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The gut brain axis : impact of dietary fiber on a murine model of multiple sclerosis Robinson, Hannah 2019

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THE GUT BRAIN AXIS: IMPACT OF DIETARY FIBER ON A MURINE MODEL OF MULTIPLE SCLEROSIS  by  Hannah Robinson  B.Sc., The University of California, Santa Barbara, 2016  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  MASTER OF SCIENCE in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Microbiology and Immunology)   THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)   April 2019   © Hannah Robinson, 2019  ii The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, a thesis/dissertation entitled:  The Gut Brain Axis: Impact of Dietary Fibers on a Murine Model of Multiple Sclerosis   submitted by Hannah Robinson in partial fulfillment of the requirements for the degree of Master of Science in Microbiology and Immunology  Examining Committee: Dr. Lisa Osborne Supervisor  Dr. Marc Horwitz Supervisory Committee Member  Dr. Sean Crowe Supervisory Committee Member  Additional Examiner   Additional Supervisory Committee Members:  Supervisory Committee Member  Supervisory Committee Member   iii Abstract  Multiple Sclerosis (MS) is a chronic autoimmune disease of the central nervous system (CNS) that causes demyelination of neurons, neurodegeneration and progressive disability. The exact cause of Multiple Sclerosis remains unknown however, susceptibility to MS is influenced by genetics and environmental factors, such as diet. As zero-fiber diets have been associated with exacerbated disease in inflammatory disease models, we investigated dietary fiber’s impact on the murine model of MS, experimental autoimmune encephalomyelitis (EAE). We demonstrated that standard fiber diets (5%) do not offer protection against EAE when compared to zero-fiber diets, whereas a diet high in the soluble fiber, guar gum (30%), inhibited disease progression and prevented lymphocytic CNS infiltration. Other soluble fibers: pectin, resistant starch and inulin did not offer the same protection – providing evidence that the types of dietary fiber have differential effects on the immune system and neuroinflammation.    iv Lay Summary  Dietary fiber, a key dietary component that is indigestible by humans, is processed by microbes living in our intestine. Microbial digestion of fiber leads to production of molecules that have been shown to promote the development and/or function of anti-inflammatory immune cells. Furthermore, dietary fiber has been linked to beneficial roles in a variety of  inflammatory diseases, however its role in multiple sclerosis (MS) – an autoimmune disease which results in neurodegeneration of the central nervous system (CNS) – remains unknown. I investigated whether a zero-fiber diet would exacerbate disease in a murine model of MS and no differences were observed in severity of disease. However, when I used a diet that contained supraphysiological amounts of the dietary fiber guar gum, it was observed that guar gum offered protection from paralysis and immune cell infiltration into the CNS.  Therefore, fiber’s role – specifically guar gums – in disease in MS patients should be investigated.    v Preface All experimental protocols were approved by the Animal Care Committee of UBC (A15-0122 and A17-0037) and experiments performed in accordance with guidelines set by Canadian Council for Animal Care.  Biosafety protocol B15-0113 was approved by UBC’s Biosafety Committee.  Figures from Chapter 1 were modified from a literature review in preparation for submission: Impact of environmental influences on the microbiome and autoimmune neuroinflammation by HG Robinson, NM Fettig & LC Osborne. A version of Figures 3.1-3.7 from Chapter 3 were modified from a primary research article in preparation for submission: Impact of Dietary Soluble Fiber on Autoimmune Neuroinflammation by HG Robinson, R Simister, JR Allanach, AJ Sharon, BK Hardman, NM Fettig, N Saleh, K Doshi, S Crowley, BA Vallance, MS Horwitz, S Crowe & LC Osborne.  I will be primary author and will write the manuscripts. For the research article, I conceived of the project with LC Osborne and conducted data collection and analysis for all in vivo experiments, with technical support from JR Allanach, AJ Sharon, BK Hardman, NM Fettig and N Saleh. Sample preparation was performed by R Simister and sequenced at the Pharmacological Sciences core facilities at UBC. Sequence analysis was performed by R Simister, K Doshi and myself. S Crowley and BA Vallance provided technical expertise for analysis of colonic mucus widths.        vi Table of Contents  Abstract .................................................................................................................................... iii Lay Summary ............................................................................................................................ iv Preface ....................................................................................................................................... v Table of Contents ...................................................................................................................... vi List of Tables ........................................................................................................................... viii List of Figures ........................................................................................................................... ix List of Symbols ........................................................................................................................... x List of Abbreviations ................................................................................................................. xi Acknowledgements ................................................................................................................... xv Chapter 1: Introduction ........................................................................................................ 1 1.1 Multiple Sclerosis .................................................................................................... 1 1.2 EAE: a murine model of human MS ........................................................................ 2 1.3 Immune cells and pathways involved in EAE and MS ............................................. 3 1.4 The microbiota in MS .............................................................................................. 7 1.5 Probiotics and EAE/MS ......................................................................................... 10 1.6 Types of dietary fiber and their implications in the pathogenesis of EAE ............... 13 1.7 Hypothesis ............................................................................................................. 15 Chapter 2: Methods ............................................................................................................ 16   vii 2.1 Mouse strains, housing and diets ............................................................................ 16 2.2 Experimental autoimmune encephalomyelitis model .............................................. 16 2.3 Tissue and single cell preparation .......................................................................... 17 2.4 Cell stimulation and staining .................................................................................. 18 2.5 Flow cytometry ...................................................................................................... 19 2.6 Histology ............................................................................................................... 19 2.7 RNA extraction and cDNA synthesis ..................................................................... 19 2.8 Gene expression measured by qPCR ...................................................................... 20 2.9 16S sequencing ...................................................................................................... 20 Chapter 3: Results ............................................................................................................... 24 3.1 Zero-fiber diets do not exacerbate disease when compared to standard fiber diets .. 24 3.2 Guar gum offers ameliorative properties against EAE disease development ........... 30 3.3 The impact of dietary soluble fibers on the gut ....................................................... 41 Chapter 4: Discussion ......................................................................................................... 46 References ................................................................................................................................ 50 Appendix – Diet specifications ................................................................................................. 65     viii List of Tables Table 1.1 Microbial populations altered in MS patients and their inflammatory implications. ...... 9 Table 1.2 Impact of probiotics on the pathogenesis of EAE in murine models and multiple sclerosis. ................................................................................................................................... 12 Table 2.1 Buffers ...................................................................................................................... 21 Table 2.2: Antibodies ................................................................................................................ 23    ix List of Figures Figure 1.1: Immune cell pathways in the exacerbation and protection against neurodegeneration in murine EAE. ........................................................................................................................... 4 Figure 3.1: Zero-fiber diets did not exacerbate EAE disease. ..................................................... 25 Figure 3.2: Standard-fiber diets did alter EAE associated T cells. .............................................. 27 Figure 3.3: Zero-fiber diet impacts intestinal microbiota and mucus .......................................... 29 Figure 3.4: Supraphysiologic doses of guar gum offers protection against EAE. ........................ 32 Figure 3.5: Supraphysiologic doses of guar gum prevents T cell CNS infiltration. ..................... 34 Figure 3.6: Supraphysiologic doses of guar gum does not alter splenic T cell activation or adhesion molecules. .................................................................................................................. 36 Figure 3.7: Supraphysiologic amounts of soluble fibers cause a shift in the gut microbiome. ..... 40 Figure 3.8: The impact of soluble fibers on the gut. ................................................................... 44    x List of Symbols α  Alpha   β Beta γ Gamma µ Micron % Percent I One II Two   xi List of Abbreviations ACC  Animal care committee ACK  Ammonium-Chloride-Potassium BBB  Blood brain barrier BFA  Brefeldin A BREG  Regulatory B cell CD  Cluster of differentiation cDNA  Complementary deoxyribonucleic acid CFA  Complete Freud’s adjuvant CDM  Center for Disease Modeling CHS  Clinical health score CNS  Central nervous system CTL  Cytotoxic T lymphocyte DC  Dendritic cell DNA  Deoxyribonucleic acid DMEM Dulbecco's Modified Eagle Medium EAE  Experimental autoimmune encephalomyelitis EDTA  Ethylenediaminetetraacetic acid ELISA  Enzyme-linked immunosorbent assay FACS  Flow cytometry FBS  Fetal bovine serum   FcR  Fc receptor FITC  Fluorescein Isothiocyanate   xii Fp  Fecal pellet Foxp3  Forkhead box P3 GM-CSF Granulocyte-macrophage colony-stimulating factor GPR  G-coupled protein receptor HEPES Hydroxyethyl piperazineethanesulfonic acid HLA  Human Leukocyte Antigen IFA  Incomplete Freud’s adjuvant IFN  Interferon Ig  Immunoglobulin  IL  Interlukin LCFA  Long chain fatty acid LFA  Lymphocyte function-associated antigen Ly6C  Lymphocyte Ag 6C Ly6G  lymphocyte Ag 6G M1  Pro-inflammatory macrophage  M2  Anti-inflammatory macrophage MAPK14 Mitogen-activated protein kinase 14 MBF  Modified Barrier Facility MCP  Monocyte chemoattractant protein MFI  Mean fluorescence intensity MHC  Major histocompatibility complex MHI  Mental health inventory MIP  Macrophage inflammatory protein   xiii MNV  Murine norovirus MOG  Myelin oligodendrocyte glycoprotein MS  Multiple sclerosis NCS  Neo-natal calf serum NK  Natural killer PBS  Phosphate buffered saline PB  Pacific blue PE-Cy7 Phycoerythrin-Cyanine7 PE  Phycoerythrin PCS  Physical component summary PGE  Prostaglandin E PMA  Phorbol 12-myristate 13-acetate RPMI  Roswell Park Memorial Institute RNA  Ribonucleic acid RRMS  Relapse-remitting multiple sclerosis SCFA  Short chain fatty acid Sp.  species SPMS  Secondary progressive multiple sclerosis TCR  T cell receptor TGF  Transforming growth factor TH1  T helper 1 cell TH17  T helper 17 cell TH2  T helper 2 cell   xiv TNF  Tumor necrosis factor TREG  Regulatory T cell UBC  University of British Columbia VLA  Very late antigen   xv Acknowledgements  I’d like to thank the University of British Columbia, facility staff and funding sources for making this research project possible. I owe my gratitude to Dr. Lisa Osborne, whose patience and support made my Master’s degree a compelling experience and has inspired me to further my education. I appreciate my committee members Dr. Sean Crowe and Dr. Marc Horwitz, whose feedback has propelled me to be a better scientist. To my lab mates from the Osborne, Horwitz and Crowe lab, thank you for all your help with my experiments – the size of them require a team to complete them and you are the best team that I could have asked for. To my fellow UBC students and friends, I am thankful for your support and community. I will always remember the joyous memories made from softball pitches to adventures in the mountains.  1 Chapter 1: Introduction  1.1 Multiple Sclerosis Multiple Sclerosis (MS) is a chronic autoimmune disease characterized by neurodegeneration due to immune-mediated degradation of the myelin sheath surrounding neuronal axons of the central nervous system (CNS).  This damage leads to improper neuronal signaling and a various range of motor and/or cognitive side effects, such as impaired balance, vision, learning and long-term memory1–3. Relapsing and remitting MS (RRMS) is the most common form of disease and presents as alternating periods of symptoms and remission4.  A less common form of MS, primary progressive, presents as a continuous increase in symptoms with no remission5.  RRMS patients can develop secondary progressive MS (SPMS) where periods of remission disappear and neurodegeneration continues without reprieve6. Although MS remains incurable, a number of treatment options exist that can limit progression, reduce symptoms and/or increase quality of life for people living with MS. Supporting the hypothesis that MS is an immune-mediated disease, the majority of these treatment options either ablate immune cells or interrupt signaling pathways necessary for immune function.  Susceptibility to MS is influenced by both genetic and environmental factors, including diet, infection history, and geographic location7–9.  Recently, the gut microbiome, the collection of viruses, archaea, bacteria, fungi and protists that inhabit the intestines has been linked to both disease progression and prevention10,11.  Multiple studies have observed that adult and pediatric MS patients have microbial dysbiosis11–13, with certain species similarly reduced in other inflammatory conditions, such as inflammatory bowel disease14,15.  Although each study concludes that MS patient microbiomes are distinct from healthy controls, there are few   2 consistencies in the types of microbes that have been found that differ between control and MS subjects. It remains unknown whether these changes are causative or occur in response to disease development. Because the microbiome regulates the immune system16, these data provoke the hypothesis that microbial community composition and function may alter the immune system and contribute to either MS susceptibility or progression.  1.2 EAE: a murine model of human MS There are many ways to model neuroinflammation that mimics MS in mice, including experimental autoimmune encephalomyelitis (EAE), coronavirus-induced demyelination disease17, adoptive T cell transfer18 (passive disease) and toxic demyelination upon cuprizone administration19. These models, along with human studies, indicate that both innate and adaptive immune cell lineages are involved in neurodegeneration.  My thesis uses EAE, which induces myelin-specific autoimmunity by administration of myelin-derived peptides or proteins along with an adjuvant and reagents that help bypass the blood brain barrier (BBB)20,21.   Neuroinflammation in EAE is associated with changes in multiple immune cell types and their functions. The innate immune cells involved in disease include macrophages, dendritic cells and NK cells. Both T and B cells of the adaptive immune system contribute to disease. The common thread between both immune categories is that there are pro-inflammatory and anti-inflammatory versions of these cells that either contribute to the pathogenesis or protection against disease22,23. In the following sections, we will investigate these cell types’ roles in EAE and Multiple Sclerosis in more detail.     3 1.3 Immune cells and pathways involved in EAE and MS 1.3.1 B cells Early studies of the role of immune cells in MS implicated plasma cells (antibody secreting B cells) as major contributors to disease pathogenesis due to autoantibodies found in brain lesions24 and myelin-specific antibodies present in the cerebral spinal fluid of MS patients25. In addition, plasma cells have been shown to induce demyelination via the complement system26,27 and may contribute to disease via interactions with FcRs on pro-inflammatory microglia28. However, recent clinical trials have demonstrated that treatment with Rituximab (a-CD20), a reagent that depletes immature and mature B cells but leaves antibody-producing plasma B cells intact can reduce CNS lesions in MS patients. These data indicate that although some populations of B cells contribute to disease progression, autoantibody-secreting plasma cells are not the prime pathogenic B cell subset in MS29. Notably, recent data suggests that IL-10+ plasma cells producing commensal-specific IgA were found in the CNS in the context of neuroinflammation and were associated with protection from disease30.  Independent of antibody production, B cells influence disease pathogenesis through antigen presentation and cytokine secretion. Using a mouse model where MHCII was deleted specifically in B cells, one study determined that B cell-intrinsic MHCII antigen presentation was essential for a B cell dependent EAE disease progression31. Furthermore, B cell subsets have been categorized as either protective or pathogenic, defined by their cytokine secretion profile, where B cells secreting IL-6 or GM-CSF have been shown to be pathogenic32–34, but secretion of IL-10 or IL-35 is associated with protection32,35–37 (Fig 1.1).  A subset of IL-10-producing B cells called regulatory B (BREG) cells are protective in EAE45.    4  Figure 1.1: Immune cell pathways in the exacerbation and protection against neurodegeneration in murine EAE.  Cytokine-mediated influence of B cells, T cells and several innate cells involved in disease pathogenesis. Pro-inflammatory pathways are denoted by arrowheads, anti-inflammatory/myelin-protective pathways are denoted by the inhibitor lines.  1.3.2 T cells The cell type most commonly associated with EAE and MS are T cells (CD3+ lymphocytes): they are the disease drivers in MOG35-55 induced EAE and the cell type used to passively induce EAE. The main T cell subsets that modulate disease pathogenesis in T cell-mediated EAE are CD4+ T helper type 1 (TH1) cells (identified by IFNγ production), CD4+ T   5 helper type 17 (TH17) cells (identified by IL-17 production), and cytotoxic CD8+ T lymphocytes (CTLs)38–41, which exacerbate disease, and regulatory T cells (TREG) cells (identified by Foxp3 transcription) limit disease severity44.  Adoptive T cell transfers of myelin specific TH1 and TH17 cells have been used to determine the relative contributions of each cell type in the development of EAE42,43. Although it remains unclear whether TH1 cells are sufficient to induce disease, they contribute to the development of neuroinflammation by facilitating the translocation of TH17 cells across the blood brain barrier in an IFNγ-dependent manner42–45. In contrast, TH17 cell adoptive transfer can consistently initiate disease, suggesting that TH17 cells are the main T cell subset involved in promoting EAE development. Similarly, in MS, TH17 cells are consistently elevated in MS patients and are thought to be important contributors to disease progression46.  The role of  TH1 cells in MS patients remains unclear as one study observed similar amounts in healthy controls46 but an independent study found that TH1 cells from MS patients were more likely to be MOG-specific47,48. Further, reports of highly inflammatory IFNγ-producing TH17 cells isolated from MS patients indicate that these cells have reduced gene expression of anti-inflammatory IL-10, which serves to counteract the production and inflammatory effects of IL-649. Along with the toxicity of IL-17 producing T cells, CD8 T cells and GM-CSF expressing helper T cells have been linked to disease pathogenesis in both murine EAE and MS patients50–54, in which downstream effects of GM-CSF have the potential to regulate of DCs55 and macrophages56,57.  TREG cells function as the main T cell subset responsible for reducing the inflammatory effects of effector T cells58. Additionally, natural killer T (NK-T) cells have been shown to protect against EAE through dampening TH1 cell cytokine secretion and shifting the immune system to a TH2 response59,60. MS patients have demonstrated reduced TREG cell function, characterized by a decreased ability to prevent effector cell proliferation in vitro61,62,   6 indicating that immune dysregulation in MS occurs on both pro-inflammatory and anti-inflammatory fronts.   1.3.3 Macrophages, dendritic and NK cells Just as adaptive immune cells have dual role in EAE, macrophages/microglia and dendritic cells (DCs) can be proinflammatory mediators of disease and anti-inflammatory inhibitors of disease63.  The macrophage pro-inflammatory (M1) and anti-inflammatory (M2) phenotypes resemble the DC subsets, determined by cytokine secretion profiles, however the terminology for these DC subsets have yet to be established. DCs contribute to pathogenicity in EAE through antigen presentation and cytokine production. Similar to B cells, CD11c+ DCs play a role in disease pathogenesis through antigen presentation. Using a T cell dependent model, restricting MHCII expression to DCs was sufficient to elicit EAE disease and allow blood-brain barrier penetration. Both B cells and DCs play an integral role in models that rely on different cell types, which cell types more better represent the pathogenesis of neurodegeneration in MS patients is still up for debate31,64. Supporting a role for DCs in the initiation of autoimmune neuroinflammation, DCs are known to  promote differentiation of TH17 cells in vivo through trans-presentation of IL-665. Furthermore, IL-6 deficient mice were resistant to EAE induction and had decreased numbers of infiltrating macrophages and CNS-resident microglia66. Moreover, one study administered the MS drug resveratrol to mice induced with EAE and observed there was a decrease in IL-6 expression in macrophages67,68, potentially reducing disease via this mechanism. In both DCs and M1 macrophages, IL-6 plays a pivotal role in disease pathogenesis. The homeostasis of M1/M2 cells is important in disease severity – an increased abundance of M1 macrophages promoted paralysis relapses in SJL mice with induced EAE, while M2   7 macrophages induced recovery69. Moreover, IL-10 secreting DCs70,71, M2 macrophages and microglia can promote remyelination72,73. Another innate immune cell type involved in disease are NK cells, which are thought to be protective in EAE, however conflicting data makes their role in disease pathogenesis unclear and an avenue for further exploration74,75. Each innate and adaptive immune cell type has a subset analogous to each other in cytokine secretion profiles with similar roles in disease progression.  This suggests that the specific cell type has less of an effect on disease than their downstream cytokine production.   1.4 The microbiota in MS The bacteria that occupy a host’s external surfaces, including the skin, gut, lung and urogenital tract – have been linked to susceptibility to inflammatory diseases76–78. In MS patients, the relationship between the host microbiota and disease has not been fully uncovered. However, several studies have uncovered that MS patients have altered microbiomes compared to healthy  controls11,12,79,80 (Table 1.1). In one study, MS patients’ gut microbiomes had altered genera within the following phylums: increased Bacteroidetes: Pedobacter and Flavobacterium; decreased Bacteroidetes: Parabacteroides; increased Firmicutes: Blautia and Dorea; decreased Actinobacteria: Adlercreutzia and Collinsella; increased Proteobacteria: Mycoplana and Pseudomonas and decreased Proteobacteria: Haemophilus12. In other studies, the genera Methanobrevibacter and Akkermansia were increased while Butyricimonas was reduced in MS patients11,13.  The inconsistent microbial differences in MS patients across studies could potentially be due to the different locations, diets, sample processing, taxonomic database and other parametes81,82. Whether the altered microbial populations in the MS gut microbiome contribute to disease or are a response to a dysregulated immune system remains unknown,   8 however correlations to other diseases could provide some clues. Faecalibacterium, Prevotella, Lachnospiraceae, and Akkermansia, which are altered in MS patients, are similarly affected in the neurodegenerative disease, Parkinson’s disease. These microbial similarities could suggest that the inflammatory and/or metabolic conditions in Parkinson’s and MS patients are similar and lead to an analogous change in the gut ecosystem that allows these bacteria population dynamics to change.  Alternatively, it could suggest that people with these microbiome profiles predispose them to either condition. Evidence for the latter is that Akkermansia (and Methanobrevibacter) was positively correlated with a gene associated with neuronal apoptosis in Parkinson’s disease (MAPK14) , whereas Butyricimonas was negatively associated11,83. Despite microbial consistencies between Parkinson’s disease and MS, their compositions of Blautia, Dorea and Ruminococcaceae differ. While the significance of the microbiome in MS remains unclear, its role in EAE is better established – in broad-spectrum oral antibiotic treated and germ-free conditions, mice were resistant to EAE development and autoimmune demyelination was significantly reduced84–86. Microbe Population change in RRMS patients Immunological Implications Associations in other diseases Pedobacter Increased  ---- Increased in Vitamin D cell receptor knock out mice87  Increased in children with Autism88 Reduced in colorectal cancer patients89 Mycoplana Increased  ---- ---- Blautia Increased Butyrate (Treg and M2 inducer) producing microbe in mice16,90,91. Increase in Blautia hydrogenotorophica and a decrease in Blautia wexlerae in obese patients92  Genera and species Blautia glucerasea decreased in Parkinson’s patients93,94  Decreased in children with autism88  Decreased Blautia hansenii in Crohn’s disease patients95   Dorea Increased ---- Increased in Trichuris suis infection96  Decreased in Parkinson’s patients93,94  Reduced in patients with food sensitization97 Pseudomonas Increased  ---- Increased in children with Autism98 Methanobrevibacter Increased ---- Methanobrevibacter smithii decreased in obesity, increased in anorexia99   9 Akkermansia Increased ---- Akkermansia muciniphila, used as a probiotic, improved metabolic health in obese patients100  Increased in Parkinson’s patients93  Increased in children with Autism98 Butyricimonas Decreased Butyrate (TREG cells and M2 inducer) producing microbe in mice16,90,91. Increased in Vitamin D cell receptor knock out mice87 Collinsella Decreased ---- Increased on colorectal cancer tissues101  Increased in symptomatic atherosclerosis patients102 Slackia Decreased Slackia equolifaciens sp. nov. can produce antioxidants that can induce apoptosis in tumor cells101,103,104 Increased on colorectal cancer tissues101 Prevotella Decreased ---- Decreased in Parkinson’s patients94  Increased in Vitamin D cell receptor knock out mice87  Reduced in autistic children105  Prevotella copri increases susceptibility to arthritis106 Faecalibacterium Decreased (vitamin D insufficient)80 Butyrate (TREG cells and M2 inducer) producing microbe in mice16,90,91. Decreased in Parkinson’s patients94  Decreased in children with Autism98  Decreased Faecalibacterium prausnitzii in Crohn’s disease and ulcerative colitis patients95,107  Increased in allergic asthma patients108 Desulfovibrionaceae  Increased (pediatric) ---- Increased abundance in mice with impaired glucose intolerance109 Lachnospiraceae Decreased (pediatric) ---- Decreased in Parkinson’s patients93  Reduced in patients with colorectal cancer110 Ruminococcaceae Decreased (pediatric) ---- Decreased Ruminococcus callidus and increased Ruminococcus bromii in Parkinson’s patients94  Increased Ruminococcus lactaris in rheumatoid arthritis patients111  Decreased in children with Autism98  Decreased Ruminococcus gnavus  and Ruminococcus torques in Crohn’s disease patients95 Clostridia XIVa and IV Clusters112 Decreased Induces colonic TREG cells113,114 Clostridium leptum decreased in patients with Crohn’s disease and ulcerative colitis107  Clostridium is reduced in patients with food sensitization97 Bacteroides112 Decreased Bacteroides fragilis increased TREG cells and IL-10 productiton115 Decreased Bacteroides vulgatus and Bacteroides caccae in Crohn’s disease patients95   Table 1.1 Microbial populations altered in MS patients and their inflammatory implications.    10 1.5 Probiotics and EAE/MS Probiotics have demonstrated therapeutic potential in both neuroinflammatory murine and human studies. In murine models, administration of Lacotobacillus and Bifidobacterium can reduce EAE clinical scores, however the efficacy of probiotic treatment depends on the specific species and strain10,116,117 (Table 1.2). Specifically, probiotic administration of Lactobacillus plantarum DSM 15312 and L. paracasei DSM 13434 were able to reduce EAE clinical scores and lower the percentage of CD4 T cell infiltration into the CNS.  Additionally, L. paracasei DSM 13434 increased levels of IL-10 and the TH2 associated cytokine, IL-4116. Moreover, mice induced with EAE and gavaged with the probiotic mixture of the Lactobacillus strains L. paracasei DSM 13434 and L. plantarum DSM 15312/15313 had lower clinical scores that were associated with reduced levels of IL-17 in the CNS and IFNg in the spleen (ELISA of MOG35-55 stimulated spleen supernatant), as well as an increased frequency IL-10+ TREG cells in the CNS116. Furthermore, a probiotic mixture containing L. casei, L. acidophilus, L. reuteri, B. bifidum, and Streptococcus thermophilus decreased EAE clinical scores and increased IL-10 and IL-4-producing T cells118. In contrast, a probiotic mixture used in an EAE guinea pig model, composed of B. breve and L. casei did not impact disease pathogenesis117. The efficacy of probiotic administration was demonstrated in a rat EAE model, which revealed that probiotic administration of Enterococcus faecium strain L-3 ameliorated disease to the same extent as the MS drug glatiramer acetate119,120. Furthermore, probiotic administration of Bacteroides fragilis reduced EAE disease via microbial metabolite polysaccharide A and increased IL-10 production from Foxp3+ TREG cells115. Collectively, these murine studies demonstrate that probiotics, depending on the species and strain, are effective at dampening EAE clinical scores.   11 In one small human trial investigating the efficiency of probiotic administration in MS patients, a combination of L. acidophilus, L. casei, B. bifidum and L. fermentum decreased the Expanded Disability Status Scale (a scoring system of disease in MS patients)121. Another small MS patient study found that taking a probiotic mixture consisting of Lactobacillus, Bifidobacterium and Streptococcus (L. paracasei DSM 24734, L. plantarum DSM 24730, L. acidophilus DSM 24735, L. delbruckei DSM 24734, B. longum DSM 24736, B. infantis DSM 24737, B. breve DSM 24732, and S. thermophilus DSM 24731) for two months decreased the relative abundance of disease-associated genera Akkermansia and Blautia, and reduced peripheral monocytes122.  Human trials have yielded promising data, however cohort sizes have been relatively small and have thus made drawing firm conclusions difficult. Studies with larger and geographically distinct cohorts would help determine the benefits of probiotics.  Probiotic treatment Clinical Scores Immunological impact Inducing agent Disease host EAE animal models L. paracasei DSM13434116 Decrease in clinical scores    IL-4, IL-10 and TGF-β1 secretion from T cells in vitro    Percentage of CD4+ T cells in CNS and IFNγ and TNFα secretion from  T cells in vitro MOG35-55  C57/BL6J mice L. plantarum DSM1512116 Decrease in clinical scores    TREG in mesenteric lymph nodes     MOG35-55 C57/BL6J mice L. plantarum DSM1513116 Decrease in clinical scores    IL-27 in blood serum       Percentage of CD4+ T cells in CNS MOG35-55 C57/BL6J mice L. paracasei DSM13434 and L. plantarum DSM1512 + DSM1513116 Decrease in clinical scores   Spleen culture IL-10 secretion in vitro, Foxp3 expression in cerebellum and percentage of Foxp3+CD4+ T cells in MLNs and spleens   Spleen culture IFN-γ, TNF-α and IL17 secretion in vitro MOG35-55 C57/BL6J mice L. casei, L.acidophilus L reuteni, B. bifidum, S. thermophius118 Decrease in disease incidence and clinical scores   IL-4 and IL-10 secretion from T cells in vitro   Lymphocyte and T cell infiltration, percentage of Gr1+ MOG35-55 C57/BL6J mice   12  and/or CD11b+ monocytes and CD4+ T cells in the spinal cord and IFNγ, TNFα and IL-17 secretion from T cells in vitro E. coli MG1655119 Decrease in incidence ---- MOG35-55 C57/BL6J male mice E. coli Nissle 1917119 Decrease in clinical scores   Number of peripheral MOG-specific T cells and draining LN Foxp3+CD4+ T cells and IL-10 secretion from T cells in vitro   Number of spinal MOG-specific T cells and IFNγ, GM-CSF, IL-17 and TNFα secretion from T cells in vitro MOG35-55 C57/BL6J male mice Bacteroides fragilis115 Decrease   Percentage of Foxp3+ regulatory T cells in cervical LNs and expression of: GATA-3, IL-10, SMAD-3 in the brain   Expression of RORγt and IL-17 in the brain PLP139-151 SJL mice Enterococus faecium L-3120 Decrease   Percentage of: CD3+, CD3+CD8a+ in blood peripheral MOG-specific T cells   Percentage of CD3-CD161a+ cells in blood (161a activates leukocyte effector functions) and spinal MOG-specific T cells Encephalitogenic mixture Wistar rats B. breve Yakult and L.casei Shirota117 No effect ---- Spinal cord homogenate or MBP LEW/CrlCrl rats and guinea pig (Slc:Hartley)  Multiple Sclerosis patients  L. acidophilus, L. casei, B. bifidum, L. fermontum121 Decrease in   EDSS    C-reactive protein, serum insulin ---- RRMS patients Lactobacillus, Bifidobacterium, Streptococcus122 Not provided   Peripheral monocytes, mean CD80 MFI on classical monocytes, HLA-D MFI on DCs, MS risk HLA-DQA1 allele expression  ---- RRMS patients  Table 1.2 Impact of probiotics on the pathogenesis of EAE in murine models and multiple sclerosis.    13 1.6 Types of dietary fiber and their implications in the pathogenesis of EAE 1.6.1 Types of dietary fiber Dietary fiber is a carbohydrate polymer that in indigestible by the host, and is rather processed by the host’s microbiome. It is often used as a broad term, however there many different types of fiber, split into four main categories: water soluble and highly fermentable fibers, intermediately soluble and fermentable, insoluble and slowly fermentable and insoluble and not readily fermentable123. The main water soluble and highly fermentable fibers: inulin, guar gum, pectin and resistant starch are degraded by microbial enzymes to produce short chain fatty acids (SCFAs) such as acetate, propionate and butyrate. These SCFAs are recognized by the G-protein coupled host cell receptors GPR41, GPR43 and GPR109A. GPR41 is expressed on polymorphonuclear and dendritic cells and has affinity for propionate>butyrate>acetate. GPR43 is expressed on polymorphonuclear cells, monocytes and mast cells and responds equally to acetate, butyrate and propionate. Finally, the butyrate receptor GPR109A is expressed on DCs, macrophages and epithelial cells124,125. Interactions with these receptors can lead to downstream immunological modulation; for example, SCFAs can increase TREG cells  in a GPR43  dependent manor – demonstrating the potential of SCFAs to dampen inflammatory conditions126. When present in the diet, fermentable fibers can influence SCFA levels locally in the colon and systemically127. In a study where patient fecal samples were isolated and cultured under anaerobic conditions, supplementation with either resistant starch, inulin, pectin or guar gum was sufficient to increase acetate production128. However, under the same conditions butyrate production was only increased upon supplementation with guar gum or inulin and propionate production only increased with guar gum supplementation128. Thus, in healthy patient microbiome cultures, guar gum was able to have the most diverse impact on SCFA production.   14 However in vivo, several studies have demonstrated that a 30% pectin diet increases propionate127,129.  This discrepancy could be potentially due to microbial differences or the lack of biofeedback from the host in the in vitro study. As for guar gum, in vivo, it was able to increase all three SCFAs, reflecting the in vitro data130. In contrast to the water-soluble fibers, the water-insoluble fiber cellulose is broken down into long chain fatty acids (LCFAs)131. The ability of dietary fiber to alter immune modulatory molecules demonstrates its importance in immune health and its potential as a supplementary therapy to dampen inflammatory diseases.  1.6.2 Dietary fiber and EAE Recently, many studies have explored diet as a prospective source of disease amelioration. An EAE study demonstrated diets consisting of supraphysiologic amounts of pectin, a water soluble and fermentable dietary fiber, reduced disease severity in mice. However, the mechanism of protection was not elucidated129,131. Moreover, diets supplemented with cellulose, a water insoluble fiber, protected mice from EAE in a spontaneous murine MS model by shifting the mice’s immune system towards TH2 immunity via LCFAs131. As dietary fibers are only digested by the gut microbiome, fiber’s capability to influence disease deepens the tie between the microbiome and disease – determining whether protection is due to disease ameliorating SCFAs162 or another microbial metabolite is a promising field to explore.  Differences in the effectiveness of dietary alterations between murine models and human studies demonstrates the importance of translating EAE ameliorative conditions to MS patients to see if the dietary intervention can remain efficacious and improve quality of life.    15 1.7 Hypothesis The overarching hypothesis of my thesis is that dietary soluble fiber plays a protective role in the murine MS model (EAE), however the extra-intestinal nature of EAE requires supraphysiologic amounts of fiber.  I aimed to determine the effects of soluble fiber on EAE induced murine cohorts. First, I tested the hypothesis that suboptimal levels of dietary fiber exacerbate EAE pathogenesis. To do this, mice were fed diets containing no fiber (0%), a low-fiber diet that mimics the average Canadian fiber diets (2.5%), or a standard fiber diet (5%) that mimics the fiber concentrations recommended by nutritionists14. Then, I tested the hypothesis that supraphysiologic amounts of soluble, fermentable fibers (30% pectin, guar gum, inulin or resistant starch) can limit EAE pathogenesis. In all studies, I monitored clinical scores following EAE induction. At harvest, I analyzed the TH1, TH17 and TREG compositions in spleens, brains and spinal cords. Finally, I analyzed fecal samples and colon sections to determine how distinct dietary fibers impact microbiota community composition and gut structure. Collectively, information gained from this research program provided a better understanding of how fiber impacts the immune system in the context of neuroinflammation.    16 Chapter 2: Methods 2.1 Mouse strains, housing and diets Mice were housed at the University of Columbia (UBC) in the Center for Disease Modeling (CDM) which is a pathogen and murine norovirus-free facility in nonexperimental mice.  C57BL/6 mice (from 6-12 weeks old) were either bred in the facility or ordered from Jackson Laboratories. Female mice and bedding were rotated between cages to maintain a similar basal microbiome.  For experiments using male mice, only bedding was exchanged between cages.  Mice were kept on a 12 hour day/night cycle. Experimental protocols were approved by the UBC ACC and the Biosafety Committee. Diets of 0% dietary fiber 9GKZ, 2.3% dietary fiber 9GKY (1:1 cellulose and guar gum), 5%  dietary fiber (1:1 cellulose/guar gum) 9GQP and 5% fiber (cellulose) 57W5 and 30% dietary fibers of inulin, guar gum, resistant starch and pectin (5BX1, 5BSE, 5BAC and 5BSX, respectively) were ordered from Test Diet. Mice fed 30% guar gum diets received special care – food pellets were wet daily/bi-daily in a petri dish or placed in hydrogel to encourage weight stabilization as if this step was excluded, mice lost significant weight. Diets were isocaloric and contained similar levels of macro- and micronutrients other than fiber to ensure that differences observed during disease were due to the change in dietary fiber. Diet specs can be found in Appendix A.  Mice were fed diets two weeks prior to intervention and throughout the remainder of each experiment.  2.2 Experimental autoimmune encephalomyelitis model 2.2.1 EAE induction Two weeks after dietary intervention, mice received a 100µL rear subcutaneous injection of an emulsion containing 200µg neuro-antigen MOG35-55 (specific to C57BL/6 mice)   17 (GenScript, catalog #: RP10245) resuspended in 1mL PBS and 400µg of non-viable M. tuberculosis H37 Ra (BD Difco™ Adjuvants) resuspended in 1.1mL of incomplete Freud’s adjuvant (IFA) (BD Biosciences, catalog #: DF0639606), making complete Freud’s adjuvant (CFA). Mice were also given a 200µL intraperitoneal injection of 200ng of pertussis toxin resuspended in PBS on the day of induction and a second injection of the same reagents, 48 hours later132. 2.2.2 Murine scoring Mice were scored on a 5 point scale132 with 0 = no paralysis, 0.5 = partial tail tonicity lost, 1 = complete tail tonicity lost, 2 = loss in coordinated movement/hind limb paresis, 2.5 = one hind limb paralyzed. 3 = both hind limbs paralyzed, 3.5 = weakness in forearms, 4 = forelimbs paralyzed and 5 = moribund. Mice in high fiber diet experiments were scored for stool consistency: 0 = normal fecal pellet, 1 = no fecal pellet/softer than normal stool, 2 = soft stool and 3 = diarrhea. Cumulative scores were calculated by adding the daily scores for each individual mouse from onset of disease to endpoint.  2.2.3 Murine monitoring Mice were weighed and scored for EAE every 24-72 hours after being placed on the diet up until day 6 post EAE induction, then mice were monitored daily. Once mice reached a CHS or EAE score of 2, food pellets and hydrogel were placed on the cage floor.   2.3 Tissue and single cell preparation Cardiovascular perfusion was performed with at least 15 mL of sterile 4°C PBS (Sigma-Aldrich, catalog #: D8537) immediately after sacrifice. Brains, spinal cords and spleens were extracted for either flow cytometry or histology. For histological analysis, brains and spinal   18 cords were kept in 10% formaldehyde and paraffin embedded, sectioned at stained by Wax-It (UBC). For flow cytometric analysis, brains and spinal cords were placed in PBS or FACs buffer minus EDTA (buffer recipes can be found in Table 2.1) and stored on ice.  Brains, spinal cords and spleens were mashed through a 70 µm nylon mesh filters (Falcon, catalog #: CA21008-952) to create single cell suspensions. Spleens were subjected to ACK lysis buffer to remove red blood cells. Brain and spinal cord leukocytes were enriched by Percoll gradient centrifugation. Single cells were suspended in 40% Percoll (Sigma-Aldrich, catalog #: 17-0891-01) (see Table 2.1)  and spun with no deceleration for 15 min at 1400 rpm. The SN and fat layer were vacuumed off and the remaining leukocytes washed prior to further analysis. 2.4 Cell stimulation and staining  2-4 million cells from spleens and the entire sample of brains and spinal cords were resuspended in 300µL simulation media (1:1000 BFA (Sigma-Aldrich, catalog #: B6542-5MG), 1:1500 Golgi stop (BD Bioscience, catalog #: 554724), 1:1000 PMA (Sigma-Aldrich, catalog #: P8139) and 1:1000 ionomycin (final concentration of 1ng/mL) (Sigma-Aldrich, catalog #: I0634)) for 3-5 hours at 37°C in a 96 well round bottom plate.  After stimulation, cells were washed in PBS and resuspended in 100µL live/dead stain at 1:600 (Thermo Fisher, catalog #: L34957) in PBS for 15 minutes at 4°C in the dark.  Cells were washed 1.5 times with PBS and resuspended in the surface stain (1:200-300 antibodies in FACs buffer) for 30 minutes in the dark at 4°C.  Cells were washed 1.5 times with FACs buffer and resuspended in Fix/perm solution (Thermo Fisher: eBioscience, catalog #: 00-5521-00) and kept at 4°C for 20-30 minutes.  Cells were washed 1.5 times in perm wash buffer (1:10 dilution of perm was concentrate: dH2O) (BD Biosciences, catalog #: 554723) and resuspended in the intracellular stain (1:100 antibodies in perm wash buffer) for 30 minutes at room temperature (see Table 2.2 for antibodies). Cells   19 were washed 1.5 times in in perm wash buffer and resuspended in 250 µL of FACs buffer prior to flow cytometric analysis. 2.5 Flow cytometry Stained samples were run on a BD LSR II equipped with four lasers (BD Bioscience – blue (488nm), yellow/green (561nm), violet (405nm) and red (633nm)). FlowJo (version 10.1r3, Tree Star Inc.) was used during data analysis, in which all dead cells (positive for live/dead stain) were excluded from analysis.  2.6 Histology 2.6.1 Alcian blue and PAS staining  After extraction (colonic slices were taken from the distal colon – cuts were made around the second to last fecal pellet – still intact), tissues were kept in methacarn for 4-24 hours and then sent to Wax-It for paraffin embedding and staining133. Horizonal cross-sections 5 µm thick through the fecal pellet were taken in duplicate for each sample, 10 µm apart. 2.6.2 Mucus measurements Slides obtained from Wax-it were imaged on a ZEISS Axio Observer using ZEN 2.3 software. Mucus widths were measured with the ‘line’ tool – three measurements were taken per colonic slice (two slices per sample)134,135, resulting in a total of 6 measurements per mouse. The average of these 6 measurements are presented as a representation of mucus width per mouse.  2.7 RNA extraction and cDNA synthesis Colon tissues from the proximal colon (the second cm below the cecum) that were stored in RNAlater (Thermo Fisher, catalog #: AM7021) were thawed and added to 600µL of β-mercaptoethanol and TissueLyser II (1:100) (Qiagen, catalog #: 85300) and homogenized with beads at a frequency of 30beats/second for 6 minutes. RNA was extracted using Ambion   20 PureLinkTM RNA Mini Kit, according to the protocol and cDNA synthesized using SuperscriptII reverse transcriptase and random hexamers (Life Technologies, catalog #: N8080127).  2.8 Gene expression measured by qPCR Gene expression was measured using PowerUp SYBR Green Master Mix and the supplier’s protocol was followed (Applied Biosystems) on a Quant Studio 3 Real-Time PCR System.  The gene expression of TNF-α (QuantiTech: Mm_Tnf_1_SG) was measured and normalized to hprt (QuantiTech: Mm_Hprt_1_SG).  2.9 16S sequencing All fecal pellets were stored on dry ice and then at -80°C for long-term storage. RNA from fecal pellets was extracted as per protocol from the DNeasy PowerSoil Kit (Qiagen, catalog #: 12888-100). Purity for each sample and negative controls was checked by nanodrop.  PCR quantification was performed by Rachel Simister from the Crowe Lab, UBC. Samples were sent for 16S rRNA gene profiling down to the genera level. SOBS (observed richness - # of OTUs) were calculated using the estimated OTUs. Data analysis was done in collaboration with Rachel Simister using the Mothur progam136.   Buffer Components Wash buffer 10mL of Neo-natal calf serum (NCS) in DMEM or RPMI Complete tissue culture media (CTCM) 450 mL of RPMI or DMEM 50 mL of 100% Fetal bovine serum (FBS) 5 mL of 200µM L-Glutamine 5 mL of 5000U/mL Penicillin-streptomycin 12.5 mL of 1M HEPES  500 µL of 55mM β-mercaptoethanol FACs buffer 500 mL of PBS 20 mL of 100% neonatal calf serum (NCS) 2 mL of 500mM EDTA ACK lysis buffer 1L dH2O 8.29 g of NH4Cl 1 g KHCO3   21 0.0367 g of EDTA 40% Percoll For 150mLs: 6 mL 10x PBS 54 mL Percoll 90 mL serum-free media MacConkey agar plates 1 L of  dH2O 50g of MacConkey agar 10g of Difco™ Granulated agar Mathacarn 60% methanol  30% chloroform  10% glacial acetic acid Table 2.1 Buffers     Antibody Channel Supplier/catalog Concentration IL-17a APC  APC eBioscience™, catalog #: 17-7177-81 1/100 Foxp3 (Ax700) Ax700 eBioscience™, catalog #: 56-5773-82 1/100 CD44 APC-Cy7 Biolegend, catalog #: 103028 1/300 CD3 Pacific Blue eBioscience™, catalog #: 48-0033-82 1/300 CD19 PE-Cy7 eBioscience™, catalog #: 25-0193-82 1/300 CD8α PE Texas red Invitrogen, catalog #: MCD0817 1/300 CD25 PE eBioscience™, catalog #: 12-0251-82 1/300   22 IFNγ PerCP Cy5.5 eBioscience™, catalog #: 45-7311-82 1/100 IL-17a  PE Texas red Biolegend, catalog #: 506938 1/100 Foxp3  Ax 488 eBioscience™, catalog #: 53-5773-82 1/100 CD45.2 Ax700 Biolegend, catalog #: 109822 1/300 CD3 PerCP Cy5.5 Biolegend, catalog #: 100218 1/300 B220 APC-Cy7 eBioscience™, catalog #: 47-0452-82 1/300 CD8α e650 BD Horizon™, catalog #: 563234 1/300 CD4 PE-Cy7 eBioscience™, catalog #: 25-0041-82 1/300 IFNγ PE Biolegend, catalog #: 505808 1/100 IL-10 APC Biolegend, catalog #: 505010 1/100 TCR-β PerCP Cy5.5 Biolegend, catalog #: 109228 1/300 IFNγ Pacific Blue eBioscience™, catalog #: 48-7311-82 1/100 CD11c e650 eBioscience™, catalog #: 64-0114-82 1/300 CD11b PE Texas red Biolegend, catalog #: 101256 1/300 CD103 FITC eBioscience™, catalog #: 11-1031-82 1/300   23 Ly6G Pacific Blue BD Horizon™, catalog #: 560603 1/300 Ly6C PE-Cy7 BD Pharmingen™ catalog #: 560593 1/300 NK1.1 PE eBioscience™, catalog #: 12-5941-63 1/300  Table 2.2: Antibodies   24 Chapter 3: Results  3.1 Zero-fiber diets do not exacerbate disease when compared to standard fiber diets 3.1.1 Zero-free diets displayed no significant differences in disease severity Canadians have one of the highest rates of MS and on average do not meet the fiber consumption recommendations, however the impact of this nutritional deficit on MS susceptibility remains unclear137,138. Furthermore, past literature has demonstrated that zero-fiber diets exacerbated inflammatory conditions127,135. Therefore, we sought to test the hypothesis that zero-fiber diets would lead to exacerbated disease in the murine model of MS, EAE.  To test this hypothesis, we selected diets mimicking the recommended amounts of fiber for Canadians according to the Government of Canada138 (5%), the average amount of fiber that Canadians actually consume (2.5%) and zero-fiber diets. To determine whether the type of dietary fiber differentially impacts disease, I used two standard fiber diets that contained either cellulose, a non-readily fermented fiber, or a mixture of cellulose and the highly fermentable fiber guar gum.  Mice were fed diets consisting of 5% cellulose (standard), 5% cellulose and guar gum (1:1) (standard C+G) , 2.3% cellulose and guar gum (low-fiber) (1:1) and 0% fiber (zero-fiber) diets two weeks prior to EAE induction and throughout the course of disease. Mice were induced by a subcutaneous injection containing 400µg of M. tuberculosis and 200µg MOG35-55 peptide. Mice also received an intraperitoneal injection of 200ng of pertussis toxin on day 0 and day 2 to permeabilize the BBB.  Disease severity in the context of ascending paralysis was measured for 15-25 days, with clinical scores in our facility starting to appear between days 6-9 post-induction. No significant differences in disease paralysis were observed across standard, standard C+G,  low-fiber and zero-fiber diets. Clinical scores and disease incidence were recorded from   25 five separate experiments (the standard cellulose diet was used in 4 experiments and standard cellulose and guar gum diet was used in 2 with a total n=5, however results were consistent so no additional repeats were performed). Clinical scores and the percentage of mice that developed disease were pooled together and no differences were observed (Fig 3.1A-B). Experiments were performed in two facilities to investigate whether the diets would have differential effects in another facility with a more diverse microbiota, however the data remained consistent. These data demonstrate that zero-fiber diets displayed no beneficial or detrimental effects on disease onset, severity or progression when compared to diets mimicking the recommended and actual consumption of dietary fiber in Canadian populations.  Figure 3.1: Zero-fiber diets did not exacerbate EAE disease.  (A) Mice fed diets two weeks prior to induction, and then monitored for levels of paralysis. (B) The fraction of mice that developed disease each day post induction was compiled from five independent experiments.   A BEAE incidence (%)25050751005 10 15 20 2505 10 15 20 250EAE clinical score1023455% Cellulose and Guar gum5% Cellulose2.3% Cellulose and Guar gum0% FiberDays post immunization Days post immunization  26 3.1.2 Immune populations in the CNS and periphery were unaffected by low- or zero-fiber diets Even though no differences in paralysis was observed, we wanted to address whether the lack of fiber impacted EAE pathogenic immune populations. Mice from 3.1.1 that were fed either standard cellulose, standard cellulose and guar gum, low fiber or zero fiber diets and induced with EAE were sacrificed on day 15-25 post induction and brains, spinal cords and spleens were extracted for tissue processing and staining (some tissues were taken for histology instead of immune cell analysis). Processed tissues were stained with antibodies, run through a flow cytometer and analyzed to determine the cell counts and frequencies of pathogenic IFN- γ (TH1) and IL-17a (TH17) producing CD4+ T cells, as well as the protective CD4+ Foxp3+ TREG cells. Across all diets, no differences in CD4+ T cell frequencies or counts were observed in the brain or spinal cord (Fig 3.2A-C). In addition to quantifying the total numbers of pathogenic and protective cells in the tissues, we wanted to investigate cellular differentiation and function, using transcription factor expression and cytokine production, to determine if the individual cells from zero-fiber diet fed mice had a more pathogenic phenotype.  To do so, an analysis of expression level per cell was performed. However, the mean fluorescence intensity (MFI) of  Foxp3, IFNγ, IL-17a in TREG, TH1 and TH17 cell subsets were consistent between standard, low and zero-fiber diet treated mice (Fig 3.2C). These data are consistent with the lack of differences in clinical scores between diets, demonstrating that a lack of dietary fiber does not significantly influence disease scores or the priming or function of immune cells involved in EAE pathogenesis.    27  Figure 3.2: Standard-fiber diets did alter EAE associated T cells. (A-C) T cell profiles from EAE mice were analyzed in spinal cord and brain tissues through Ab staining and flow cytometry. The following antibodies were used: CD3 to isolate T cells, CD4 to select T helper cells and either IFNγ, IL-17a or Foxp3 to identify the T cell as a TH1, TH17, or TREG cell, respectively. Statistics were calculated using one-way ANOVA. ACB5% Cellulose + Guar gum 5% Cellulose2.3% Cellulose + Guar gum 0% fiberSpinal Cord# of IFNɣ+ cells102103104105102103104105101 102103104# of IL-17a+ cells# of Foxp3+ cells02040608001020300102030Freq of IFNɣ+ cellsFreq of IIL-17a+cellsFreq of Foxp3+cellsBrainCD4+ CD3+ T cellsCD4IFNɣIL-17aFoxp3Brain29.6 +/- 16.325.3 +/- 5.932.7 +/- 8.6 26.8 +/- 8.17.8 +/- 1.9 8.9 +/- 6.7 14.6 +/- 10.1 9.4 +/- 6.622.3 +/- 7.3 10.3 +/- 9.815.0 +/- 9.916.0 +/- 10.4MFI 5,067 MFI 8,589 MFI 8,299 MFI 9,836MFI 6,945 MFI 12,080 MFI 9,340 MFI 8,936MFI 1,984 MFI 1,887 MFI 1,839 MFI 1,624TH1 TH17 TREGFigure 1: Standard-fiber diets did not protect mice from EAE A) Mice fed diets two weeks prior to induction, and then monitored for levels of paralysis: 1– tail paralysis, 2 – one hind limb paralyzed, 3 – both hind limbs paralyzed, 4 – front and hind limb paralysis, 5 – moribund. c) T c ll profiles from EAE mice were analyzed in brain tissues through flow cytometry and staining of CD3 to isolate T cells, CD4 to select for T helper cells and either IFN-γ, IL-1a or Foxp3 to identify the T cell as Th1, Th17 or Treg cell, respectively.   28  3.1.3 Microbiome differences between standard- and zero-fiber diet fed mice  As dietary fiber modulates the microbiome135, we hypothesized our diets containing varying amounts and types of fiber would significantly alter the microbial populations in the gut. We took fecal samples from mice before and after dietary intervention, along with samples after disease progression to determine whether dietary fiber altered the gut microbiome in naïve and EAE conditions. 16S rRNA gene profiling was performed to profile the microbial community down to the genera level. Samples from naïve and EAE mice fed the four diets were plotted on an ordination (NMDS) plot that used rank orders to visualize community differences in reduced dimensions. No obvious differences were seen across diets on the NMDS plot (Fig 3.3A), however when pulling out the standard C+G and zero-fiber diet fed mice, it became apparent that these two diets clustered away from each other (Fig 3.3B). This suggests that there was a difference in their core microbiomes. In the absence of fiber, mucus becomes the main food source for microbes (thinning the mucus layer) and consequently microbes that can utilize mucus as a food source gain a competitive advantage in the gut and thus increase in frequency135. As expected, in naïve mice the relative abundance of Akkermansia – a genus containing a mucus consuming species – increased when fed a zero-fiber diet (Fig 3.3C-D). This was consistent with a thinner mucus layer in mice fed a zero-fiber diet compared to controls receiving a standard-fiber diet (Fig 3.3E). Collectively, these results indicate that our zero-fiber diets impacted the gut microbiome in naïve conditions yet failed to alter the clinical or immunological outcomes following EAE induction.      29  Figure 3.3: Zero-fiber diet impacts intestinal microbiota and mucus. (A-B) The non-metric multidimensional scaling (NMDS) axis were used to plot the location of each naïve and EAE induced mouse sample.  The lowest stress for the NMDS axis is 0.0754833 and R2 for 10020304050Relative % 16S rRNA read abundanceGenera: Akkermansia5% Cellulose0% Fiber-0.2 0.2 0.4 0.6 0.8-0.20.4-0.4-0.3-0.10.10.20.3Ordination plotAxis 1Axis 2ADBOrdination plotAxis 1Axis 2Pre-diet Post-diet-0.2 0.2 0.4 0.6 0.8-0.20.4-0.4-0.3-0.10.10.20.3*manuscript_PCA naives+takedown dayNaive 0% Naive 2.3% Cellulose + Guar gumNaive 5% celluose Naive 5% Cellulose + Guar gumEAE 0% EAE 2.3% Cellulose + Guar gumEAE 5% celluose EAE 5% Cellulose + Guar gummanuscript_PCA naives+takedown dayNaive 0% Naive 2.3% Cellulose + Guar gumNaive 5% celluose Naive 5% Cellulose + Guar gumEAE 0% EAE 2.3% Cellulose + Guar gumEAE 5% celluose EAE 5% Cellulose + Guar gum5% Cellulose + guar gum5% Cellulose2.3% Cellulose + guar gum0% FiberNaïve EAEmanuscript_PCA naives+takedown dayNaive 0% Naive 2.3% Cellulose + Guar gumNaive 5% celluose Naive 5% Cellulose + Guar gumEAE 0% EAE 2.3% Cellulose + Guar gumEAE 5% celluose EAE 5% Cellulose + Guar gummanuscript_PCA naives+takedown dayNaive 0% Naive 2.3% Cellulose + Guar gumNaive 5% celluose Naive 5% Cellulose + Guar gumEAE 0% EAE 2.3% Cellulose + Guar gumEAE 5% celluose EAE 5% Cellulose + Guar gum5% Cellulose + guar gum0% FiberNaïve EAEmanuscript_PCA naives+takedown dayNaive 0% Naive 2.3% Cellulose + Guar gumNaive 5% celluose Naive 5% Cellulose + Guar gumEAE 0% EAE 2.3% Cellulose + Guar gumEAE 5% celluose EAE 5% Cellulose + Guar gummanuscript_PCA naives+takedown dayNaive 0% Naive 2.3% Cellulose + Guar gumNaive 5% celluose Naive 5% Cellulose + Guar gumEAE 0% EAE 2.3% Cellulose + Guar gumEAE 5% celluose EAE 5% Cellulose + Guar gum5% Cellulose 0% Fiber100 μm 100 μmE10020304050Relative % 16S rRNA read abundanceGenera: Akkermansia5% Cellulose + guar gum0% FiberCPre-diet Post-diet**  30 configuration is 0.97. (C-D) The relative percent of 16S rRNA read abundance of the genera Akkermansia for each naïve mouse placed on standard, standard C+G or zero-fiber diets were plotted before and after two weeks of dietary intervention. (E) Colonic slices from the second to last fecal pellet were taken from naïve mice post dietary intervention. Sections were stained with alcian blue dye and then imaged and analyzed using Zen 2.3 software. Statistics were calculated using t tests on prism. For *, p<0.05; **, p<0.005.  3.2 Guar gum offers ameliorative properties against EAE disease development While there was no protective effect of a standard amount of dietary fiber, we then considered if due to the peripheral nature of EAE, more drastic diets were required to cause change in disease progression. To address the hypothesis that supraphysiologic amounts of dietary soluble fiber can impact the pathogenesis of EAE, diets were designed containing 30% of four soluble fibers: inulin, guar gum, resistant starch and pectin. This supraphysiologic amount of fiber would be hard to achieve through dietary sources, yet is a common dose in murine studies evaluating the role of fermentable fibers in host physiology127,129. These studies have demonstrated that a diet rich in pectin decreased allergic airway disease through a reduction of IL-17 and IL-4 transcription in the lungs, while the same diet exacerbated disease in a serum induced arthritis model. Furthermore, the high pectin diet increased propionate and acetate production and increased Bacteroidaceae abundance in the gut. A recent paper observed that mice fed this high pectin diet were protected from EAE, however the mechanism of action was not elucidated and only one fiber type was investigated129. My project aimed to test the hypothesis that supraphysiologic doses of single sources of fermentable fibers could   31 differentially impact EAE disease outcome and immune cell development and function, as well as the microbiota composition.   3.2.1 Clinical Scores and EAE incidence decreased in guar gum fed mice When mice were fed the soluble fiber diets (30% inulin, guar gum, resistant starch and pectin), the standard cellulose and zero-fiber diets containing similar vitamin, protein and fat proportions, only mice fed 30% guar gum were significantly and reproducibly protected from disease when compared to the standard and zero-fiber diets (Fig 3.4A-E). In mice receiving either a standard diet or zero-fiber diets, clinical signs of paralysis developed 7-9 days post-induction (Fig 3.4A). In contrast, disease onset was delayed until days 13-15 post-induction in mice receiving the 30% guar gum diet (Fig 3.4A). Moreover, the percentage of guar gum-fed mice that developed disease was reduced by nearly 3-fold compared to mice on a standard or zero-fiber diet (Fig 3.4C).  At the day of sacrifice (day 15 post-induction), mice fed guar gum had a decreased score distribution and lower cumulative disease score (Fig 3.4D-E). This data supports the hypothesis that supraphysiologic amounts of dietary fiber can protect mice from EAE and that there are differential effects based on the fiber type. Notably, the 30% pectin diet did not offer any protection from EAE onset or severity. This is in contrast to published data. The reason for these differences is unclear, but may be due to facility-dependent microbiota effects129. These data provoke the hypothesis that guar gum may influence EAE pathogenesis directly through immunomodulation or via changes to the microbiota.    32  Figure 3.4: Supraphysiologic doses of guar gum offers protection against EAE.  (A-B) Mice fed diets two weeks prior to induction, and then monitored for levels of paralysis.  Stats were analyzed by two-way ANOVA. */$, p< 0.05; **/$$, p<0.005; ***/$$$, p<0.001. * represents comparison between 30% Guar gum and standard cellulose diets. $ represents comparison between 30% Guar gum and zero-fiber diets.  Representative of three independent experiments. (C) The fraction of mice that developed disease within each dietary group was compiled from three independent experiments over the course of 15 days. (D) Cumulative scores of mice from each dietary group across three independent experiments were calculated.  Statistics were calculated by t-test. ****, p<0.0001.  3.2.2 Lymphocytic infiltration into the CNS was inhibited in guar gum fed mice Brains, spinal cords and spleens were taken to analyze EAE pathogenic and protective T cell profiles.  The total number of pathogenic IFNγ-producing TH1 and IL-17a-producing TH17 cells that infiltrated into the brain was significantly reduced in the guar gum fed mice as CED2 7 9 11 13 15 2 7 9 11 13 15EAE Clinical Score012345Days post immunization Days post immunizationBA         EAE Clinical Score2 7 9 11 13 15EAE incidence (%)25050751005% Cellulose0% Fiber30% Inulin30% Resistant Starch30% Pectin30% Guar GumScore distribution at endpointCumulative score5% Cellulose0% Fiber30% Inulin30% Resistant Starch30% Pectin30% Guar Gum0510152025050751000<11-22-3********5% Cellulose30% Inulin30% Resistant Starch30% PectinDays post immunization01234530% Guar gum5% Cellulose0% Fiberellulose Fiber Guar Gum0 2 6 7 8 9 10 11 12 13 14 1501234Copy of EAE clinical scores_HGR_32 Inulin Resi tant starch Pectin Cellulose30% Inulin30% Guar gum30% Resistant starch30% Pectin5% Cellulose0% Fiber Cellulose Fiber0% Inulin30% Resistant Starch30% Pectin30% Guar Gum$$$$$$$****$$$$****************$$$$$$$$$$$$$$$$$  33 compared to mice fed standard and zero-fiber diets (Fig 3.5A). These data could have suggested that the mechanism of disease reduction was due to diminished CNS recruitment of pathogenic cells, however the number of protective Foxp3+ TREG cells found in the CNS was also decreased, indicating a general inhibition of lymphocytic infiltration into the CNS. The number of TH1 cells in the spinal cord was decreased in guar gum fed mice, however there were no significant differences in TH17 cell or TREG cells despite a trend in TREG cell reduction (Fig 3.5A). Interestingly, of the few T cells that did enter the CNS of guar gum fed mice, there was no impairment of TH1 or TH17 differentiation, indicated by a similar frequency of IFNg- or IL-17a-producing CD4+ T cells compared to standard diet fed mice (Fig 3.5B-C). However, there was a reduction in the frequency of protective TREG cells in the brains of guar gum fed mice (not seen in the spinal cords) (Fig 3.5B,C). This reduction could be explained by the reduced number of infiltrating T cells in this group, likely leading to a reduced amount of local inflammatory cytokine production, thereby limiting the need for immune regulation by TREG cells.  While the T cell CNS infiltration was altered in guar gum fed mice, some of those mice didn’t develop disease and would therefore bring down the average immune infiltration.  It begs the question of whether the immune response in guar gum mice that developed disease was similar or altered compared to standard-diet fed mice that developed disease. To test if guar gum altered immune inflammation once EAE developed, mice that had an EAE score of zero were removed from the analysis. Of the mice that developed disease, there was a non-significant trend in guar gum fed mice to have lower numbers of TH1 and TREG cells in the brain (Fig 3.5D). This suggests that the guar gum fed mice that develop disease have decreased clinical pathogenic immune presentation compared to the control diet fed mice.  However, due to the low number of guar gum-fed mice that developed disease and could be included in this analysis, these results   34 should be interpreted with caution. Notably, one sample from a guar gum mouse that developed disease had much higher T cell infiltration than other mice in the same group, but this mouse did not exhibit the highest clinical score. More experiments would be necessary to determine whether that mouse was an outlier, or if more guar gum diet fed mice that developed disease would demonstrate similar levels of T cell infiltration.   Figure 3.5: Supraphysiologic doses of guar gum prevents T cell CNS infiltration. (A-B) T cell profiles from EAE mice were analyzed in brain and spinal cords through flow cytometry and BA5% Cellulose30% Guar GumCD4IFNɣ IL-17 Foxp3MFI 3,072  MFI 2,580  *9.2 +/- 4.2 ***11.0 +/- 7.631.1 +/- 5.332.7 +/- 10.0 8.6 +/- 5.8 29.0 +/- 5.9MFI 13,698   MFI 17,871  MFI 17,376 MFI 10,742 TH1 TH17 TREGBrainSpinal Cord# of IFNɣ+cells# of IFNɣ+cells# of IL-17a+cells10005000 0500100015002000# of Foxp3+cells 10005000# of IL-17a+cellsTH1 TH17 TREGCD4+ CD3+ T cells5% Cellulose 0% Fiber 30% Guar gum*** *****TREGFreq of CD4 T cells020201000TH1Freq of CD4+ T cells nsnsTH17100nsns30205% Cellulose 0% Fiber 30% Guar gum02×10304×1036×1038×1032×1031×1033×10301×1035×1021.5×103**nsns*ns15105806040Freq of CD4+ T cellsCDTREG# of Foxp3+CD4 T cells01×1045×1031.5×104 p = 0.0515 p = 0.06962×1031×1033×1030TH1# of IFNɣ+CD4+ T cellsnsnsTH17# of IL-17a+CD4+ T cells2×1030nsns1×1048×1036×1034×1035% Cellulose 0% Fiber 30% Guar gumSpinal cordsBrains – excluding mice that failed to get disease# of Foxp3+cells  35 antibody staining of CD3 to isolate T cells, CD4 to select T helper cells and either IFNγ, IL-17a or Foxp3 to identify the T cell as a TH1, TH17, or TREG cell, respectively. The median cytokine concentration per cell was calculated using MFI in FlowJo. Brains were representative of three independent experiments, 4-5 mice per cohort. Spinal cord populations were compiled from two independent experiments. (C) T cell frequencies from spinal cords were compiled from two independent experiments with similar lymphocytic infiltration. (D) Brain T cell immune profiles from mice that developed disease were compiled from two independent experiments. Statistics were calculated by t-test. For *, p< 0.05; **, p<0.005; ***, p<0.001.  One explanation for the decrease in pathogenic T cells in the brains of guar gum-fed mice could be an impairment of the initial inflammatory immune reaction driven by EAE induction.  Alternatively, the autoimmune reaction could occur in the guar gum mice, however something is preventing the pathogenic cells from crossing the blood brain barrier.  To test the first hypothesis, I analyzed the differentiation of splenic TH1, TH17, and TREG cells at day 15. This analysis revealed no differences in total numbers compared to standard- or zero-fiber diet fed mice (Fig 3.6A), suggesting that guar gum, standard- and zero-fiber diet fed mice have similar immune reactions post induction and there is some factor preventing infiltration of autoimmune cells into the CNS in guar gum-fed mice. However, as day 15 is past the peak of EAE peripheral response, it could be too late to detect the early activation stage of EAE in the spleen.  Spleens taken at day 9 post EAE induction, thought to be the peak of peripheral EAE activation139, revealed no differences in TH1, TH17, or TREG cell counts (Fig 3.6B). This confirmed that the T cell peripheral immune response to EAE induction is the same in the standard diet and 30% guar gum diet fed mice. In addition, the median levels of the adhesion markers VLA-4 and LFA-1   36 (measured by CD49d and CD11a, respectively) were investigated at day 9 by MFI to determine if guar gum prevented CNS immune infiltration by reducing adherence to the endothelial BBB. However, no significant differences in the levels of these markers on activated (selected by CD44) T helper cells across diets were observed (Fig 3.6C). Together, these data suggest that guar gum ameliorates disease progression through inhibition of cellular infiltration into the CNS through an as yet unidentified mechanism.   Figure 3.6: Supraphysiologic doses of guar gum does not alter splenic T cell activation or adhesion molecules.  (A-B) T cell profiles from EAE mice on either day 15 or 9 were analyzed in spleen tissues through flow cytometry and antibody staining of CD3 to isolate T cells, CD4 to select T helper cells and either IFNγ, IL-17a or Foxp3 to identify the T cell as a TH1, TH17, or TREG cell, respectively. Representative of three independent experiments, 4-5 mice per cohort. (C) Mice fed standard or 30% guar gum diets were sacrificed on day 9 post EAE induction and spleens were taken for adhesion marker analysis. Day 15 is representative of three experiments # of IFNɣ+cells# of IL-17a+cells# of Foxp3+cells01×1063×10602×1054×1056×1058×1052×10603×1061×1064×1061×1062×106BCSpleen – CD44+CD4+ T cellsCD49d MFI1500300025002000CD11a MFI2500400035003000# of IFNɣ+cells# of IL-17a+cells# of Foxp3+cells01×1063×10602×1064×1066×1068×1062×10601×1063×1062×1065% Cellulose 0% Fiber 30% Guar gumA TH1 TH17 TREGCD4+ CD3+ T cellsSpleenDay 15Day 9  37 and day 9 is representative of one experiment. Statistics were calculated by t-test. For *, p< 0.05; **, p<0.005; ***, p<0.001.  3.2.3 The impact of soluble dietary fibers on the microbiome Because we used diets with large amounts of fiber that readily provides substrates for the gut microbiome, we investigated how the microbial dynamics were altered from diet to diet in naïve and EAE conditions using 16S rRNA gene profiling.  As dietary fiber impacts the gut microbiome, we hypothesized that each diet would differentially impact the microbiome in naïve and EAE conditions, in particular, questioning if there was a microbial difference in guar gum fed mice that could explain its mechanism of protection127,135. To test this, we first used an ordination plot to see the general microbial landscape of each diet represented in an easy to read two dimensional fashion. In the ordination plot (using NMDS values), the high fiber diets separate from both the standard and zero-fiber diets in both naïve and EAE conditions (Fig 3.7A).  Except for the 30% resistant starch diet fed mice, EAE and naïve samples place similarly on the plot.  In the 30% soluble fiber mice, most of the microbes were derived from the phylum Bacteroidetes in both naïve and EAE conditions (Fig 3.7B).  On the other hand, in naïve conditions standard diet fed mice had an increased abundance of Firmicutes (the genera Tyzerella) and zero-fiber diet fed mice had an increased abundance of Verrucomicrobia (the genera Akkermansia) (Fig 3.7B, D-F), confirming the results found in Fig 3.3 C. Furthermore, when we looked into the microbial community richness using the SOBS index, mice fed a standard fiber diet had the greatest diversity, indicating that a narrower variety of microbes exist efficiently in the absence of fiber or in the presence of a supraphysiologic amount of a select dietary fiber.  Alternatively, those extreme conditions may provide a competitive advantage for   38 select subsets of microbes. Consistent with these observations, in another study that investigated the effect of exposure to a high concentration (26%) of cellulose, a non-fermentable dietary fiber, the microbiome was composed of a reduced number of OTUs131, suggesting that a wide variety of fibers (both fermentable and non-fermentable) in supraphysiologic amounts limit the variety of microbes that can coexist in the gut.  This decrease in diversity in the high fiber diets can partially be explained by increase in the phylum Bacteroidetes. In standard fiber-diet fed mice, Bacteroidetes comprised an average of 45% of the 16S read abundance, but this increased to an average of 82% in each of the high fiber diet fed mice. Within Bacteroidetes, differences between the high fiber diets can be detected at the genera level. In naïve conditions, mice fed 30% guar gum, resistant starch or pectin had increased representation of Bacteroides, whereas inulin fed mice had high representation of Muribaculaceae (Fig 3.7D).  However, upon EAE induction, inulin fed mice shift to a Bacteroides majority. With these results, no obvious differences in guar gum fed mice’s microbiome was observed, suggesting that either guar gum does not protect against EAE in a microbiome dependent manner or that taxonomy needs to be resolved to a species or strain level to reveal differences in community composition. Microbiomes of MS patients have been reported to have increased proportional abundance of Akkermansia, however the question of whether this dysbiosis existed before disease development and contributed to their susceptibility or resulted from disease remains unknown.  Investigating microbiome structures in our EAE model before and after disease could provide answers to this question. After EAE induction, the diversity of the standard diet fed mice was decreased (Fig 3.6C), most likely due to the decrease in Firmicute abundance and increase in Verrucomicrobia abundance. On the genera level, Akkermansia was increased upon EAE induction in standard diet fed mice, which aligns with clinical studies that have observed an   39 increase in abundance of Akkermansia in MS patients11. As Akkermansia abundance was increased in zero-fiber diet fed mice compared to standard fiber diet fed mice, yet disease did not differ between the two, it suggests that the increase in Akkermansia abundance is a result of and not a contributor to disease. Yet, in the 30% fiber diets, Akkermansia was not increased after EAE induction – potentially because the amount of fiber overpowers the microbial changes from EAE or the standard diet best mimics the dietary conditions in the MS patients studies, however dietary data was not reported.  Altogether, our standard fiber diets were altered after EAE induction to better resemble the microbiomes of MS patients through an increase of Akkermansia, suggesting that this change was a result of disease, rather than a causative agent. Further, the development of disease in some of the high-fiber diets without an associated increase in Akkermansia suggests that this is not a requirement for nor broadly applicable biomarker of autoimmune neuroinflammation.    40  Figure 3.7: Supraphysiologic amounts of soluble fibers cause a shift in the gut microbiome. 16S rRNA gene profiling was performed to analyze microbiome composition down to the genera level. (A) Naïve (from control mice and experimental mice before induction) and EAE (experimental mice post induction) samples were pooled together in an ordinance plot using Naive inulinNaive guar gum Naive Resistant StarchNaive pectin Naive 5% celluose Naive 0% EAE inulin EAE guar gum EAE Resistant Starch EAE pectin EAE 5% celluose EAE 0% Figu e 4: Guar gum alters the gut microbiome  a) PCA analysis b) diversity c) chia pl t d) stool icrobial dynamics at day -14, 0 and 15 days post EAE induction e) blank microbe or expand for multiple  R2 of nmds = 0.94 and lowest stress 0.10Ordination plotAxis 1Axis 2A0.60.40.2-0.2-0.4-0.60.60.40.2-0.2-0.4-0.65% Cellulose0% Fiber30% Guar Gum30% Inulin30% Resistant Starch30% PectinNaive inulinNaive guar gum Naive Resistant StarchNaive pectin Naive 5% celluose Naive 0% EAE inuli  EAE guar gum EAE Resistant Starch EAE pectin EAE 5% celluose EAE 0% sobs25020015010050C5% Cellulose0% Fiber30% Guar Gum30% Inulin30% Resistant Starch30% PectinNaïve EAE5% Cellulose0% Fiber30% Guar Gum30% Inulin30% Resistant Starch30% PectinNaïve EAEB5% Cellulose0% Fiber30% Guar Gum30% Inulin30% Resistant Starch30% Pectin5% Cellulose0% Fiber30% Guar Gum30% Inulin30% Resistant Starch30% PectinVerrucomicrobiaNaïve EAETenericutesProteobacteriaFirmicutesBacteroidetesActinobacteriaOther***$$$$****$$$$$$$$@@@@@@$@@@@@@@@**** *******@@@@@@@$ $$$$$******** *Genus: Akkermansia6040200DGenus: Bacteroides1007550250Relative % 16S rRNA Read Abundance 1.510.50Genus: Tyzzerella6040200Genus: Muribaculaceae_geEF G$ gg, @ p, * 5%D – significant F – 5% (*) and differences from 5% (@) and inulin ($) pectin (*)E – 5% (@) and inulin ($)F  5% (*)************************$$$$$$$$$$$$ $$$$@@$$$$$$$$@@@@****$$$$@@@@$$$$@@@@$$$$@@@@p = 0.051** **@@@@$$$$$$$$$*********@@@@ @@@@@@@@@@@@$$$$@@@@$$$$ $$$$********************@@@@@@@@@@@@@@@@@@@@@$$$***$********Relative % 16S rRNA Read Abundance *  41 NMDS with a lowest stress level of 0.10 and R2 for configuration of 0.94. (B) Relative percent of 16S rRNA read abundance at the phylum level was calculated. (C) The richness of the microbiomes of mice before and after EAE induction were plotted in a box plot. Statistics were calculated using t-tests where one symbol represents p< 0.05, two symbols represent p<0.005, three symbols represent p<0.001 and four symbols represent p<0.0001. */$/@ represent comparisons across diets restricted to either naïve or EAE conditions and a * with a line denotes a change amongst the same diet post EAE induction for (C-G). * represents comparison against the standard diet, $ represents comparison against the 30% guar gum diet and @ represents comparison against the 30% pectin diet. (D-G) Plots represent the relative abundance of microbes at the genera level, where statistics were calculated using one-way ANOVA. (D) $ represents comparison against the 0% fiber diet and @ represents comparison against the standard diet (E) @ represents comparison against the standard diet, $ represents comparison against the 30% inulin diet and * represents comparison against the 30% pectin diet. (F) * represents comparison against the standard diet. (G) @ represents comparison against the 30% inulin diet and $ represents comparison against the 30% pectin diet. Representative of one experiment, with n = 4-5.  3.3 The impact of dietary soluble fibers on the gut  When intestinal barrier integrity is altered, it can leave the host more susceptible to gastrointestinal pathogens135.  One agent that is essential for the maintenance of the mucus layer is dietary fiber – in the absence of fiber, the mucus thins from degradation by the microbiome135. We therefore hypothesized that in the presence of supraphysiologic doses of soluble fibers, the mucus layer would become wider and in the absence of fiber, the mucus layer would become   42 thinner. To test this, mucus layers stained by alcian blue were measured using ZEN software. However to our surprise, it was observed that the mucus layers of the colons from standard diet fed mice was thicker than those in the mice that were fed 30% diets (inulin displayed a trend in decreased thickness) (Fig 3.8A-B). This suggested that perhaps a standard amount of cellulose is a better nutrient source for a wide variety of microbes, supported by the diversity plot in Fig 3.7C, which allows microbes to restrict their utilization of the mucus layer as their major nutrient source. Furthermore, perhaps in the select high fiber diet fed mice, the gut microbes that are not able to readily digest that fiber turn to the mucus layer as a food source, resulting in thinner mucus layers. One study found that a high fiber diet rich in a variety of different fiber types led to an increased mucus layer thickness135. This, combined with my findings provides evidence that large amounts of one type of fiber does not provide the nutrients necessary to support a diverse microbiome, however a diet consisting of a wide variety fibers can.   To determine whether the zero, standard or high-fiber diets I had tested had an impact on colonic immune cell infiltration, a veterinary pathologist (Dr. Ian Welch, DVM) performed histologic examination of prepared slides. This analysis revealed no differences in colonic immune cell infiltration across all diets (Fig 3.8A). However, gene expression analysis revealed diminished expression of the pro-inflammatory cytokine TNFa in guar gum fed mice compared to standard diet fed mice (Fig 3.8C). Interestingly, in both dietary conditions, the relative expression TNF-α decreased in response to EAE induction.  However as the mouse with the highest EAE score in the guar gum cohort had the highest relative TNF- α expression, this suggests that this effect is more of a product of the induction process, rather than disease onset.  As these supraphysiologic doses of fiber displayed an impact on the microbiome and gut integrity, we further investigated the impact that guar gum had on intestinal homeostasis via   43 stool consistency. Mice fed 30% guar gum diets developed diarrhea (Fig 3.8D). However, this effect could be due to the mice eating softer food because immersing the guar gum pellets in hydrogel was necessary to encourage the food consumption.  To control for this, standard fiber diets were immersed in hydrogel, however only the mice fed standard fiber diets pulled out the pellets, limiting the softness of the standard pellets.  In the future, diets could be softened before being fed to mice and therefore would determine if guar gum truly leads to uncomfortable side effects or is merely a result of diet administration.   44  Figure 3.8: The impact of soluble fibers on the gut. (A) Colonic slices from the second to last fecal pellet were taken from naïve mice post dietary intervention. Sections were stained with alcian blue dye and then imaged and analyzed using Zen 2.3 software. The mucus was measure using the line tool for a total of six measurements per mouse and averaged in (B). (C) qPCR for TNF-α was run on proximal colonic samples from naïve and EAE induced mice and was Figure 3: Guar gum causes gut CHANGES– colonic integrity and stool consistency a) Stool scores were marked s 0 – solid stool, 2 – soft stool and 4 – watery stool. b) colonic sections were taken at day 15 post induction and in control mice and stained with H&E to investigate lymphocyte infiltration c) Colonic sections with Alcian blue stain to investigate mucus layer width d) colonic RNA for genes blank, blank and bl nk (foxp3, IL-17, IFN-ɣ) e) immune cells in the colon/cecum - Th1/Th17/Treg STA S BY T TESTStool consistency432100 10 20 30Days post dietary interventionMucus thickness (μm) 3020100Naive inulinNaive guar gum Naive Resistant StarchNaive pectin Naive 5% celluose Naive 0% EAE inulin EAE guar gum EAE Resistant Starch EAE pectin EAE 5% celluose EAE 0% 5% Cellulose0% Fiber30% G Gum30% Inulin30% Resistant Starch30% PectinNaïve Naive inulinNaive guar gum Naive Resistant StarchNaive pectin Naive 5% celluose Naive 0% EAE inulin EAE guar gum EAE Resistant Starch EAE pectin EAE 5% celluose EAE 0% i  inulini  g ar gum aive R sistant StarchNaive pectin i  5  celluose i  0%  inulin  guar gum  R sistant Starch EAE pectin  5  celluose  0% Naïve 5  C l loseNaïve 30% G ar GumNaive inulinNaive guar gum Naive Resistant t rNaive pectin Naive 5% celluose Naive 0% EAE inulin EAE guar gum EAE Resistant Starch EAE pectin EAE 5% celluose EAE 0% 5% Cellulose0% Fiber30% Guar Gum30% Inulin30% Resistant Starch30% Pectin**** **** **** ****Relative TNF-α expression1.510.50Naive inulinNaive guar gum Naive Resistant StarchNaive pectin Naive 5% celluose Naive 0% EAE inulin EAE guar gum EAE Resistant Starch EAE pectin EAE 5% celluose EAE 0% i  inulini  g ar gum i  R sistant StarchNaive pectin i  5  celluose i  0%  inulin  guar gum  R sistant Starch EAE pectin  5  celluose  0% Naïve 5  C l loseNaïve 30  G ar GumNaive inulinNaive guar gum Naiv  Resistant StarchNaive pectin Naive 5% celluose Naive 0% EAE inulin EAE guar gum EAE Resistant Starch EAE pectin EAE 5% celluose EAE 0% iv  in linguar gum Resistant Starchi  p ctin Naive 5% celluose 0in lin guar gum Resistant Starch pect n EAE 5% celluose 0EAE 5% C ll loseEAE 30  G ar Gum****** * *100 μm100 μm100 μm100 μm100 μm100 μmABC D  45 normalized to the housekeeping gene, hprt. (D) Stool consistency was measured by 0=normal fecal pellet, 1= no fecal pellet or mildly soft fecal pellet, 2=soft fecal pellet or 3=diarrhea.       46 Chapter 4: Discussion In this study I aimed to determine how different types of fibers in varying amounts impacted the pathogenesis of EAE and what effects those diets had on the gut at the microbiome and histological level. I discovered that mice fed zero-fiber diets did not have exacerbated EAE disease when compared to mice fed standard fiber diets. Upon this discovery, I decided to investigate how supraphysiologic amounts of soluble fiber impacted the course of disease.  I used inulin, guar gum, resistant starch and pectin diets because soluble fibers are digested into SCFAs which, in turn, can have modulatory effects on the host’s immune system (increase TREG cells) 127,128,140.  I discovered that one of those soluble fiber diets had a significant impact on the course of disease – guar gum.  Guar gum decreased disease severity and the average cumulative score and furthermore, disease onset was significantly delayed. On an immunological level, I discovered that guar gum inhibited T cell infiltration into the brain and TH1 infiltration into the spinal cord.  Peripheral levels of these cells remained the same in the spleens across guar gum and control diets, which suggests that the initial autoimmune reaction occurred in guar gum fed mice, however by some unknown mechanism, lymphocyte infiltration into the CNS was inhibited. In the future, determining if guar gum inhibits disease by dampening the pathogenicity of T cells could be conducted by using the model of passive EAE induction. In this model, encephalogenic T cells are elicited by EAE immunization and then isolated at the peak of the T cell response (day 9) and transferred into new, naïve hosts where the T cells home to the CNS and induce disease. My data has already shown that initial T cell priming is similar between standard and 30% guar gum fed mice, thus immunizing and isolating T cells from these mice would allow one to test the hypothesis that T cells transferred from guar gum-fed mice are impaired in their ability to home to the CNS or bypass the BBB. Furthermore, investigating the   47 impact of guar gum on other disease associated immune populations in the periphery and CNS, such as DCs and macrophages, could provide more immunological clues as to guar gum’s mechanism of protection. Alternatively, guar gum may have an effect on the BBB that limits T cell infiltration of the CNS. The findings that pectin did not offer any significant and reproducible protective effects contradicts a previous study that demonstrated that pectin, at the same level, was able to dampen disease129.  However this study was conducted at a different facility and as the microbiome plays an integral role in the development of EAE, perhaps microbiome differences led to the varied results141.   One important thing to note about these diets is that 30% of a diet is an extreme proportion and would be difficult to replicate in human diets as Canadians are recommended to have 5% of their diets consisting of dietary fiber.  Other studies that have used these fiber diets have not discussed the limitations of these supraphysiologic fiber diets and their translation to patients.  However, there are differences in dietary fiber requirements between mice and people – one study used a murine diet made from a mouse’s natural food sources that consisted of approximately 15% dietary fiber135.  This demonstrates that the diets used in our study were approximately twice as much as the natural murine diet.  Taking this into consideration, if these results were translated into patient studies, a 10% fiber diet would be easier to achieve than a 30% fiber diet.  Maintaining a 10% guar gum diet would be difficult in human trials, however given the drastic protection against disease, if translated to MS, could be worth it to those patients. As this diet would most likely be used after disease establishment, investigating whether a switch to the guar gum after one disease period in a relapse/remitting murine model would better determine translatability to patient trials.  However, given the fact that guar gum inhibits disease by preventing immune translocation into the CNS, rather than inhibiting the   48 initial peripheral activation response suggests that guar gum might prevent further disease by inhibiting immune cell translocation into the CNS after disease development. On a microbial level, zero fiber diets displayed a higher abundance of the genera Akkermansia, which contains species that can utilize the mucus layer as a nutrient source. Because zero-fiber diets lack a major microbial food source, microbes turn to the mucus layer as a nutrient source and therefore the mucus layer became thinner135. At the microbial level, the supraphysiologic soluble fiber diets separated from each other and the standard/zero-fiber diets on an ordination plot. On the phylum level, these high fiber diets had increased Bacteroidetes, suggesting that this phylum best utilizes soluble fibers. Interestingly, my data suggests that the other microbes in the guts of mice fed high fiber diets were not able to rely on the soluble fiber as the nutrient source – mucus layer thickness was decreased in these mice.  I hypothesize that the microbes that cannot utilize the soluble fibers as a nutrient source degrade the mucus layer, thereby making it thinner.  Having a thinner mucus layer can leave a host more susceptible to gut pathogens, and therefore it is important to take into consideration the side effects of a single high fiber diet135. Similar to germ-free mice, which demonstrate that the inability to digest dietary fiber increases cecum size, the cecums from supraphysiologic fiber diet fed mice were increased (data not shown) – supporting the hypothesis that the microbes are not fully utilizing the soluble fibers.  Overall, these data provide important contributions with applications for patient trials that want to utilize diets as a supplementary intervention to modulate disease.  As multiple sclerosis does not have a cure and no drug successfully prevents symptoms, an additional therapeutic intervention that could improve the quality of life would be an important field to investigate.  Furthermore, my research has targeted the long asked question of whether microbes that are   49 increased in MS patients are linked to disease pathogenesis or are a reaction to systemic changes caused by disease.  Akkermansia, previously shown to have species increased in MS patients, was increased in zero-fiber diets compared to standard fiber diets, yet disease pathogenesis between the two cohorts was the same. This demonstrates that an increase in Akkermansia does not predispose these mice to more severe disease. Moreover, in standard diet fed mice, Akkermansia was increased after EAE induction, aligning with the MS patient study. Although the 30% fiber diet fed mice did not experience this change after EAE induction, this phenomenon could be explained if the pressure caused by the supraphysiologic doses of soluble fibers on microbial communities was greater than the pressure from EAE induction to alter Akkermansia, indicating that diet has more of an impact on the gut microbiome than EAE induction. Together, these data suggest that in standard diet conditions, the increase in Akkermansia was a reaction to disease, rather than contributing to it. In the future, gavaging mice with Akkermansia and other microbes altered in MS patients would determine the roles that they have in the pathogenesis of EAE.  In summary, my thesis provided novel discoveries of the roles of dietary fibers in standard and supraphysiologic levels in the pathogenesis of EAE and the potential of guar gum as a dietary supplemental therapeutic worth investigating in MS patients. 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Sci. 108, 4615–4622 (2011).            65 Appendix – Diet specifications Arginine, % 0.70Biotin, ppm 0.2Calcium, % 0.54Chloride, % 0.21Choline Chloride, ppm 1,250Cobalt, ppm 0.0Copper, ppm 6.0Fat, % 7.2Fiber (max), % 22.8Folic Acid, ppm 2.1Histidine, % 0.52Iodine, ppm 0.21Iron, ppm 38Isoleucine, % 0.96Leucine, % 1.73Lysine, % 1.45Magnesium, % 0.05Manganese, ppm 11Methionine, % 0.52Pantothenic Acid, ppm 16Phenylalanine, % 0.96Phosphorus, % 0.42Potassium, % 0.36Protein, % 18.0Pyridoxine, ppm 5.8Riboflavin, ppm 6.7Selenium, ppm 0.24Sodium, % 0.15Thiamin, ppm 4.8Threonine, % 0.77Tryptophan, % 0.22Tyrosine, % 1.01Valine, % 1.14Vitamin A, IU/g 4.3Vitamin B-12, mcg/kg 28Vitamin D-3 (added), IU/g 1.0Vitamin E, IU/kg 81.6Vitamin K, ppm 0.75Zinc, ppm 35Niacin, ppm 30Ascorbic Acid, ppm 3.6Cystine, % 0.37MineralsVitamins2/3/2018Fluorine, ppm 1.0Chromium (added), ppm 1.0N U T R I T I O N A L   P R O F I L E 11.  Formulation based on calculated values from the latest ingredient analysis information.  Since nutrient composition of natural ingredients varies and some nutrient loss will occur due to manufacturing processes, analysis will differ accordingly.  Nutrients expressed as percent of ration on an As-Fed basis except where otherwise indicated.  2.  Energy (kcal/gm) - Sum of decimal fractions of protein, fat and carbohydrate x 4,9,4 kcal/gm respectively.D E S C R I P T I O NModification of TestDiet® AIN-93G Semi-Purified Diet, 57W5, with no cellulose and 30% FiberSym.Intended for rodents in a laboratory setting.F E E D I N G   D I R E C T I O N SFeed ad libitum.  Plenty of fresh, clean water should be available at all times.I N G R E D I E N T S (%)Carbohydrates, % 64.6Energy (kcal/g) 3.95Molybdenum, ppm 0.142CAUTION: Perishable - store properly upon receipt.For laboratory animal use only; NOT for human consumption.Mod TestDiet® 57W5 w/ No Cellulose, 30% FiberSymCholesterol, ppm 05BACProduct Forms Available* Catalog #*Other Forms Available On RequestProtein 18.2Fat (ether extract) 16.4Carbohydrates 65.4%kcalFrom:0.7210.6462.584Linoleic Acid, % 3.58Linolenic Acid, % 0.55Arachidonic Acid, % 0.00Omega-3 Fatty Acids, % 0.55Total Saturated Fatty A 1.10Total Monounsaturated Fatty Acids, % 1.56Glycine, % 0.39Serine, % 1.10Aspartic Acid, % 1.29Glutamic Acid, % 4.08Alanine, % 0.55Proline, % 2.36Taurine, % 0.00Polyunsaturated Fatty Acids, % 3.85CAUTION:  Contains a new animal drug for investigational use only in laboratory research animals or for tests in vitro.  Not for use in humans.Storage conditions are particularly critical to TestDiet® products, due to the absence of antioxidants or preservative agents.  To provide maximum protection against possible changes during storage, store in a dry, cool location.  Storage under refrigeration (2° C) is recommended.  Maximum shelf life is six months.  (If long term studies are involved, storing the diet at -20˚ C or colder may prolong shelf life.)  Be certain to keep in air tight containers.NOTE: When assayed, actual levels may vary from calculated values.*See page 2 for Expanded Ingredient Listingsw w w . t e s t d i e t .  c o m 30.0000Resistant Wheat Starch20.0000Casein - Vitamin Tested14.7486Corn Starch13.2000Maltodextrin10.0000Sucrose7.0000Soybean Oil3.5000AIN 93G Mineral Mix1.0000AIN 93G Vitamin Mix0.3000L-Cystine0.2500Choline Bitartrate0.0014t-Butylhydroquinone1818357-2041/2" Pellet, Irradiated  66 Arginine, % 0.70Biotin, ppm 0.2Calcium, % 0.51Chloride, % 0.21Choline Chloride, ppm 1,250Cobalt, ppm 0.0Copper, ppm 6.0Fat, % 7.0Fiber (max), % 30.0Folic Acid, ppm 2.1Histidine, % 0.52Iodine, ppm 0.21Iron, ppm 38Isoleucine, % 0.96Leucine, % 1.73Lysine, % 1.45Magnesium, % 0.05Manganese, ppm 11Methionine, % 0.52Pantothenic Acid, ppm 16Phenylalanine, % 0.96Phosphorus, % 0.32Potassium, % 0.36Protein, % 18.0Pyridoxine, ppm 5.8Riboflavin, ppm 6.7Selenium, ppm 0.24Sodium, % 0.12Thiamin, ppm 4.8Threonine, % 0.77Tryptophan, % 0.22Tyrosine, % 1.01Valine, % 1.14Vitamin A, IU/g 4.0Vitamin B-12, mcg/kg 28Vitamin D-3 (added), IU/g 1.0Vitamin E, IU/kg 81.6Vitamin K, ppm 0.75Zinc, ppm 35Niacin, ppm 30Ascorbic Acid, ppm 0.0Cystine, % 0.37MineralsVitamins2/3/2018Fluorine, ppm 1.0Chromium (added), ppm 1.0N U T R I T I O N A L   P R O F I L E 11.  Formulation based on calculated values from the latest ingredient analysis information.  Since nutrient composition of natural ingredients varies and some nutrient loss will occur due to manufacturing processes, analysis will differ accordingly.  Nutrients expressed as percent of ration on an As-Fed basis except where otherwise indicated.  2.  Energy (kcal/gm) - Sum of decimal fractions of protein, fat and carbohydrate x 4,9,4 kcal/gm respectively.D E S C R I P T I O NModification of TestDiet® AIN-93G Semi-Purified Diet, 57W5, with no cellulose and 30% guar gum.Intended for rodents in a laboratory setting.F E E D I N G   D I R E C T I O N SFeed ad libitum.  Plenty of fresh, clean water should be available at all times.I N G R E D I E N T S (%)Carbohydrates, % 38.2Energy (kcal/g) 2.88Molybdenum, ppm 0.142CAUTION: Perishable - store properly upon receipt.For laboratory animal use only; NOT for human consumption.Mod TestDiet® 57W5 w/ No Cellulose, 30% Guar GumCholesterol, ppm 05BSEProduct Forms Available* Catalog #*Other Forms Available On RequestProtein 25.0Fat (ether extract) 22.0Carbohydrates 53.0%kcalFrom:0.7210.6331.528Linoleic Acid, % 3.58Linolenic Acid, % 0.55Arachidonic Acid, % 0.00Omega-3 Fatty Acids, % 0.55Total Saturated Fatty A 1.05Total Monounsaturated Fatty Acids, % 1.54Glycine, % 0.39Serine, % 1.10Aspartic Acid, % 1.29Glutamic Acid, % 4.08Alanine, % 0.55Proline, % 2.36Taurine, % 0.00Polyunsaturated Fatty Acids, % 3.78CAUTION:  Contains a new animal drug for investigational use only in laboratory research animals or for tests in vitro.  Not for use in humans.Storage conditions are particularly critical to TestDiet® products, due to the absence of antioxidants or preservative agents.  To provide maximum protection against possible changes during storage, store in a dry, cool location.  Storage under refrigeration (2° C) is recommended.  Maximum shelf life is six months.  (If long term studies are involved, storing the diet at -20˚ C or colder may prolong shelf life.)  Be certain to keep in air tight containers.NOTE: When assayed, actual levels may vary from calculated values.*See page 2 for Expanded Ingredient Listingsw w w . t e s t d i e t .  c o m 30.0000Guar Gum20.0000Casein - Vitamin Tested14.7486Corn Starch13.2000Maltodextrin10.0000Sucrose7.0000Soybean Oil3.5000AIN 93G Mineral Mix1.0000AIN 93G Vitamin Mix0.3000L-Cystine0.2500Choline Bitartrate0.0014t-Butylhydroquinone1818358-2031/2" Pellet, Iirradiated  67 Arginine, % 0.70Biotin, ppm 0.2Calcium, % 0.51Chloride, % 0.21Choline Chloride, ppm 1,250Cobalt, ppm 0.0Copper, ppm 6.0Fat, % 7.0Fiber (max), % 30.0Folic Acid, ppm 2.1Histidine, % 0.52Iodine, ppm 0.21Iron, ppm 38Isoleucine, % 0.96Leucine, % 1.73Lysine, % 1.45Magnesium, % 0.05Manganese, ppm 11Methionine, % 0.52Pantothenic Acid, ppm 16Phenylalanine, % 0.96Phosphorus, % 0.32Potassium, % 0.36Protein, % 18.0Pyridoxine, ppm 5.8Riboflavin, ppm 6.7Selenium, ppm 0.24Sodium, % 0.12Thiamin, ppm 4.8Threonine, % 0.77Tryptophan, % 0.22Tyrosine, % 1.01Valine, % 1.14Vitamin A, IU/g 4.0Vitamin B-12, mcg/kg 28Vitamin D-3 (added), IU/g 1.0Vitamin E, IU/kg 81.6Vitamin K, ppm 0.75Zinc, ppm 35Niacin, ppm 30Ascorbic Acid, ppm 0.0Cystine, % 0.37MineralsVitamins2/3/2018Fluorine, ppm 1.0Chromium (added), ppm 1.0N U T R I T I O N A L   P R O F I L E 11.  Formulation based on calculated values from the latest ingredient analysis information.  Since nutrient composition of natural ingredients varies and some nutrient loss will occur due to manufacturing processes, analysis will differ accordingly.  Nutrients expressed as percent of ration on an As-Fed basis except where otherwise indicated.  2.  Energy (kcal/gm) - Sum of decimal fractions of protein, fat and carbohydrate x 4,9,4 kcal/gm respectively.D E S C R I P T I O NModification of TestDiet® AIN-93G Semi-Purified Diet, 57W5, with no cellulose and 30% pectin.Intended for rodents in a laboratory setting.F E E D I N G   D I R E C T I O N SFeed ad libitum.  Plenty of fresh, clean water should be available at all times.I N G R E D I E N T S (%)Carbohydrates, % 38.2Energy (kcal/g) 2.88Molybdenum, ppm 0.142CAUTION: Perishable - store properly upon receipt.For laboratory animal use only; NOT for human consumption.Mod TestDiet® 57W5 w/ No Cellulose, 30% PectinCholesterol, ppm 05BSXProduct Forms Available* Catalog #*Other Forms Available On RequestProtein 25.0Fat (ether extract) 22.0Carbohydrates 53.0%kcalFrom:0.7210.6331.528Linoleic Acid, % 3.58Linolenic Acid, % 0.55Arachidonic Acid, % 0.00Omega-3 Fatty Acids, % 0.55Total Saturated Fatty A 1.05Total Monounsaturated Fatty Acids, % 1.54Glycine, % 0.39Serine, % 1.10Aspartic Acid, % 1.29Glutamic Acid, % 4.08Alanine, % 0.55Proline, % 2.36Taurine, % 0.00Polyunsaturated Fatty Acids, % 3.78CAUTION:  Contains a new animal drug for investigational use only in laboratory research animals or for tests in vitro.  Not for use in humans.Storage conditions are particularly critical to TestDiet® products, due to the absence of antioxidants or preservative agents.  To provide maximum protection against possible changes during storage, store in a dry, cool location.  Storage under refrigeration (2° C) is recommended.  Maximum shelf life is six months.  (If long term studies are involved, storing the diet at -20˚ C or colder may prolong shelf life.)  Be certain to keep in air tight containers.NOTE: When assayed, actual levels may vary from calculated values.*See page 2 for Expanded Ingredient Listingsw w w . t e s t d i e t .  c o m 30.0000Pectin20.0000Casein - Vitamin Tested14.7486Corn Starch13.2000Maltodextrin10.0000Sucrose7.0000Soybean Oil3.5000AIN 93G Mineral Mix1.0000AIN 93G Vitamin Mix0.3000L-Cystine0.2500Choline Bitartrate0.0014t-Butylhydroquinone1818359-2031/2" Pellet, Irradiated  68 Arginine, % 0.70Biotin, ppm 0.2Calcium, % 0.51Chloride, % 0.21Choline Chloride, ppm 1,250Cobalt, ppm 0.0Copper, ppm 6.0Fat, % 7.0Fiber (max), % 26.4Folic Acid, ppm 2.1Histidine, % 0.52Iodine, ppm 0.21Iron, ppm 38Isoleucine, % 0.96Leucine, % 1.73Lysine, % 1.45Magnesium, % 0.05Manganese, ppm 11Methionine, % 0.52Pantothenic Acid, ppm 16Phenylalanine, % 0.96Phosphorus, % 0.32Potassium, % 0.36Protein, % 18.0Pyridoxine, ppm 5.8Riboflavin, ppm 6.7Selenium, ppm 0.24Sodium, % 0.14Thiamin, ppm 4.8Threonine, % 0.77Tryptophan, % 0.22Tyrosine, % 1.01Valine, % 1.14Vitamin A, IU/g 4.0Vitamin B-12, mcg/kg 28Vitamin D-3 (added), IU/g 1.0Vitamin E, IU/kg 81.6Vitamin K, ppm 0.75Zinc, ppm 35Niacin, ppm 30Ascorbic Acid, ppm 0.0Cystine, % 0.37MineralsVitamins2/3/2018Fluorine, ppm 1.0Chromium (added), ppm 1.0N U T R I T I O N A L   P R O F I L E 11.  Formulation based on calculated values from the latest ingredient analysis information.  Since nutrient composition of natural ingredients varies and some nutrient loss will occur due to manufacturing processes, analysis will differ accordingly.  Nutrients expressed as percent of ration on an As-Fed basis except where otherwise indicated.  2.  Energy (kcal/gm) - Sum of decimal fractions of protein, fat and carbohydrate x 4,9,4 kcal/gm respectively.D E S C R I P T I O NModification of TestDiet® AIN-93G Semi-Purified Diet, 57W5, with no cellulose and 30% inulin.Intended for rodents in a laboratory setting.F E E D I N G   D I R E C T I O N SFeed ad libitum.  Plenty of fresh, clean water should be available at all times.I N G R E D I E N T S (%)Carbohydrates, % 67.0Energy (kcal/g) 4.03Molybdenum, ppm 0.142CAUTION: Perishable - store properly upon receipt.For laboratory animal use only; NOT for human consumption.Mod TestDiet® 57W5 w/ No Cellulose, 30% InulinCholesterol, ppm 05BX1Product Forms Available* Catalog #*Other Forms Available On RequestProtein 17.9Fat (ether extract) 15.7Carbohydrates 66.4%kcalFrom:0.7210.6332.680Linoleic Acid, % 3.58Linolenic Acid, % 0.55Arachidonic Acid, % 0.00Omega-3 Fatty Acids, % 0.55Total Saturated Fatty A 1.05Total Monounsaturated Fatty Acids, % 1.54Glycine, % 0.39Serine, % 1.10Aspartic Acid, % 1.29Glutamic Acid, % 4.08Alanine, % 0.55Proline, % 2.36Taurine, % 0.00Polyunsaturated Fatty Acids, % 3.78CAUTION:  Contains a new animal drug for investigational use only in laboratory research animals or for tests in vitro.  Not for use in humans.Storage conditions are particularly critical to TestDiet® products, due to the absence of antioxidants or preservative agents.  To provide maximum protection against possible changes during storage, store in a dry, cool location.  Storage under refrigeration (2° C) is recommended.  Maximum shelf life is six months.  (If long term studies are involved, storing the diet at -20˚ C or colder may prolong shelf life.)  Be certain to keep in air tight containers.NOTE: When assayed, actual levels may vary from calculated values.*See page 2 for Expanded Ingredient Listingsw w w . t e s t d i e t .  c o m 30.0000Inulin20.0000Casein - Vitamin Tested14.7486Corn Starch13.2000Maltodextrin10.0000Sucrose7.0000Soybean Oil3.5000AIN 93G Mineral Mix1.0000AIN 93G Vitamin Mix0.3000L-Cystine0.2500Choline Bitartrate0.0014t-Butylhydroquinone1818360-2031/2" Pellet, Irradiated  69 Arginine, % 0.70Biotin, ppm 0.2Calcium, % 0.51Chloride, % 0.22Choline Chloride, ppm 1,250Cobalt, ppm 0.0Copper, ppm 6.0Fat, % 7.1Fiber (max), % 2.3Folic Acid, ppm 2.1Histidine, % 0.52Iodine, ppm 0.21Iron, ppm 39Isoleucine, % 0.96Leucine, % 1.73Lysine, % 1.45Magnesium, % 0.05Manganese, ppm 11Methionine, % 0.52Pantothenic Acid, ppm 16Phenylalanine, % 0.96Phosphorus, % 0.32Potassium, % 0.36Protein, % 18.3Pyridoxine, ppm 5.8Riboflavin, ppm 6.7Selenium, ppm 0.24Sodium, % 0.13Thiamin Hydrochloride, ppm 6.1Threonine, % 0.77Tryptophan, % 0.22Tyrosine, % 1.01Valine, % 1.14Vitamin A, IU/g 4.0Vitamin B-12, mcg/kg 29Vitamin D-3 (added), IU/g 1.0Vitamin E, IU/kg 81.6Vitamin K, ppm 0.75Zinc, ppm 35Niacin, ppm 30Ascorbic Acid, ppm 0.0Cystine, % 0.37MineralsVitamins11/28/2016Fluorine, ppm 1.0Chromium (added), ppm 1.0N U T R I T I O N A L   P R O F I L E 11.  Formulation based on calculated values from the latest ingredient analysis information.  Since nutrient composition of natural ingredients varies and some nutrient loss will occur due to manufacturing processes, analysis will differ accordingly.  Nutrients expressed as percent of ration on an As-Fed basis except where otherwise indicated.  2.  Energy (kcal/gm) - Sum of decimal fractions of protein, fat and carbohydrate x 4,9,4 kcal/gm respectively.D E S C R I P T I O NModification of TestDiet® AIN-93G Purified Growth Diet 57W5 with 2.3% fiber. Fiber provided by equal amounts of cellulose and guar gum. Comparable to SF13-055.F E E D I N G   D I R E C T I O N SFeed ad libitum.  Plenty of fresh, clean water should be available at all times.I N G R E D I E N T S (%)Carbohydrates, % 65.9Energy (kcal/g) 4.01Molybdenum, ppm 0.142CAUTION: Perishable - store properly upon receipt.For laboratory animal use only; NOT for human consumption.w w w . t e s t d i e t .  c o m AIN-93G w/ 2.3% fiber from cellulose and guar gumCholesterol, ppm 09GKYProduct Forms Available* Catalog #*Other Forms Available On RequestProtein 18.3Fat (ether extract) 15.9Carbohydrates 65.8%kcalFrom:0.7320.6382.636Linoleic Acid, % 3.58Linolenic Acid, % 0.55Arachidonic Acid, % 0.00Omega-3 Fatty Acids, % 0.55Total Saturated Fatty A 1.05Total Monounsaturated Fatty Acids, % 1.54Glycine, % 0.39Serine, % 1.10Aspartic Acid, % 1.29Glutamic Acid, % 4.08Alanine, % 0.55Proline, % 2.36Taurine, % 0.00Polyunsaturated Fatty Acids, % 3.78CAUTION:  Contains a new animal drug for investigational use only in laboratory research animals or for tests in vitro.  Not for use in humans.Storage conditions are particularly critical to TestDiet® products, due to the absence of antioxidants or preservative agents.  To provide maximum protection against possible changes during storage, store in a dry, cool location.  Storage under refrigeration (2° C) is recommended.  Maximum shelf life is six months.  (If long term studies are involved, storing the diet at -20˚ C or colder may prolong shelf life.)  Be certain to keep in air tight containers.NOTE: When assayed, actual levels may vary from calculated values.42.4486Corn Starch20.0000Casein - Vitamin Tested13.2000Maltodextrin10.0000Sucrose7.0000Soybean Oil3.5000AIN 93G Mineral Mix1.1500Powdered Cellulose1.1500Guar Gum1.0000AIN 93 Vitamin Mix0.3000L-Cystine0.2500Choline Bitartrate0.0014t-Butylhydroquinone1817770-2031/2" Pellet, Irradiated  70 Arginine, % 0.70Biotin, ppm 0.2Calcium, % 0.50Chloride, % 0.17Choline Chloride, ppm 1,250Cobalt, ppm 0.0Copper, ppm 6.0Fat, % 7.0Fiber (max), % 0.0Folic Acid, ppm 2.1Histidine, % 0.52Iodine, ppm 0.21Iron, ppm 37Isoleucine, % 0.96Leucine, % 1.73Lysine, % 1.45Magnesium, % 0.05Manganese, ppm 11Methionine, % 0.52Pantothenic Acid, ppm 16Phenylalanine, % 0.96Phosphorus, % 0.32Potassium, % 0.36Protein, % 17.9Pyridoxine, ppm 5.8Riboflavin, ppm 6.7Selenium, ppm 0.24Sodium, % 0.11Thiamin Hydrochloride, ppm 4.8Threonine, % 0.77Tryptophan, % 0.22Tyrosine, % 1.01Valine, % 1.14Vitamin A, IU/g 4.0Vitamin B-12, mcg/kg 28Vitamin D-3 (added), IU/g 1.0Vitamin E, IU/kg 81.6Vitamin K, ppm 0.75Zinc, ppm 35Niacin, ppm 30Ascorbic Acid, ppm 0.0Cystine, % 0.37MineralsVitamins5/2/2017Fluorine, ppm 1.0Chromium (added), ppm 1.0N U T R I T I O N A L   P R O F I L E 11.  Formulation based on calculated values from the latest ingredient analysis information.  Since nutrient composition of natural ingredients varies and some nutrient loss will occur due to manufacturing processes, analysis will differ accordingly.  Nutrients expressed as percent of ration on an As-Fed basis except where otherwise indicated.  2.  Energy (kcal/gm) - Sum of decimal fractions of protein, fat and carbohydrate x 4,9,4 kcal/gm respectively.D E S C R I P T I O NModification of TestDiet® AIN-93G Purified GrowthDiet, 57W5, with no added fiber or starch. Comparable to SF09-028.Intended for rodents in a laboratory setting.F E E D I N G   D I R E C T I O N SFeed ad libitum.  Plenty of fresh, clean water should be available at all times.I N G R E D I E N T S (%)Carbohydrates, % 68.9Energy (kcal/g) 4.10Molybdenum, ppm 0.142CAUTION: Perishable - store properly upon receipt.For laboratory animal use only; NOT for human consumption.AIN-93G without fiber or starchCholesterol, ppm 09GKZProduct Forms Available* Catalog #*Other Forms Available On RequestProtein 17.4Fat (ether extract) 15.4Carbohydrates 67.2%kcalFrom:0.7150.6302.757Linoleic Acid, % 3.58Linolenic Acid, % 0.55Arachidonic Acid, % 0.00Omega-3 Fatty Acids, % 0.55Total Saturated Fatty A 1.05Total Monounsaturated Fatty Acids, % 1.54Glycine, % 0.39Serine, % 1.10Aspartic Acid, % 1.29Glutamic Acid, % 4.08Alanine, % 0.55Proline, % 2.36Taurine, % 0.00Polyunsaturated Fatty Acids, % 3.78CAUTION:  Contains a new animal drug for investigational use only in laboratory research animals or for tests in vitro.  Not for use in humans.Storage conditions are particularly critical to TestDiet® products, due to the absence of antioxidants or preservative agents.  To provide maximum protection against possible changes during storage, store in a dry, cool location.  Storage under refrigeration (2° C) is recommended.  Maximum shelf life is six months.  (If long term studies are involved, storing the diet at -20˚ C or colder may prolong shelf life.)  Be certain to keep in air tight containers.NOTE: When assayed, actual levels may vary from calculated values.*See page 2 for Expanded Ingredient Listingsw w w . t e s t d i e t .  c o m 67.9486Dextrose20.0000Casein - Vitamin Tested7.0000Soybean Oil3.5000AIN 93G Mineral Mix1.0000AIN 93 Vitamin Mix0.3000L-Cystine0.2500Choline Bitartrate0.0014t-Butylhydroquinone1817771-2031/2" Pellet, Irradiated  71 Arginine, % 0.70Biotin, ppm 0.2Calcium, % 0.51Chloride, % 0.22Choline Chloride, ppm 1,250Cobalt, ppm 0.0Copper, ppm 6.0Fat, % 7.1Fiber (max), % 5.0Folic Acid, ppm 2.1Histidine, % 0.52Iodine, ppm 0.21Iron, ppm 40Isoleucine, % 0.96Leucine, % 1.73Lysine, % 1.45Magnesium, % 0.05Manganese, ppm 11Methionine, % 0.52Pantothenic Acid, ppm 16Phenylalanine, % 0.96Phosphorus, % 0.32Potassium, % 0.36Protein, % 18.3Pyridoxine, ppm 5.8Riboflavin, ppm 6.7Selenium, ppm 0.24Sodium, % 0.13Thiamin Hydrochloride, ppm 6.1Threonine, % 0.77Tryptophan, % 0.22Tyrosine, % 1.01Valine, % 1.14Vitamin A, IU/g 4.0Vitamin B-12, mcg/kg 29Vitamin D-3 (added), IU/g 1.0Vitamin E, IU/kg 81.6Vitamin K, ppm 0.75Zinc, ppm 35Niacin, ppm 30Ascorbic Acid, ppm 0.0Cystine, % 0.37MineralsVitamins11/18/2014Fluorine, ppm 1.0Chromium (added), ppm 1.0N U T R I T I O N A L   P R O F I L E 11.  Formulation based on calculated values from the latest ingredient analysis information.  Since nutrient composition of natural ingredients varies and some nutrient loss will occur due to manufacturing processes, analysis will differ accordingly.  Nutrients expressed as percent of ration on an As-Fed basis except where otherwise indicated.  2.  Energy (kcal/gm) - Sum of decimal fractions of protein, fat and carbohydrate x 4,9,4 kcal/gm respectively.D E S C R I P T I O NTestDiet® AIN-93G Growth Purified Diet is the growth diet for rodents recommended by the American Institute of Nutrition.  It is formulated to substitute for the previous version (AIN-76A) to improve animal performance.F E E D I N G   D I R E C T I O N SFeed ad libitum to mice and rats.  Plenty of fresh, clean water should be available at all times.I N G R E D I E N T S (%)Carbohydrates, % 63.2Energy (kcal/g) 3.90Molybdenum, ppm 0.142CAUTION: Perishable - store properly upon receipt.For laboratory animal use only; NOT for human consumption.w w w . t e s t d i e t .  c o m AIN-93G Growth Purified Diet (also known as #5801-G)Cholesterol, ppm 057W5Product Forms Available* Catalog #*Other Forms Available On RequestProtein 18.8Fat (ether extract) 16.4Carbohydrates 64.9%kcalFrom:0.7310.6372.528Linoleic Acid, % 3.58Linolenic Acid, % 0.55Arachidonic Acid, % 0.00Omega-3 Fatty Acids, % 0.55Total Saturated Fatty A 1.05Total Monounsaturated Fatty Acids, % 1.54Glycine, % 0.39Serine, % 1.10Aspartic Acid, % 1.29Glutamic Acid, % 4.08Alanine, % 0.55Proline, % 2.36Taurine, % 0.00Polyunsaturated Fatty Acids, % 3.78CAUTION:  Contains a drug or compound for investigational use only in laboratory research animals or in vitro.  Not for human use.Storage conditions are particularly critical to TestDiet® products, due to the absence of antioxidants or preservative agents.  To provide maximum protection against possible changes during storage, store in a dry, cool location.  Storage under refrigeration (2° C) is recommended.  Maximum shelf life is six months.  (If long term studies are involved, storing the diet at -20˚ C or colder may prolong shelf life.)  Be certain to keep in air tight containers.39.7486Corn Starch20.0000Casein - Vitamin Tested13.2000Maltodextrin10.0000Sucrose7.0000Soybean Oil5.0000Powdered Cellulose3.5000AIN 93G Mineral Mix1.0000AIN 93 Vitamin Mix0.3000L-Cystine0.2500Choline Bitartrate0.0014t-Butylhydroquinone75971/2" Pellet18103931/2" Pellet, Irradiated18165301/2" Pellet, Irradiated, 5 Kg Heat Seal1810538Meal  72  Arginine, % 0.70Biotin, ppm 0.2Calcium, % 0.51Chloride, % 0.22Choline Chloride, ppm 1,250Cobalt, ppm 0.0Copper, ppm 6.0Fat, % 7.1Fiber (max), % 5.0Folic Acid, ppm 2.1Histidine, % 0.52Iodine, ppm 0.21Iron, ppm 39Isoleucine, % 0.96Leucine, % 1.73Lysine, % 1.45Magnesium, % 0.05Manganese, ppm 11Methionine, % 0.52Pantothenic Acid, ppm 16Phenylalanine, % 0.96Phosphorus, % 0.32Potassium, % 0.36Protein, % 18.3Pyridoxine, ppm 5.8Riboflavin, ppm 6.7Selenium, ppm 0.24Sodium, % 0.13Thiamin Hydrochloride, ppm 4.8Threonine, % 0.77Tryptophan, % 0.22Tyrosine, % 1.01Valine, % 1.14Vitamin A, IU/g 4.0Vitamin B-12, mcg/kg 28Vitamin D-3 (added), IU/g 1.0Vitamin E, IU/kg 81.6Vitamin K, ppm 0.75Zinc, ppm 35Niacin, ppm 30Ascorbic Acid, ppm 0.0Cystine, % 0.37MineralsVitamins4/12/2017Fluorine, ppm 1.0Chromium (added), ppm 1.0N U T R I T I O N A L   P R O F I L E 11.  Formulation based on calculated values from the latest ingredient analysis information.  Since nutrient composition of natural ingredients varies and some nutrient loss will occur due to manufacturing processes, analysis will differ accordingly.  Nutrients expressed as percent of ration on an As-Fed basis except where otherwise indicated.  2.  Energy (kcal/gm) - Sum of decimal fractions of protein, fat and carbohydrate x 4,9,4 kcal/gm respectively.D E S C R I P T I O NModification of TestDiet® AIN-93G Semi-Purified Diet, 57W5, with 5% Fiber (50% from cellulose and 50% from guar gum).Intended for rodents in a laboratory setting.F E E D I N G   D I R E C T I O N SFeed ad libitum.  Plenty of fresh, clean water should be available at all times.I N G R E D I E N T S (%)Carbohydrates, % 63.2Energy (kcal/g) 3.90Molybdenum, ppm 0.142CAUTION: Perishable - store properly upon receipt.For laboratory animal use only; NOT for human consumption.Mod TestDiet® AIN-93G w/ 5% Fiber from Cellulose & Guar GuCholesterol, ppm 09GQPProduct Forms Available* Catalog #*Other Forms Available On RequestProtein 18.8Fat (ether extract) 16.4Carbohydrates 64.9%kcalFrom:0.7310.6372.528Linoleic Acid, % 3.58Linolenic Acid, % 0.55Arachidonic Acid, % 0.00Omega-3 Fatty Acids, % 0.55Total Saturated Fatty A 1.05Total Monounsaturated Fatty Acids, % 1.54Glycine, % 0.39Serine, % 1.10Aspartic Acid, % 1.29Glutamic Acid, % 4.08Alanine, % 0.55Proline, % 2.36Taurine, % 0.00Polyunsaturated Fatty Acids, % 3.78CAUTION:  Contains a new animal drug for investigational use only in laboratory research animals or for tests in vitro.  Not for use in humans.Storage conditions are particularly critical to TestDiet® products, due to the absence of antioxidants or preservative agents.  To provide maximum protection against possible changes during storage, store in a dry, cool location.  Storage under refrigeration (2° C) is recommended.  Maximum shelf life is six months.  (If long term studies are involved, storing the diet at -20˚ C or colder may prolong shelf life.)  Be certain to keep in air tight containers.NOTE: When assayed, actual levels may vary from calculated values.*See page 2 for Expanded Ingredient Listingsw w w . t e s t d i e t .  c o m 39.7486Corn Starch20.0000Casein - Vitamin Tested13.2000Maltodextrin10.0000Sucrose7.0000Soybean Oil3.5000AIN 93G Mineral Mix2.5000Powdered Cellulose2.5000Guar Gum1.0000AIN 93 Vitamin Mix0.3000L-Cystine0.2500Choline Bitartrate0.0014t-Butylhydroquinone1818039-2031/2" Pellet, Irradiated

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