HT-29
53 cells.
Effects of TAAR1 partial agonists RO5263397 on HT-29 cells 54 Metabolites from cultured cheese and probiotics elicit a response in HEK-
54
TAAR1-Cre-Luciferase reporter cells
FIGURES AND TABLES 55
DISCUSSION 63
MATERIALS AND METHODS 66
NCBI Data Analysis 66
Cell Culture 66
Drug Treatments 67
RNA Extraction, PCR and qRT-PCR 67
Immunostaining 68
Western Blotting 69
Inter-epithelial Distance 70
Transwell FITC-Dextran Flux 70 Chapter 3: TAAR1 activation modulates inflammatory cytokine production in
71
BMDM
BACKGROUND 72
RESULTS 74
TAAR1 is expressed in mouse BMDM and can be upregulated by TYR. 74 Additive inflammatory response in polarized BMDMs in response to LPS
74 and TYR. The TAAR1 antagonist EPPTB attenuates the upregulation of TAAR1 and
75 inflammatory gene expression. Differentiation of BMDM and treatment with TYR alters the phenotype of
76 mouse BMDM
FIGURES AND TABLES 77
DISCUSSION 82
MATERIALS AND METHODS 85
Animal husbandry 85
Cell Culture 85
Drug Treatments 86 xi
RNA Extraction, PCR and qRT-PCR 86
Immunostaining 87 Chapter 4: Dextran sulfate sodium induced colitis is attenuated in TAAR1-/-
88 mice.
BACKGROUND 89
RESULTS 91
TAAR1 is expressed in the gastrointestinal tract of mice. 91
TAAR1-/- colon are phenotypically different from C57BL/6 colon. 91
TAAR1-/- mouse is protected from the onset of an acute DSS colitis model 92 TAAR1-/- mice have shorter lesions and signs of healing compared to
92
C57BL/6 animals. TAAR1 is expressed in human colon, and is increased in intensity in UC
93 disease biopsies
FIGURES AND TABLES 94
DISCUSSION 99
MATERIALS AND METHODS 102
Animal Husbandry 102
TAAR1-/- generation and genotyping 102
Imaging of mouse intestine 103
Tissue Staining: Immunofluorescence 103
RNA Extraction and Analysis 103
Tissue Staining: Hematoxylin & Eosin 104
Tissue Staining: Alcian Blue 104
Quantification Techniques 105
DSS-induced colitis and scoring 105
US Biomax Tissue Array 106
Chapter 5: Conclusions and future studies 108
5A. Dissertation Discussion 109
5B. Future studies 115
5C. Concluding Remarks 118 References 119
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Statement of the problem
Gastrointestinal inflammatory disorders, including ulcerative colitis (UC), involve breakdown of the lining in the colon and hyper-activation of immune cells. Pathogenesis of UC remains poorly understood, though emerging evidence suggests that there is a dynamic interplay of environmental stimuli and stress to exacerbate symptomology in a genetically susceptible individual. Current therapeutic options for UC remain limited, and include dietary modifications, attempts at modifying the microbiome, immunosuppression, and in severe cases surgical removal of necrotic tissue. Despite the understanding of environmental stimuli in UC pathogenesis, there is a gap in knowledge in the field on how the gastrointestinal tract interacts with these stimuli. The work completed in this dissertation aimed to identify a trace amine receptor, Trace Amine Associated Receptor-1 (TAAR1), as a novel integrator of environmental stimuli (e.g. diet, microbiome) in the onset and progression of UC. Tyramine (TYR), a trace amine, is at high levels in foods that trigger bouts of UC, and levels of TYR are elevated in the fecal matter of UC patients. Here, I tested the hypothesis that TAAR1 in intestinal immune and epithelial cells can mediate the effects of TYR in the gastrointestinal tract (GIT), which causes exacerbation of UC. The data generated in this dissertation provides the first evidence that: 1)
TYR levels in the GIT can propagate ulcerative colitis; 2) TYR can regulate tight junction mRNA and protein expression in epithelial cells; 3) TAAR1 activation by TYR can modulate the expression of pro-inflammatory cytokines from macrophages; and 4) TAAR1 is expressed in the
GIT and the onset of dextran sulfate sodium induced colitis is attenuated in TAAR1-/- mice.
Taken together, this thesis reveals a new mechanism that drives UC pathology and a new pharmacological target for combating UC.
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Review of the Literature
Gastrointestinal biology
The gastrointestinal tract (GIT) is a heterogeneous layer of tissue comprised of smooth muscle, neuronal cells, immune cells, and epithelial cells. Maintenance of gastrointestinal (GI) homeostasis is dynamic and involves the regulation of the epithelial cell monolayer to protect the underlying immune cells to prevent excessive inflammation (Rao & Wang, 2010). The wall of the GIT is comprised of four layers: the mucosa, the submucosa, muscularis propria, and serosa. Dependent on the functional needs of the tissue throughout the GIT, the thickness and exact composition of each layer of the wall is variable. The mucosa, consisting of three layers, is perhaps the most regulated and segmentally differentiated layer of the intestinal tract.
Comprised of a network of immune and epithelial cells, the main function of the mucosa is to maintain intestinal homeostasis in response to luminal antigens (Rao & Wang, 2010). The intestinal epithelium, often referred to as either colonocytes or intestinal epithelial cells (iECs), is a single layer of cells that protects the underlying lamina propria and muscle layers from lumenal contents. Due to the nature of the GIT, the cells at the top of the crypts are constantly being replenished by the stem cells at the base of the crypts. Colonocytes (or enterocytes) are the most common cell type in the intestine and are responsible for absorption of nutrient and absorption of water. Epithelial cells interfacing with the lumen of the GIT are protected by a thin layer of mucous derived from specialized goblet cells deeper in the crypts. (Allaire et al., 2018;
Lameris et al., 2013; Y. Z. Zhang & Li, 2014). Finally, enteroendocrine cells, including enterochrommafin cells, modulate hormone levels and interactions with underlying neuronal cells in the gut (Allaire et al., 2018).
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Figure 2. General colon anatomy. General Colon Anatomy A) Graphical representation of the wall of the colon. Green stars highlight the mucosa, submucosa, muscularis the serosa and the lumen. IBD results in breakdown of mucosa and epithelial barrier in the intestine. B) Anatomy of the wall of colon demonstrating the relative organization of the lining of the colon and relative thickness of the mucosa, submucosa, muscularis and serosal layers. C) Healthy colon histology demonstrating epithelial cells, goblet cells and mucus in the mucosa layer, small muscularis mucosae (blue star) separating mucosa and submucosa (green star).
Barriers in the body are regulated by membrane junctions of epithelial and specialized endothelial cells. In the GIT, the epithelial barrier is important for the first line of defense of the immune system, protecting delicate internal structures from pathogens and food antigens in the lumen. The mucous layer produced by the goblet cells protects the colonocytes and serves as the first line of defense of the food and pathogenic organisms that protect the underlying muscle and immune cells from hyperactivation and degradation. Anchoring the adjacent epithelial cells together are complex networks of integral and associated membrane proteins, all of which play a role in anchoring the plasma membrane to the stable actin cytoskeleton. These tight junction proteins include Occludin, Claudins, Zonula Occludens (ZO), and Junction Adhesion Molecules
(JAMs). Heterogeneous expression of these proteins regulates the relative permeability of the tissue, and is naturally varied throughout the length of the GIT under different physiological and pathological conditions (Lu, Ding, Lu, & Chen, 2013) (Figure 3).
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Figure 3. Tight junctions and macrophages in the GIT. Left, an epithelial monolayer and regulation by claudin, occludin, ZO-1 and F-actin interactions. Right, a dysregulated epithelial monolayer indicative of a „leaky gut‟ caused by breakdown of claudin, occludin, and ZO-1 interactions with F-actin. Degradation of the monolayer allows the transgression of lumen contents to the underlying lamina propria. Left, a normal healthy mucosa, with balanced secretion of cytokines by tissue resident macrophages. Right, inflammation of the lamina propria caused by aberrant activation of tissue resident macrophages from lumen contents, resulting in an increase in proinflammatory cytokines from tissue resident immune cells, and infiltration of peripheral immune cells to the site of injury. Colonic mucosa Additionally, the intestinal lamina propria hosts various immune cells which integrate signals from the colonocytes and any pathogens that breach the epithelial monolayer. The variety of immune cells found in the lamina propria includes the innate immune system
(dendritic cells, macrophages, mast cells), as well as the adaptive immune system (naïve T- cells, Th17 cells, Th1 cells, Treg cells, B cells and plasma cells). Briefly, dendritic cells in the lumen can modulate T cell differentiation (Johansson-Lindbom et al., 2005); (Sun et al., 2007), activation and „class switching‟ in naïve B cells (Mora et al., 2006), and differentiate into antigen presenting cells producing a concerted immunological response to phagocytosed pathogens
(Lombardi & Khaiboullina, 2014). Intestinal macrophages have diverse functions to mediate intestinal homeostasis. Severe insults to the mucosal layer results in an inflammatory response, and in susceptible individuals, progressive uncontrolled inflammation ultimately can cause Page 4
disease by promoting breakdown of the epithelial barrier (Figure 3). Because of their unique capacity to regulate both pro and anti-inflammatory cytokines, macrophages are often thought to be “gatekeepers” of inflammatory bowel diseases (Grimm et al., 1995). An in-depth discussion on macrophage functions in the GIT will be discussed in a later section.
Inflammatory Bowel Diseases: Ulcerative Colitis
Human incidence/symptomology Inflammatory bowel diseases (IBD) – including Crohn‟s disease (CD) and ulcerative colitis (UC) – result from prolonged inflammation of the gastrointestinal tract, affecting approximately 2 million Americans in 2017 (S. C. Ng et al., 2018). Symptoms of CD and UC are similar including severe nausea, bloody loose stools, boating and severe cramping, malnutrition due to a decreased absorbance of nutrients, and weight loss. While CD and UC are classified as “IBD” and are oftentimes considered one disease due to their shared symptoms (D. H. Kim &
Cheon, 2017), there are distinct molecular and pathological differences between the two disease states. In CD, the localization and depth of inflammation in the intestines are markedly more severe in comparison to UC. CD can affect any portion of the GIT spanning from the esophagus to the rectum and often causes discontinuous damage whereas UC is localized to the superficial layers of the colon (Waugh et al., 2013; Y. Z. Zhang & Li, 2014).
Breakdown of epithelial barrier in UC UC lesions are limited to the mucosal layer of the colon, and are seen as disruption of crypts, crypt abscesses and a depletion of goblet cell. These pathogenic defects result in a decrease in mucous production and subsequent loss of the protective mucous layer atop the colonocytes, allowing degradation of the tight junctions in the epithelial lining (Waugh et al.,
2013; Y. Z. Zhang & Li, 2014). As briefly mentioned, tight junctions in the colonocytes are crucial for the protection of the mucosal immune cells from pathogens (Anderson & Van Itallie,
1995; Gonzalez-Mariscal, Tapia, & Chamorro, 2008; Ulluwishewa et al., 2011). As a result of chronic inflammation, the tight junctions between colonocytes are degraded, allowing the
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passage of food products and lumen microflora to interact with the immune cells in the lamina propria (Figure 3) (Lee, 2015).
Normal regulation and maintenance of the epithelial barrier is complex and requires continual regeneration of the epithelial cells in contact with luminal antigens, and complex coordination of the tight junction protein (TJP) barriers to prevent paracellular transport. The
TJP are multi-protein complexes responsible for anchoring adjacent cells to the actin cytoskeletal components (Lee, 2015). Phosphorylation and dephosphorylation by cAMP/PKA and Ca2+/PKC are known to alter the localization and intracellular linkage of both transmembrane and peripheral membrane proteins. In healthy cells, ZO proteins (ZO-1, -2, and
-3) anchor the Filamentous Actin (F-Actin) cytoskeleton to two families of integral proteins,
Claudin and Occludin(Anderson & Van Itallie, 1995; Atkinson & Rao, 2001; Fujibe et al., 2004;
Gonzalez-Mariscal et al., 2008; Landy et al., 2016; Lee, 2015; Ulluwishewa et al., 2011; Van
Itallie & Anderson, 2006; Willott et al., 1993). Claudin is a key component of tight junctions and is considered the backbone of the cellular interaction. (Atkinson & Rao, 2001; Fujibe et al.,
2004; Lee, 2015; Van Itallie & Anderson, 2006; Willott et al., 1993). Specific protein kinase phosphorylation events aid in the trafficking of membrane proteins in and out of the membrane, allowing for the regulation of TJs in neighboring cells. PKA mediated phosphorylation of
Occludin is responsible for the internalization of the transmembrane protein, allowing degradation of the Occludin-ZO-F-actin membrane anchors (Figure 4.). Conversely, PKC mediated phosphorylation of claudin localizes claudin to the membrane, whereas dephosphorylation of claudin internalizes the transmembrane protein, degrading tight junction protein complexes (Figure 4).
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Figure 4. Tight junction protein regulation by PKA/PKC. Tight junction protein formation is due to specific phosphorylation and dephosphorylation events. PKC dependent phosphorylation of claudin anchors claudin in the cell membrane and crosslinking of claudins between cells. Dephosphorylation of claudin results in internalization of protein causing degradation of tight junctions. PKC dependent phosphorylation of Zonula Occludin proteins causes destabilization of anchorage to the F-Actin cytoskeleton, causing degradation of TJs. (Anderson & Van Itallie, 1995; Gonzalez-Mariscal et al., 2008; Ulluwishewa et al., 2011). PKA dependent phosphorylation of occludin destabilizes complexes formed with a neighboring cell and disrupts the TJs.
Macrophages in UC While the colon epithelium can secrete some cytokines in response to antigens
(Bahrami, Macfarlane, & Macfarlane, 2011; Kuhn, Manieri, Liu, & Stappenbeck, 2014; Parikh,
Salzman, Kane, Fischer, & Hasselgren, 1997), it is largely accepted that it is the dynamic interplay of mucosal epithelial cells and immune cells that is fundamental in driving the progression of UC and UC flares (Belkaid & Hand, 2014). In healthy individuals, when the antigens are adequately cleared, the macrophages secrete cytokines halting further inflammation. In UC, the macrophages fail to attenuate inflammatory cascades, which results in chronic inflammation in the lamina propria. This inflammation causes severe tissue damage and is thought to be responsible for symptoms of UC (Bain & Mowat, 2014). Tissue resident macrophages have altered recognition receptors (only some express CD14 and CD11c responsible for detecting LPS (Bain et al., 2013) as well as „toleration‟ of food derived or commensal antigens that they encounter (Kuhl, Erben, Kredel, & Siegmund, 2015; Rugtveit,
Bakka, & Brandtzaeg, 1997). Given these unique characteristics, macrophages are considered
„gate-keepers‟ of disease in the lamina propria, mediating the effects of specific antigens that they encounter. Monocytes, differentiating to macrophages, are polarized into the classical M1-
M2 paradigm, with M1 macrophages considered pro-inflammatory and M2 macrophages
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considered anti-inflammatory (Mantovani et al., 2004). In UC, peripherally infiltrating macrophages in the mucosa produce an array of pro-inflammatory cytokines including IL-6, IL-
1β, TNF-α, IL-23, IL-8 and IFNγ (Schenk, Bouchon, Seibold, & Mueller, 2007), as well as CCL2 a monocyte chemoattractant protein (Kamada et al., 2008). Monocytes and macrophages are directly responsible for barrier defects in UC, and reside in the inflamed mucosa (Lissner et al.,
2015) as macrophages aberrantly respond to bacteria in the lumen (Rahman et al., 2010).
Macrophage function is regulated by many intracellular signaling cascades, including cAMP/PKA and Ca2+/PKC pathways. Both cAMP/PKA and Ca2+/PKC pathways in macrophages have been intimately studied. Oftentimes, cAMP/PKA signaling has been described as suppressive of normal cellular functions, whereas heightened Ca2+/PKC pathways induce cellular activation. In response to cAMP/PKA and Ca2+/PKC signaling, macrophages become activated, and increase the release of proinflammatory cytokines (Foey &
Brennan, 2004; Hadjimitova, Bakalova, Traykov, Ohba, & Ribarov, 2003; Kontny, Ziolkowska,
Ryzewska, & Maslinski, 1999; Shishodia, Shrivastava, & Sodhi, 1998).
Emerging knowledge in UC Although much has been learned about the cellular and pathological sequela that unfolds during the manifestation of IBD the exact etiology remains elusive. The current working hypothesis driving research in the field is that in a genetically susceptible patient, the intestine is vulnerable to environmental triggers, and more prone to react to alterations in the content and microbial composition of the intestine, resulting in a continued state of inflammation. Because of this over-reaction to antigens resulting in perpetual inflammation, IBDs are often considered autoimmune diseases, despite the overwhelming prevalence of the disease in industrialized countries (Das & Biancone, 2008; Kaplan, 2015; Siew C. Ng et al., 2017), implicating diet and lifestyle as major contributors.
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Figure 5. Westernization and dietary correlates of disease. A. The global incidence of IBD in 2015, from Kaplen et al. demonstrates that countries that are „westernized‟ have a higher incidence of IBD than countries than countries that are less developed. B. Recommendations from the Crohn‟s and Colitis foundation to alleviate symptoms of IBD. (Crohns and Colitis Foundation, 2013; Kaplan, 2015)
Dietary correlates of disease Of the commonly accepted environmental triggers, the antigens that are known to increase the global risk of IBD includes the consumption of milk and cheeses, processed animal protein, high fat diets (Jowett et al., 2004) (Figure 5) and fermented food products which are most commonly linked to IBD Foundation (Crohns and Colitis Foundation, 2013)- commonly referred to as the „Western Diet‟ . Even with the correlation of known food products in exacerbation of disease, a large gap in knowledge in studying IBD is that there is no known consensus molecular target(s) for these environmental triggers (Turpin, Goethel, Bedrani, &
Croitoru Mdcm, 2018).
Microbiome’s role in disease The human gut microbiome is described as the community of microorganisms that take residence in the gastrointestinal tract. Physicians and researchers have recognized the importance of replenishing the normal microflora during antibiotic treatments since the early
1970‟s (Williams, 1973) and 1980‟s (Burros, 1982) though it wasn‟t until the late 1990‟s that researchers began recognizing the importance of the gut microbiome to human health
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(Meijnikman, Gerdes, Nieuwdorp, & Herrema, 2017). Since the identification of the importance of the microbiome in human health, a new field of research has emerged focusing on identifying beneficial commensal organisms and determining how population shifts of these microorganisms may be causative for a spectrum of diseases, including GI diseases.
Currently the gut microbiome has been categorized into three main „categories‟ of species: symbiotic organisms, opportunistic pathogens, and pathogenic bacteria. Symbiotic organisms play a major role in the nutritional needs of humans, as well as a crucial role in the maturation of the immune system and immune cell regulation (Shen et al., 2018). In a healthy individual Bifidobacterium are the first microorganisms to colonize a newborn‟s intestine
(Ventura, Turroni, Lugli, & van Sinderen, 2014). Through maturation, many Bacteroides and
Peptococcus organisms, as well as Akkermansia muciniphila (A. muciniphila) colonize the intestine maintaining intestinal homeostasis by producing nutrients and stimulating generation of the protective mucous layer atop colonocytes (Bajer et al., 2017; Shen et al., 2018). Despite being opportunistic pathogens, certain microorganisms including Enterococcus and
Enterobacter species are generally considered symbiotic, or innocuous to human health (Shen et al., 2018). Finally, there are members of the microbiome that exist in very low levels, and are purely pathogenic such as Proteus, Pseudomonas, and Clostridium species. These species are present in healthy individuals in low concentrations, and are outcompeted for survival, but a sudden loss in diversity can result in a pathogenic response from these organisms (Shen et al.,
2018). The relative abundance of species in the human microbiome can be observed in Figure
6.
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Figure 6. Phylogenetic abundance of microorganisms in a healthy microbiome. ‘Common genera of the human gut microbiota. Genus abundance variation box plot for the 30 most abundant genera of the human gut microbiota as determined by metagenomic sequencing of human fecal samples. Genera are colored by their respective phylum (see inset for color key). Inset shows phylum abundance box plot (source: Figure 1b (Guarner, 2015; Manimozhiyan et al., 2011)‟
Role in disease Emerging evidence suggests that the gut microbiome has significant impact on human health, including maturation of the immune system by release of biologically active compounds to regulate inflammation. Of these biologically active compounds, monoamine production is widely studied (Lyte & Brown, 2018) as is the production of short chain fatty acids (SCFAs).
SCFAs such as acetic acid, butyric acid, and propionic acid serve as food sources for microbes, as well as immunomodulatory effects in the development of the immune system and butyrate is a primary fuel source for the colonocytes of the GIT (Al-Lahham, Peppelenbosch, Roelofsen,
Vonk, & Venema, 2010; Shen et al., 2018).
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Figure 7. Phylogenetic microbial diversity in healthy and ulcerative colitis microbiome compared to probiotic diversity. Relative abundance in microorganisms in the human healthy microbiome (Ma, Yu, Zhao, Zhang, & Zhang, 2018) and patients with ulcerative colitis (Hansen et al., 2012). A summary of ingredients in 15 of the most popular over-the-counter probiotic supplements analyzed with PhyloT (Letunic & Bork, 2016) and summarized by relative abundance.
In UC, it is widely accepted that a loss of diversity in the microbiome leads to disease, though the exact microorganisms that propagate disease are variable between patients.
Specifically, pathogenic organisms including Helicobacter (Mannion, Shen, & Fox, 2018),
Salmonella (Gradel et al., 2009), Escherichia, Clostridium (Reddy & Brandt, 2013), Listeria
(Huijsdens et al., 2003) and Yersina (Saebo, Vik, Lange, & Matuszkiewicz, 2005) species are elevated in patients with UC, whereas the protective organisms such as A. muciniphilia are decreased. Klebsiella pneumonia, Morganella morganii and Escherichia coli (E. Coli) have previously been implicated in IBD (Campieri & Gionchetti, 2001; Kaur, Vadivelu, &
Chandramathi, 2018; McLaughlin et al., 2010; Walmsley, Anthony, Sim, Pounder, & Wakefield,
1998). A summary of the relative diversity in the microbiome in healthy patients and diseased patients is demonstrated in Figure 7.
Current treatment options for UC Based on the current understandings and prevalence of transmural inflammation in UC, most treatment strategies are focused on decreasing inflammation to induce remission of the disease. There are three main treatment options for UC: aminosalicylate (5-ASA) (e.g. Page 12
sulfasalazine or mesalamine), gluococorticoids, and monocolonal antibodies (anti- TNFα, CD3,
IFNy, ICAM1, IL6) (Meier & Sturm, 2011). Typically the first line therapy with the goal of triggering maintenance and remission begins with the 5-ASAs such as sulfasalazine, or mesalamine. 5-ASA therapies are a class of drugs that prevent leukocyte recruitment in the colon epithelium and have been in practice since the 1940s rectally, or oral formulations which began emerging in the early 2000s. While 5-ASA drugs are commonly used for minor inflammation, they are relatively ineffective for severe inflammation. When inflammation is non- responsive to 5-ASA treatment, or is refractory to treatment, it is considered severe. In a severe state of inflammation, the glucocorticoid steroids, including prednisone or prednisolone, are used to rapidly induce remission by immunosuppression. Glucocorticoids, while effective in treating acute flares with severe inflammation, are not used to maintain remission. Still, use of glucocorticoids has been used in active cases of the disease since the 1950s. Recently approved biological therapeutics are focused on taming the augmented immune response, though they are designed as specific monoclonal antibodies to target elevated cytokines in disease. Currently, Food and Drug Administration (FDA) approved therapeutics include anti-
TNFα and anti-IFNγ treatments, with anti-IL6 therapies currently in clinical trials (Shah & Mayer,
2010).
Emerging treatment: dietary modifications There is a growing awareness that diet and lifestyle are implicated in disease activation and progression, and there is a general lack of pharmacological interventions available for combating the disease. Non-pharmacological treatments of IBD-related diseases are becoming more popular in patient and physician populations, as can be seen with any web-search for “IBD
Treatment.” With the recent publications implicating dietary triggers of disease, and dietary modifications based on scientific studies such as the Low-FODMAP (Fermentable
Oligosaccharides, Disaccharides, Monosaccharides, and Polyols) diet (J. S. Barrett et al., 2010;
Ong et al., 2010; Shepherd, Parker, Muir, & Gibson, 2008; Witkowski, Witkowski, Gagliani, &
Huber, 2018), clinicians have a greater opportunity to explore non-pharmacological therapies in
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otherwise „healthy‟ individuals. Furthermore, with the apparent resolution of symptoms in certain populations that take-on severe dietary changes, physicians have begun questioning on a biological level whether or not diet can affect the manifestation of IBD. Several of the diets recommended for IBD have their own recommendations for food modifications, and each diet has apparent benefit for numerous populations of patients (Knight-Sepulveda, Kais, Santaolalla,
& Abreu, 2015). Despite the emerging usage of dietary modifications to treat IBDs, the
American College of Gastroenterology only recommends nutritional modifications during exacerbation periods to combat absorptive issues. Enough research has emerged that the
National Institute for Diabetes and Digestive and Kidney Diseases have published guidelines for physicians treating IBDs to follow (Hawkey & ProQuest (Firm), 2012). However, many patients struggle with maintaining consistent dietary modifications, and experience disease recurrence from “trigger” food exposures.
Emerging treatment: Alternative therapeutics Another mechanism that is currently employed to restore dysbiosis in UC has been
probiotic treatment (Kruis et al., 2004). Despite the push for an increase in probiotic
therapeutics in UC, only one commercially available probiotic has resulted in significant
improvement in UC symptoms, produced by Alfasigma, VSL#3 (Sood et al., 2009). Still,
additional species of microorganisms have been found to mediate remission, including
Faecalibacterium prausnitzii (F. prausnitzii ) (Shen et al., 2018) and E. coli Nissle 1917 (Kruis
et al., 2004). Both VSL#3 and E coli Nissle 1917 produced remission with comparable rates to
the first line 5-ASA treatments. Certain studies have found beneficial effects of probiotics
occurring by their ability to regulate the mucosal barrier function by promoting secretion of
mucins (Kabeerdoss et al., 2011) and improving tight junction function (Karczewski et al.,
2010). Despite their purported benefits, in certain patient populations there have been reports
of adverse reactions with probiotic therapies. Certain probiotics have been found to increase
peristalsis- changing stool consistency and frequency, exacerbating diarrhea, and increase in
disease severity (Agathou & Beales, 2013). Additionally, in some studies despite efforts to
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restore the dysregulated microbiome, there was no difference in recovery in patients that were administered Lactobacillus and Bifidobacterium species and the control group (Wildt,
Nordgaard, Hansen, Brockmann, & Rumessen, 2011). Interestingly, probiotic species diversity remains limited (Figure 7) compared to the diversity typically expected in a healthy microbiome.
Given the correlation of a dysregulated microbiome and disease, and perhaps the limited efficacy of probiotics, fecal microbiota transplantation has also been explored as a means to modulate dysbiosis in diseased patients (Shen et al., 2018). Fecal microbiota transplantation
(FMT) has been successfully used in treating antibiotic induced Clostridium difficile infections, with a low frequency of adverse effects (Gough, Shaikh, & Manges, 2011). The primary goal of
FMT in patients with UC is to restore dysbiosis by increasing the species diversity and decreasing opportunistic pathogenic colonization of the GIT, aiming to normalize both immune inflammatory responses, dysregulation of neurotransmitters, and restoring the energy metabolism balance through diversifying the gut (Shen et al., 2018). Currently, FMT as a therapeutic option in patients has limitations. Efficacy in patient populations ranges on a success rate of 20-92% of patients recovering from UC flares, with an average success rate of
30.4% (Shen et al., 2018). Additional studies have identified a need for continuous FMT, with remission of UC not occurring until after 4 months of treatment (Witkowski et al., 2018).
Perhaps one of the major limitations of FMT is that there is no criteria for donor acquisitions, often time leading to familial donors with little to no screening occurring (Shen et al., 2018).
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Biogenic Amines in the GIT
Concentrations of Biogenic Amines in the GIT
Serotonin Biogenic amines are a broad spectrum of biologically active monoamines. It is widely understood that the GIT is an abundant source of classical biogenic amines including serotonin, dopamine, epinephrine, and norepinephrine. Emerging research into these classical biogenic amines in the GIT have focused on parsing out the role that microbiota have in regulating their synthesis, as well as their direct actions on GI epithelium, enterochromaffin cells, immunological cells and neuronal cells in the GIT (Lyte & Brown, 2018; Pugin et al., 2017; Raab et al., 2016; van Kessel et al., 2019), though direct influence of microbial-associated metabolic activity has been poorly evaluated. 5HT is perhaps the best studied biogenic amine in the gut as it is known to regulate gastric emptying, intestinal peristalsis, nausea and emesis, intestinal secretion, and colonic tone (Berger, Gray, & Roth, 2009). In mice, in the lumen contents of the colon, 5HT can be found at levels ranging from 40ng/g to 300ng/g of protein, and at 1-12.5 μg/g of tissue (Hata et al., 2017; Yano et al., 2015). In the human metabolome, concentrations of serotonin in the lumen/feces are confirmed, but have not been meaningfully quantified (Barrios, Spector, &
Menni, 2016; Loftfield et al., 2016; Xu et al., 2015), but is found in tissue homogenates averaging at 40,000pmol/g tissue, while patients with inflamed and non-inflamed UC have levels of 5HT at 8,000 and 22,000 pmol/g tissue respectively (Magro et al., 2002)
Dopamine and Norepinepherine Dopamine presence in the intestine is elusive, though there have been recent studies suggesting that microbiome species are capable of modulating dopamine synthesis in certain disease states (van Kessel et al., 2019). Dopamine has been detected in the feces of healthy patients using LC/MS, though there was no quantifiable measure reported (Gao, Pujos-Guillot,
& Sebedio, 2010). In tissue, dopamine is reportedly 130pmol/g tissue in healthy controls, compared to patients with inflamed and non-inflamed UC having levels of dopamine at concentrations of 50pmol/g and 70 pmol/g respectively (Magro et al., 2002). Epinephrine and norepinephrine both have a role in modulating absorption rates in the body, and exogenous Page 16
administration of epinephrine results in alterations in glucose concentrations when coadministered with thyroid hormones (Olaleye & Elegbe, 2005). Epinephrine and norepinephrine have not been identified in the fecal metabolome of human patients (Wishart et al., 2013) although there are reports of healthy patients having on average 875pmol/g of norepinephrine per tissue and 15pmol/g epinephrine in the intestine. In patients with ulcerative colitis, these values are markedly and significantly altered, as inflamed patients have 325pmol/g norepinephrine and 20pmol/g epinephrine per tissue whereas in non-inflamed patients with UC, norepinephrine exists at 650pmol/g and epinephrine at 10pmol/g tissue (Magro et al., 2002).
Trace amine levels in the GIT Trace amines are classically defined as any monoamine with a physiological level less than 100ng/g of tissue weight (Boulton, 1974) though oftentimes higher levels are subsequently identified in new tissue assessments of particular amines. In multiple metabolomic studies investigating trace amine levels in healthy patients, tyramine (TYR) was found at detectable levels in both the feces and urine of over 3030 healthy volunteers, although the exact values were not quantified (Azario et al., 2017; De Angelis et al., 2016; Di Cagno et al., 2011; Gao et al., 2009; Goedert et al., 2014; Loftfield et al., 2016; Su et al., 2016; Yen et al., 2015).
Interestingly, TYR is not absorbed from the colon but is absorbed from the small intestine (Coyle
& Boyd, 1932; Tchercansky, Acevedo, & Rubio, 1994). In one study, β-phenylethylamine (PEA) levels were quantified in fecal/luminal contents at concentrations of 10nmol/g in healthy patients
(Turroni et al., 2016) correlating to a passage of 1020 – 4070 nmol per day (Loftfield et al.,
2016), 28842642). TYR levels in the feces have also been found to be significantly higher in children with Celiac‟s disease compared to healthy children, though the exact concentrations were not reported (Celiac D: 1.34% - 3.21% (median 2.81%) compared to 0.74 - 7.87%
(median 1.88%) (Di Cagno et al., 2011). Additionally, trace amines including caverdine and spermine have been found in levels of 1.25 – 75 μg/μl of luminal contents (Gao et al., 2010;
Weiss et al., 2004).
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Figure 8. Tyrosine decarboxylase from the small intestine of humans produces TYR ex vivo by action of tyrosine decarboxylase. “Bacteria in jejunal content decarboxylate levodopa to dopamine coinciding with their production of TYR ex vivo. A Decarboxylation reaction for tyrosine and levodopa. B. From left to right coinciding bacterial conversion of tyrosine (TYR) to TYR (TYRM) and 1 mM of supplemented levodopa (LD) to dopamine (DA) during 24 h of incubation of jejunal content. The jejunal contents are from four different rats ranked form left to right based on the decarboxylation levels of tyrosine and levodopa, showing that tyrosine decarboxylation is coinciding with levodopa decarboxylation. C. Gut bacteria harboring tyrosine decarboxylases are responsible for levodopa decarboxylation. Aligned genomes of E. faecium, E. faecalis, and L. brevis. The conserved tdc-operon is depicted with tdc gene in orange.”(van Kessel et al., 2019)and D. Enzymatic mechanism of tyrosine TYR transformation requires coexpression of the tyrosine transporter in the membrane of bacteria (Wolken, Lucas, Lonvaud-Funel, & Lolkema, 2006). Modified and reproduced from van Kessel et al 2019 (van Kessel et al., 2019), with permission.
Trace amine presence in trigger foods Natural fermentation processes present in various microbes have been monopolized in food production common to the Western diet. Foods including pickles, sauerkraut, and olives have been fermented to extend the shelf life of vegetables, while sausages and cheeses often are considered novelty foods(Bugda Gwilt, 2018). Many beverages including beers, wines, and other alcoholic beverages rely heavily on the natural fermentation pathways in microorganisms to mass manufacture and produce an array of products for human consumption (Andreoletti et al., 2011; Bargossi et al., 2017; Benkerroum, 2016; Bonnin-Jusserand, Grandvalet, Rieu,
Weidmann, & Alexandre, 2012; Bover-Cid & Holzapfel, 1999; Broadley et al., 2009; Bugda
Gwilt, 2018; Burdychova & Komprda, 2007; Kabeerdoss et al., 2011; Luthy & Schlatter, 1983;
Marcobal, De las Rivas, Landete, Tabera, & Munoz, 2012; Ohta, Takebe, Murakami, Page 18
Takahama, & Morimura, 2017; Pereira, Matos, San Romao, & Crespo, 2009; Pessione et al.,
2009; Pozo-Bayon, Monagas, Bartolome, & Moreno-Arribas, 2012; Shalaby, 1996; Stratton,
Hutkins, & Taylor, 1991; Torriani et al., 2008; Ventura et al., 2014). As such, organizations including the World Health Association, Center for Disease Control and Prevention, the United
States FDA, and United States Department of Agriculture have heavily researched the affects that fermented food products have in human health. There are published guidelines for safe- food manufacturing to regulate biologically active compounds in human health(Andreoletti et al.,
2011). Accordingly, the food industry uses fermentation to create and enhance various food products, and assays microbial activity as an indicator of food spoilage. Trace amines are commonly found in food products as a byproduct of normal microbial processes, and are exploited in the food industry to indicate food freshness (Benkerroum, 2016; Bover-Cid &
Holzapfel, 1999; Food and Drug Administration, 2010; Pessione et al., 2009; Torriani et al.,
2008). To date, the most understood biogenic amine consumed by humans is histamine.
Typically thought of as an indicator of aging in food, from 1977 to 1981 there were over 500 cases of “food poisoning” from excessive histamine in numerous fish products (Shalaby, 1996).
After investigations, the FDA issued regulatory recommendations to keep levels of histamine below 500ppm in fermented food products (Food and Drug Administration, 2010). Given the adverse health risks of histamine, other biogenic amines including β-PEA, TYR, caverdine, spermine and others have been explored as indicators of spoilage in the manufacturing process of food, although there has been little to no guidance from regulatory bodies on the levels of these biogenic amines in food (Andreoletti et al., 2011; Food and Drug Administration, 2010).
Tyramine, a biogenic amine that is now known to be a full agonist at Trace Amine
Associated Receptor 1 (TAAR1), gained the attention of physicians and pharmacists in the
1970s with the use of monoamine oxidase inhibitors (MAO-I) to treat depression. Monoamine oxidase is an enzymatic system responsible for the oxidative deamination of monoamines to their corresponding aldehyde or ketone, and ammonia (Price & Smith, 1971). Monoamine oxidase substrates include serotonin, norepinephrine, dopamine, as well as trace amines
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including TYR and β-PEA. Patients receiving concurrent MAO-I treatment and consuming large quantities of foods containing trace amines would often experience a hypertensive crisis due to the inhibition of normal transformation of these biologically active compounds (Sathyanarayana
Rao & Yeragani, 2009). While consumption of these trace amines are typically harmless in a healthy individual, inhibition of the degradation of these molecules would have severe biological effects in human health including hypertension, migraine, headaches, and other neurological problems (Price & Smith, 1971; Shalaby, 1996; Stratton et al., 1991) in the presence of an
MAO-I. This provided some of the first evidence that trace amines in food products were capable of eliciting a biological response in humans, although the mechanism by which this response occurs remains poorly understood.
Trace amine production by microorganisms
Trace amine production in food Production of many fermented food products relies on the use of a variety of bacterial species. While the fermentation pathways in these microorganisms are primarily dependent on degradation of carbohydrates such as fructose, sucrose, glucose, into lactic acid, the microorganisms also cause extensive proteolysis which forms simple peptides and free amino acids, which may undergo further transformation by microbial enzymatic systems (Singh,
Kumar, Mittal, & Mehta, 2017). In specific microorganisms, in response to a change in pH of the environment, an enzymatic system including a tyrosine transporter and a tyrosine decarboxylase enzyme will upregulate expression. As shown in Figure 8, the tyrosine transporter (TyrP) transports tyrosine into the microorganism. Once inside the microorganism, tyrosine is rapidly decarboxylated to TYR by the bacterial tyrosine decarboxylase (TyrDC), where it is then exported from the microorganism by TyrP (Wolken et al., 2006). Previous phylogeny studies (Bonnin-Jusserand et al., 2012) have indicated that the amino acid sequence of the TyrDC and TyrP are very highly conserved among Lactobacillus strains as deduced indirectly from DNA studies and not by direct amino acid analysis of proteins. While the entire
Lactobacillus species are considered producers of TYR (Pessione et al., 2009), only specific
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Lactobacillus species are found in food products, and some have been found to survive transit through the gastrointestinal tract (Pugin et al., 2017). Bacterial jejunal contents were found to coincide with production of TYR in the presence of tyrosine decarboxylase ex vivo as monitored by High Performance Liquid Chromatography with Electrochemical Detection, and monitoring chromatograms and function of conversion of tyrosine to TYR (Fernandez de Palencia et al.,
2011; van Kessel et al., 2019) (Figure 8). TYR plasma levels can reach levels of 0.2μM after ingestion of 200mg of TYR in healthy individuals (Vandenberg, Blob, Kemper, & Azzaro, 2003).
Microbiome Biogenic amines are produced by the decarboxylation of amino acids occurring within bacteria in fermented foods and the microbiome by specific enzymatic systems, as described above. Levels of these biogenic trace amines were quantified in the feces of human volunteers that were cultured ex vivo (Pugin et al., 2017; van Kessel et al., 2019). It is worth noting that these quantifications were the potential for human fecal microbes to produce biogenic amines as specific gram (+) and gram (-) lysates were isolated and cultured individually.
A new and emerging field of microbiology focuses on the roles of microorganisms in the gut, as well as probiotics, to synthesize ligands capable of activating mammalian receptors.
Interestingly, germ free mice have significantly higher levels of tyrosine in the plasma (1.44 fold higher) implicating the gut microbiota in the metabolism and processing of free tyrosine in the body (Wikoff et al., 2009). Additionally, germ free mice have lower levels of TYR compared to
„chow-fed‟ controls and „ex-germ free‟ littermates, as well as significantly higher levels of tyrosine compared to their siblings (Matsumoto et al., 2012). In bacteria, biogenic amine production is essential for growth and proliferation as they serve as a defense mechanism used by bacteria to withstand acidic stress. For example, a trace amine, spermine, has been shown to inhibit pro-inflammatory cytokine secretion in PBMC‟s (peripheral blood mononuclear cell)
(Bridoux et al., 1997). Biogenic amine-producing bacteria are mostly facultative anaerobes and belong to the phyla Firmicutes and Proteobacteria (Letunic & Bork, 2016; Pessione et al., 2009).
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Taken together, these data implicate gastrointestinal microbiota in the regulation of TYR levels in the gastrointestinal tract (Broadley et al., 2009; Pugin et al., 2017; van Kessel et al., 2019).
Studies dating back to the 1980‟s explored the role of TYR on global health and found no significant effects of these trace amines on inducing symptoms such as headache, dizziness and discomfort, though they fail to report whether or not they studied gastrointestinal effects
(Luthy & Schlatter, 1983). Emerging evidence suggests that trace amine levels are elevated in ulcerative colitis (Li et al., 2011; Robinson et al., 2016; Santoru et al., 2017; M. Zhang et al.,
2017), with TYR levels being higher in patients with ulcerative colitis, and β-PEA levels elevated in patients with Crohn‟s disease (Santoru et al., 2017). TYR has been demonstrated to have effects on contractility in the GIT (Broadley et al., 2009), with varying toxic effects on colon cell health, although whether or not it has a direct effect on cell health, or exacerbate injury to already compromised cells remains to be fully understood (Biaggini et al., 2017; Del Rio et al.,
2017; Linares et al., 2016).
Trace amine associate receptor 1
History of receptor Trace amines are structurally related to classical biogenic amine neurotransmitters, although they bind their own receptor, Trace Amine Associated Receptor 1 (TAAR1). TAAR1 is a G protein-coupled receptor that was deorphanized in 2001 (Borowsky et al., 2001; Bunzow et al., 2001) and has been widely studied as a major regulator of dopamine and neuropsychiatric disorders, and drugs of abuse (Miller, 2011). Biogenic trace amines such as TYR activate
TAAR1 at nM affinities (Panas et al., 2012; Xie et al., 2007), and brief description of TAAR1 ligands and their affinities can be found in Table 1 (Borowsky et al., 2001; Bradaia et al., 2009;
Chiellini et al., 2012; Hoefig et al., 2015; Peleg-Raibstein, Knuesel, & Feldon, 2008; Pugin et al.,
2017; Revel, Meyer, et al., 2012; Revel, Moreau, et al., 2012; Revel et al., 2013; Sotnikova et al., 2010) .
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Ligands for the receptor TYR has been widely studied for its roles in neuropsychiatric disorder and its ability to induce a pressor effect in patients taking MAO-Is as antidepressants. TYR has been studied since the late 1800‟s as it was found in elevated levels in patients with neuropsychiatric illness.
Perhaps because of the focus of TYR in psychiatric illness over prior decades, the discovery of
TAAR1 in 2001 led to a body of research studying the effects of TAAR1 in modulating monoaminergic signaling in the brain. From this dogmatic impetus, Hoffman La Roche developed a library of compounds specific for TAAR1, and spearheaded current ongoing clinical trials investigating novel TAAR1-targetted drugs for the treatment of schizophrenia. EC50 and
Ki values for TAAR1 compounds are outlined in Table 1. Interesting, many of the endogenous compounds that are known TAAR1 agonists are also known gut metabolites.
Table 1. TAAR1 Ligand potency at mTAAR1 and hTAAR1 and presence in the gut Activity mTAAR1 hTAAR1 Gut Metabolite? EC50 (nm) Ki (nm) EC50 (nm) Ki (nm) Ref Tyramine Agonist 280 380 990 34 yes Borowsky et al., 2001 β-phenylethylamine Agonist 200 310 260 8 yes Borowsky et al., 2001 Tryptamine Agonist 2700 1400 2100 yes Borowsky et al., 2001 Octopamine Agonist 20000 4029 493 unclear Borowsky et al., 2001 Serotonin Agonist >50000 >50000 6000 yes Borowsky et al., 2001 Dopamine Agonist 12000 1600 422 yes van Kessel et al., 2019 Histamine Agonist 5000 3107 yes Pugin et al., 2017 Borowsky et al., 2001; 3-Methoxytyramine Agonist 700 unclear Sotnikova et al., 2010 Chiellini et al., 2012.; 3-Iodothyronamine Agonist 90 1700 yes Hoefig et al., 2015 RO5212773 (EPPTB) Antagonist 28 0.9 7500 >5000 - Bradaia et al., 2009 RO5166017 Agonist 3.3 1.9 55 31 - Peleg-Raibstein et al., 2008 RO5256390 Agonist 2 4.4 16 24 - Revel et al., 2013
RO5203648 Partial 4 0.5 30 6.8 - Revel, Meyer et al., 2012 RO5263397 Partial 1.3 0.9 17 4.1 - Revel et al., 2013
RO5073012 Partial 23 3.2 25 5.8 - Revel, Moreau et al., 2012
Agonists Agonists at TAAR1 are diverse and include a spectrum of biogenic amines, trace amines, and drugs of abuse, such as methamphetamine (Borowsky et al., 2001; Bunzow et al.,
2001; Xie & Miller, 2009). TAAR1 is the primary human receptor activated by the trace amines including tyramine, β-PEA, tryptamine, and octopamine (Borowsky et al., 2001; Bradaia et al.,
2009; Chiellini et al., 2012; Hoefig et al., 2015; Peleg-Raibstein et al., 2008; Pugin et al., 2017;
Revel, Meyer, et al., 2012; Revel, Moreau, et al., 2012; Revel et al., 2013; Sotnikova et al., Page 23
2010). Not only are trace amines primary agonists at the receptor, biogenic amines including serotonin, dopamine and histamine can elicit a response at physiological concentrations, as can the thyroid hormone derivative, 3-iodothyronamine (T1AM). T1AM has emerged as a compound of specific interest due to its reported potency as the most potent endogenous ligand for TAAR1 (Scanlan et al., 2004). The compounds generated by Roche encompass agonists
(RO5166017, RO5256390), partial agonists (RO5203648, RO5263397, RO5073012), and a single antagonist (RO5212773, EPPTB). The Roche agonist, RO5166017, was studied as a treatment for schizophrenia (Peleg-Raibstein et al., 2008) by monitoring the locomotor stimulating effects of psychostimulant drugs. Additionally, it was also found to increase glucose- dependent insulin secretion in β-cells of the pancreas (Raab et al 2016 ). RO5256390 is an analog of RO5166017, and was also studied as a new class of compounds for treatment of schizophrenia (Revel et al., 2013)
Antagonists Currently, the only known antagonist for TAAR1 is RO5212773, commonly referred to as
EPPTB (Bradaia et al., 2009). EPPTB is a mouse-specific compound that has limited use in vivo due to its poor pharmacokinetic profile, and poor absorption from the intestines (Bradaia et al., 2009). Still, this compound can be used experimentally in vitro.
Partial Agonists There are several TAAR1 partial agonists that were generated by Roche, including
RO5263397 (Revel et al., 2013), RO52603648 (Revel, Moreau, et al., 2012), and
RO5073012(Revel, Meyer, et al., 2012). TAAR1 is constitutively active, and partial agonists have been observed to behave as antagonists in vitro and agonists in vivo. In vitro stimulation with RO5263397 and RO52603648 showed increased firing of dopamine neurons that was characteristic of treatment with EPPTB, though the in vivo results showed full agonist activity at the receptor(Revel, Meyer, et al., 2012; Revel, Moreau, et al., 2012; Revel et al., 2013).
RO5203648 was studied for its effects in decreasing impulsivity in ADHD and drug addiction
(Espinoza et al., 2015). The partial agonist RO5263397 has purportedly been able to reverse
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weight gain seen with antipsychotic for schizophrenia (Revel, Moreau, et al., 2012), attenuate the rewarding effects of cocaine and methamphetamine (Thorn et al., 2014), as well as improve the negative symptoms of schizophrenia (Revel, Moreau, et al., 2012). RO5263397 was advanced to clinical trials as an antipsychotic compound for the treatment of schizophrenia, however, when the phase 1 trials commenced a small population of African individuals were experiencing toxicity to the compound due to a liver enzyme polymorphism that prevented the degradation of the drug (Fowler et al., 2015).
Intracellular signaling TAAR1 has been widely studied for its activation by trace amines. TAAR1 is a Gαs coupled receptor that increases cAMP, phosphorylated PKA levels and downstream CREB transcription upon stimulation (Barak et al., 2008; Borowsky et al., 2001; Bunzow et al., 2001;
Panas et al., 2012). In addition to Gαs intracellular signaling, TAAR1 activation can lead to an increase in PKC phosphorylation and downstream NFAT transcription in lymphocytes (Panas et al., 2012), and CREB (Sotnikova et al., 2010; Sriram et al., 2016). TAAR1 signaling can also lead to the increased activation of GIRK channels by the βy subunits of G proteins in ventral tegmental area brain slices (Bradaia et al., 2009). Heterodimerization with the D2 receptor can regulate dopamine receptor signaling in the brain, by the β-arrestin/Akt/GSK3 pathway
(Espinoza et al., 2015; Espinoza et al., 2011; Harmeier et al., 2015; Revel et al., 2013). In addition to G-protein coupled signaling, TAAR1 activation can also cause activation of ERK
(Sotnikova et al., 2010).
Anatomical Localization of TAAR1
TAAR1 tissue expression has been reported for a number of species including mouse, rat, and rhesus monkey brains (Borowsky et al., 2001; Bunzow et al., 2001; Di Cara et al., 2011;
Lindemann et al., 2008; Miller et al., 2005; Xie & Miller, 2009; Xie et al., 2007). In the brain,
TAAR1 is primarily found in monoaminergic regions. In addition to brain dopaminergic pathways TAAR1 has also been identified in peripheral organs (e.g. liver, kidney, intestine,
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immune cells) though few studies have been conducted studying the effects of TAAR1 in these systems. Functional TAAR1 was found in almost all peripheral immune cells (Babusyte,
Kotthoff, Fiedler, & Krautwurst, 2013; Panas et al., 2012; Wasik, Millan, Scanlan, Barnes, &
Gordon, 2012), with evidence that TAAR1 can modulate not only intracellular signaling (Panas et al., 2012), but also some normal immune cell functions such as chemotaxis and expression of some cytokines (Babusyte et al., 2013). Limited studies have identified the presence of TAAR1 in the stomach and the intestine (Chiellini et al., 2012; Ito et al., 2009; Ohta et al., 2017; Raab et al., 2016), but function remains largely unexplored. Currently, the only publication studying the specific TAAR1-mediated effects of TYR in the GIT have shown that dietary trace amines can act as agonists for hTAAR1 in epithelial cells derived from GI tissue (Ohta et al., 2017). Also, one study has shown that TAAR1 is expressed in the gastrointestinal tract of C57BL/6 mice (Ito et al., 2009). A summary of the known peripheral TAAR1 localization is found in Figure 9.
Figure 9. Peripheral localization of TAAR1, as characterized by the human protein atlas and unpublished data from the Miller Lab. A. CD68 positive macrophages(red) co-stained with TAAR1 (green), B. Western blot data from Panas et al on PBMC‟s from rhesus monkeys showing positive TAAR1 staining in TAAR1-transfected HEK cells, with low expression in unstimulated PBMC, and increased expression in PHA stimulated PBMC. C. Screenshot from “The Human Protein Atlas” cataloging protein and RNA expression in human body systems as analyzed from autopsy samples. D. Localization of TAAR1 (green) in rhesus monkey thalamus. E. Localization of TAAR1 in rhesus monkey ileum (green). F. IHC staining of healthy female and male colon sections showing localization of TAAR1 (brown) in the colon, in the peripheral cells of the colonic crypts. (Uhlen et al., 2015)
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While the effects of TAAR1 signaling have yet to be studied in macrophages, Babusyte et al studied the effects that trace amine activation of TAAR1 have in human B-Cells, T-Cells, and polymorphonuclear cells (PMN) (Babusyte et al., 2013). This publication was the first and only publication demonstrating that trace amines can serve as chemoattractive agents, and affect cytokine production and gene expression in immune cells.
To date, TAAR1 expression in the gut has not been systematically studied, and there have been no functional studies on the role of TAAR1 in modulating the effects of trace amines on the epithelial barrier in the gastrointestinal tract in inflammatory bowel diseases such as UC and CD. Trace amines have defined sources in the gastrointestinal tract, originating from mammalian amino acid degradation by aromatic amino acid decarboxylases (Lauweryns & Van
Ranst, 1988), contamination of prepackaged and fermented food products, and the organisms in the microbiome.
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Chapter One: Design and validation of custom TAAR1 antibody
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BACKGROUND
Since the cloning of TAAR1 in 2001 there have been challenges in the field in using standard molecular technologies to detect TAAR1 mRNA and determine protein localization
(Berry, Gainetdinov, Hoener, & Shahid, 2017; Liu & Li, 2018; Nelson, Tolbert, Singh, & Bost,
2007). This handicap is not uncommon to G-protein coupled receptors and has been widely reviewed in the literature (Bordeaux et al., 2010). Despite challenges in specificity of antibodies for TAAR1, researchers have been able to delineate TAAR1 localization (Bunzow et al., 2001;
Miller et al., 2005; Raab et al., 2016; Xie, Vallender, et al., 2008), signaling cascades (Barak et al., 2008; Borowsky et al., 2001; Miller et al., 2005; Panas et al., 2012; Xie & Miller, 2008; Xie,
Westmoreland, & Miller, 2008) and carry pharmaceutical compounds to clinical trials (Roche and Sunovion). Still, the development of commercially available antibodies, especially for mouse TAAR1 (mTAAR1), has been slow, and while primate TAAR1 antibodies have been developed and successfully used for immunofluorescence(Sriram et al., 2016) and Western analyses (Panas et al., 2012), there remains only one advertised mouse-specific antibody from
Alomone labs.
DNA, RNA, and protein sequence information for thousands of organisms have been decoded and catalogued in the United States National Library of Medicine at the NIH since
1988. These sequences can be found as raw data through various search platforms, and have progressed to the modern-day basic local alignment search tools (BLAST) algorithms and software developed by David Lipman in 1990 (Altschul, Gish, Miller, Myers, & Lipman, 1990).
The National Center for Biotechnology Information (NCBI) consists of databases allowing for researchers to deposit genomic data, and now, microarray and RNA-Seq data to the NLM ("The
NCBI Handbook," 2002). Computational bioinformatics is a growing field with precise algorithms being developed to streamline data analysis, and an increased presence of open source software for data access (e.g. UniProt, Ensemble), data analysis (e.g. R statistical
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programming) and sequence analysis (e.g. Chromaseq, Clustal Omega, Jalview) being widely available to the scientific community.
Experimental validation of commercially available antibodies in vitro and ex vivo revealed significant nonspecific staining in samples that were indeed TAAR1 null. To understand the consistently observed non-specificity of TAAR1 antibodies in mouse samples, including the commercially available mouse TAAR1 antibody generated by Alomone labs open access bioinformatics platforms were used to compare TAAR family protein homology in human and mouse proteomes. Additionally, to broaden the experimental modus available to the TAAR1 research community, an antibody with high specificity and more validity than commercially available antibodies was generated. The custom TAAR1 antibody (D274) was designed to have cross species specificity (e.g., human, mouse), with limited cross reactivity to other proteins as well as mTAAR and hTAAR family members. This informatics specificity was then confirmed both in vitro and ex vivo to validate the use of D274 in mouse and human. Generation of an antibody that has interspecies specificity and which recognizes an extracellular epitope fills a major gap in the field and facilitates the understanding of TAAR1 and TAAR receptor family members in normal and pathological physiology.
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RESULTS
Specificity of commercially available antibodies to mouse and human TAAR1 To determine the specificity of antibodies to mouse TAAR1 (mTAAR1) in the colon, sections from C57BL/6 and TAAR1-/- mice were stained with various commercially available antibodies
(Figure 10). Antibodies previously validated in primates such as Imgenex IMG-7155 (Panas et al., 2012) demonstrates strong and pronounced staining of TAAR1 in the colon epithelium of
C57BL/6 mice, with faint staining in the epithelium of the TAAR1-/- mouse. A similar phenomenon is observed using the Thermo Fisher PA141477 antibody on colon sections
(Figure 10B). Here, a markedly decreased staining of the colonocytes is observed in C57BL/6 with even more diffuse staining in the TAAR1-/- sections. Due to the limitations of the antibodies raised against hTAAR1 in mouse, and the apparent non-specificity of the antibodies, the commercially advertised mTAAR1 specific antibody was purchased from Alomone labs, and validated in mouse intestines (Figure 10C), and spleen sections (Figure 10D). Upon first use of the antibody, there appeared to be specific staining in the colon of the C57BL/6 mouse. To further validate the specificity of the staining in the mouse, spleen sections were also incubated with the Alomone antibody, given the presence of TAAR1-expressing T-Cells and B-Cells
(Panas et al., 2012; Sriram et al., 2016), as a positive control for the antibody. Similar to the other commercially available antibodies used, there was specific staining in the C57BL/6 mouse spleen, with a decrease in, but presence of nonspecific staining of the TAAR1-/- mice in spleen sections (Figure 10D).
The diffuse staining of the C57BL/6 mouse with Thermo Fisher PA1-41477 antibody begged the question: is the Thermo Fisher antibody mouse specific? To determine the specificity of the
Thermo Fisher-anti-TAAR1 antibody to both mouse and human for future studies, untransfected
HEK cells (TAAR1-negative by PCR) and HEK cells transduced with hTAAR1 or mTAAR1 were immunostained with the Thermo Fisher antibody (Figure 11). Using lot no. PH1889029A, a strong membranous staining for TAAR1 in the hTAAR1 transduced HEK cells is very apparent
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(Figure 11B) but the staining of the mTAAR1 transduced HEK cells is not as discernable (Figure
11C), and resembles the untransfected HEK cells (Figure 11A). Unfortunately, this specific stain absolved with the use of a new lot of antibody (Lot no. RG2232021C), and the staining in
Figure 11B was subsequently not reproduced (Figure 11D-F). With use of the new lot of the antibody a strong non-specific signal is observed in untransfected HEK cells (Figure 11D) and with strong non-specific punctate staining co-localizing to the nucleus in hTAAR1 (Figure 11E) and mTAAR1 (Figure 11F).
Specificity of TAAR1 antibodies determined bioinformatically. To determine the non-specificity of the antibody sequences, protein sequence analysis was performed using the published antigenic determinants in the literature for Imgenex, PA41477 and Alomone Labs anti-TAAR1 antibodies. Sequences for human TAAR1 and mouse TAARs
1-9 were downloaded from UniProt and sequences were aligned with Clustal Omega open access software and viewed in Jalview v.2.10.5 to determine physiochemical properties of amino acid residues (Figure 12A). Hydrophobic residues are colored salmon whereas hydrophilic residues are green, aromatic are orange, positive and negatively charged residues are blue and red respectively. Conformationally „special‟ residues include proline and glycine which are pink, and cysteine is yellow. This color scheme is applied to the antibodies from
Alomone labs (Figure 12B), Imgenex (Figure 12C) and Thermo Fisher (Figure 12D) to determine homology to mTAAR1, and mTAARs 2-9. As seen in figure 12B, the Alomone Labs antibody is homologous to mTAAR1, with slight conservation of sequences through mTAAR2-9.
The Imgenex antibody (Figure 12C) is almost 100% homologous to all mTAARs (1-9), as is the
Thermo Fisher antibody (Figure 12D). Homologies of additional commercially available antibodies to mTAARs 1-9 are outlined in Table 1.
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D274: A novel TAAR1 antibody. Commercially available TAAR1 antibodies are generally considered of poor quality and poor selectivity, hindering research progress in the field (Berry et al., 2017), therefore an interspecies, extracellular loop custom antibody, D274 was generated. Using commercially available TAAR1 antibodies there is consistent nonspecific staining in TAAR1-/- mice (Figure 10) as well as in various cell lines that are PCR-negative for TAAR1 (data not shown). Analysis of the amino acid sequences of hTAAR1 and mTAAR1 demonstrates divergence of D274 to other mTAAR receptor family members and 79% sequence identity between mTAAR1 and hTAAR1
(Figure 13), indicating that D274 most likely does not cross react with other TAAR family members. To validate the specificity of the antibody, the specificity of D274 to mTAAR1 and hTAAR1 was explored in transfected HEK293 cells (Panas et al., 2012) using D274. A robust
TAAR1 staining was observed in transfected cells as well as a slight nonspecific staining in non- transfected HEK, similar to observations seen when using other TAAR1 antibodies (Figure 14).
To further validate the specificity of D274 in mouse non-polarized C57BL/6 and TAAR1-/- BMDM were stained with D274. Use of D274 resulted in a robust staining of TAAR1 intracellularly with slight membrane staining in C57BL/6 mice compared to absent staining in TAAR1-/- BMDM
(Figure 14). This observation is consistent with our previous studies on TAAR1 localization in cells.
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FIGURES AND TABLES
Figure 10. Antibody validation of commercially available antibodies with immunohistochemistry (IHC). IHC on C57BL/6 and TAAR1-/- intestines (A-C) and spleen (D) using commercially available A. Imgenex Antibody, B. Thermo Fisher antibody and C&D. Alomone Labs antibody. A. Strong staining in the lumen epithelial cells in the C57BL/6 intestine, and faint, non specific staining in the TAAR1-/- intestines. B. Thermo Fisher antibody produces weak staining in C57BL/6 intestines that is similarly observed in the TAAR1-/- intestines. C. Mouse TAAR1 specific Alomone Labs antibody produces diffuse TAAR1 staining in the C57BL/6 intestines, with faint staining in the TAAR1-/- intestines. D. Validation of the specificity of Alomone Labs mTAAR1 antibody in spleen sections from C57BL/6 and TAAR1-/- sections demonstrates strong specific staining in the immune cells in the C57BL/6, but demonstratable staining in the TAAR1-/- spleen as well.
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Figure 11. Thermo Fisher antibody specificity. HEK cells and HEK cells stably expressing hTAAR1 (B&E) or mTAAR1 (C&F) were cultured and stained using two different lots of the Thermo Fisher PA41477 antibody. Here in panels A-C, cells were stained with lot no. PH1889029A, and in panels D-F cells were stained with lot no. RG2232021C. A. There is slight non-specific staining of the HEK cells, but B. distinctive membranous staining of hTAAR1 in transfected HEK cells. In C there is slight staining of mTAAR1, though no specific staining. An experimental replicate of the staining, using the new lot of PA1-41477 on D. HEK, E. hTAAR1-HEK cells, and F. mTAAR1-HEK cells. In both E and F there is a complete loss any resemblance of specific staining observed with lot no 12345, and observe non-specific punctate staining of the nuclei. The control stain demonstrates no staining aside from background.
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Figure 12. Multiple Sequence Alignment of published antigenic determinants of commercially available antibodies with human TAAR1 (hTAAR1) and mouse TAAR (mTAAR) family members. A. Table outlining ZAPPO identity assignments grouping amino acid residues by physiochemical properties. B. Sequence alignment of the supposed mouse specific TAAR1 antibody produced by Alomone Labs. The antigenic determinant is not significantly homologous to other TAAR family members, though the physical properties of the amino acid residues are. C. Alignment of IMGENEX antibody, previously demonstrated to be specific for rhTAAR1 as previously published by the Miller lab (panas). Imgenex antibody has homology to all mouse TAAR family members. D. Alignment of Thermo Fisher commercially available antibody predicted antigenic determinant to mouse TAAR1 sequences. Thermo Fisher sequence has homology to all mouse TAAR1 sequences.
Table 2: Commercially available antibodies and reactivity to mTAAR family members
Confirmed mTAAR family Manufacturer Catalog Number Species Reactivity Antigenic Determinant experimentally reactivity? Imgenex / Novus IMG-71855 H ,M, R, NHP Recombinant Protein: MVRSAEHCWYFGEVFC yes yes Biologics Synthetic peptide; residues 200-230 of human TAAR1: Thermofisher PA1-41477 H, NHP yes yes PGSIMLCVYYRIYLIAKEQARLISDANQK A portion of amino acids 200-230 of hTAAR1: Novus NBP2-24714SS H, M, R no yes PGSIMLCVYYRIYLIAKEQARLISDANQK Alomone Labs ATR-021 H, M, R Peptide: KMVLFGKIFQKDS yes slight Thermofisher PA5-77759 H, M, R Peptide: KMVLFGKIFQKDS no slight LKGVEELYRSQVSDLGGCSPFFSKVSGVLAFMTSFYIPGSVMLF Santa Cruz sc-514311 M yes yes VYYRIYFIAKGQARSINRTNVQVGLEGKSQAPQSKETKAA Synthetic peptide: Thermofisher OSR00119W M no yes IPGSVMLFVYYRIYFIAKGQARSINRTNVQVGLEGKSQAPQSKET
Thermofisher PA5-23141 H, NHP Synthetic peptide (230 KLQIGLEMKNGISQSKERKA 249) no slight Synthetic peptide: Thermofisher OPA1-15207 H no yes KLHYYRIYLIAKEQARLISDANQKLQIGLEMKNGISQSKERKAVKT
Thermofisher PA5-34235 H Synthetic peptide: MVRSAEHCWYFGEVFCKI no yes Synthetic peptide: Thermofisher PA5-33066 H yes YYRIYLIAKEQARLISDANQKLQIGLEMKNGISQSKERKAVKT 19 amino acid peptide from: MBU Intl MC-2041 H, NHP no yes YYRIYLIAKEQARLISDANQKLQIGLEMKNGISQSKERKAVK Novus NBP2-38762 H KEQARLISDANQKLQIGLEMKNGISQSKER no yes A portion of amino acids 225-250 of hTAAR1: Novus NBP2-24720SS H, NHP no yes SDANQKLQIGLEMKNGISQSKERKAV Abcam ab150646 H, Cat, Pig, E Synthetic peptide: MVRSAEHCWYFGEVFCKI no yes
Abcam ab189016 H, NHP, Cat, Synthetic peptide: MMPFCHNIINISCVKNNWSNDVRAS no slight
Sigma HPA055614 H Synthetic peptide: KEQARLISDANQKLQIGLEMKNGISQSKER no yes Sigma SAB2900032 H, NHP, Rab, E Synthetic peptide: LQIGLEMKNGISQSKERKAV no yes Species reactivity key: H- Human, M- Mouse, R- Rat, NHP-Non human primate, Rab-Rabbit, E-Elephant
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Figure 13. Multiple Sequence Alignment of protein sequence of D274 antibody with human TAAR1 (hTAAR1) and mouse TAAR (mTAAR) family members. A. Amino acid sequences and predicted amino acid sequences for hTAAR and mTAAR family members were retrieved from NCBI GenBank. Multiple sequences were aligned using the Clustal Omega software by percent identity, and viewed using Jalview 2.1 software(Waterhouse, Procter, Martin, Clamp, & Barton, 2009). D274 amino acid homology to mouse TAARs highlighted in gray.
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Figure 14. Immunofluorescence of D274 on HEK cells, HEK cells transfected with mTAAR1 or hTAAR1, and C57BL/6 and TAAR1-/- BMDM. A. Immunofluorescence images of HEK cells and TAAR1-/-BMDM showing minor background staining in HEK cells with no TAAR1 transfected and BMDM that do not have TAAR1 expression. B. HEK cells transfected with mTAAR1 and C57BL/6 BMDM demonstrating membranous and intracellular TAAR1 staining. C. HEK cells transfected with hTAAR1 demonstrating membranous and intracellular TAAR1 staining. Page 39
Table 3. Bioinformatics analysis determined commercially available antibody cross- reactivity to human TAAR family members
Confirmed Manufacturer Catalog Number Species Reactivity Antigenic Determinant experimentally hTAAR family reactivity? Imgenex / Novus IMG-71855 H ,M, R, NHP Recombinant Protein: MVRSAEHCWYFGEVFC yes no Biologics Synthetic peptide; residues 200-230 of human TAAR1: Thermofisher PA1-41477 H, NHP yes no PGSIMLCVYYRIYLIAKEQARLISDANQK A portion of amino acids 200-230 of hTAAR1: Novus NBP2-24714SS H, M, R no slight PGSIMLCVYYRIYLIAKEQARLISDANQK Alomone Labs ATR-021 H, M, R Peptide: KMVLFGKIFQKDS yes yes Thermofisher PA5-77759 H, M, R Peptide: KMVLFGKIFQKDS no yes LKGVEELYRSQVSDLGGCSPFFSKVSGVLAFMTSFYIPGSVMLF Santa Cruz sc-514311 M yes no htaar 2-9 specificity VYYRIYFIAKGQARSINRTNVQVGLEGKSQAPQSKETKAA Synthetic peptide: Thermofisher OSR00119W M no no htaar 2-9 specificity IPGSVMLFVYYRIYFIAKGQARSINRTNVQVGLEGKSQAPQSKET
Thermofisher PA5-23141 H, NHP Synthetic peptide (230 KLQIGLEMKNGISQSKERKA 249) no slight Synthetic peptide: Thermofisher OPA1-15207 H no no KLHYYRIYLIAKEQARLISDANQKLQIGLEMKNGISQSKERKAVKT
Thermofisher PA5-34235 H Synthetic peptide: MVRSAEHCWYFGEVFCKI no yes Synthetic peptide: Thermofisher PA5-33066 H slight YYRIYLIAKEQARLISDANQKLQIGLEMKNGISQSKERKAVKT 19 amino acid peptide from: MBU Intl MC-2041 H, NHP no slight YYRIYLIAKEQARLISDANQKLQIGLEMKNGISQSKERKAVK Novus NBP2-38762 H KEQARLISDANQKLQIGLEMKNGISQSKER no no A portion of amino acids 225-250 of hTAAR1: Novus NBP2-24720SS H, NHP no no SDANQKLQIGLEMKNGISQSKERKAV Abcam ab150646 H, Cat, Pig, E Synthetic peptide: MVRSAEHCWYFGEVFCKI no yes Abcam ab189016 H, NHP, Cat, Synthetic peptide: MMPFCHNIINISCVKNNWSNDVRAS no no Sigma HPA055614 H Synthetic peptide: KEQARLISDANQKLQIGLEMKNGISQSKER no slight Sigma SAB2900032 H, NHP, Rab, E Synthetic peptide: LQIGLEMKNGISQSKERKAV no slight Species reactivity key: H- Human, M- Mouse, R- Rat, NHP-Non human primate, Rab-Rabbit, E-Elephant
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DISCUSSION
The lack of a robust antibody for TAAR1 has hindered progress in TAAR1 research.
Oftentimes, sensitivity of primary antibodies is overshadowed by the apparent non-specific binding of the antibody to tissue, or the endogenous Fc receptors in the tissue samples, resulting in extensive blocking protocols or antigen retrieval techniques to optimize the staining protocol (Buchwalow, Samoilova, Boecker, & Tiemann, 2011). In the intestine, endogenous Fc receptors are not the main limitation in immunofluorescent staining, but it is the extensive mucous layer that lines the epithelium. Mucous in the intestine produces significant background fluorescence at wavelengths nearing 488 nm, proving challenging to determine specific binding of the Alexa-fluor 488 secondary antibodies. Because of the potential for non-specific binding of the primary antibodies and the fluorescent activity of the mucous in the intestine that is routinely observed (data not shown), the specificity of commercially available antibodies was determined using DAB peroxidase kits. Regardless of the detection methods employed with the commercially available antibodies, a similar staining pattern was observed in the intestine of the
C57BL/6 and TAAR1-/- mouse (Figure10A-C). Initial studies with the Thermo Fisher antibody
PA1-41477 suggested there may be potential for use in the mouse model (data not shown).
Accordingly, the lack of staining with the chromogen detection kit prompted further exploration of the specificity of the antibody to mTAAR1. To determine the specificity of the Thermo Fisher antibody, immunostaining was performed on HEK cells transfected to express hTAAR1 and mTAAR1. The initial data using this system did not provide enough evidence to determine if
PA1-41477 was specific to mTAAR1 (Figure 11C), despite confirmation that the HEK-cells were expressing mTAAR1 (data not shown). To validate the antibody further the experiment was repeated - although with a different lot of antibody as initial stocks were depleted. Interestingly, use of the new lot of antibody produced completely non-specific staining on the HEK-hTAAR1 and HEK-mTAAR1 cells, tending to be colocalized with the DAPI stain in the nucleus (Figure
11E & F). Here, it was concluded that the PA1-41477 polyclonal antibody was not a valid tool to continue pursuing for experiments.
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The IHC analysis of the Imgenex antibody (Figure 10A) and the „mouse specific‟ antibody from Alomone Labs prompted further exploration to understand why these antibodies generated less intense staining in the TAAR1-/- compared to the C57BL/6 intestines, despite apparent specificity when using NCBI blast against the antigenic determinant. A bioinformatics proteomics approach was implored to identify cross reactivity of the commercially available antibodies to other TAAR family members. Previous work from the Miller lab in 2010 (Vallender,
Xie, Westmoreland, & Miller, 2010) identified that there was a pattern of duplication and conservation across Taar family members, and the goal of this analysis was to determine the relationship of the TAAR protein homology, rather than genomic homology as described by
Vallender et al in 2010. Protein sequences for hTAAR1 and mTAAR1-9 were then downloaded from UniProt, a searchable database of curated NCBI sequences cross linked with NCBI accession numbers for validation. ClustalOmega open source software was used to attain multiple sequence alignments and the data was downloaded into a user-friendly viewer, Jalview
2.0 to process the sequences. A comparison of mTAAR sequences was performed, identifying conservation between mTAAR family members, with some areas of divergence. Published antibody sequences were attained from manufacturer‟s websites and used to determine antibody homology to other TAAR family members. Commercial antibodies against hTAAR1 sequences including Imgenex and Thermo Fisher PA1-41477 had almost 100% homology to mTAARs 2-9, accounting for the apparent non-specific binding observed in the TAAR1-/- tissue
(Figure 10). Despite the sequence of the Alomone Labs antibody appearing to be sequence- specific to mTAAR1, Zappo analysis of the sequence suggests that there is the potential for cross reactivity of the antibody as observed while staining the TAAR1-/- spleen. Interestingly, this same phenomenon is not seen in human TAAR family members, perhaps accounting for the ability to generate some specific antibodies to hTAAR1 (Table 3). It is notable that more
TAAR1 family members are expressed in mouse than in human (Lindemann et al, 2008).
D274 is a polyclonal antibody raised in rabbit, and was designed to target the third extracellular loop of the TAAR1 protein, matching the raw amino acid sequence 274-290 in
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human TAAR1. This antibody was custom manufactured by Rockland Immunochemicals
(Limerick, PA). Furthermore, the reactivity of this antibody to both hTAAR1 and mTAAR1 in transfected HEK cells was validated by immunostaining. The observed staining is consistent with previous reports of TAAR1 localization in cells (Sriram et al., 2016; Xie, Vallender, et al.,
2008). To validate the specificity of the D724 in mouse systems, BMDM from C57BL/6 and
TAAR1-/- animals were used and there was no non-specific staining in the TAAR1-/- mouse
BMDM. Cultured BMDM lack the specific tissue IgGs that may cross react with the antibody, reduces the need for antigen retrieval methods, and allows for normal blocking mechanisms.
Additional studies validating the D274 antibody, including stainings of mouse and human tissues, will be described in detail in Chapter 4.
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MATERIALS AND METHODS
Cell culture TAAR1-Transfected HEK Cells A stably transfected HEK293 cell line was used in this study. Previously, HEK293 cells were stably transfected with hTAAR1 (Panas et al., 2012). Geneticin (G418) was used for selection and maintenance of selection of stable cell lines. HEK293 cells were grown in DMEM supplemented with 10% FBS, 1% Anti/Anti and 1% non-essential amino acids.
Bone marrow derived macrophages Bone marrow-derived macrophages (BMDMs) were collected from both hind limbs of C57BL/6 as previously described (Zanoni, Ostuni, & Granucci, 2009). Briefly, mouse femur and tibia were removed from both hind limbs, cleaned of muscle, sterilized with 70% ethanol, and kept in
BMDM isolation media (RPMI + 10% FBS + 1% Anti/Anti (Thermo Fisher, Waltham,
Massachusetts) until isolation of cells. To isolate cells, each bone was briefly soaked in 70% ethanol, then flushed with PBS, and filtered through a 70μm cell strainer, followed by a 40μm strainer, and centrifuged at 1100 RPM for 5 minutes. Cell pellets were resuspended in conditioned media to stimulate differentiation into BMDMs, (RPMI + 10% L929-conditioned media + 1% Pen/Strep, (Thermo Fisher, Waltham Massachusetts) and allowed to differentiate for approximately 6 days. To polarize cells, 1 ng/ul lipopolysaccharide (LPS)(Sigma Aldrich,
Natick, MA) was added to culture media for 24 hours.
All cells were maintained in a 37°C incubator, with humidity and 5%CO2.
Immunohistochemistry
Tissues were fixed in Formalin and embedded in paraffin wax. Tissue blocks were cut to 5μM thickness and mounted on a glass slide. Sections were deparaffinized and permeabilized with
TBS+0.1% Triton-X100, blocked with 3% BSA+1.5%skim milk-TBS-0.01% tween for 1 hour at room temperature and incubated with primary antibodies as outlined in Table 2. Sections were washed with TBS-0.01% tween 4x5 minutes, 3 times, and incubated with an HRP-conjugated, goat anti-rabbit igg h-l (Catalog no. NC1517638, Thermo Fisher Scientific, Waltham MA) for 1 hour. Slides were washed with TBS-0.01% tween 4x5 minutes and were developed using Page 44
Abcam DAB Substrate Kit (Catalog no. ab64238, Abcam, Cambridge MA) according to manufacturer‟s instructions. Slides were treated with chromogen for 10 minutes, rinsed, counterstained with hematoxylin, dehydrated, and covered with Cytoseal-60 (Catalog no. 23-
244257, Thermo Fisher Scientific, Waltham, MA).
Immunostaining
Cells were fixed with 4% paraformaldehyde for 10 minutes at 4°C, and permeabilized in 0.1% triton-x100 in TBS for 15 minutes at room temp. Cells were blocked with 3% BSA+1.5% skim milk-TBS-0.01% tween for 1 hour at room temperature and incubated with antibodies outlined in
Table 2. Cells were washed 4x5 minutes in TBS-0.01% tween at room temperature (RT) and incubated with Alexafluor-647 (Thermo Fisher, Waltham, MA) for 45 minutes at RT. Cells were washed 4x5 minutes in TBS-0.01% tween at RT, dipped in Hoescht (1:10,000 in dH2O) briefly washed in TBS and cover slipped with Prolong Diamond Antifade Mountant (P36970, Thermo
Fisher, Waltham, MA). Individual image channels were captured on a Zeiss Axio Observer fluorescent microscope (Carl Zeiss) at 63x under oil, and individual channels were colorized with non-conventional colors – DAPI colored blue, Alexafluor-647 colored green – after grey- scale imaging of individual channels using ZenPro2 software.
Protein Sequence Analysis
TAAR family protein sequences were downloaded from UniProt (UniProt, 2019). Multiple sequences were aligned using the Clustal Omega software by percent identity, and viewed using Jalview 2.1 software (Waterhouse et al., 2009).
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Custom Antibody
D274 is a polyclonal antibody raised in rabbit, and was designed to target the third extracellular loop of the TAAR1 protein, matching the raw amino acid sequence 274-290 in human TAAR1.
This antibody was custom manufactured by Rockland Immunochemicals (Limerick, PA).
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Chapter Two: Expression, activation, and modulation of TAAR1 in intestinal epithelial cells (IECs)
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BACKGROUND
Gastrointestinal inflammatory disorders, including ulcerative colitis (UC) are plagued by chronic inflammation and degradation of the intestine epithelial cell (iEC) barrier. The iEC barrier is critical for protection of the delicate stem cells and the underlying mucosa of the intestine from lumen pathogens (Anderson & Van Itallie, 1995; Fujibe et al., 2004; Lee, 2015;
Van Itallie & Anderson, 2006). Proper maintenance and regulation of the tight junctions in epithelial cells in the GIT is important to prevent intestinal inflammation. Current efforts are attempting to understand the cause of the epithelial barrier defects in UC, and determine if the epithelial cells are being damaged by lumen contents, or inflammatory responses from the underlying immune cells.
In a healthy individual, the epithelial cell barrier if formed and dynamically regulated by a network of tight junction proteins (TJPs). These tight junction proteins consist of integral proteins such as Claudin proteins or Occludin that are anchored to the actin cytoskeleton of the cell by membrane associated proteins, including Zonula Occludens (ZO) proteins. It is established that the expression of specific TJP is altered in UC and variances in TJP expression results in altered absorption and transit over the epithelial barrier (Gunzel & Yu, 2013). In UC,
TJPs are variable in expression. For example, ZO-1 is stably expressed, while Occludin is markedly lower and Claudin-1 and Claudin 2 are increased (Landy et al., 2016). The regulation of TJP expression and localization is dynamic, and can be modulated in part by PKA and PKC phosphorylation of proteins (Figure 4). Disruption of the TJs in UC can result from a dephosphorylation of Claudin the translocation of proteins from the membrane to the cytoplasm whereas PKC dependent phosphorylation of claudin will result in trafficking to the membrane.
Conversely, phosphorylation of ZO-1 or Occludin by PKC or PKA, respectively, results in internalization of the proteins from the membrane disrupting their anchorage to the actin cytoskeleton and driving degradation of the monolayer.
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Previous work from the Miller lab has focused on Trace Amine Associated Receptor 1
(TAAR1), a G protein-coupled receptor that upon activation by trace amines can signal through both cAMP/PKA/CREB and Ca2+/PKC/NFAT pathways (Panas et al., 2012). Metabolomic studies have demonstrated elevated levels of fecal tyramine, a trace amine, in patients with active ulcerative colitis (Santoru et al., 2017). TYR occurs naturally in the body as a byproduct of tyrosine metabolism, and it is found in certain food products and produced by a variable subset of common microbiome organisms (Bonnin-Jusserand et al., 2012). Both “trigger” foods which are often rich in tyramine, and alterations to the microbiome are common drivers of UC flares (Burdychova & Komprda, 2007; Pozo-Bayon et al., 2012; Roig-Sagues, Hernandez-
Herrero, Lopez-Sabater, Rodriguez-Jerez, & Mora-Ventura, 1997). Indeed, the food industry measures TYR levels in food as a proxy for bacterial growth and an indicator of food spoilage
(Burdychova & Komprda, 2007; Roig-Sagues et al., 1997). TYR has been studied for greater than a century, though it was not until 2001 that a GPCR for TYR and other trace amines was identified (Borowsky et al., 2001; Bunzow et al., 2001). Since its identification, Trace Amine
Associated Receptor 1 (TAAR1) has mainly been studied for its roles in monoaminergic regulation in the brain, while few studies have investigated its role in the periphery (Babusyte et al., 2013; Ohta et al., 2017; Panas et al., 2012; Sriram et al., 2016). There have been no investigations on TAAR1 in the lower GIT, where TYR traverses with stool at higher than normal levels in UC (Santoru et al., 2017). It is unknown where TAAR1 is expressed; cell type, localization and function in the lower intestine are all unknown. TAAR1 as a potential mediator of TYR biological action in lower GIT remains completely unstudied.
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RESULTS
TAAR1 mRNA and protein is expressed in HT-29 cells.
Trace amines exist in the gastrointestinal epithelium as a byproduct of normal host and microbial metabolic pathways, as well as in high concentrations in certain food products.
Previous work has identified toxicity of the trace amine, tyramine, on intestinal epithelial cells ex vivo, though no work has been done to delineate the mechanism of its toxicity (Del Rio et al.,
2017; Linares et al., 2016). First, TAAR1 mRNA expression levels in intestine epithelial cell lines were assessed by searching the NCBI curated GEO datasets. Applicable data was downloaded and TAAR1 mRNA expression was observed in HT-29 colon epithelial cells, consistently at a range from 15% to 22.5% relative to GAPDH expression levels (GeoData
Accession Numbers GDS4511, GDS4397, GDS3330, GDS4700; Figure 15A). Next TAAR1 mRNA expression was experimentally confirmed in total RNA extracted from cultured HT-29 cells using RT-PCR (Figure 15B). Further, TAAR1 protein expression was confirmed in HT-29 cells. Finally, TAAR1 protein expression was confirmed by Western blot, and the 3 independent
HT-29 cell cultures are represented with GAPDH as a loading control (Figure 15C). Finally, to identify TAAR1 localization in vitro, cells were stained with the custom D274 antibody to visualize TAAR1 protein by immunofluorescence (Figure 15 D,E). In HT-29 cells stained with the D274 custom antibody TAAR1 is seen to be colocalized with the tight junction proteins ZO-1
(Figure 15D) and Occludin (Figure 15E). Accordingly, consistent expression of TAAR1 in HT-29 cells is observed on the gene transcript and protein level.
TAAR1 expression is upregulated by full agonist treatment in HT-29 cells, but not partial agonist treatment.
Previous studies (Sriram et al., 2016; Stavrou et al., 2018) have demonstrated that TAAR1 mRNA can undergo agonist mediated upregulation. To determine the effects of TAAR1 agonists on TAAR1 expression, the effects of the full agonist TYR (TYR) and the partial agonist
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RO5263397 (RO) were assessed in vitro. Initial studies comparing TAAR1 expression in HT-29 cells treated with 1μM TYR show a significant increase in TAAR1 gene expression compared to vehicle treatment alone (Figure 16A). Furthermore, in TYR treated cells, a significant 3-fold increase is seen in TAAR1 protein levels (normalized to GAPDH)(Figure 16B). To assess whether the TAAR1 partial agonist RO5263397 would cause upregulation of TAAR1, HT-29 cells were stimulated with 1μM TYR, or 470nM RO5263397 (EC50=47nM). Both mRNA and protein expression was monitored (Figure 16 C&D), demonstrating a significant increase in
TAAR1 mRNA expression in TYR-treated cells compared to vehicle, but no significant effect of
RO5263397 on TAAR1 expression in HT-29 cells compared to vehicle (Figure 16C). While TYR consistently induces a significant upregulation of TAAR1 protein expression compared to vehicle, there was no significant effect of RO5263397 on TAAR1 expression levels compared to vehicle (Figure 16D). Accordingly, the TAAR1 full agonist TYR caused upregulation of TAAR1 expression on the mRNA and protein levels in HT-29 cells, but the partial agonist RO5263397 did not.
ZO-1 gene expression in CACO-2 cells is mediated by TYR exposure, but TAAR1 expression is not.
TAAR1 expression in the iEC, CACO-2, was also assessed using available datasets from NCBI
GeoData. Data is represented as relative TAAR1 expression to GAPDH. In 6 of the 7 applicable datasets, TAAR1 mRNA was not detected (Figure 17A). To confirm the biased expression of TAAR1 in HT-29 cells, but not CACO-2 cells, RT-PCR and qRT-PCR analyses were performed on total RNA extracts from CACO-2 cultures. Despite the apparent null detection in NCBI Geo datasets, TAAR1 mRNA is detectable in CACO-2 cells despite not being upregulated with TYR treatment (Figure 17B). TAAR1 protein expression in CACO-2 cells was determined by immunostaining with the D274 antibody, demonstrating membrane localization in some CACO-2 cell cultures (Figure 17C). Finally, ZO-1 mRNA levels were analyzed in CACO-2
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cells with vehicle and TYR stimulation. TYR treatment of CACO-2 cells produces significant upregulation of ZO-1 mRNA, compared to vehicle (Figure 17D).
Tight junction protein gene expression and protein levels are modified by TYR in
HT-29 cells
Current efforts in understanding the pathogenesis of inflammatory disorders of the GIT are focused on controlling the inflammatory cascades triggered by intestinal immune cells. These intestinal immune cells are activated by substances that pass through a degraded epithelial barrier, though no treatments are directly focused on preventing the breakdown of the epithelial barrier in the gut. Owing to previous metabolomic studies identifying TYR in the stool of patients with UC, expression of tight junction protein (Claudin-1, ZO-1, and Occludin) gene transcripts in response to TYR treatment were monitored using qRT-PCR. In HT-29 cells treated with TYR there is a significant increase in gene expression for Claudin-1 and ZO-1 gene expression (Figure 18A, B). Conversely, TYR treatment resulted in a significant decrease in
Occludin gene expression in HT-29 cells treated with TYR compared to vehicle (Figure 18C).
TJP levels were monitored by Western blot in HT-29 cells treated with vehicle and TYR. Here,
Claudin-1 protein levels are significantly decreased in HT-29 cells (Figure 18D), though there is an increase in both ZO-1 (Figure 18E) and Occludin (Figure 18F) protein levels in response to
TYR treatment.
Increased inter-epithelial cellular distance and increase in permeability in response to TYR in HT-29 cells
Altered expression of tight junction proteins often leads to a dysregulated epithelial barrier. To determine the effects of TYR treatment on the epithelial barrier, monolayer health was assessed in differentiated monolayers by immunofluorescence by quantifying the average inter-epithelial cellular distance. A distance of „0‟ was used for cellular membranes that are touching, and the
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raw distance between cellular membranes that were not touching was measured, as previously described (Barry, Wang, & Leckband, 2015)(Figure 19B). TYR stimulation resulted in a significant increase in the inter-epithelial gap distance between cells (Figure 19A,B). In addition to the measured distance between cells, the number of cellular membranes that were in contact with neighboring cells in vehicle-treated cells compared to TYR was quantified. There is a significant increase in the number of cells in the monolayer that are separated when exposed to
TYR compared to vehicle-treated monolayers (Figure 19B). Finally, to demonstrate that this increase in inter-epithelial cell distance was transferrable to a functional defect in the monolayers HT-29 cells were grown to monolayers on transwells and FITC-dextran flux across the transwell membrane was quantified. The schematic representation of this assay is described in Figure 19C. TYR-treated monolayers display a non-significant increase in FITC- dextran flux across the transwell membrane compared to vehicle (Figure 19D).
Inhibition of PKA and PKC pathways blocks the effects of TYR in HT-29 cells.
Currently the only known TAAR1 antagonist is mouse specific (Table 1)(Bradaia et al., 2009).
To circumvent the use of the absence of an hTAAR1 antagonist, specific PKA and PKC pathway inhibitors were used to characterize TYR effects on TAAR1 pathways. Here, HT29 cells were treated with vehicle or TYR, pretreated with RO32-0432 (PKCi), a PKC inhibitor or H-
89, a PKA pathway inhibitor, or pretreated with PKCi and treated with TYR (Figure 20 A&C) or pretreated with H-89 or pretreated with H-89 and treated with TYR (Figure 20 B&D). Inhibition of PKC activation had no significant effect on TYR mediated upregulation of TAAR1 mRNA
(Figure 20A), though PKA inhibition did significantly decreased TYR mediated upregulation of
TAAR1 mRNA (Figure 20B). Conversely, PKC inhibition had a significant effect on TYR mediated ZO-1 gene expression (Figure 20C), while H-89 had no significant effect on ZO-1 gene expression (Figure 20D).
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Effects of TAAR1 partial agonists RO5263397 on HT-29 cells To determine the therapeutic potential of RO5263397 in mediating epithelial monolayer health and integrity, Claudin-1, Occludin, and ZO-1 gene expression was monitored by qRT-PCR. As previously seen, TYR produced a significant increase in claudin-1 gene expression compared to vehicle, as did RO5263397 (RO), (Figure 21A). RO5263397 had no significant effect on occludin gene expression compared to TYR or vehicle (Figure 21B), though it did cause a significant upregulation of ZO-1 gene expression compared to baseline (Figure 21C).
Immunofluorescent analysis of epithelial cell monolayer health showed that HT-29 cells treated with RO5263397 experienced a significant increase in the number of touching cells (Figure 21
D-E). Finally, RO5263397 caused a significant increase in FITC dextran flux across an HT-29 monolayer, an effect that was significantly decreased with co-stimulation of RO5263397 and
TYR (Figure 21F).
Metabolites from cultured cheese and probiotics elicit a response in HEK-TAAR1- Cre-Luciferase reporter cells Given the abundance of Lactobacillus bacteria in cheese and in probiotics, determining the effects that microbial metabolites have on TAAR1 activation was investigated to provide confirmation that bacterial products can directly activated TAAR1. To investigate the intracellular signaling pathways triggered by commonly consumed biogenic amine-forming bacteria, cultures of two probiotics, VSL#3 and DigestiviT, and two cheese products, parmesan cheese and goat cheese were screened by use of stably expressing hTAAR1 cells transduced with Cre-Luc Cignal lenti reporter (Qiagen). Microbial byproducts activation of Cre-Luc reporter cells was compared to the known TAAR1 agonist TYR and partial agonist RO5263397. As previously reported, TYR acts as a full agonist, and RO5263397 acts as a partial agonist for cAMP/PKA/CREB responsivity (Figure 22A). There was a significant increase in luminescence of parmesan and goat cheese culture extracts compared to vehicle treatment (Figure 22B).
Similarly, culture extracts derived from both probiotics tested, digestiviT and VSL#3 elicited a significant increase in luminescence compared to vehicle treatment (Figure 22C).
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FIGURES AND TABLES
Figure 15. TAAR1 mRNA and protein are expressed in HT-29 cells. A. Geodata analysis of TAAR1 mRNA expression in HT-29 control samples from 4 independent data sets, normalized to levels of GAPDH mRNA. TAAR1 mRNA expression relative to GAPDH mRNA expression ranges from 15% to 22.5%. B. RT-PCR analysis of TAAR1 mRNA expression in HT-29 cells, compared to GAPDH mRNA expression in HT-29 cells. TAAR1 mRNA was detected in 4 independent HT-29 cultures, with GAPDH as a house keeping gene. No presence of genomic DNA contamination in RNA samples was observed utilizing gDNA detection primers. C. Representative Western blot analysis of TAAR1 protein in HT-29 cells compared to GAPDH. D. Immunofluorescence staining of TAAR1 using D274 antibody co-stained with Occludin antibody. TAAR1 staining is primarily localized to the cytoplasm, with few instances of TAAR1 protein expression on the membrane. E. Immunofluorescence staining of TAAR1 co-stained with ZO-1 antibody, demonstrating intracellular TAAR1 staining and membranous staining of TAAR1.
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Figure 16. TAAR1 upregulation in HT-29 cells by full agonist TYR, but not partial agonist RO5263397. A. QRT-PCR analysis of TAAR1 in HT-29 cells that were treated with vehicle and TYR. Treatment with TYR produced a significant increase in TAAR1 gene expression compared to vehicle (n=9; p<0.001, Student‟s t-test.) B. Western blot analysis of TAAR1 expression in HT-29 cells, with vehicle and TYR. Treatment with TYR caused a significant increase in TAAR1 protein expression compared to vehicle treated cells (n=9; p<0.01; Students t-test). C. Analysis of TAAR1 gene expression in HT-29 cells treated with TYR, or the partial TAAR1 agonist, RO. TYR produced a significant increase in TAAR1 gene expression compared to vehicle, whereas RO did not elicit a significant increase in compared to vehicle. (n=3; p<0.01, One-Way ANOVA with Bonferroni correction). D. Quantification of relative protein expression in HT-29 cells treated with TYR and RO. TYR produced a significant increase in TAAR1 protein expression compared to vehicle treatment. Treatment with RO5263397 had no effect on TAAR1 protein expression in HT-29 cells. (n=3, *=p<0.05, One Way ANOVA with Bonferroni correction).
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Figure 17. Expression and regulation of gene expression in CACO-2 cells by TYR. A. Geodata analysis of TAAR1 expression in CACO-2 control samples from 7 independent data sets, normalized to GAPDH. TAAR1 gene expression relative to GAPDH gene expression ranges from 0% to 1.9%. B. qRT-PCR quantification of TAAR1 gene expression in cultured CACO-2 cells treated with TYR or vehicle. (n=3; *=p<0.05, **=p<0.01, ***=p<0.001; Student‟s t-test). C. Immunostain with of TAAR1 protein expression in CACO-2 cells, to confirm baseline expression of TAAR1 in on the protein level. D. qRT-PCR analysis of TAAR1 gene expression in cultured CACO-2 cells treated with TYR demonstrates that ZO-1 mRNA is significantly upregulated upon treatment with TYR (n=3, p<0.01, Student‟s t-test).
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Figure 18. TYR alters expression of tight junction proteins in HT-29 cells. QRT-PCR and protein quantification in HT-29 cells treated with vehicle or TYR. A-C. qRT-PCR quantification of Claudin-1, ZO-1 and Occludin gene expression in HT-29 cells treated with vehicle or TYR. A-B. Treatment with TYR elicited a significant increase in Claudin-1 and ZO-1 gene expression compared to vehicle(n=9; *=p<0.05, **=p<0.01, ***=p<0.001; Student‟s t-test). C. TYR treatment resulted in a significant decrease in Occludin expression compared to vehicle treated cells. (n=9; *=p<0.05, **=p<0.01, ***=p<0.001; Student‟s t-test). D-F. Western blot quantification of Claudin-1, ZO-1, and Occludin gene expression in HT-29 cells treated with vehicle or TYR. D. TYR resulted in a significant decrease in Claudin-1 protein expression compared to vehicle n=9; *=p<0.05, **=p<0.01, ***=p<0.001; Student‟s t-test). E-F. TYR treatment resulted in a significant increase in both ZO-1 and Occludin protein expression in HT-29 cells compared to vehicle. (n=9; *=p<0.05, **=p<0.01, ***=p<0.001; Student‟s t-test).
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Figure 19. TYR can increase inter-epithelial cellular distance and mediate permeability in HT-29 cells. A. Immunofluorescent staining of ZO-1 (red) in HT-29 monolayers treated with vehicle or TYR. B. Quantification of inter-epithelial gap distance between neighboring HT-29 cells. Treatment with TYR significantly increases the presence of and distance in gaps in confluent HT-29 monolayers compared to vehicle treatment alone (n=3; *=p<0.05, **=p<0.01, ***=p<0.001; Student‟s t-test). C. Schematic representation of transwell permeability assay using FITC-Dextran. D. Quantification of FITC-dextran flux to the basolateral chamber in the transwells over time. Treatment with TYR yields a non-significant increase in flux of FITC-dextran compared to vehicle treatment (n=3; *=p<0.05, **=p<0.01, ***=p<0.001; Student‟s t-test).
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Figure 20. PKC inhibitors block TYR mediated upregulation of ZO-1 mRNA whereas PKA inhibitors block TYR mediated upregulation of TAAR1 HT29 cells were treated with vehicle, TYR, pretreated with RO32-0432 (PKCi), or pretreated with PKCi and treated with TYR (A&C) or pretreated with H-89 or pretreated with H-89 and treated with TYR (B&D). A. PKC inhibition had no significant effect on TYR mediated upregulation of TAAR1 mRNA (n=3, OneWay ANOVA), though B. Inhibition of PKA significantly decreased TYR mediated upregulation of TAAR1 mRNA (p<0.01, n=3, One Way ANOVA). C. Inhibition of PKC activation had a significant effect on TYR mediated ZO-1 gene expression (p<0.01), while D. H-89 had no significant effect on ZO-1 gene expression.
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Figure 21. Effects of RO5263397 on TJP mRNA expression, inter-epithelial cellular distance and permeability in HT-29 cells. A. RO5263397 (RO) significantly upregulates Claudin-1 and B. ZO-1 compared to vehicle, and has no effect on C. Occludin gene expression (n=3 *=p<0.05, **=p<0.01, ***=p<0.001, One Way ANOVA with Bonferroni Correction). D. Measure of inter-epithelial cellular distance as quantified by ZO-1 (red) membrane staining. RO significantly alters the inter-epithelial cellular distance compared to both TYR and vehicle (n=3, χ2- test, p<0.001). E. Treatment of HT-29 monolayers with RO compound compared to vehicle, and RO+TYR treatment. RO significantly increases the flux of dextran across the membrane, compared to RO+TYR treatment and vehicle (n=3, One Way Anova with Bonferroni correction).
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Figure 22. Bacteria culture supernatants from common probiotics and cheeses elicit a cAMP/CRE response in HEK-TAAR1 cells. Raw RLU increase in TAAR1-HEK-CRE-LUC cells after stimulation with A. vehicle,TYR, RO5263397 (RO) or sonicated bacterial supernatants from cultured B. parmesan and goat cheese, or C. commercially available probiotics – digestiviT or VSL#3. A. TYR significantly increases in luminescence compared to vehicle, and RO produces a non-significant increase in luminescence compared to vehicle, that is significantly less than TYR responsivity. B. Bacterial supernatants form parmesan and goat cheese both caused a significant increase in relative luminescence compared to vehicle (p<0.01 and p<0.001, OneWay ANOVA, n=3 samples of HEK-CRE cells). C. Both digestiviT and VSL#3 elicited a significant increase in luminescence compared to vehicle (p<0.01 and p<0.001, OneWay ANOVA, n=3 samples of HEK-CRE cells).
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DISCUSSION
Although research is highly limited on TAAR1 in the periphery, emerging studies have suggested a role of TAAR1 in immune cells (Babusyte et al., 2013; Panas et al., 2012) and in stomach motility (Broadley et al., 2009). To address the role that TAAR1 has in the GIT, TAAR1 expression, upregulation, and TAAR1 signaling pathways were monitored in response to
TAAR1 ligands in HT-29 cells. TAAR1 mRNA and protein expression were validated in both
CACO-2 and HT-29 cells, though it was only HT-29 cells that demonstrated an increase in
TAAR1 expression after stimulation by TYR. TAAR1-agonist induced upregulation is a phenomenon that has previously described in the literature (Sriram et al., 2016; Stavrou et al.,
2018). TYR is officially classified as a trace amine as it is found at picomolar levels in the brain, but emerging metabolomic studies have identified concentrations of TYR in the GIT, nearing millimolar concentrations in some instances. In this regard, TYR levels have been identified in higher levels in the fecal content of patients with UC, compared to healthy controls (Santoru et al., 2017), suggesting that any endogenous TAAR1 may be upregulated in the colonocytes, where TYR is not absorbed (Coyle & Boyd, 1932). TAAR1 upregulation appears to be attenuated by inhibition of PKA pathway activation, and inhibition of PKC pathway activation yields no effect. In the same regard, the partial agonist RO5263397 failed to increase TAAR1 protein expression, but elicited a partial response in the HEK-TAAR1-Cre reporter cells. Taken together, these data implicate TAAR1 activation of the cAMP/PKA/CREB pathway in the upregulation of TAAR1 protein. Given the expression of TAAR1 in the HT-29 cell line and the correlation of elevated TYR in metabolomic studies, TJP mRNA and protein expression were monitored in differentiated HT-29 cell monolayers. Somewhat consistent with observed variances in human disease, TYR causes altered expression of TJP mRNA and protein expression. There is a consistent observation that TYR causes an increase in ZO-1 mRNA and protein expression, whereas mRNA and protein expression of claudin and occludin are contradictory. In HT-29 cells treated with TYR there is increased claudin-1 mRNA expression with a decrease in Claudin-1 protein expression while the same treatment results in decreased
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occludin mRNA and increased occludin protein expression. The discordance between claudin-1 and occludin mRNA and protein expression levels is somewhat surprising, but may be due to the subtle difference in the formation of differentiated monolayers. Determination of
„differentiated‟ HT-29 cells in vitro depends on galactose rich media, monolayer formation, and the human eye to determine that the phenotype of cells in a culture dish all possess the same phenotype. This experimental variance may account for the differences that are observed in protein levels and mRNA levels in these TJPs. Another limitation in using the HT-29 cell line is that the cells are malignant adenocarcinoma cells, not „healthy‟ epithelial cells. Despite having a differentiated phenotype structurally and presence of villi that is similar to primary colonocytes,
HT-29 cells have an impaired glucose metabolism, and cannot fully recapitulate the phenotype of colonocytes as they express hydrolases typically found in the small intestine (Rao & Wang,
2010). Metabolic or phenotypic variance may account for the altered gene expression and protein expression seen when monitoring the tight junction proteins occludin and claudin.
Lastly, there are many examples in biology of changes in levels of mRNA expression that are not correlated to coordinated changes in protein expression, and vice versa, as a gene's mRNA level does not usually predict its protein level (Lundberg et al., 2010).
Much of the literature is conflicting on the exact pathology of tight-junction protein up- and down-regulation, but altered expression of TJPs tends to be of pathological significance. In
UC, symptomology can be augmented by a functional defect in the epithelial monolayer. Here, the health and integrity of in vitro monolayers was monitored in response to TYR. Inter- epithelial gap distance was monitored in the HT-29 cells by assessing ZO-1 staining in cells.
Analysis of 12 images per treatment group revealed a significant increase in the average interepithelial gap distance in response to the TAAR1 agonist TYR and partial agonist
RO5263397. To validate this observation, the functional permeability using FITC-dextran flux across a transwell membrane was also assessed. TYR appears to cause an increase in FITC- dextran flux across the membrane compared to vehicle, as does RO5263397, however this trend did not achieve significance. The lack of significance between treatment groups could be
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due to the low sample size (n=3), and a power analysis of the means suggests that increasing the sample size will increase the experimental validity for this readout.
Finally, given the prevalence of biogenic amine-producing bacteria that transit the GIT and from the human microbiome, there is a possibility for these metabolites to elicit a response from TAAR1. Two commercially available probiotic strains as well as two generic cheese types were cultured, and metabolites from these cultures were isolated and analyzed for their potential to elicit a response from hTAAR1. Both the probiotic supplements and the dietary components significantly increased the activity of TAAR1 compared to vehicle, similarly to the responsivity of
TYR. Taken together, these data suggest that TAAR1 activation is a mechanism for microbiomic constituents to promote cellular signaling. This presents a new avenue for developing pharmacological agents for GIT disorders, and potential off-label use of TAAR1- targeted drugs in clinical trials by both Hoffman La Roche and Sunovion Pharmaceuticals.
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MATERIALS AND METHODS
NCBI Data Analysis
The NCBI Gene Expression Omnibus (GEO) includes the GEO profiles database that curates gene expression profiles from GEO Datasets searchable by keywords (T. Barrett et al., 2013).
Datasets were searched for “HT-29” and “CACO-2” to find applicable datasets for intestinal epithelial cells. Each dataset was searched using the official gene symbol “TAAR1” and
“GAPDH.” All datasets with untreated HT-29 and CACO-2 samples were exported, and TAAR1 expression was normalized to GAPDH expression.
Cell Culture
HT-29 Cells were purchased from ATCC, and cultured in McCoy‟s 5A media with 10% FBS and
1% Anti/Anti (Thermo Fisher). Monolayers are formed by culturing in low-glucose DMEM supplemented with galactose. Monolayers were formed after approximately two weeks in culture. CACO-2 cells were purchased from ATCC and cultured in DMEM supplemented with
10% FBS and 1%Anti/Anti (Thermo Fisher). Cells were briefly grown to confluency and used for downstream applications. All cells were maintained at 37°C in a humidified incubator with 5%
CO2 and were fed every 2 days.
TAAR1-HEK-Cre-Luciferase Reporter Cells HEK293 cells stably transfected with human TAAR1 and then transduced with a Lenti-Cre-
Luciferase reporter construct (Qiagen) was utilized in this study. Geneticin (G418) and/or puromycin were used for selection and maintenance of selection of stable cell lines. HEK293 cells were grown in DMEM supplemented with 10% FBS, 1% Anti/Anti and 1% non-essential amino acids.
Bacterial Culture Two cheeses, parmesan and goat cheese, generic brand, (Whole Foods Market, Boston, MA) and commercially available probiotics representing commonly used organisms in probiotic therapeutics were obtained, and capsules were broken open and cultured in Luria Broth (LB) at
37°C with continuous agitation, for 4 days. Bacterial suspensions were centrifuged at 5,000 x g Page 66
for 10 minutes at 4°C. Supernatants were harvested, heat inactivated at 95°C for 5 minutes, and sonicated for 20 minutes in a water bath sonicator to cleave any proteins that remained in suspension. Sonicates were then centrifuged at 12,000RPM for 10 minutes at 4°C to pellet debris. From here, 10μl of supernatant was added to HEK-TAAR1-Cre-Luc cells and monitored for cAMP activation.
Drug Treatments
TYR (Sigma Aldrich, Natick, MA) was dissolved in PBS to make a stock concentration of 100
μM. Stock solution was diluted in cell culture media to a final concentration of 1 μM for all treatments. Roche compound: RO5263397, “(S)-4-(3-Fluoro-2-methyl-phenyl)-4,5-dihydro- oxazol-2-ylamine” (RO) was dissolved in ethanol to a final concentration of 861μM and added to cells at a final concentration of 470 nM .
RNA Extraction, PCR and qRT-PCR
Cell pellets were resuspended in 1ml of Trizol reagent, thoroughly mixed, and incubated for 5 minutes at 4°C. Following, 200ul of chloroform was added and samples were shaken vigorously, incubated for 5 minutes at room temperature, then spun for 5 minutes, at 4°C, at
12,000RPM. The aqueous layer of the sample was collected and added to 1ml of ice cold isopropanol. Samples were placed at -20°C for 1 hour and spun for 15 minutes at 4°C at
12,000RPM. The supernatant was removed, and the pellet was washed with 70% ethanol made with RNase free water. Samples were spun again at 5 minutes, at 4°C, at 12,000RPM.
The pellet was washed a second time with 70% ethanol and spun at 7500RPM for 5 minutes at
4°C. RNA pellets were allowed to air dry for 45 minutes and resuspended in RNase free water.
RNA concentration was calculated using a nanodrop spectrophotometer and the 260/280 ratios were determined for each samples. For RT-PCR, samples with 260/280 ratios near 2.0 were converted to cDNA using the Qiagen RT-PCR Kit (Item No. 205313; Qiagen, Germantown MD), according to the manufacturer‟s instructions. Upon conversion to cDNA, samples were diluted
1:1 with nuclease free water, and quantitative real-time PCR (qRT-PCR) reactions were Page 67
performed using SYBR Green Master Mix (Item No. 436765, Thermo Fisher, Waltham, MA) and run on a Stratagene Mx3005p (Agilent, Santa Clara, CA) Fold gene expression was determined using the ∆∆CT method and normalized to vehicle treatment. Primers outlined in
Table 4 were used for qRT-PCR reactions.
Table 4. Human Primers for qRT-PCR Gene Species Application Sequence GAPDH Human q/PCR* 5‟- ACCACAGTCCACGCCATCAC -3‟ 5‟- TCCACCACCCTGTTGCTGTA -3‟ TAAR1 Human q/PCR 5‟- CTTCTGGGGTGTCTGGTCAT -3‟ 5‟- AACAGCAGGGACACTCCAAC -3‟ Zona Occludens -1 Human q/PCR 5‟-TGCCATTACACGGTCCTCTG -3‟ 5‟-GGTTCTGCCTCATCATTTCCTC -3‟ Occludin Human q/PCR 5‟-GTCCAATATTTTGTGGGACAAGG -3‟ 5‟-GGCACGTCCTGTGTGCCT -3‟ Claudin -1 Human q/PCR 5‟-ttcgtacctggcattgactgg-3‟ 5‟- ttcgtacctggcattgactgg -3‟ *q/PCR indicates standard PCR and qPCR platform
Immunostaining
Cells were fixed with 4% paraformaldehyde for 10 minutes at 4°C, and permeabilized in 0.1% triton-x100 in TBS for 15 minutes at room temp. Cells were blocked with 3% BSA+1.5%skim milk-TBS-0.01% tween for 1 hour at room temperature and incubated with an anti-TAAR1 antibody (D274) overnight at 4°C. Cells were washed 4x5minutes in TBS-0.01%tween at RT and incubated with Alexafluor-647 (Thermo Fisher, Waltham, MA) for 45 minutes at RT. Cells were washed 4x5minutes in TBS-0.01%tween at RT, dipped in Hoechst (1:10,000 in dH2O) briefly washed in TBS and cover slipped with Prolong Diamond Anti-fade Mountant (P36970,
Thermo Fisher, Waltham, MA). Individual image channels were captured on a Zeiss Axio
Observer fluorescent microscope (Carl Zeiss) at 63x under oil, and individual channels were colorized with non-conventional colors – DAPI colored blue, Alexafluor-647 colored green – after grey-scale imaging of individual channels using ZenPro2 software. Antibodies for experiments are outlined in Table 5.
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Table 5. Antibodies and Dilutions for Chapter 2 experiments Protein Antibody Species Reactivity Dilution Occludin Invitrogen™ Alexa Fluor® 488, Clone: OC-3F10 Human, Mouse IF: 1:500 Claudin-1 Invitrogen: 37-4900 Human, Mouse Western: 1:1000; IF: 1:500 TAAR1 Imgenex Human Western: 1:1000 D274 Rockland Immunochemicals Human, Mouse IF: 1:500 GAPDH Thermo Fisher: MA515738HRP Human, Mouse Western: 1:2000 DAPI/Hoechst Thermo Fisher: 62249 Human, Mouse IF: 1:10,000
Western Blotting
Protein was isolated from both HT-29 cells and U937 cells by Triton-X Lysis buffer with Halt
Protease/Phosphatase inhibitor cocktail. Briefly, adherent HT-29 cells were lysed directly on the culture dish with 1ml of Lysis buffer. The suspension cell line U937 were centrifuged at 1100
RPM for 8 minutes at 4°C, and pellets were directly lysed with lysis buffer. For both cell types, lysates were sonicated for 5 minutes in a water bath sonicator, spun at 12,000 RPM to pellet debris, and the supernatant was collected. A BCA Assay using the Pierce BCA Assay Kit was performed to calculate the protein concentration in each sample. For western blot, 20μg of protein lysate with 4X Reducing LDS were heated to 75°C for 7 minutes, cooled to 4°C for 2 minutes, and spun again at 12,000 RMP to pellet extraneous debris. The supernatant was loaded onto a 4-12% Nupage Bis-Tris Gel (Item no, NP0322, Invitrogen) and run for 10 minutes at 80V, 15 minutes at 120V and up to 45 minutes at 160V. Protein was transferred to a 0.2 μM
PVDF membrane blocked for 1 hour at room temperature in 5% BSA or non-fat dry milk.
Primary antibodies were diluted to varying concentrations, outlined in Table 5. Membranes were washed 4x5 minutes each with TBS-0.1% Tween at RT, hybridized with HRP-Conjugated secondary antibodies for 1 hour at RT, and washed again 4x5 minutes in TBS-0.1% Tween.
Detection of proteins was determined using Pierce™ ECL Western Blotting Substrate, and blots were exposed on a Biorad Imager. To ensure equal loading of protein blots were stripped using
Restore Western Reagent and re-probed with GAPDH (Item no. G9295, Sigma, Natik, MA)
Antibodies and dilutions for experiments are outlined in Table 5.
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Inter-epithelial Distance
Inter-epithelial gap distance was monitored in ZO-1 stained HT-29 monolayers in response to vehicle, TYR and RO526339 treatments. Protocols were slightly modified from previously published methodologies (Barry et al., 2015). Z-stacks were captured for HT-29 monolayers
(0.5μm consecutive stacks, 10 slices minimum) and processed to one concerted image using
ZenPro2 software, or Photoshop FileStack Script (Lo Celso et al., 2009). Compressed Z-stacks were opened with FIJI/Object J and analyzed. Cells that were sharing a membrane were counted as a distance of 0μM, and cells that were not sharing any membrane features with the adjoining cell were measured and calibrated to the scale bar in the image. A minimum of 700 cells were counted from images captured on n=3 monolayer of N=4 images per group. Data is represented as mean ±SEM.
Transwell FITC-Dextran Flux
Corning costar 6.5mm diameter and 5μM pore transwells (Item no 3421Thermo Fisher,
Waltham, MA) were coated with Rat tail collagen-I (Gibco Item no. A1048301, Thermo Fisher,
Waltham, MA). Collagen was purchased as a 3mg/ml suspension, and diluted to a working concentration of 50μg/ml by adding 7.8ul of stock in 600μl sterile water. The apical side of the transwell was coated with 50μl of the working concentration, and incubated for 2 hours at 37°C.
The remaining liquid in each transwell was aspirated from the apical chamber, and allowed to dry under the tissue culture hood. Media was added to the apical and basolateral compartments and incubated at 37°C for 1 hour prior to seeding of cells. Cells were seeded at a density of 5x105 cells/transwell, and allowed to polarize for 8 days prior to treatment. Cells were then stimulated with 1μM tyramine, RO526339, and vehicle for 24 hours prior to FITC- dextran assay. Flux of FITC-dextran 40kDa (Item no. 501526773, Thermo Fisher, Waltham,
MA) was monitored as previously described (Puzan, Hosic, Ghio, & Koppes, 2018), for 20 minutes and compared to standard curve on a Perkin Elmer EnSight Plate reader.
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Chapter 3: TAAR1 activation modulates inflammatory cytokine production in BMDM
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BACKGROUND
Ulcerative colitis (UC) is a chronic relapsing inflammatory condition of the gastrointestinal tract (GIT) thought to be triggered by a dysregulated immune response in genetically susceptible patients to commensal microbiomic bacteria and certain dietary triggers
(Matricon, Barnich, & Ardid, 2010). Recent metabolomic studies in human patients with UC seeking to understand the metabolomic pathways of the microbiome involved in the propagation of UC found elevated tyramine (TYR) levels in patients with UC, compared to controls (Santoru et al., 2017). TYR occurs naturally in the body as a byproduct of tyrosine metabolism, is found in certain food products and is produced by a variable subset of common microbiome organisms
(Bonnin-Jusserand et al., 2012). Bacteria common to the human microbiome possess tyrosine decarboxylase enzymatic systems that are capable of synthesizing TYR from tyrosine (van
Kessel et al., 2019). Tyramine has been studied for greater than a century, though it was not until 2001 that a receptor for tyramine and other trace amines was identified (Borowsky et al.,
2001; Bunzow et al., 2001). Since its identification, Trace Amine Associated Receptor 1
(TAAR1) has mainly been studied in the brain, while few studies have investigated its role in the immune cells (Babusyte et al., 2013; Panas et al., 2012; Raab et al., 2016; Sriram et al., 2016;
Sriram, Haldar, Cenna, Gofman, & Potula, 2015). There have been no investigations on
TAAR1‟s role in the lower GIT, where tyramine traverses with stool at higher than normal levels in UC in human patients and mouse models of colitis (Robinson et al., 2016; Santoru et al.,
2017). The sources of tyramine in the mouse GIT were found to be from both dietary sources and components of the host microbiome (Carpene, Schaak, Guilbeau-Frugier, Mercader, &
Mialet-Perez, 2016; Matsumoto et al., 2012).
In the GIT, resident macrophages are the „first responders‟ recruiting peripherally circulating blood cells to aid in the clearance of pathogens and potentiation of inflammation.
Macrophages in the gastrointestinal tract are continuously integrating microbiomic, pathogenic, and dietary stimuli to maintain health of the epithelial barrier in the colon. In response to
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environmental stimuli, macrophages secrete cytokines to promote inflammation, e.g. IL-6, IL-1β, and TNF-α, which are key regulators in driving the recruitment of the innate and adaptive immune responses in UC (Rugtveit, Nilsen, et al., 1997; Stevens et al., 1992). Recent reports investigating the role of TAAR1 in immune cell function demonstrate that TAAR1 mRNA is detected in up to 80% of monocytes, and a recent meta-analysis from our lab reported that
TAAR1 expression was highest in macrophages, among other cells (Fleischer, Somaiya, &
Miller, 2018). Furthermore, TAAR1 has been previously implicated in altering cytokine release from B-cells and T-cells (Babusyte et al., 2013) in response to trace amines, presumably through the activation of intracellular cAMP/PKA and Ca2+/PKC signaling cascades which are known to be activated downstream of TAAR1 activation (Panas et al., 2012). PKA and PKC pathways are known to have immunomodulatory effects and alter cytokine release (Foey &
Brennan, 2004; D. Kim et al., 2016; Kontny et al., 1999). Currently, it is unclear whether or not macrophage function is modulated by TAAR1 activation.
The present study aimed to demonstrate TAAR1 expression in non-polarized and LPS- polarized bone marrow-derived macrophages (BMDM) derived from mice, assess its function and its potential to serve as a novel therapeutic target for the treatment of gastrointestinal disorders. TAAR1 expression in BMDM was confirmed and found to mediate effects of TYR on macrophage function. Activation of TAAR1 by TYR upregulated both TAAR1 expression as well as proinflammatory cytokine gene expression in non-polarized and LPS-polarized BMDM, suggesting a feed-forward mechanism by which TYR could drive elevations in proinflammatory cytokine secretion by macrophages in the GIT. Finally the mouse specific TAAR1 antagonist was explored to demonstrate that TYR is acting on TAAR1 to modulate alterations in BMDM cytokine expression. Establishing TAAR1 as a mediator of the innate immune response reveals a new therapeutic target for treating UC.
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RESULTS
TAAR1 is expressed in mouse BMDM and can be upregulated by TYR.
In response to pathogenic signals transgressing the epithelial barrier tissue resident macrophages become activated driving inflammation and recruit peripheral immune cells which further infiltrate the epithelial barrier in the GIT. Previous work has identified TAAR1 expression in mixed populations of immune cells but no studies to date have identified TAAR1 in mouse
BMDM. To determine TAAR1 expression patterns in BMDM mouse bone marrow stem cells were differentiated to BMDM and monitored TAAR1 gene expression in non-polarized and LPS- polarized BMDM. Using qRT-PCR detectable levels of TAAR1 were identified in both non- polarized and LPS-polarized BMDM (Figure 23). Furthermore, LPS-polarized BMDM treated with TYR showed significant upregulation of TAAR1 gene expression whereas TYR or LPS- treatment alone did not significantly augment TAAR1 expression in non-polarized BMDM.
Accordingly, these data suggest that mouse BMDM express TAAR1 and polarized BMDM upregulate TAAR1 expression in the presence of TYR (Figure 23).
Additive inflammatory response in polarized BMDMs in response to LPS and TYR.
Proinflammatory cytokines are implicated in driving inflammation and promoting immune cell infiltration in the epithelial barrier of the GIT. Inflammatory cytokine production in macrophages can be stimulated by PKA and PKC activation, both of which are activated by TYR activation of
TAAR1. Owing to previous metabolomic studies identifying elevated TYR in the stool of patients with UC, gene transcripts for inflammatory cytokines (IL-6, IL-1β, TNFα) as well as the inducible nitric oxide synthase (iNOS) enzyme were quantified by qRT-PCR in non-polarized
BMDM, non-polarized BMDM treated with TYR, LPS-polarized BMDM or LPS-polarized BMDM treated with TYR (Figure 24). Gene transcripts for IL-6, IL1-, TNF and iNOS gene expression were confirmed in all treatment groups of BMDM. LPS-polarized BMDM treated with TYR
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showed significant upregulation of IL-6 gene expression compared to vehicle treatment in non- polarized BMDM, TYR treatment in non-polarized BMDM, or untreated polarized BMDM (Figure
24A). Significant upregulation of IL-1β gene expression was observed in non-polarized BMDM treated with TYR, as well as in LPS-polarized BMDM, and LPS-polarized BMDM treated with
TYR. Furthermore, LPS-polarized BMDM treated with TYR show significant increases in IL-1β gene expression compared to LPS-polarized BMDM alone (Figure 24B). TNFα gene expression was not significantly upregulated in non-polarized BMDM treated with TYR, but was significantly upregulated in LPS-polarized BMDM compared to vehicle. Treatment of LPS- polarized BMDM with TYR significantly upregulated TNFα gene expression compared to all treatment groups (Figure 24C). Finally, iNOS gene expression was only upregulated in the
LPS-polarized BMDM treated with TYR compared to vehicle; with no significant upregulation of iNOS in any other treatment group (Figure 24D).
The TAAR1 antagonist EPPTB attenuates the upregulation of TAAR1 and inflammatory gene expression.
TYR is a specific TAAR1 agonist, and in this regard I sought to determine if the upregulation of
TAAR1, iNOS, and proinflammatory cytokine gene expression by TYR was TAAR1-mediated. A major limitation in TAAR1 research has been the absence of available TAAR1 antagonists for humans; only one antagonist -EPPTB- exists and it is mouse specific (Bradaia et al., 2009).Here pharmacological inhibition of TAAR1 was explored using EPPTB to determine whether the upregulation of proinflammatory gene expression by TYR was TAAR1-mediated in mouse
BMDM. LPS-polarized BMDM were treated with TYR, EPPTB or pretreated with EPPTB followed by TYR. In LPS-polarized BMDM treated with TYR upregulation of TAAR1, IL-6, IL-1β, and TNF-α (Figure 25 A-E) gene expression was confirmed as previously seen in Figures 23 and 24. EPPTB pretreatment significantly attenuated the TYR-mediated upregulation of
TAAR1, IL-6, IL-1β, and TNF-α gene expression (Figure 25A-D), but not iNOS gene expression
(Figure 25E). Pre-treatment of LPS-polarized BMDM with EPPTB alone had no significant effect
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on TAAR1 (Figure 25A), IL-6 (Figure 25B), IL-1β (Figure 25C), TNFα (Figure 25D), or iNOS
(Figure 25E) gene expression in cells.
Differentiation of BMDM and treatment with TYR alters the phenotype of mouse
BMDM
Macrophages act as a first line of defense in the gastrointestinal tract, often switching phenotypes and promoting inflammation in response to inflammatory stimuli. To determine the effects of TYR on BMDM phenotype, non-polarized BMDM, non-polarized BMDM treated with
TYR, LPS-polarized BMDM, and LPS-polarized BMDM treated with TYR were cultured and immunostained with the previously validated D274 custom antibody (Chapter 1). All treatment groups stained positive for TAAR1 protein expression. Non-polarized BMDM appear to have primarily intracellular TAAR1 expression (Figure 26A), whereas some TAAR1 staining becomes localized to the cellular membrane of cells upon treatment with TYR (Figure 26B). LPS- polarized BMDM tend to have higher expression of TAAR1 intracellularly and on the cellular membrane of BMDM (Figure 26C), and TYR treatment of LPS-polarized BMDM appears to upregulate the expression of TAAR1 and alter cellular shape (Figure 26D-E). Accordingly, the average diameter of BMDMs from all treatment groups was quantified to determine the loss of elongated cellular forms into round- and –activated cells. There is a significant decrease in the average cell diameter with a loss of the elongated projections characteristic of non-activated
BMDM (Figure 26F).
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FIGURES AND TABLES
Table 6. Primers used for qRT-PCR analysis.
Gene RefSeq Forward Sequence Reverse Sequence mTAAR1 NM_053205.1 5‟- GGGTACTGGCGTTCATGACT -3‟ 5‟- CGCCCACCATGATCCCTAAG -3‟ mIL-6 NM_031168.2 5‟- CCGGAGAGGAGACTTCACAG -3‟ 5‟- GGAAATTGGGGTAGGAAGGA -3‟ mIL-1β NM_008361.4 5'- GCACTACAGGCTCCGAGATGAAC -3' 5'- TTGTCGTTGCTTGGTTCTCCTTGT -3' mTNF-α NM_001278601.1 5‟- TACTGAACTTCGGGGTGATTGGTCC -3‟ 5‟- CAGCCTTGTCCCTTGAAGAGAACC -3‟ miNOS NM_001313922.1 5'- CGAAACGCTTCACTTCCAA -3' 5'- TGAGCCTATATTGCTGTGGCT -3' mIL-10 NM_010548.2 5'- ATAACTGCACCCACTTCCCA -3' 5'- GGGCATCACTTCTACCAGGT -3'
Figure 23. TAAR1 is expressed in mouse BMDM and is upregulated by TYR. QRT-PCR analysis of TAAR1 in mouse BMDM that were treated with vehicle, TYR, LPS, LPS+TYR. Treatment with either TYR or LPS produced a non-significant increase in TAAR1 gene expression compared to vehicle. Treatment with TYR in the presence of LPS significantly increased TAAR1 gene expression in comparison to vehicle, TYR, and LPS alone. (n=9, ##: p<0.01, one-way ANOVA with Bonferroni post-test.)
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Figure 24. TYR-dependent upregulation of IL-6, IL-1β, TNF-α and iNOS. BMDM were treated with vehicle, TYR, LPS and LPS+100nM TYR, and monitored for inflammatory cytokine and iNOS gene expression. A. IL-6 gene expression was significantly upregulated by TYR in LPS-polarized BMDM. B. IL-1 gene expression was significantly upregulated by LPS, TYR in non- polarized BMDM and LPS-polarized BMDM compared to vehicle treatment. C. TNF-α gene expression was significantly upregulated by LPS compared to vehicle and by TYR in LPS-polarized BMDM. D. iNOS gene expression was significantly upregulated by TYR in LPS-polarized BMDM. (n=9, #: p<0.05; ## p<0.01, ### p<0.001 one-way ANOVA with Bonferroni post-test.)
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Figure 25. Upregulation of TAAR1 and inflammatory cytokine gene expression is attenuated by the TAAR1-antagonist EPPTB. BMDM were treated with vehicle, LPS, LPS+EPPTB, LPS+TYR, or LPS+TYR+EPPTB and monitored for TAAR1, inflammatory cytokine, and iNOS gene expression. A. TAAR1 gene expression was significantly upregulated by TYR treatment in LPS-polarized BMDM compared to vehicle, LPS-polarized BMDM and LPS-polarized BMDM treated with EPPTB alone. Pretreatment with EPPTB followed by TYR treatment significantly diminished the TYR mediated upregulation of TAAR1 in mouse BMDM. B. IL-6 gene expression was significantly upregulated in LPS-polarized BMDM treated with TYR, and the upregulation was blocked by pre-treatment with EPPTB. C. IL-1β gene expression was significantly upregulated in LPS-polarized BMDM treated with TYR compared to vehicle, LPS-polarized and LPS-polarized BMDM treated with EPPTB. Pretreatment with EPPTB attenuated the TYR mediated upregulation of IL-1β in LPS-polarized BMDM. D. TNF-α gene expression was significantly upregulated by TYR in LPS-polarized BMDM compared to vehicle. TYR mediated upregulation of TNF-α was attenuated by pretreatment of LPS-polarized BMDM with EPPTB. E. iNOS gene expression was significantly upregulated in LPS- polarized BMDM, LPS-polarized BMDM treated with EPPTB, LPS-polarized BMDM treated with TYR, and LPS-polarized BMDM pretreated with EPPTB and treated with TYR. Pretreatment of LPS-polarized BMDM with EPPTB had no significant effect on iNOS gene expression compared to LPS-polarized BMDM treated with TYR. (n=3, #: p<0.05; ## p<0.01, ### p<0.001 one-way ANOVA with Bonferroni post-test.)
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Figure 26. TAAR1 protein expression in mouse BMDM and phenotypic alterations in response to 100nM tyramine. Immunofluorescence staining of adherent, day 7 derived mouse BMDM with D274 antibody (green) and DAPI (blue). A. Vehicle treated BMDM with intracellular expression of TAAR1. B. Non-polarized BMDM treated with TYR with intracellular, and extracellular expression of TAAR1. C. Expression of TAAR1 in LPS-polarized BMDM. D. Expression of TAAAR1 in LPS-polarized BMDM treated with TYR. E. Quantifiction of TAAR1 protein expression by histogram quantification in BMDM. TAAR1 expression is significantly upregulated in LPS-polarized BMDM treated with TYR. F. Quantification of diameter changes in mouse BMDM in response to TYR, LPS-polarization, and LPS-polarized cells treated with TYR. Compared to vehcile, all groups experience siginifiacnt decreases in diameter of mouse BMDM. LPS-polarized BMDM treated with TYR are significantly smaller than LPS-polarized BMDM alone. (n=3 mice, 4 images per animal, #: p<0.05; ## p<0.01, ### p<0.001 one-way ANOVA with Bonferroni post- test.)
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Key: Tyramine traverses the epithelial barrier Chemo-attraction of immune cells to tyramine in lumen Pathogenic or Commensal Microbes
Tissue Resident Macrophage
Inflammatory (M1) Macrophage
Peripherally Infiltrating Monocyte/Macrophage
Inflammatory Cytokines
T-cells and B-cells Activation of tissue resident macrophages TAAR1
↑IL-1β ↑IL-6 ↑TNF-α
blood vessel Recruitment of peripheral macrophages, T-Cells and Inflamed Mucosa B-Cells.
Figure 27. Proposed mechanism for TAAR1 mediated inflammatory response in the gastrointestinal mucosa by tyramine. TYR traverses the GIT at significantly elevated levels in patients with UC compared to healthy controls (Santoru et al., 2017). In a patient with UC, TYR accumulates in the intestinal lumen and diffuses across the epithelial barrier activating underlying immune cells. Translocation of pathogenic lumen microbes primes tissue resident macrophages and TYR activation of TAAR1 yields an increase in secretion of IL- 1β, IL-6, and TNF-α. Upregulation of inflammatory cytokine secretion propagates the inflammatory response, activating tissue resident macrophages (and T-cells, dendritic cells, etc), recruiting peripheral macrophages, T-Cells and B-Cells. Elevated levels of TYR in the lumen serves as a chemoattractant for immune cells, and T-Cells, B-cells, and macrophages migrate towards the lumen, causing further breakdown of the epithelial barrier propagating underlying tissue inflammation.
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DISCUSSION
Macrophages are one of the most abundant leukocytes in the intestinal mucosa, and play an essential role in homeostasis of the intestine. Aberrant activation of macrophages has been implicated in the onset and progression of UC, and current therapeutics aim to attenuate the augmented cytokine production from these cells. To date, there have been no studies on the effects of TYR on macrophage function and the potential for TYR to mediate gastrointestinal inflammation, despite reports that TYR is elevated in the fecal content in patients with UC. TYR is a high affinity agonist for TAAR1, which signals through cAMP/PKA and Ca2+/PKC pathways
(Panas et al., 2012). Based on previous reports of TAAR1 expression in immune cells(Babusyte et al., 2013; Fleischer et al., 2018; Panas et al., 2012; Raab et al., 2016; Sriram et al., 2016;
Sriram et al., 2015), the expression and regulation of TAAR1 in BMDM was explored. For the first time, TAAR1 expression is identified in mouse BMDM and is inducible upon stimulation with
TYR. This agonist-induced upregulation of the receptor mirrors studies exploring TAAR1 in other cell types (Sriram et al., 2016; Stavrou et al., 2018).
After differentiation of bone marrow hematopoeitc cells to macrophages, cells require signals to determine which polarization state they will terminally differentiate to. Non-polarized
BMDM are differentiated cells that are not activated, whereas LPS-polarized cells resemble the
M1 polarization that is classified as pro-inflammatory. Interestingly, M1 macrophages are known to contribute to the onset and progression of experimental dextran sulfate sodium colitis models (Chang et al., 2013). Because of this the ability of TYR to elicit an inflammatory response from BMDM was explored, a phenomenon that may occur in the colon of patients with
UC. Accordingly, expression of TAAR1 was monitored, as well as expression of IL-6, IL-1β, and
TNF-α cytokine gene expression which has previously been shown to be upregulated by cAMP/PKA and Ca2+/PKC pathways. As an internal control, the upregulation of iNOS was explored as it is known to be elevated in patients with UC, but primarily upregulated by TLR4 and JAK/STAT signaling cascades, which are not implicated in TAAR1 signaling. The data
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revealed that TYR treatment in LPS-polarized macrophages causes significant increases in IL-
6, IL-1β and TNFα cytokine gene expression, indicating that TYR may be responsible for driving inflammation in UC. The effects of TYR on BMDM were seen with a submaximal TYR dose
(100nM), as previously described (Borowsky et al., 2001; Simmler, Buchy, Chaboz, Hoener, &
Liechti, 2016; Xie & Miller, 2008). To determine if TYR effects on BMDM were TAAR1 mediated, the only TAAR1 antagonist currently available, EPPTB was used (Bradaia et al.,
2009; Stalder, Hoener, & Norcross, 2011). Pretreatment with EPPTB attenuated the TYR- mediated upregulation of IL-6, IL-1β and TNF-α cytokine gene expression, whereas there was no attenuation of iNOS gene expression. These data suggest that TYR is activating TAAR1 in
BMDM, promoting inflammatory cascades.
Finally, to confirm TAAR1 protein expression in BMDM was done so using the D274 as described in Chapter 1. Here non-polarized BMDM with and without TYR, and LPS-polarized
BMDM with and without TYR were stained to determine the protein expression patterns in
BMDM. Similarly to the gene expression studies monitoring TAAR1 upregulation, TYR upregulated TAAR1 protein expression in mouse BMDM, and TYR appears to alter the phenotype of the LPS-polarized BMDM. To quantify this switch in phenotype the diameter of cells was measured and counted to determine the number of cells per treatment group that have lost their spiny projections. Upon stimulation with TYR, there was an observed significant cellular rounding, as well as a complete loss of spiny projections. While these findings are descriptive, future studies exploring TYR on LPS-polarized BMDM will seek to identify whether
TYR initiates pyroptosis, a form of programmed cell death commonly observed in the inflammasome in colitis(Cookson & Brennan, 2001).
Owing to previous reports that TAAR1 activation can elicit chemotactic responses from immune cells (Babusyte et al., 2013), and preliminary data from the Miller lab (data not shown) there is evidence that TAAR1 may be acting as an integrator of dietary and microbiomic signals.
As outlined in Figure 27, TYR originating in the lumen of the GIT, either originating from consumed food or secreted by the microbiota, has the potential to transgress the epithelial
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barrier and cause an increase in production of proinflammatory gene expression in macrophages. This increase in proinflammatory cytokine gene expression will recruit peripheral immune cells, as well as further activate both tissue and infiltrating macrophages to the site of inflammation. Babusyte et al demonstrate that polymorphonuclear cells chemotax towards trace amines as nanomolar affinities(Babusyte et al., 2013). As previously demonstrated, TYR levels are elevated in the fecal content of patients with UC. Linares et al have previously shown cytotoxic effects of TYR on colon epithelial cells in vitro, but to date, there have been no studies on the effects of TYR on BMDM. Due to the high levels of TYR in the lumen of the GIT, infiltrating immune cells may chemotax towards biogenic amines in the lumen promoting breakdown of the tight junctions as they permeate the epithelial cell barrier. Accordingly, the use of TAAR1 antagonists (or partial agonists) would attenuate this feed-forward mechanism in the lumen, potentially serving as a novel therapeutic approach to attenuate inflammation while reducing the need for intensive monoclonal anti-TNFα therapies.
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MATERIALS AND METHODS
Animal husbandry
C57BL/6 purchased from the Jackson Laboratories. Mice used in these studies were 8 week old males purchased at three independent times. TAAR1-/- mice were bred at Northeastern
University as previously described (Lynch et al., 2013). All animal care was in accordance with the Guide for the Care and Use of Laboratory Animals (Council, 2011) and all procedures were conducted in accordance with the Animal Experimentation Protocol (17-0825) approved by the
Northeastern University IACUC.
Cell Culture
Bone marrow-derived macrophages (BMDMs) were collected from both hind limbs of C57BL/6 as previously described (Zanoni et al., 2009). Briefly, mouse femur and tibia were removed from both hind limbs, cleaned of muscle, sterilized with 70% ethanol, and kept in BMDM isolation media (RPMI + 10% FBS + 1% Anti/Anti (Thermo Fisher, Waltham, Massachusetts) until isolation of cells. To isolate cells, each bone was briefly soaked in 70% ethanol, then flushed with PBS, and filtered through a 70μm cell strainer, followed by a 40μm strainer, and centrifuged at 1100 RPM for 5 minutes. Cell pellets were resuspended in conditioned media to stimulate differentiation into BMDMs, (RPMI + 10% L929-conditioned media + 1% Pen/Strep,
(Thermo Fisher, Waltham Massachusetts) and allowed to differentiate for approximately 6 days.
To polarize cells, 1 ng/ul lipopolysaccharide (LPS)(Sigma Aldrich, Natick, MA) was added to culture media for 24 hours. These cells were then used for the various experiments described below.
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Drug Treatments
TYR (Sigma Aldrich, Natick, MA) was dissolved in PBS to make a stock concentration of 100
μM. Stock solution was diluted in cell culture media to a final concentration of 100 nM. The
TAAR1 antagonist EPPTB N-(3-Ethoxy-phenyl)-4-pyrrolidin-1-yl-3-trifluoro-methyl-benzamide) supplied by F. Hoffmann-La Roche Ltd, Pharma Research (CH-4070 Basel, Switzerland) was dissolved in DMSO to a final concentration of 10μM (Stalder et al., 2011) . From this stock,
600ul were dissolved in 3ml PBS and mixed to make a homogenous solution, and added to tissue culture dishes for a final concentration of 1uM EPPTB (DMSO 0.02% (v/v).
RNA Extraction, PCR and qRT-PCR
Cell pellets were resuspended in 1ml of Trizol reagent, thoroughly mixed, and incubated for 5 minutes at 4°C. Following, 200ul of chloroform was added and samples were shaken vigorously, incubated for 5 minutes at room temperature, then spun for 5 minutes, at 4°C, at
12,000RPM. The aqueous layer of the sample was collected and added to 1ml of ice cold isopropanol. Samples were placed at -20°C for 1 hour and spun for 15 minutes at 4°C at
12,000RPM. The supernatant was removed, and the pellet was washed with 70% ethanol made with RNase free water. Samples were spun again at 5 minutes, at 4°C, at 12,000RPM.
The pellet was washed a second time with 70% ethanol and spun at 7500RPM for 5 minutes at
4°C. RNA pellets were allowed to air dry for 45 minutes and resuspended in RNase free water.
RNA concentration was calculated using a Nanodrop spectrophotometer and the 260/280 ratios were determined for each samples. For RT/PCR, samples with 260/280 ratios near 2.0 were converted to cDNA using the Qiagen RT-PCR Kit (Item No. 205313; Qiagen, Germantown MD), according to the manufacturer‟s instructions. Upon conversion to cDNA, samples were diluted
1:1 with nuclease free water, and quantitative real-time PCR (qRT-PCR) reactions were performed using SYBR Green Master Mix (Item No. 436765, Thermo Fisher, Waltham, MA) and run on a Stratagene Mx3005p (Agilent, Santa Clara, CA) Fold gene expression was
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determined using the ∆∆CT method and normalized to vehicle treatment. Primers outlined in
Table 6 were used for qRT-PCR reactions.
Immunostaining
Cells were fixed with 4% paraformaldehyde for 10 minutes at 4°C, and permeabilized in 0.1% triton-x100 in TBS for 15 minutes at room temp. Cells were blocked with 3% BSA+1.5%skim milk-TBS-0.01% tween for 1 hour at room temperature and incubated with an anti-TAAR1 antibody (D274) overnight at 4°C. Cells were washed 4x5minutes in TBS-0.01%tween at RT and incubated with Alexafluor-647 (Thermo Fisher, Waltham, MA) for 45 minutes at RT. Cells were washed 4x5minutes in TBS-0.01%tween at RT, dipped in Hoescht (1:10,000 in dH2O) briefly washed in TBS and cover slipped with Prolong Diamond Antifade Mountant (P36970,
Thermo Fisher, Waltham, MA). Individual image channels were captured on a Zeiss Axio
Observer fluorescent microscope (Carl Zeiss) at 63x under oil, and individual channels were colorized with non-conventional colors – DAPI colored blue, Alexafluor-647 colored green – after grey-scale imaging of individual channels using ZenPro2 software.
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Chapter 4: Dextran sulfate sodium induced colitis is attenuated in TAAR1-/- mice.
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BACKGROUND
Trace amine-associated receptors (TAARs) including TAAR1 have been studied since their deorphanization in 2001 for their roles modulating monoaminergic signaling in the central nervous system. Few studies have identified roles for TAAR1 in the periphery including one study demonstrating TAAR1 localization in the stomach (Broadley et al., 2009). Ligands for
TAAR1 include biogenic trace amines which have been studied for greater than a century as a correlate of neuropsychiatric disorders. Additionally, with the industrialization of the food industry, assays have been developed to determine the levels of biogenic trace amine (e.g. tyramine, β-PEA) in food products as a marker of microbial contamination by Lactobacillus and
Enterococcal fermentation.
Although the trace amine TYR first garnered the attention of physicians in the 1950s, with the advent of monoamine oxidase inhibitors (MAO-I) and the potential for TYR to cause a severe hypertensive crisis, TYR and β-PEA have been recently explored for their activity at
TAAR1 in mammalian systems (Price & Smith, 1971). Typically, TYR is absorbed, or degraded by monoamine oxidase enzymes in the small intestine, though in inflammatory states (or inpatients taking MAO-Is) TYR can transit through the GIT to the colon where it is not absorbed or metabolized by humans, but secreted in the feces (Coyle & Boyd, 1932; Santoru et al., 2017).
With the advent of the human microbiome project, a new field has developed studying the „metabolome‟ in patients with GI inflammatory disorders including IBD, irritable bowel syndrome, and celiacs disease. Perhaps most interesting, in 2017, a metabolomic study identified elevated levels of the trace amines β-PEA and TYR in the fecal content of patients with Crohn‟s disease and ulcerative colitis, respectively. TYR and β-PEA have a wide spectrum of sources in the GIT: 1) normal mammalian amino acid metabolism; 2) ingestion of food products rich in tyramine; and 3) metabolic by products from the gut microbiome or probiotics.
Indeed, TYR and β-PEA are produced in cultures when the microorganisms are stressed in an acidic environment (pH 5.5-6.0), and the pH of the human GIT is markedly decreased in
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inflammatory bowel diseases. Additionally, van Kelssen et al demonstrated that microbes isolated from human small intestine and grown in culture can produce TYR, among other trace amines (van Kessel et al., 2019).
Despite understandings of UC disease correlates, treatment options for patients with UC remain limited and are focused on the suppression of the immune system in an effort to alleviate the inflammatory symptoms and heal the gut. TAAR1 has yet to be systematically studied in the
GIT of mouse or human populations in normal or diseased states.
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RESULTS
TAAR1 is expressed in the gastrointestinal tract of mice.
To determine if TAAR1 was expressed in the colon of healthy C57BL/6 mice TAAR1 gene expression in total RNA extracts from the colon of WT and KO mouse was monitored. TAAR1 mRNA was found to be expressed in the colon of WT animals, and not in the colon of KO animals (Figure 29E). To understand the cellular localization of TAAR1 in the GIT Swiss rolled colons from C57BL/6 and TAAR1 KO mice were immunostained using the previously discussed
D274 custom antibody (Chapter 1). Here, TAAR1 is found to be expressed in the cells closest to the lumen in the colon of C57BL/6 mice co-localizing with Occludin staining in the epithelial cells (Figure 29A), compared to no expression in the TAAR1-/- mice (Figure 29B). Due to tissue autofluorescence of the colon as an artifact of mucin fluorescence at 488nm, TAAR1 staining was validated against secondary only antibody incubation to determine background fluorescence in C57BL/6 colon sections, and the „TAAR1‟ staining in Figure 29A was determined to be specific (Figure 29C). Finally, to fully characterize TAAR1 expression in the
GIT of C57BL/6 mice, small intestine sections were immunostained revealing TAAR1 specific staining at both the tips of the villi and the base of the crypts (Figure 29D). These data demonstrate that TAAR1 is expressed in the colon of mice, with co-localization with occludin stain in the epithelial cells nearest the lumen.
TAAR1-/- colon are phenotypically different from C57BL/6 colon.
A hallmark of gastrointestinal inflammation in mouse models of colitis includes shortened colon length, decreased crypt height, and a loss of goblet cells in the colon. Here, to determine if the
TAAR1-/- mice had any baseline inflammatory conditions the GIT of C57BL/6 and TAAR1-/- littermates was characterized. C57BL/6 colon and TAAR1-/- colon have no signs of blood or inflammation upon examination (Figure 30A), though the TAAR1-/- colon are significantly longer than the C57BL/6 colon (Figure 30B). Additionally, the TAAR1-/- tissue sections have retained
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crypts, similarly to the C57BL/6 crypts (Figure 30C), but the TAAR1-/- crypts are significantly taller than WT crypts. Finally, there was a retention of goblet cells in the TAAR1-/- mice (Figure
30E) but there are significantly more goblet cells per 10uM of tissue in the TAAR1-/- mouse
(Figure 30F), and that the goblet cells in the TAAR1-/- mice are significantly larger than the crypts of the WT mice (Figure 30G). Taken together, there are no observable signs of inflammation in the TAAR1-/- colon, compared to the C57BL/6 mice, but the TAAR1-/- colon differ phenotypically with regard to colon length.
TAAR1-/- mouse is protected from the onset of an acute DSS colitis model
To assess the role of TAAR1 in intestinal health, 8- to 10-week old C57BL/6 and TAAR1-/- littermates were administered 3% DSS in drinkng water for 10 days. TAAR1-/- mice appear more resistant to disease severity compared with C57BL/6 littermate controls (Figure 31). With respect to body weight, the C57BL/6 experienced an increased loss, and an earlier onset of weight loss compared to TAAR1-/- mice (Figure 31A). There was no significant difference in
DSS body score (observing 10 key features of health including activity levels, stool consistency, and alertness (Figure 28) in C57BL/6 vs. TAAR1-/- groups (Figure 31A). Grossly, while both the
C57BL/6 and TAAR1-/- animals experienced a significant loss in colon length compared to healthy groups, the TAAR1-/- mouse colon remained significantly longer than the colon of
C57BL/6 mice (Figure 31 B&C).
TAAR1-/- mice have shorter lesions and signs of healing compared to C57BL/6 animals.
Histologically, C57BL/6 mice have significantly longer lesions compared to TAAR1-/- mice
(Figure 32). Additionally, C57BL/6 mice have severe transmural inflammation characterized by infiltration of inflammatory cells in the mucosa, severe crypt erosion characterized by long erosive lesions and complete loss of crypts, disruption of the outer muscle layers, a complete
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loss of goblet cells and no signs of healing over erosions. The severity and extent of the tissue damage was less severe in the TAAR1-/- mice, as can be seen with the shorter lesions in the
TAAR1-/-, dilation of crypts rather than the complete loss, mild submucosal inflammation and absence of disruption of the outer muscle layers, and signs of healing of the epithelial cells in the mucosa.
TAAR1 is expressed in human colon, and is increased in intensity in UC disease biopsies
To assess TAAR1 expression differences between healthy human colon samples and UC human colon tissues, I surveyed a tissue mircroarray containing 24 specimens from 4 healthy donors, and 20 samples with inflammatory conditions of the colon (4 chronic esocolitis, 4 chronic UC, 4 CD, 4 polypus and 4 adenocarcinomas)(Figure 33C). TAAR1 expression was positively identified in both healthy and inflamed tissue (Figure 33A &B). Interestingly, TAAR1 staining appeared more intense in the patients with chronic UC, and histogram quantification to
%DAPI area suggests that TAAR1 expression is elevated in the epithelium of patients with UC
(Figure 33D).
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FIGURES AND TABLES
Figure 28. TAAR1 is expressed in the colon and small intestine of C57BL/6 mice. A. C57BL/6 colon sections stained with Occludin (red), TAAR1 D274 (green) and DAPI (blue). TAAR1 specific staining is localized in the epithelial cells along the lumen (arrowheads) with faint staining in the epithelial cells in the deeper epithelial cells (stars). B. TAAR1-/- colon sections stained with Occludin (red), TAAR1 (green), and DAPI (blue). No TAAR1 specific staining exists in the TAAR1-/- colon section. C. Staining of C57BL/6 colon with AlexaFluor-647 secondary only (green) and DAPI, shows minimal background staining in the colon. D. C57BL/6 small intestine (6 stitched images) stained with TAAR1 (D274) to confirm the expression of TAAR1 in the villi and deep crypts. E. RT-PCR analysis of TAAR1 transcript levels in mouse tissue of C57BL/6 and TAAR1-/- shows identification of TAAR1 transcript in liver, kidney, colon spleen and heart of the C57BL/6 mouse, with no TAAR1 gene expression in the TAAR1-/-, with GAPDH as a loading control for cDNA loading.
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Figure 29. TAAR1-/- colon are phenotypically different from C57BL/6 colon. A. Representative image of C57BL/6 and TAAR1-/- colon from 8-week old littermates. B. Average colon length of TAAR1-/- mice is significantly longer (8cm vs 6cm) than C57BL/6 colon length in 8-week old littermates (n=12, Student‟s t-test, ***p<0.001). C. Representative cross section of colon from C57BL/6/ and TAAR-/- litter mates. D. Average crypt height analysis in healthy 8 –week old litter C57BL/6 and TAAR1-/- littermates. TAAR1-/- mice have significantly increased crypt height compared to their C57BL/6 littermates. E. Representative images of Alcain blue goblet cell stain in healthy C57BL/6 and TAAR1-/- littermates. F&G. Quantification of the number of and size of goblet cells in healthy littermates from C57BL/6 and TAAR1-/- mice. F. TAAR1-/- mice have significantly greater number of goblet cells per tissue area compared to C57BL/6 controls, and G. have a significant increase in the average diameter of goblet cells in the colon. Page 95
Figure 30. TAAR1-/- mice are protected from adverse effects of DSS induced colitis. A. Comparison of body weight and DSS body score in acute DSS model. TAAR1-/- mice retain their body weight until day 5, while C57BL/6 littermates demonstrate weight loss as early as day 3. TAAR1-/- mice tent to mirror C57Bl/6 mouse body score variance throughout the duration of the study, though surviving mice retain a lower DSS body score compared toC57BL/6 mice. B. Representative images measuring colon length in C57BL/6 and TAAR1-/- control and DSS treated mice. C. Quantification of average colon length in DSS studies. TAAR1-/- mice have a significant „retention‟ of colon length (6.5cm) when subjected to DSS, compared to C57Bl/6 litter mates (5cm). C57BL/6 and TAAR1-/- mice administered DSS have a significantly shorter colon compared to healthy littermate controls.
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Figure 31. TAAR1-/- mice have significantly shorter lesions compared to C57BL/6 mice. Top, representative H&E images from C57BL/6 mice. Long lesions (4290 μM) with few signs of regeneration, transmural inflammation and complete loss of normal crypt and goblet cell architecture. Bottom, representative H&E images from TAAR1-/- mice with DSS colitis. Lesions in TAAR1-/- are significantly shorter than C57BL/6 lesions (2622 μM), with incomplete loss of crypt architecture including goblet cells, and less severe transmural inflammation and signs of regeneration.
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Figure 32. Expression of TAAR1 in the colon of healthy and diseased human colon tissue. A. Healthy human colon stained with TAAR1 (Green) and dapi (Blue) and H&E image of corresponding sections from USbiomax website available to customers. TAAR1 is localized in the epithelial cells of the crypts of patients with UC. B. UC diseased human colon tissues stained with TAAR1 (green) and DAPI (blue), and corresponding H&E image from US Biomax. C. Tissue array outline as demonstrated on usbiomax.com. D. Quantification of fluorescent intensity of healthy human colon vs. chronic UC patients normalized to DAPI area (n=4 images, per n=3 patients, Student‟s t-test, p<0.05).
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DISCUSSION
The Western diet contains high levels of trace amines, which are known ligands for
TAAR1. There are currently no studies on the effects of TAAR1 in the onset and progression of colitis in vitro, or in human populations. Previous characterizations of the TAAR1-/- mouse have reported no overt phenotype, but this analysis precluded the intestine. A comparative analysis of C57BL/6 and TAAR1-/- GIT samples were analyzed to determine the role of TAAR1 in the GIT and in the onset and progression of colitis, and identify for the first time TAAR1 expression in the colon of mouse and human colon samples. Observation of TAAR1-/- mice compared to
C57BL/6 controls demonstrates a unique colon phenotype, typically observed in germ free mice
(Grover & Kashyap, 2014). Compared to C57BL/6, TAAR1-/- mice have an elongated colon as well as an increase in the crypt height and an overall increase in the number of goblet cells in the crypts and a significant variance in size of goblet cells. Despite the observable differences between C57BL/6 and TAAR1-/- mice there are no hallmarks of an inflammatory phenotype at baseline and so my work provides an ethical justification to progress to studying the role of
TAAR1 in an in vivo colitis pilot study.
The mechanism of DSS is poorly understood, but the basis of the model relies on DSS- mediated disruption of the intestinal epithelial barrier. This model emulates the innate immune driven responses seen in human colitis patients. The entry of luminal bacteria is due to the barrier loss in the colon. In dextran sulfate sodium, a sulfated polysaccharide is administered in the drinking water of mice, and is toxic to the colon epithelium, resulting in injury, barrier dysfunction and immunological responses in affected areas. DSS does not directly cause intestinal inflammation that is common to UC, but the chemically induced injury results in transgression of the lumen contents to the lamina propria, triggering an immune response from resident cells (Kawada, Arihiro, & Mizoguchi, 2007).
This pilot study implored an acute stimulation with 3% DSS in the drinking water to characterize the onset of colitis in TAAR1-/- mice. Upon stimulation with DSS in C57BL/6 and
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TAAR1-/- mice, the TAAR1-/- mice appear to be protected from the onset and severity of DSS- induced colitis. This is first seen in the onset of weight loss in the TAAR1-/- beginning at day 5, whereas their C57BL/6 littermate controls experience onset of weight loss as early as day 2.
This, however, is not reflected in the DSS body score as the KO mice become inactive as early as day 2, affecting the average body score early in the experiment (data not shown).
Conversely, TAAR1-/- mice experience onset of visual rectal bleeding on average at day 5 whereas C57BL/6 mice show rectal bleeding as early as day 3 of treatment with DSS (data not shown). Similarly, TAAR1-/- mice have a retained colon length compared to C57BL/6 littermates, suggesting that they do not have the same severity of inflammation as the C57BL/6 mice. This is confirmed with histological scoring of the TAAR1-/- lesions ex vivo, as can be seen by the incomplete loss of crypt morphology and instead dilation of crypts, mild submucosa inflammation, no inflammatory disruption of the outer muscle layers, and signs of healing of the muscoal layer.
TAAR1 has been previously described in the stomach of humans, though there have been no systematic studies investigating the expression and localization of TAAR1 in the colon.
To determine the relevance of the TAAR1-/- model to human disease, tissue arrays were purchased from US Biomax and stained and quantified TAAR1 expression in healthy human biopsies and human biopsies with chronic UC. TAAR1 expression was confirmed in the healthy human biopsies as well as in the biopsies from patients with chronic UC. TAAR1 is found in the
GIT of humans, found in epithelial cells, and at a greater intensity in UC patients than in healthy patients. Fluorescent intensity of the TAAR1 staining was quantified to the percent intensity of
DAPI area for 4 independent biopsies, and revealed that TAAR1 expression is elevated in the patients with chronic UC compared to healthy controls. This is remarkable, as TAAR1 has not been previously studied in humans with UC, nor has it been characterized in the epithelial cells of the colon. An increase in TAAR1 expression levels may have many implications in the pathogenesis and progression of UC. Metabolomic studies have previously identified elevated trace amines including TYR in the feces of patients with UC. The source of TYR has yet to be
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confirmed as has the implication that TYR may be causative in disease. In chapter two, TAAR1 expression in intestine epithelial cell lines was found to be stimulated by TYR treatment, a phenomenon that may be seen in vivo. Additionally, TYR is known to be produced by the microbiome, be elevated in foods that aggravate UC flares, and occur as a normal byproduct of tyrosine metabolism in mammalian cells. My data provides preclinical evidence that TAAR1 should be explored for a role in onset and aggravation of UC. TAAR1 expression upregulation has also demonstrably been upregulated by immune cell activation by PMA (Panas et al.,
2012). This phenomenon has not been observed in other cell types, but may suggest that an increase in inflammation or inflammatory signals may cause an increase in TAAR1 expression in additional body systems, and may provide an explanation for an upregulation of TAAR1 in human biopsies with UC. With this preclinical data, and the use of an antibody that appears functional in mouse tissue, my research provides several new avenues to explore the function of
TAAR1 in GIT disorders, and to determine if TAAR1 is a pharmacological target for GI diseases.
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MATERIALS AND METHODS
Animal Husbandry
Eight to ten week old male wild type C57BL/6 mice were ordered from the Jackson Laboratories
(Bar Harbor, ME). C57BL/6 mice were allowed to acclimate to the colony and were paired with
TAAR1-/- mice to establish het x het crosses. Littermates from het x het crosses were utilized for the outlined experiments, and then euthanized with CO2 followed by cervical dislocation. All animal experiments were approved under protocol 17-0825, amendment 17-0825R-A1 and conducted in accordance with DLAM Institutional Regulations Protocol at Northeastern
University, Boston MA.
TAAR1-/- generation and genotyping
TAAR1-/- mice were originally gifted from Lundbeck Research USA, Inc. (Paramus, NJ) with a mixed strain background consisting of 75%-C57BL/6J and 25%-129S1/Sv described in [1].
Briefly, 3.6kb of the TAAR1 gene has been deleted, including the entire TAAR1 coding sequence (single exon), 1.0 kb of upstream sequence, and 1.3 kb of downstream sequence.
Further, in-house, TAAR1 heterozygotes were backcrossed 9 generations to wildtype C57BL/6 from Charles River Laboratories (Wilmington, MA). Ninth generation heterozygote pups were then mated to generate TAAR1 knockout mice with a strain background of 99.95%-C57BL/6.
Het x het crosses to generate C57BL/6 and TAAR1-/- littermates are used to maintain the colony.
Mice were ear tagged with a punch biopsy tool, and tissue was used for genotyping as previously described. Briefly, DNA was extracted using KAPA Extraction Kit (Catalog no. ) and
PCR was performed using KAPA genotyping master mix. Genotyping primers are outlined in
Table 8. Confirmation of results was obtained by sending samples to Transnetyx for validation of in house assay, as previously described.
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Imaging of mouse intestine
Mouse intestine were isolated from the abdomen of C57BL/6 and TAAR1-/- mice and placed in ice cold PBS. Fascia and connective tissue were gently removed from the outer layers of the intestine and were gently spread on a Nalgene dissection board with a metric ruler for image captures. Images were captured by a 12 megapixel dual pixel,with f/1.7 1.4-micron pixels optical image stabilization lens, at 4034x3024px resolution.
Tissue Staining: Immunofluorescence
Mouse colon were flushed with formalin and filled with 0.25%agarose in formalin. Tissue were rolled into Swiss rolls (spirals) placed in cassetes and fixed in formalin for 36 hours. Samples were then embedded in paraffin wax, and tissue blocks were cut to 5μM thickness and mounted on a glass slide. Sections were deparaffinized and permeabilized with TBS+0.1% Triton-X100, blocked with 3% BSA+1.5%skim milk-TBS-0.01% tween for 1 hour at room temperature and incubated with D274. Sections were then washed with TBS-0.01% tween 4x5 minutes, 3 times, and incubated with Donkey anti Rabbit Alexa-fluor 647 and Anti Occludin. Antibodies for experiments are outline in Table 7.
Table 7. Antibodies for experiments Protein Antibody Species Reactivity Dilution Occludin Invitrogen™ Alexa Fluor® 488, Clone: OC-3F10 Human, Mouse IF: 1:500 D274 Rockland Immunochemicals Human, Mouse IF: 1:500 DAPI/Hoechst Thermo Fisher: 62249 Human, Mouse IF: 1:10,000
RNA Extraction and Analysis
Mouse tissues were snap frozen in Eppendorf tubes and stored at -80°C until time of extraction.
Tissues were ground to a fine powder at -80°C, resuspended in 1ml Trizol reagent, thoroughly mixed and incubated for 5 minutes at 4°C. Samples were then loaded to a 5Prime Phase Lock
Gel (Quantabio, Item No. NC1093153; Beverly, MA), incubated for 5 minutes at room temperature, then spun for 5 minutes, at 4°C, at 12,000RPM. The aqueous layer of the sample was collected and added to 0.500 ml of ice cold isopropanol. Samples were placed at -20°C for
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1 hour and spun for 5 minutes at 4°C at 12,000RPM. The supernatant was removed, and the pellet was washed with 70% Ethanol made with RNase free water. Samples were spun again at
5 minutes, at 4°C, at 12,000RPM. The pellet was washed a second time with 70% ethanol and spun at 7500RPM for 5 minutes at 4°C. RNA pellets were allowed to air dry for 45 minutes and resuspended in RNase free water. RNA concentration was calculated using a Nano-drop and the 260/280 ratios were determined for each samples. For RT/PCR, samples with 260/280 ratios near 2.0 were converted to cDNA using the Qiagen RT-PCR Kit (Item No. 205313;
Qiagen, Germantown MD), according to the manufacturer‟s instructions. Upon conversion to cDNA, samples were diluted 1:1 with nuclease free water. GAPDH primers were used as a positive control in the RT-PCR reactions to confirm the integrity of the cDNA. All primers used in experiments are outline in Table 8.
Table 8. Mouse Primers for PCR Gene Species Application Sequence TAAR1 Mouse genotyping 5'- CAGTTAAAATAACCTTGGGAGTATTC -3' 5'- CAATGCTTCTCCAGAACTCATAGTC -3' Neomycin resistance 5'- ATCGCCTTCTTGACGAGTTC -3' TAAR1 Mouse q/PCR 5‟- GGGTACTGGCGTTCATGACT -3‟ 5‟- CGCCCACCATGATCCCTAAG -3‟ GAPDH Mouse q/PCR 5‟- CCCAGGGACCTCTCTCTAATC -3‟ 5‟- ATGGGCTACAGGCTTGTCACT -3‟
Tissue Staining: Hematoxylin & Eosin
Five micron sections were taken from Swiss rolled colon. Tissue sections were stained with hematoxylin as previously described (Wu & Gussoni, 2011) and images were captured on a
KEYENCE, BZ-X710 All-in-one fluorescence microscope.
Tissue Staining: Alcian Blue
Five micron sections were cut from Swiss rolled colon, deparaffinized, and cleared in distilled water. Sections were then soaked in Alcian-blue staining solution (Item no TMS010C, Thermo
Fisher, Waltham, MA) for 30 minutes, rinsed for 2 minutes in distilled water, and counter stained with nuclear fast red (Catalog no. R5463200500, Thermo Fisher, Waltham, MA). Sections were Page 104
dehydrated and cover slipped with Cytoseal-60 (Catalog no. 23-244257, Thermo Fisher
Scientific, Waltham, MA).
Quantification Techniques
All values for intestinal length were measured using Fiji is just ImageJ (FIJI) and normalized to the metric ruler in each image. Goblet cell count and crypt height were analyzed with FIJI, using the ObjectJ plug-in.
DSS-induced colitis and scoring
Sterile powdered DSS was dissolved in autoclaved drinking water by dissolving DSS powder until a clear solution is reached to a final concentration of 3% wt /volume (MW 36,000–50,000).
100ml of DSS water was administered at a time, and replenished with freshly prepared DSS containing water every 2 days. DSS water was volumetrically measured to monitor intake of
DSS. Mice were treated with DSS for a maximum of 10 days. On day 10 of the experiment, mice will be euthanized by CO2 asphyxiation followed by cervical dislocation. Mice were assessed daily for pathology and onset of colitis as outlined in Figure 28 (D. H. Kim & Cheon,
2017). Scoring was performed only by KBG, and images of mice were captured periodically for comparison between days.
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Figure 33. Score sheet for the assessment of mice with intestinal pathology in DSS- Induced Colitis.
US Biomax Tissue Array
Consecutive sections of human colon tissue array CO245 were purchased from US Biomax.
Sections were deparaffinized, and immunostained as described above. Quantification of fluorescent intensity was performed using FIJI software, and histogram normalized to %area of the DAPI stain.
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Chapter 5: Conclusions and future studies
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5A. Dissertation Discussion
The primary focus of TAAR1 research since its deorphanization in 2001 has been on its role as a monoamine neuromodulator. To date, the work in this dissertation is the only study that explores the presence and/or a functional role of TAAR1 in the colon. The data generated in this dissertation provides evidence that TAAR1 may be functionally relevant in both epithelial cells and macrophage homeostatic mechanisms in the colon mucosa. Current emphases from funding foundations such as the Crohn‟s and Colitis Foundation aim to identify a pharmacological target for environmental triggers of UC. In this regard, TYR levels are reported to be elevated in fecal content of patients with UC suggesting a high level of colonic tyramine.
The trace amine TYR is historically reported to exist in the body at picomolar levels, which is intriguing because TAAR1 requires nanomolar levels to elicit a response. Emerging evidence suggests TYR levels in the GIT are much higher than in other body systems and perhaps the phrasing “trace amine” is a misnomer.
There is a general acceptance in the TAAR1 field that there are no commercially available antibodies that can be used with reliability and specificity in mouse systems. Because of this, understandings in peripheral localization and subcellular expression remains poorly understood in mouse systems. Proteomics analysis of the mTAAR family reveals that there is significant homology between mTAAR1 and mTAARs 2-9, and further analysis identified the potential for cross reactivity of commercially available tools and mTAARs 2-9 (Chapter 1). The same issues with antibody specificity and reactivity have not been observed, and proteomics analysis of the hTAAR family members reveals that there is more divergence in protein sequence than is seen in mouse. To overcome the obstacle of mTAAR homology, a custom antibody was designed against the third extracellular loop of the human TAAR1 sequence, corresponding to amino acids 274-290. Bioinformatics analysis suggests low sequence homology against other proteins in NCBI protein blast (data not shown) in both mouse and human, as well as low sequence homology against mTAAR and hTAAR family members (Chapter 1). Furthermore, validation of
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the antibody in vitro (Chapter 1) and eventually in vivo (Chapter 4) enables further studies into
TAAR1 peripheral localization and eventually functional relevance in body systems.
Using D274, hTAAR1 was successfully identified in human epithelial cell lines including
HT-29 and CACO-2 cells. Expression of TAAR1 in HT-29 cells was validated by qRT-PCR as well as Western blot analysis with the commercially available antibody from Imgenex that has previously been validated by the Miller lab (Panas et al., 2012). Identification of TAAR1 in intestine epithelial cell lines was exciting as TYR was previously studied and revealed to have functional relevance in HT-29 cells (Del Rio et al., 2017; Linares et al., 2016). To further understand the roles that TYR and TAAR1 have in these intestinal epithelial cell lines, mRNA and protein levels of TAAR1 as well as the TJPs were studies in response to tyramine and the
TAAR1 specific partial agonist RO5263397 (Chapter 2). In line with previous reports (e.g.
(Sriram et al., 2016), TYR stimulation of HT-29 cells resulted in upregulation of TAAR1 mRNA and protein, a phenomenon that was not observed with partial agonist treatment. Agonist mediated upregulation of TAAR1 is not fully understood in any body system, though in the GIT could bear functional relevance as a chemosensor for immune cell activation as previously implied (Babusyte et al., 2013; Panas et al., 2012), microbes or altered monoamine levels. This apparent function of TAAR1 in intestinal epithelial cells revealed new experimental strategies to understand how elevated levels of trace amines (Santoru et al., 2017) may contribute to UC pathogenesis or progression. A hallmark of UC is the so-called “leaky-gut,” a controversial misnomer that is used to describe the breakdown of the GIT epithelium in disease states. This breakdown of the GIT epithelium is in part due to the dysregulation of TJP expression and localization at the cellular membrane (Figure 4), leading to progression of inflammation in the underlying immune cells (Figure 3). To understand if TAAR1 has a role in the regulation of
TJPs, both mRNA and protein expression were analyzed in response to TYR and RO5263397 treatment of HT-29 cells. TYR appears to upregulate ZO-1 mRNA and protein, and has discordant effects on the expression of occludin and claudin-1 mRNA and protein expression in these cells. These discordant levels of occludin and claudin-1 may be an artifact of the
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experimental system, or may suggest that TAAR1 is not involved in the regulation of protein expression in these cells, at least acutely in an in vitro model. Despite an inconclusive effect of
TYR on occludin and claudin-1 expression, quantification of monolayer health and subsequent functional assays reveal an interesting phenomenon: both TYR and RO5263397 appear to disrupt the monolayer integrity and increase permeability. RO5263397 and other TAAR1 partial agonists are reported to have paradoxical agonist activity in vivo, which may account for the unexpected response of the functional assays to TAAR1 ligands. My data provides the first evidence of a receptor mediated mechanism for TYR in intestine epithelial cells, and provides a new impetus for investigating TAAR1 as a therapeutic target for attenuating dysregulation of
TJPs in the GIT.
In addition to dysregulation of TJPs in the GIT, a hallmark of ulcerative colitis is macrophage-mediated inflammatory cascades potentiating chronic inflammation of the colon.
TYR has demonstrably elicited chemotaxis from a subset of human immune cells in a TAAR1- dependent fashion (Babusyte et al., 2013). To understand the mechanism by which macrophages interacting with TYR in the lumen may propagate inflammatory cascades, an ex vivo model using mouse BMDM was explored. Ex vivo stimulation of BMDM with TYR augments IL-6, IL-1β, and TNF-α inflammatory cytokine gene expression. Similarly to HT-29 cells, TYR stimulation of BMDM upregulates TAAR1 expression and co-stimulation by TYR and
LPS elicits a significant upregulation of TAAR1 expression. A limitation in TAAR1 research is that the only available TAAR1 antagonist, EPPTB, is mouse specific. Accordingly, the use of
BMDM from C57BL/6 mice permits the use of a pharmacological antagonist to TAAR1.
Pretreatment of BMDM with EPPTB attenuated TAAR1 gene expression upregulation, as well as IL-6, IL-1β, and TNF-α gene expression upregulation. Inhibition of proinflammatory cytokine gene expression in BMDM makes EPPTB and other TAAR1 antagonists, once developed, useful for assessing TAAR1 mediation of localized immunosuppression in the GIT. EPPTB has a poor pharmacokinetic profile(Bradaia et al., 2009), rendering its use for in vivo studies targeting the central nervous system impractical; however, based on the ex vivo model of
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inflammatory actions of TAAR1 in BMDM, use of EPPTB in studying GIT inflammation in vivo may be remarkable. Studies ex vivo in BMDM provide some evidence for TAAR1 being a potential pharmacological target that may mediate inflammation in UC. Novel drugs with poor absorption such as EPPTB may offer physicians and patients a new and suitable treatment avenue devoid of systemic immunosuppression.
In the last chapter of this study, effects of TAAR1 in the GIT were explored in vivo, comparing C57BL/6 and TAAR1-/- intestinal morphology and responsivity in an in vivo colitis model. Previous reports characterizing the TAAR1-/- mouse report that there is no overt phenotype (Lindemann et al 2008), though these initial reports excluded intestinal phenotypes.
To characterize the expression of TAAR1 in C57BL/6 intestine, colon sections were isolated from C57BL/6 and TAAR1-/- littermates. Immunostaining with D274 produced a specific staining in the epithelial cells near the lumen of the colon, the tips of the villi in the small intestine, and near the glandular cells deep in the crypts of the small intestine. Further characterization of the
TAAR1-/- colon sections revealed morphological differences in crypt height, crypt width, goblet cell count and goblet cell size. The huge variation in the sizes and numbers of goblet cells observed in the TAAR1-/- tissue could be attributed to a proliferative defect, migratory defect in the terminally differentiating cells, or a differentiation defect in stem cells. Despite these morphological differences, there was no overt inflammatory phenotype which provided the basis for studying the onset and progression of acute DSS-induced colitis. Surprisingly, TAAR1-/- mice appear to be protected from the pathological features of DSS, retaining crypt height in non- affected regions, colon length, body weight, and in inflammatory lesions, demonstrating dilation of crypts, incomplete ablation of goblet cells, and signs of healing. The attenuation of colitis in
TAAR1-/- mice begs the question: is there substantial evidence that TAAR1 is involved in the onset and progression of UC? The attenuation of colitis in TAAR1-/- mice could very well be attributed to a loss of a „microbial‟ sensor in the GIT and a retained epithelial barrier, though there are no signs of a retained epithelial barrier histologically. TAAR1 expression in C57BL/6 mice could also be upregulated by the inflammatory signals from resident immune cells in the
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GIT. Identifying co-localization of macrophage markers, such as IBA-1 and TAAR1 in the GIT is proving challenging as the robust macrophage markers are typically raised in rabbit, whereas the D274 antibody is also raised in rabbit. Consecutive sections prove challenging as well as the standard thickness (5μM) of the section may lead to the loss of the immune cell, or TAAR1 staining in the consecutive sections. Attempts to identify the cell type that TAAR1 is localized in using occludin have been inconclusive as well, and further investigation of the literature suggests that occludin is differentially expressed in macrophages (Van den Bossche et al.,
2012).
Figure 34. TAAR1 co-localization with occludin in DSS-induced colitis lesion in C57BL/6 colitis lesions
Finally, the finding that humans express TAAR1 in their epithelium is remarkable and has yet to be described. Furthermore, the identification of elevated TAAR1 expression levels in the epithelial cells in UC biopsies could have major implications. First, it may suggest that
TAAR1 may mechanistically be involved in the onset and progression of disease, and second, that TAAR1 may be upregulated by microbial components as indicated in vitro in Chapter 2. Page 113
Finally, previous data generated from the Miller lab (Panas et al., 2012), as well as others
(Sriram et al., 2016) suggests that inflammatory modulators (e.g. PHA) can significantly upregulate TAAR1 expression. This phenomenon has not yet been described in epithelial cells though the effect may be the same. Further, this work is also significant as Hoffman LaRoche and Synovian Pharmaceutials has led the development of novel TAAR1-targetted drugs for the treatment of schizophrenia, which are now in clinical trials. These drugs may inadvertently carry the potential to remit (or exacerbate) UC flares, a property that insofar may be unrecognized.
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5B. Future studies Taken together, this dissertation reveals preclinical data suggestive of a novel mechanism for propagation of UC. This dissertation reveals a new avenue to identify pharmacological compounds for combating UC, and potentially a broad spectrum of gastrointestinal disorders. Still, while the work in this dissertation is promising, it has not fully validated TAAR1 as a therapeutic target for UC. To properly identify TAAR1 as a viable therapeutic target, work with human populations should be systematically explored on the RNA and protein level. Biopsies from colonoscopies in patients with UC and samples deemed healthy should be explored for altered TAAR1 expression. Additionally, intestine stem cells can be isolated from human patients to monitor the effects of hTAAR1 on epithelial cell health ex vivo to understand the effects of TYR on human primary cells. Additionally, the apparent
„proliferative‟ defects observed in the TAAR1-/- intestine suggest TAAR1 may not be a viable therapeutic target, as TAAR1 may be involved in normal differentiation and morphological organization of the GIT, making the observed attenuation of colitis in vivo an artifact of a much greater phenotype.
Much work remains to successfully understand the role of TAAR1 in the GIT and elucidate its function as a novel therapeutic target. Additional studies to further understand the effects of TYR in HT-29 cells could prove beneficial. While HT-29 cells tend to be used for IBD studies in vitro, effects of TAAR1 activation in colorectal cancer would be beneficial.
Preliminary studies on the effects of
TYR on HT-29 cell division with an Figure 35. MTT Proliferation Assay in HT-29 Cells. HT-29 cells were treated with 1μM TYR or vehicle for 24 MTT assay suggest that TYR activation hours. After 24 hours, cell counts were determined. Pilot study, n=1 replicate, 11 samples per group. Student’s t-test, p<0.05. of TAAR1 may decrease proliferation of
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cells (Figure 35). A recent metaanalysis by Fleischer et al suggests that higher expression of
TAAR1 correlates to a longer median survival for colorectal cancer. This phenomenon remains unexplored.
Additional studies can also be performed on HT-29 cells to elucidate the role of TAAR1 in IBD. There is potential to explore the use of CRISPR to knockdown TAAR1 in HT-29 cells.
Alternatively, ex vivo mouse or human stem cells can be isolated from the intestine and grown in organoids, or in transwell differentiation models (Puzan et al., 2018) to monitor the effects of
TAAR1 in primary cell models.
Immunological studies are another promising avenue to pursue in the future. In addition to the effects of TYR on the M1 phenotype that is intimately described in Chapter 3, the effects of TYR on the myriad of M2 macrophages can provide insight into the immunological functions of TAAR1 in macrophage polarization and regulation. My ongoing studies with collaborators at
The University of Groningen are aiming to understand the effects of TYR in immune cell polarization in human monocytes.
Based on previous reports of TYR in immune cells provoking a chemotactic response in
T-cells and B-cells, preliminary studies were conducted to assess the chemotactic ability of
C57BL/6 and TAAR1-/- BMDM to TYR, compared to the chemotactic factor N-Formylmethionyl- leucyl-phenylalanine (fMLP). C57BL/6
BMDM demonstrate a normal chemotactic Chemotaxis of BMDM to TYR
15 n.s. C57BL/6 response 10-fold increase in migration to ** *** TAAR1 -/- ** n.s. fMLP compared to vehicle, whereas TYR 10 n.s. * appears to be a moderate macrophage 5
chemoattractant with a 5-fold increase in to Vehicle change Fold 0 chemotaxis over vehicle (Figure 36). The fMLP TYR fMLP TYR vehicle vehicle chemotactic response of TAAR1-/- BMDM C57BL/6 TAAR1-/- Figure 36. Transwell chemotaxis assay. C57BL/6 -/- was also assessed to fMLP and tyramine, and TAAR1 chemotaxis to fMLP, TYR and vehicle. (n=3, Two-way ANOVA.) and the TAAR1-/- mice have a significant
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defect in chemotactic responses to fMLP compared to C57BL/6. This preliminary data is supportive of the hypothesis outlined in Figure 27, as TYR is a chemoattractant for C57BL/6
BMDM. Still, the lack of responsivity of TAAR1-/- BMDM to fMLP has not been fully explored or explained. The apparent defect of normal immune cell function in the TAAR1-/- immune macrophages could be suggestive of a compensatory mechanism of the immune cells, a defect in differentiation, or an innate immunological function of TAAR1 in macrophages that has yet to be fully described. Future studies can utilize the TAAR1-/- BMDM to elucidate the role of TAAR1 in normal macrophage function that may be affected by its absence. Experiments in RNA-seq of C57BL/6 compared to TAAR1-/- BMDM could provide a starting point for this investigation.
Finally, perhaps the most interesting avenue for exploration is the function of TAAR1 in the GIT. Preliminary data generated in Chapter 4 is suggestive of a role of TAAR1 in proliferation or differentiation of the GIT. The preliminary data in HT-29 cells (Figure 35) suggests that TYR can decrease proliferation in an iEC cell line. In the TAAR1-/- mice, the attenuation of colitis may be an artifact of a defect in proliferation or differentiation. To further study this, TAAR1-/- intestine stem cells can be isolated and cultured ex vivo, and compared to littermate controls for their ability to differentiate in vitro, monitoring signaling cascades common to differentiation (e.g. Wnt signaling), or even proliferation assays to monitor division.
Alternatively, immunostaining of the C57BL/6 and TAAR1-/- healthy intestine may provide insight to the presence or absence of a proliferative zone in the intestines. Experiments looking at Ki-
67 expression, a hallmark of dividing cells, or intestinal differentiation markers (e.g. CDX1,
KRT20 (Chan et al., 2009) can also be monitored in C57BL/6 and TAAR1-/- tissue sections.
Alternatively to protein analysis of tissue sections, RNA-seq data may provide insight to any defects in differentiation seen in the TAAR1-/- mice.
To understand the role of the microbiota in the regulation of TAAR1 expression and activation, it would be prudent to study TAAR1 expression in germ free mice or specific pathogen free mice (SPF). Interestingly, the TAAR1-/- mouse resembles a germ free mouse phenotype, as seen with the crypt architecture and apparent enlargement of the cecum and the
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intestine. Future studies will involve the comparison of TAAR1-/- mice to germ free mice, as well as a comparison of the „conventionalization‟ of germ free mice to the addition of TYR and other trace amines to the diet of germ free mice. Similar studies can be conducted in SPF mice.
Some datasets in NCBI GeoData suggest a low level of TAAR1 expression in germ free and
SPF mice, though the conflicting detection of TAAR1 in RNA-seq data may be confounding these effects.
Given the noteworthy conservation between TAAR family members in the mouse (Chapter
1), a complete knock-down of the entire TAAR family should be explored for future in vivo experiments. Despite the design of a custom antibody to mTAAR1, there may be functional relevance of mTAARs 2-9 that has yet to be fully described. To fully understand the function of the TAARs in the GIT, a promoter driven KD of the TAAR family could be explored for GIT tissue, or for specific immune cell populations. Generation of these specific mouse lines and mouse models would provide valid evidence for TAAR function in peripheral systems, a pressing need that has yet to be met.
5C. Concluding Remarks
The work reported in this dissertation aimed to further the knowledge of peripheral
TAAR1 functionality in mouse BMDM and intestine, and characterize its expression and function in peripheral systems. My data reveals that TAAR1 has an unappreciated role in the regulation of homeostasis in the GIT, providing strong evidence that TAAR1 is a microbial sensor in the
GIT. The chapters in this thesis further our understanding of the so-called „elusive trace amines‟ and their role in normal human physiology. The emergence of fecal metabolomic studies has classified trace amine levels at physiologically relevant levels for the first time. With the identification of TAAR1 expression in the GIT, epithelium, and immune cells, there presents a great opportunity to further study complex mechanisms of microbial influence on GIT development and immune cell maturation as it relates to the „hygiene hypothesis‟ for allergies and immunological disorders. Further, the work identifies TAAR1 as a much-needed novel therapeutic drug target to be further investigated for the treatment of GI disorders.
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