WO2020041581A1

WO2020041581A1 - Methods and compositions for preventing and treating inflammatory bowel disease and nonalcoholic fatty liver disease

Info

Publication number: WO2020041581A1
Application number: PCT/US2019/047692
Authority: WO
Inventors: Andrew J. Dannenberg; David C. Montrose
Original assignee: Cornell University
Current assignee: Cornell University
Priority date: 2018-08-23
Filing date: 2019-08-22
Publication date: 2020-02-27
Anticipated expiration: 2021-02-23

Abstract

The present technology relates to methods for preventing or treating inflammatory bowel disease or nonalcoholic fatty liver disease comprising administering an effective amount of a prebiotic composition (e.g., psyllium) or a probiotic composition (e.g., Lactobacillus johnsonii) to a subject in need thereof. Kits for use in practicing the methods are also provided.

Description

METHODS AND COMPOSITIONS FOR PREVENTING AND TREATING INFLAMMATORY BOWEL DISEASE AND NONALCOHOLIC FATTY LIVER

DISEASE

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

[001] This application claims the benefit of and priority to US Provisional Appl. No. 62/722,116, filed August 23, 2018, the disclosure of which is incorporated by reference herein in its entirety.

TECHNICAL FIELD

[002] The present technology relates to methods for preventing or treating inflammatory bowel disease or nonalcoholic fatty liver disease comprising administering an effective amount of a prebiotic composition ( e.g ., psyllium) or a probiotic composition (e.g., Lactobacillus johnsonii) to a subject in need thereof. Kits for use in practicing the methods are also provided.

BACKGROUND

[003] The following description of the background of the present technology is provided simply as an aid in understanding the present technology and is not admitted to describe or constitute prior art to the present technology.

[004] The incidence of inflammatory bowel diseases (IBD) has increased over the last several decades, including in countries with traditionally low incidence. Hou et al ., Am J Gastroenterol 104:2100-9 (2009); Molodecky et al., Gastroenterology 142:46-54 (2012); Yang et al., Gastroenterology l5l :el-e5 (2016). Given this increase and the severe morbidity associated with IBD, there is a critical need for novel preventive and therapeutic interventions. In association with this rising incidence of IBD, consumption of a western diet, has dramatically increased.

SUMMARY

[005] In one aspect, the present disclosure provides a method for treating inflammatory bowel disease in a subject in need thereof comprising administering to the subject an effective amount of a probiotic composition comprising bacterial cells belonging to one or more genera selected from the group consisting of Lactobacillus, Bifidobacterium, Bacteroides, Enterococcus and Clostridium. Also provided herein are methods for treating inflammatory bowel disease in a subject in need thereof comprising administering to the subject an effective amount of a probiotic composition including one or more of

Lactobacillus sp. strain 100-14, Lactobacillus sp. strain 100-100, Lactobacillus acidophilus, Lactobacillus delbrueckii, Lactobacillus fermentum, Lactobacillus sp. strain 100-93, Lactobacillus plantarum, Lactobacillus salivarius, Lactobacillus johnsonii, Lactobacillus amylovorous, Lactobacillus amylophilus, Lactobacillus brevis, Lactobacillus buchneri, Lactobacillus casei, Lactobacillus crispatus, Lactobacillus farciminis, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus hilgardii, Lactobacillus kefir anofaciens,

Lactobacillus lindneri, Lactobacillus paracasei, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus sanfranciscensis, L. salivaricus strain JCM1046, Lactobacillus casei Shirota, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus

mucosae, Lactobacillus rhamnosus GG, Bifidobacterium longum, Bifidobacterium

adolescentis, Bifidobacterium animalis, Bifidobacterium thermophiles, Bifidobacterium pseudolongum, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium bifidum, Bifidobacterium lactis, Enterococcus faecium, Clostridium disporicum , Clostridium perfiringens, Methanobrevibacter smithii, Methanosphera stadmanae, Bacteroides ovatus, Bacteroides thetaiotaomicron, Bacteroides acidifaciens, Bacteroides vulgatus, Bacteroides firagilis, Bacteroides firagilis subsp. firagilis, and Peptostreptococcus species. In some embodiments, the subject exhibits at least one symptom selected from the group consisting of abdominal pain, diarrhea, rectal bleeding, weight loss, fever, loss of appetite, dehydration, anemia and malnutrition. Additionally or alternatively, in some embodiments, the subject exhibits at least one mutation in one or more genes selected from the group consisting of NOD2, IRGM, ATG16L1, IL23R, IL10, IL10RA, 11.1 ORB, and PTPN2.

[006] In one aspect, the present disclosure provides a method for treating nonalcoholic fatty liver disease in a subject in need thereof comprising administering to the subject an effective amount of a probiotic composition comprising bacterial cells belonging to one or more genera selected from the group consisting of Lactobacillus , Bifidobacterium,

Bacteroides, Enterococcus and Clostridium. Also disclosed herein are methods for treating nonalcoholic fatty liver disease in a subject in need thereof comprising administering to the subject an effective amount of a probiotic composition including one or more of

mucosae, Lactobacillus rhamnosus GG, Bifidobacterium longum, Bifidobacterium

adolescentis, Bifidobacterium animalis, Bifidobacterium thermophiles, Bifidobacterium pseudolongum, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium bifidum, Bifidobacterium lactis, Enterococcus faecium, Clostridium disporicum , Clostridium perfiringens, Methanobrevibacter smithii, Methanosphera stadmanae, Bacteroides ovatus, Bacteroides thetaiotaomicron, Bacteroides acidifaciens, Bacteroides vulgatus, Bacteroides firagilis, Bacteroides firagilis subsp. firagilis, and Peptostreptococcus species. In some embodiments, the subject exhibits at least one symptom selected from the group consisting of fatty liver, nonalcoholic steatosis, tiredness, fatigue, including muscle weakness, discomfort or swelling in the upper abdomen, weight loss, low appetite, nausea, cirrhosis, liver failure, vomiting, and diarrhea, tarry stools, abdominal swelling and pain, jaundice, itchy skin, confusion, difficulty focusing, memory loss, and hallucinations.

[007] Additionally or alternatively, in some embodiments of the methods disclosed herein, the probiotic composition is administered orally or rectally. In any and all embodiments of the methods disclosed herein, the subject is human. In certain embodiments, the probiotic composition of the present technology is co-administered with a bile acid sequestrant, such as cholestyramine, colestipol, or colesevelam.

[008] In any and all embodiments of the methods disclosed herein, the probiotic composition is sequentially, simultaneously, or separately administered with at least one additional therapeutic agent selected from the group consisting of an anti-inflammatory agent, a corticosteroid, an aminosalicylate, an immunosuppressive agent, a tumor necrosis factor (TNF)-alpha inhibitor, a pain reliever, an iron supplement, calcium, and a vitamin D supplement. In certain embodiments, the probiotic composition is sequentially,

simultaneously, or separately administered with one or more agents selected from the group consisting of mesalamine, sulfasalazine, infliximab, adalimumab, prednisone, budesonide, 6- mercaptopurine, 5-aminosalicylic acid (5-ASA), azathioprine, cyclosporine, golimumab, balsalazide, olsalazine, methotrexate, natalizumab, vedolizumab, ustekinumab, acetaminophen, ibuprofen, naproxen sodium (Aleve), diclofenac sodium, orlistat, sibutramine, pioglitazone, rosiglitazone, metformin, atorvastatin, pravastatin, rosuvastatin, gemfibrozil, ursodiol, vitamin E, vitamin C, pentoxifylline, betaine, and losartan.

[009] Additionally or alternatively, in some embodiments of the methods disclosed herein, administration of the probiotic composition results in reduced levels of one or more bile acids compared to an untreated subject suffering from inflammatory bowel disease or nonalcoholic fatty liver disease. In some embodiments, the one or more bile acids are selected from the group consisting of glycocholic acid, glycochenodeoxycholic acid, glycodeoxycholic acid, taurocholic acid, taurochenodeoxycholic acid and taurodeoxycholic acid.

[0010] In another aspect, the present disclosure provides a method for monitoring the therapeutic efficacy of a probiotic composition in a subject diagnosed with inflammatory bowel disease or nonalcoholic fatty liver disease comprising: (a) detecting conjugated bile acid levels in a test sample obtained from the subject after the subject has been administered the probiotic composition; and (b) determining that the probiotic composition is effective when the conjugated bile acid levels in the test sample are reduced compared to that observed in a control sample obtained from the subject prior to administration of the probiotic composition, wherein the probiotic composition includes one or more of Lactobacillus sp. strain 100-14, Lactobacillus sp. strain 100-100, Lactobacillus acidophilus, Lactobacillus delbrueckii, Lactobacillus fermentum, Lactobacillus sp. strain 100-93, Lactobacillus plantarum, Lactobacillus salivarius, Lactobacillus johnsonii, Lactobacillus amylovorous, Lactobacillus amylophilus, Lactobacillus brevis, Lactobacillus buchneri, Lactobacillus casei, Lactobacillus crispatus, Lactobacillus farciminis, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus hilgardii, Lactobacillus kefir anofaciens, Lactobacillus lindneri, Lactobacillus paracasei, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus sanfranciscensis, L. salivaricus strain JCM 1046, Lactobacillus casei Shirota, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus mucosae, Lactobacillus rhamnosus GG, Bifidobacterium longum, Bifidobacterium adolescentis, Bifidobacterium animalis,

Bifidobacterium thermophiles, Bifidobacterium pseudolongum, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium bifidum, Bifidobacterium lactis, Enterococcus faecium, Clostridium disporicum , Clostridium perfiringens, Methanobrevibacter smithii, Methanosphera stadmanae, Bacteroides ovatus, Bacteroides thetaiotaomicron, Bacteroides acidifaciens, Bacteroides vulgatus, Bacteroides fragilis, Bacteroides firagilis subsp. firagilis, and Peptostreptococcus species. The test sample may be feces. Additionally or alternatively, in some embodiments, the method further comprises detecting bacterial bile salt hydrolase levels in the feces of the subject.

[0011] Also disclosed herein are kits for practicing the methods of the present technology. In one aspect, the kits of the present technology comprise a probiotic composition and instructions for using the probiotic composition to treat inflammatory bowel disease or nonalcoholic fatty liver disease, wherein the probiotic composition includes one or more of Lactobacillus sp. strain 100-14, Lactobacillus sp. strain 100-100, Lactobacillus acidophilus, Lactobacillus delbrueckii, Lactobacillus fermentum, Lactobacillus sp. strain 100-93, Lactobacillus plantarum, Lactobacillus salivarius, Lactobacillus johnsonii, Lactobacillus amylovorous, Lactobacillus amylophilus, Lactobacillus brevis, Lactobacillus buchneri, Lactobacillus casei, Lactobacillus crispatus, Lactobacillus farciminis, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus hilgardii, Lactobacillus kefir anofaciens,

mucosae, Lactobacillus rhamnosus GG, Bifidobacterium longum, Bifidobacterium adolescentis, Bifidobacterium animalis, Bifidobacterium thermophiles, Bifidobacterium pseudolongum, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium bifidum, Bifidobacterium lactis, Enterococcus faecium, Clostridium disporicum , Clostridium perfiringens, Methanobrevibacter smithii, Methanosphera stadmanae, Bacteroides ovatus, Bacteroides thetaiotaomicron, Bacteroides acidifaciens, Bacteroides vulgatus, Bacteroides firagilis, Bacteroides firagilis subsp. firagilis, and Peptostreptococcus species.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figures 1A-1B show modification of dietary carbohydrates for mice. Figure 1A shows 3 isocaloric diets that contained 64 kcal% carbohydrate: the control diet (AIN-93G), the 64% Glucose diet (containing 64 kcal% of glucose), and the 64% Fructose diet

(containing 64 kcal% of fructose). Figure IB shows 2 diets that contained 64 kcal% carbohydrate: the control diet (AIN-93G) and the 15% Fructose diet (containing 15 kcal% of fructose).

[0013] Figure 2 shows the experimental design used to test whether increased dietary fructose affects colitis severity. 8 week old male mice (n=8/group) were fed one of the three diets shown in Figure 1A for 2 weeks prior to being treated with 1% dextran sodium sulfate (DSS) in drinking water, a chemical that induces experimental colitis. The 3 diets were continued throughout the 1 week when DSS was given.

[0014] Figures 3A-3E demonstrate that a high fructose diet results in more severe experimental colitis. Male C57BL/6J mice were given the control diet, a high glucose diet (HGD), or a high fructose diet (HFrD) from Figure 1(A) for 2 weeks followed by

administration of 1% DSS in drinking water for 1 week. Indicators of disease severity were measured including weight loss, severe diarrhea, rectal bleeding, colon length, and percent colonic ulceration. P values represent comparisons of HFrD to control diet. N=8 mice per group. **P <0.01; ***P <0.001. Figure 3A shows the average body weight compared to day 0 (%) for mice fed the control diet, the HGD, or the HFrD. Figure 3B shows the percentage of mice with severe diarrhea (%) for mice fed the control diet, the HGD, or the HFrD.

Figure 3C shows the percentage of mice with severe bleeding (%) for mice fed the control diet, the HGD, or the HFrD. Figure 3D shows the colon length (cm) for mice fed the control diet, the HGD, or the HFrD. Figure 3E shows the percent colonic ulceration (%) for mice fed the control diet, the HGD, or the HFrD.

[0015] Figures 4A-4E demonstrate that feeding mice a HFrD substantially increases the expression of immune-related pro-inflammatory cytokines following DSS exposure. Relative cytokine gene expression was measured by qRT-PCR in the colons of mice given the control diet or the HFrD of Figure 1A for 2 weeks followed by 1% DSS administration in drinking water for 7 days. Mice receiving either diet and plain drinking water for 2 weeks served as controls. The mean log transformed data (S.D.) are shown for each gene and the P value was calculated by evaluating the difference in the magnitude of change of each gene between mice given HFrD compared to control diet following DSS exposure (n=6-8 per group) under a linear regression framework. Figure 4A shows a trend towards increased pro-inflammatory TNF cytokine expression in colons of mice given HFrD. Figure 4B shows increased pro- inflammatory IL1B cytokine expression in colons of mice given HFrD. Figure 4C shows increased pro-inflammatory IL6 cytokine expression in colons of mice given HFrD. Figure 4D shows increased pro-inflammatory IL17A cytokine expression in colons of mice given HFrD. Figure 4E shows increased pro-inflammatory IL22 cytokine expression in colons of mice given HFrD.

[0016] Figure 5 demonstrates that HFrD feeding enhances the number of immune cells (CD45+ cells, neutrophils, macrophages, dendritic cells and natural killer cells) in the colon during colitis. The number of immune cells in the lamina propria of colons from mice given the control diet or the HFrD from Figure 1A for 1 week followed by 1% DSS administration in drinking water for 7 days was measured by FACS. The mean log transformed data (S.D.) are shown for each cell type (n=4 per group). *P<0.05; **R<0.01; ***P<0.00l.

[0017] Figure 6 shows the experimental design used to test whether there is a dose- dependent increase in colitis severity in response to dietary fructose. The carbohydrate content of the diets was modulated to serially increase fructose levels to determine whether fructose feeding induced a dose-dependent increase in colitis severity. Diets were fed to mice for 2 weeks. Then, the mice were exposed to 1% DSS while being continued on their respective diets.

[0018] Figures 7A-7D demonstrate that the severity of colitis rises as the concentration of fructose in the diet increases. Mice were subjected to the experimental design of Figure 6. Indicators of disease severity were measured including weight loss, severe diarrhea, severe bleeding, and colon length p values represent the difference when fructose was used as a continuous variable. N=l6 mice per group. Figure 7A shows the average body weight compared to day 0 (%) for mice fed one of the four diets from Figure 6: HGD, 1 : 1 Glu:Fru,

1 :3 Glu:Fru, or HFrD. Figure 7B shows the percentage of mice with severe diarrhea for mice fed one of the four diets from Figure 6: HGD, 1 : 1 Glu:Fru, 1 :3 Glu:Fru, or HFrD.

Figure 7C shows the percentage of mice with severe bleeding for mice fed one of the four diets from Figure 6: HGD, 1 : 1 Glu:Fru, 1 :3 Glu:Fru, or HFrD. Figure 7D shows the colon length (cm) for mice fed one of the four diets from Figure 6: HGD, 1 : 1 Glu:Fru, 1 :3 Glu:Fru, or HFrD.

[0019] Figures 8A-8B demonstrate that feeding mice a diet containing 15 kcal% fructose worsens experimental colitis. Male C57BL/6J mice were given the control diet or the diet containing 15 kcal% fructose from Figure IB for 1 week followed by administration of 2% DSS in drinking water for 1 week. Indicators of disease severity were measured including colon length and percent colonic ulceration. N=l3-l4 mice per group. Figure 8A shows the colon length (cm) for mice fed either the control diet or the 15 kcal% fructose diet. Figure 8B shows the percent colonic ulceration (%) for mice fed either the control diet or the 15 kcal% fructose diet.

[0020] Figure 9 shows an exemplary representation that elevated dietary fructose increases the incidence and severity of colitis by altering the microbiota and disrupting colonic immune homeostasis. Specifically, Figure 9 shows that when exposed to a normal diet, the small intestine can absorb fructose efficiently via the GLUT5 transporter. By contrast, a high fructose diet saturates GLUT5 in the small intestine leading to elevated levels of fructose in the colon. Elevated amounts of fructose in the colon lead to a change in the microbiota that adversely affects gut barrier function leading to an inflammatory response and exacerbates colitis. Worse colitis should, in turn, increase the risk of colorectal cancer (CRC).

[0021] Figure 10 demonstrates that high fructose feeding increases colonic luminal fructose levels. 8 week old male mice were fed the control diet or the HFrD for 1 week and then fecal levels of fructose were measured.

[0022] Figure 11 shows the experimental design used to determine whether a HFrD diet alters the bacteria profile and/or luminal metabolites in mice. Mice were co-housed to homogenize the microbiota and fed the AIN-93 G diet for 2 weeks before being individually housed and fed either AIN-93 G or a HFrD for 1 week. Feces were collected as indicated to evaluate the microbiota at baseline and after treatment with control (AIN-93G) or HFrD.

[0023] Figure 12 demonstrates that HFrD feeding shifts the fecal microbiota in mice.

Male C57BL/6J mice were given AIN-93G purified diet and co-housed for 2 weeks. Mice were then individually housed and continued on AIN-93 G control diet (Cont) or switched to a HFrD for 1 week. Feces were collected following co-housing (Day 0), then again following 7 days of diet intervention (Day 7). 16S rRNA microbial sequencing was carried out on the feces and the results were plotted by principal coordinate analysis. The black oval indicates samples from mice given HFrD for 7 days.

[0024] Figure 13 demonstrates that a high fructose diet alters the abundance of bacterial species. Male C57BL/6J mice were given AIN-93G purified diet and co-housed for 2 weeks. Mice were then individually housed and continued on AIN-93 G control diet (Control) or switched to a HFrD (Fructose) for 1 week. Feces were collected following co-housing (Day 0), then again following 7 days of diet intervention (Day 7). 16S rRNA microbial sequencing was carried out on the feces. The graph represents the abundance (%) of each bacterial species in the feces. The different bacterial species listed in the legend to the right of the graph correspond to a subgroup stacked in each individual bar in the graph in the same order from top-to-bottom.

[0025] Figure 14 demonstrates that HFrD feeding alters the proportion of particular microbes. For example, the high fructose diet led to an increase in Akkermansia muciniphila (dark arrows/dark rectangle) and a decrease in Lactobacillus johnsonii (light arrows/light rectangle). Male C57BL/6J mice were given AIN-93G purified diet and co-housed for 2 weeks. Mice were then individually housed and continued on AIN-93 G control diet (Control) or switched to a HFrD (Fructose) for 1 week. Feces were collected following co- housing (Day 0), then again following 7 days of diet intervention (Day 7). 16S rRNA microbial sequencing was carried out on the feces. The graph represents the percent of total species of bacteria across samples in each group. The different species of microbes listed in the legend to the right of the graph each have a numerical key that corresponds to the same numerical value listed above the top of each bar in the graph.

[0026] Figure 15 shows the experimental design used to determine whether switching back to the control diet following HFrD feeding reverses susceptibility to colitis and results in normalization of the gut microbiota.

[0027] Figures 16A-16D demonstrate that switching back to the control diet following HFrD feeding reverses the colitis phenotype in association with normalization of the fecal microbiota. Male C57BL/6J mice were given AIN-93G control diet or HFrD for 1 week.

The cages then underwent bedding swapping for 2 weeks while being continued on control diet. Mice were then divided into 2 groups and either continued on control diet or given HFrD for 1 week. Following this period, half of the mice given HFrD were switched back to control diet for 1 week while the other 2 groups remained on their original diets. Mice were then challenged with 1% DSS for 1 week and changes in body weight, colon length, and ulceration were analyzed. ***P<0.00l. Figure 16A shows the average body weight compared to day 0 (%) for mice fed only the control diet, only HFrD, or first HFrD and then the control diet. Figure 16B shows the colon length (cm) for mice fed only the control diet, only HFrD, or first HFrD and then the control diet. Figure 16C shows the percent colonic ulceration (%) for mice fed only the control diet, only HFrD, or first HFrD and then the control diet. Figure 16D demonstrates that switching back to the control diet following HFrD normalizes the fecal microbiota. Feces were collected following the control diet after one and two weeks, following the HFrD after one and two weeks, before the switch from HFrD to the control diet, and after the switch from HFrD to the control diet. 16S rRNA microbial sequencing was carried out on the feces and the results were plotted by principal coordinate analysis. The black circle indicates samples from mice given HFrD for 1 or 2 weeks or mice before the switch from HFrD to the control diet.

[0028] Figure 17 demonstrates that switching back to the control diet following HFrD normalizes bacterial alterations. For example, switching back to the control diet following HFrD led to a decrease in Akkermansia muciniphila (dark arrows/dark rectangle) and an increase in Lactobacillus johnsonii (light arrows/light rectangle). Male C57BL/6J mice were given control (AIN-93G) purified diet for 1 week then continued on this diet for another 2 weeks, during which bedding swapping was performed to homogenize the microbiota.

Following this period, mice were divided into 3 groups and fed the following diets: 1) control for 2 weeks, 2) HFrD for 2 weeks, 3) HFrD for 1 week then switched to control diet for 1 week. Feces were collected on Week 0, 1 and 2 and subjected to 16S rRNA microbial sequencing. The graph represents the percent of total species of bacteria across samples in each group. The different species of microbes listed in the legend to the right of the graph each have a numerical key that corresponds to the same numerical value listed above the top of each bar in the graph.

[0029] Figure 18 shows the experimental design, using broad-spectrum antibiotics (Abx), to test whether reducing bacterial load attenuates the effects of a HFrD on colitis severity. Five week-old C57BL/6J WT mice were given control diet and administered drinking water containing Splenda (4g/L) (Groups 1 & 2) or control diet and water containing ampicillin (0.5g/L), metronidazole (0.5g/L), vancomycin (0.25g/L), neomycin (0.5g/L), gentamicin (0.5g/L) and Splenda (4g/L) (Groups 3 & 4) for 3 weeks (N=l0 mice per group). Feces were collected in week 3 to measure bacterial abundance by PCR to ensure that antibiotic treatment was effective. Following this treatment and while being kept on their respective drinking water formulations, Groups 2 & 4 were given a HFrD, while Groups 1 & 3 continued on the control diet for 1 week. All mice were then given 1% DSS for 7 days while continuing on their respective diets and drinking water formulations.

[0030] Figure 19 demonstrates that treatment with broad-spectrum antibiotics strongly reduces gut bacterial load. The antibiotic regimen (Abx) containing ampicillin (0.5g/L), gentamicin (0.5g/L), metronidazole (0.5g/L), neomycin (0.5g/L), vancomycin (0.25g/L), and Splenda (4g/L) was given for 3 weeks. Splenda (4g/L) was included to mask the bad taste of the antibiotics and therefore maintain adequate water consumption by mice. Control mice were given drinking water containing only Splenda. Mice were continued on their respective treatments during DSS exposure. Fecal pellets were collected to measure bacterial abundance. For bacterial qRT-PCR, buffer ASL was added to fecal samples followed by vigorous vortexing until thoroughly homogenized. DNA was extracted using the QIAamp DNA Stool Mini Kit. For each sample, 8 ng of DNA was used to carry out qRT-PCR using Fast SYBR green PCR master mix and universal bacterial primers on a StepOnePlus real time PCR system. The bar graph represents the mean Ct value following PCR for each group. A higher Ct value indicates less amplification and lower bacterial abundance. [0031] Figures 20A-20C demonstrate that antibiotic administration attenuates HFrD- mediated exacerbation of colitis. Mice were subjected to the experimental design Groups 1-4 of Figure 18. Figure 20A shows the average percent body weight by group (%) for Group 1 (W Control (Ctrl Water; Ctrl Diet)), Group 2 (W HFrD (Ctrl Water; HFrD Diet)), Group 3 (Abx Control (Abx Water; Ctrl Diet)), and Group 4 (Abx HFrD (Abx Water; HFrD Diet)). Figure 20B shows the colon length (cm) for mice from Group 1 (Ctrl Water; Ctrl Diet), Group 2 (Ctrl Water; HFrD), Group 3 (Abx Water; Ctrl Diet), and Group 4 (Abx Water; HFrD). Figure 20C shows the percent colonic ulceration (%) for mice from Group 1 (Ctrl; Water), Group 2 (HFrD; Water), Group 3 (Ctrl; Abx), and Group 4 (HFrD; Abx). **P<0.0l. ***P<0.00l.

[0032] Figures 21A-21E demonstrate that HFrD does not exacerbate colitis in germ-free (GF) mice. GF mice and specific-pathogen-free (SPF) mice, which have a normal microbiome, were fed either the control diet or the HFrD for 1 week followed by

administration of 1% DSS for 6 days. Figure 21A shows the average body weight compared to day 0 (%) for GF and SPF mice fed either the control diet or the HFrD. Figure 21B shows the percentage of mice with severe diarrhea (%) for GF and SPF mice fed either the control diet or the HFrD. Figure 21C shows the percentage of mice with severe bleeding (%) for GF and SPF mice fed either the control diet or the HFrD. Figure 21D shows the colon length (cm) for GF and SPF mice fed either the control diet or the HFrD. Figure 21E shows the percent colonic ulceration (%) for GF and SPF mice fed either the control diet or the HFrD.

[0033] Figures 22A-22C demonstrate that colon mucus thickness is reduced in HFrD fed mice. Mice were fed either a control or HFrD for 1 week and a segment of colon containing feces was harvested and immediately preserved in fresh Carnoy’s fixative

(methanokchloroforrmglacial acetic acid (60:30: 10)) for 4-5 days. Tissues were washed twice in 100% methanol followed by 2 washes in 100% ethanol. The fixed tissues were then washed in a 1 : 1 ratio of 100% ethanol: xylene followed by two washes in xylene and subsequently embedded in paraffin with a vertical orientation. Paraffin sections were cut and deposited onto glass slides. Alcian blue/periodic acid Schiff (AB/PAS) staining was performed to stain mucus. Following staining, 40-60 measurements of mucus thickness were made for each colon in a blinded manner using NIH ImageJ software and the average of the measurements was calculated to generate a value for each sample. Figure 22A shows a representative image illustrating the colon mucus thickness for a mouse fed the control diet. Figure 22B shows a representative image illustrating the colon mucus thickness for a mouse fed the HFrD. Figure 22C shows the Centered log(Mucus thickness (A.U.)) for mice fed either the control diet or the HFrD. N=20-22 mice per group. P<0.0l. Figure 22D shows the number of goblet cells in colonic sections from control or HFrD-fed mice. Colonic sections from control or HFrD-fed mice were stained with PAS/AB to identify mucus, and the number of goblet cells was calculated by determining the percent of positive staining relative to the entire mucosa. (n=l3/group). The data are presented as mean ± SD.

[0034] Figure 23 demonstrates that a HFrD causes colonic gene expression changes consistent with increased inflammation. RNA-seq was carried out on colonic mucosa from mice given the control diet or the HFrD for 1 week (n=4/group). Differentially expressed genes were input into the Gene Ontology Consortium database to determine significantly altered pathways. Size = number of genes in the gene set; ES = enrichment score; NES = normalized enrichment score; FDR q-val = false discovery rate.

[0035] Figure 24 demonstrates that fecal bile acid levels increase following HFrD feeding. Mice were co-housed for 2 weeks while being fed control diet then either continued on control diet or given HFrD for 1 week. Fecal samples were collected and shipped to

Metabolon for detection of metabolites using a database of over 3,000 named molecules. Fecal samples were lyophilized and then resuspended in water (20 pL/mg of dried sample) for homogenization. Following homogenization, 100 pL of the fecal suspensions was used for extraction. Extracts were prepared using the automated MicroLab STAR system

(Hamilton Company, Reno NV). A recovery standard was added prior to the first step in the extraction process for QC purposes. To remove protein, dissociate small molecules bound to protein or trapped in the precipitated protein matrix, and to recover chemically diverse metabolites, proteins were precipitated with methanol under vigorous shaking for 2 minutes followed by centrifugation. The resulting extract was divided into five fractions: one for analysis by EIPLC-MS/MS with positive ion mode electrospray ionization, one for analysis by EIPLC-MS/MS with negative ion mode electrospray ionization, one for LC polar platform analysis, one for analysis by GC-MS, and one sample was reserved for backup. Samples were placed briefly on a TurboVap (Zymark) to remove the organic solvent. For LC, the samples were stored overnight under nitrogen before preparation for analysis. For GC, each sample was dried under vacuum overnight before preparation for analysis. Detection of metabolites was performed using Ultrahigh Performance liquid chromatography-tandem mass spectroscopy and gas chromatography-mass spectroscopy (GC-MS). The data presented in this table show the relative fold changes in bile acid levels following HFrD feeding compared to mice fed control diet.

[0036] Figure 25 demonstrates reduced colonic Lactobacillus johnsonii bile salt hydrolase gene expression from HFrD fed mice. Mice were co-housed for 2 weeks while being fed control diet then either continued on control diet or given HFrD for 1 week. Fecal samples were collected and DNA was extracted using the QIAamp DNA Stool Mini Kit per manufacturer’s instructions. For each sample, 8ng of DNA was used to carry out qRT-PCR using Fast SYBR green PCR master mix and primers specific for the bile salt hydrolase gene of Lactobacillus johnsonii on a StepOnePlus real-time PCR system. N = 5-9 samples.

**P<0.0l.

[0037] Figures 26A-26B demonstrate that high fructose feeding increases colitis- associated tumor number. Mice were administered one injection of AOM (l2.5mg/kg) and were then given the control diet or HFrD for 1 week followed by 3 rounds of DSS

administration (each round consisted of 5 days of DSS followed by 14 days of plain drinking water) while being maintained on the respective diets. Four weeks following the last round of DSS, mice underwent colonoscopy and then were sacrificed to count tumor number.

N=l2-l6 mice per group. Figure 26A shows the colonoscopy of a mouse fed the control diet versus a mouse fed HFrD. White arrows mark tumors. Figure 26B shows a comparison of the tumor numbers for mice fed the control diet versus mice fed HFrD.

[0038] Figures 27A-27C demonstrate that a HFrD enhances chronic colitis. Mice were administered one injection of azoxymethane (AOM; l2.5mg/kg) and were then given the control diet or HFrD for 1 week followed by 3 rounds of DSS administration (each round consisted of 5 days of DSS followed by 14 days of plain drinking water) while being maintained on the respective diets. N=l2-l6 mice per group. Figure 27A shows the average body weight compared to day 0 (%) for mice fed either the control diet or HFrD. Figure 27B shows the colon length (cm) for mice fed either the control diet or HFrD. Figure 27C shows the histologic score for mice fed either the control diet or HFrD.

[0039] Figure 28 shows the cytokine expression in colons of mice fed control or HFrD during chronic colitis for IFNG, IL1B , IL6 , TNF, and IL17A.

[0040] Figures 29A-29D demonstrate that psyllium protects against experimental colitis. Male C57BL/6J mice were given the control diet or a HFrD containing cellulose or psyllium (Psy) as their fiber source for 1 week followed by administration of 1% DSS in drinking water for 1 week. Indicators of disease severity were measured including weight loss, severe bleeding, colon length, and percent colonic ulceration. N=8 mice per group. Figure 29A shows the average body weight compared to day 0 (%) for mice fed the control diet with cellulose, the control diet with Psy, HFrD with cellulose, or HFrD with Psy. Figure 29B shows the percentage of mice with severe bleeding (%) for mice fed the control diet with cellulose, the control diet with Psy, HFrD with cellulose, or HFrD with Psy. Figure 29C shows the colon length (cm) for mice fed the control diet with cellulose, the control diet with Psy, HFrD with cellulose, or HFrD with Psy. Figure 29D shows the percent colonic ulceration (%) for mice fed the control diet with cellulose, the control diet with Psy, HFrD with cellulose, or HFrD with Psy.

[0041] Figure 30 shows the experimental design for testing the tumor preventive effects of dietary fructose removal alone or in combination with 5-ASA. One injection of

azoxymethane (AOM, l2.5mg/kg) will be administered to mice. Subsequently, the mice will be fed either the control diet or HFrD for 1 week and exposed to DSS for 5 days followed by 1 week of plain drinking water. Mice will then be randomized into the following 5 groups: (1) continued on the control diet, (2) continued on HFrD, (3) switched from HFrD to the control diet, (4) continued on HFrD and given 5-ASA (75mg/kg in drinking water), and (5) switched from HFrD to the control diet and given 5-ASA. N=20 mice per group. Mice will be continued on plain drinking water for 1 week then given 2 cycles of DSS (1 cycle consists of 5 days of DSS followed by 2 weeks of plain drinking water) during which the respective diets/treatments (Groups 1 through 5) will be given.

[0042] Figures 31A-31D show the summary tables containing the numerical values corresponding to Figure 13.

[0043] Figure 32A shows the relative abundance of B. pseudolongum in feces from mice fed control or HFrD for 1 week as determined by qRT-PCR (n=8-9/group). Figure 32B shows the expression of BSH in Bifidobateriaum pseudolongum as measured by qRT-PCR in the same samples as Figure 32A.

[0044] Figures 33A-33E show that the HFrD enhances growth of Citrobacter rodentium and exacerbates infectious colitis. WT C57BL/6J mice were fed control or HFrD for 1 week followed by infection with Citrobacter rodentium and continued on their respective diets for 6 days. Figure 33A shows the abundance of C. rodentium measured in feces every 2 days following infection (N = 6/group). Figure 33B shows the clinical measurements of colitis severity that were carried out daily during the 6 day period. Figure 33C-33E show the colon lengths, severity of histological injury, and expression levels of cytokines. At the end of the experimental period, colon lengths were measured (Figure 33C), the severity of histologic injury was evaluated using a 0-4 scoring system (Figure 33D) and expression levels of cytokines were quantified (Figure 33E).

[0045] Figure 34 shows the quantitative analysis of the populations of multiple bacterial species in HFrD-fed animals relative to control diet fed animals.

DETAILED DESCRIPTION

[0046] The incidence of inflammatory bowel disease (IBD) has been increasing in the U.S. and the rest of the world over the last several decades, including in countries with

traditionally low incidence. IBD is a risk factor for the development of colorectal cancer (CRC), a risk that increases with duration of disease. In parallel with this rising incidence, consumption of a western diet, including free fructose, has also dramatically increased.

Indeed, consumption of fructose has increased by approximately 30% over the last 3 decades in the U.S. alone, with sugary beverages and processed foods as a major source. Although multiple factors potentially contribute to the increasing incidence of IBD, excess intake of fructose may be an important and unrecognized contributor. In addition to a correlation between fructose consumption and IBD incidence, current evidence suggests that diets containing high amounts of FODMAPs (fermentable, oligo-, di-, monosaccharides and polyols), which include fructose, may negatively impact the course of already established disease.

[0047] Given the rapid increase in IBD incidence and its related cancer risk, there is a critical need for effective preventive and therapeutic interventions. The present disclosure demonstrates that elevated fructose consumption worsened DSS as well as infectious models of colitis. The exacerbation of colitis was microbiota-dependent and appeared to be mediated by changes in the composition, and functional activity of the microbiota. These findings demonstrated a direct connection between dietary fructose, microbes and intestinal inflammation.

Definitions

[0048] Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this technology belongs. As used in this specification and the appended claims, the singular forms“a”,“an” and“the” include plural referents unless the content clearly dictates otherwise. For example, reference to“a cell” includes a combination of two or more cells, and the like. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, analytical chemistry and nucleic acid chemistry and hybridization described below are those well-known and commonly employed in the art.

[0049] As used herein, the term“about” in reference to a number is generally taken to include numbers that fall within a range of 1%, 5%, or 10% in either direction (greater than or less than) of the number unless otherwise stated or otherwise evident from the context (except where such number would be less than 0% or exceed 100% of a possible value).

[0050] As used herein, the“administration” of an agent or drug to a subject includes any route of introducing or delivering to a subject a compound to perform its intended function. Administration can be carried out by any suitable route, including orally, intranasally, parenterally (intravenously, intramuscularly, intraperitoneally, or subcutaneously), intratumorally, or topically. Administration includes self-administration and the

administration by another.

[0051] As used herein, a "control" is an alternative sample used in an experiment for comparison purpose. A control can be "positive" or "negative." For example, where the purpose of the experiment is to determine a correlation of the efficacy of a therapeutic agent for the treatment for a particular type of disease or condition, a positive control (a compound or composition known to exhibit the desired therapeutic effect) and a negative control (a subject or a sample that does not receive the therapy or receives a placebo) are typically employed.

[0052] As used herein, the term“effective amount” refers to a quantity sufficient to achieve a desired therapeutic and/or prophylactic effect, e.g ., an amount which results in the prevention of, or a decrease in a disease or condition described herein or one or more signs or symptoms associated with a disease or condition described herein. In the context of therapeutic or prophylactic applications, the amount of a composition administered to the subject will vary depending on the composition, the degree, type, and severity of the disease and on the characteristics of the individual, such as general health, age, sex, body weight and tolerance to drugs. The skilled artisan will be able to determine appropriate dosages depending on these and other factors. The compositions can also be administered in combination with one or more additional therapeutic compounds. In the methods described herein, the therapeutic compositions may be administered to a subject having one or more signs or symptoms of a disease or condition described herein. As used herein, a “therapeutically effective amount” of a composition refers to composition levels in which the physiological effects of a disease or condition are ameliorated or eliminated. A

therapeutically effective amount can be given in one or more administrations.

[0053] As used herein,“expression” includes one or more of the following: transcription of the gene into precursor mRNA; splicing and other processing of the precursor mRNA to produce mature mRNA; mRNA stability; translation of the mature mRNA into protein (including codon usage and tRNA availability); and glycosylation and/or other modifications of the translation product, if required for proper expression and function.

[0054] As used herein, the terms“individual”,“patient”, or“subject” are used

interchangeably and refer to an individual organism, a vertebrate, a mammal, or a human. In certain embodiments, the individual, patient or subject is a human.

[0055] As used herein, a“prebiotic composition” refers to a composition that comprises a nondigestable substance that promotes the growth of probiotic gut bacteria by providing a suitable environment in which the probiotics themselves can flourish. In some embodiments, the prebiotic composition includes one or more of psyllium, acadia gum, polydextrose, wheat dextrin, pectin, inulin, galactooligosaccharides (GOS), whole grain wheat, whole grain corn, and beta-glucans.

[0056] As used herein,“prevention” or“preventing” of a disorder or condition refers to one or more compounds that, in a statistical sample, reduces the occurrence of the disorder or condition in the treated sample relative to an untreated control sample, or delays the onset of one or more symptoms of the disorder or condition relative to the untreated control sample.

[0057] As used herein, a“sample” or“biological sample” refers to a body fluid or a tissue sample isolated from a subject. In some cases, a biological sample may consist of or comprise whole blood, platelets, red blood cells, white blood cells, plasma, sera, urine, feces, epidermal sample, vaginal sample, skin sample, cheek swab, sperm, amniotic fluid, cultured cells, bone marrow sample, tumor biopsies, aspirate and/or chorionic villi, cultured cells, endothelial cells, synovial fluid, lymphatic fluid, ascites fluid, interstitial or extracellular fluid and the like. The term "sample" may also encompass the fluid in spaces between cells, including gingival crevicular fluid, bone marrow, cerebrospinal fluid (CSF), saliva, mucus, sputum, semen, sweat, urine, or any other bodily fluids. Samples can be obtained from a subject by any means including, but not limited to, venipuncture, excretion, ejaculation, massage, biopsy, needle aspirate, lavage, scraping, surgical incision, or intervention or other means known in the art. A blood sample can be whole blood or any fraction thereof, including blood cells (red blood cells, white blood cells or leukocytes, and platelets), serum and plasma.

[0058] As used herein, the term“separate” therapeutic use refers to an administration of at least two active ingredients at the same time or at substantially the same time by different routes.

[0059] As used herein, the term“sequential” therapeutic use refers to administration of at least two active ingredients at different times. More particularly, sequential use refers to the whole administration of one of the active ingredients before administration of the other or others commences. It is thus possible to administer one of the active ingredients over several minutes, hours, or days before administering the other active ingredient or ingredients. There is no simultaneous treatment in this case.

[0060] “Treating”,“treat”, or“treatment” as used herein covers the treatment of a disease or disorder described herein, in a subject, such as a human, and includes: (i) inhibiting a disease or disorder, z.e., arresting its development; (ii) relieving a disease or disorder, z.e., causing regression of the disorder; (iii) slowing progression of the disorder; and/or (iv) inhibiting, relieving, or slowing progression of one or more symptoms of the disease or disorder. In some embodiments, treatment means that the symptoms associated with the disease are, e.g., alleviated, reduced, cured, or placed in a state of remission.

[0061] It is also to be appreciated that the various modes of treatment or prevention of medical diseases and conditions as described are intended to mean“substantial,” which includes total but also less than total treatment or prevention, and wherein some biologically or medically relevant result is achieved. The treatment may be a continuous prolonged treatment for a chronic disease or a single, or few time administrations for the treatment of an acute condition.

Inflammatory Bowel Disease

[0062] The term“inflammatory bowel disease” or“IBD” as used herein is a collective term describing inflammatory disorders of the gastrointestinal tract, the most common forms of which are ulcerative colitis and Crohn's disease. Other forms of gastrointestinal

inflammation, include diversion colitis, ischemic colitis, infectious colitis, chemical colitis, microscopic colitis (including collagenous colitis and lymphocytic colitis), atypical colitis, pseudomembranous colitis, fulminant colitis, indeterminate colitis, Behqef s disease, jejunoileitis, ileitis, ileocolitis, mucositis, radiation induced enteritis, pouchitis, and proctitis. [0063] Symptoms of IBD include, but are not limited to, abdominal pain, diarrhea, rectal bleeding, weight loss, fever, loss of appetite, and other more serious complications, such as dehydration, anemia and malnutrition. A number of such symptoms are subject to quantitative analysis ( e.g ., weight loss, fever, anemia, etc.). Some symptoms are readily determined from a blood test (e.g., anemia) or a test that detects the presence of blood (e.g., rectal bleeding). IBD diagnosis is typically determined by way of an endoscopic observation of the mucosa, and pathologic examination of endoscopic biopsy specimens.

[0064] The course of IBD varies, and is often associated with intermittent periods of disease remission (i.e., inactive disease) and disease exacerbation (i.e., active disease).

Various methods have been described for characterizing disease activity and severity of IBD as well as response to treatment in subjects having IBD. See, e.g., Table A. Treatment according to the present disclosure is generally applicable to a subject having IBD of any level or degree of disease activity.

[0065] Table A below sets forth criteria useful for assessment of disease activity in subjects with ulcerative colitis according to Truelove et al., Br MedJ 2: 1041-1048 (1955). Using these criteria, disease activity can be characterized in a subject having IBD as mild disease activity or severe disease activity. Subjects who do not meet all the criteria for severe disease activity, and who exceed the criteria for mild disease activity (as presented below in Table A) are classified as having moderate disease activity.

[0066] Other methods for assessing disease activity are known in the art, such as, for example, Crohn's Disease Activity Index (CDAI) (See Best et al. Gastroenterology 77:843- 846 (1979)). A CDAI score of less than 150 is indicative of clinical disease remission (i.e., inactive disease); a score greater than 450 is indicative of severely active disease. Nonalcoholic fatty liver disease

[0067] Nonalcoholic fatty liver disease and its progressive form, nonalcoholic

steatohepatitis (NASH) are the most common causes of chronic liver disease in industrialized countries. Nonalcoholic fatty liver disease has also been strongly associated with type II diabetes and cardiovascular diseases. Nonalcoholic fatty liver disease at the histological level comprises a spectrum of liver damage including in its common form, or simple steatosis, as well as nonalcoholic steatohepatitis (NASH), a potentially progressive form of nonalcoholic fatty liver disease defined by the presence of steatosis as well as hepatocyte ballooning, inflammation and variable degrees of fibrosis. Obesity at least doubles the prevalence of NASH and its progression to cirrhosis, liver failure and hepatocellular carcinoma.

Nonalcoholic fatty liver disease patients lack a history of excessive alcohol intake. The pathogenesis of nonalcoholic fatty liver disease is described in Tolman et al ., Ther Clin Risk Manag. 3(6): 1153-1163 (2007).

Prebiotic or Probiotic Compositions of the Present Technology

[0068] Prebiotics are selectively fermented ingredients that promote specific changes, both in the composition and/or activity of gastrointestinal microbiota such as Lactobacilli and Bifidobacteria. Prebiotics are fibers that have the following characteristics: (a) resists gastric acidity, hydrolysis by mammalian enzymes, and absorption in the upper gastrointestinal tract; (b) are fermented by the intestinal microflora; and (c) selectively stimulate the growth and/or activity of intestinal bacteria potentially associated with healthful benefits. In some embodiments, the prebiotic compositions of the present technology include one or more ingredients that are selected from the group consisting of psyllium, acadia gum, polydextrose, wheat dextrin, pectin, inulin, galactooligosaccharides (GOS), whole grain wheat, whole grain corn, and beta-glucans.

[0069] Probiotics have been defined as live microbial feed supplements that beneficially affect the host animal by improving its intestinal microbial balance. Some of the beneficial effects of probiotic consumption include improvement of intestinal health by the regulation of microbiota, and stimulation and development of the immune system, synthesizing and enhancing the bioavailability of nutrients, reducing symptoms of lactose intolerance, and reducing the risk of certain other diseases. In certain embodiments, the probiotic

compositions of the present technology include bacteria belonging to one or more genera selected from the group consisting of Lactobacillus , Bifidobacterium, Bacteroides, Enterococcus and Clostridium. Additionally or alternatively, in some embodiments, the probiotic compositions of the present technology include one or more bacterial species selected from the group consisting of Lactobacillus sp. strain 100-14, Lactobacillus sp. strain 100-100, Lactobacillus acidophilus, Lactobacillus delbrueckii, Lactobacillus fermentum, Lactobacillus sp. strain 100-93, Lactobacillus plantarum, Lactobacillus salivarius,

Lactobacillus johnsonii, Lactobacillus amylovorous, Lactobacillus amylophilus,

Lactobacillus brevis, Lactobacillus buchneri, Lactobacillus casei, Lactobacillus crispatus, Lactobacillus farciminis, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus hilgardii, Lactobacillus kefir anofaciens, Lactobacillus lindneri, Lactobacillus paracasei, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus sanfranciscensis, L.

salivaricus strain JCM1046, Lactobacillus casei Shirota, Lactobacillus

delbrueckii subsp. bulgaricus, Lactobacillus mucosae, Lactobacillus rhamnosus GG, Bifidobacterium longum, Bifidobacterium adolescentis, Bifidobacterium animalis,

Bifidobacterium thermophiles, Bifidobacterium pseudolongum, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium bifidum, Bifidobacterium lactis, Enterococcus faecium, Clostridium disporicum , Clostridium perfiringens, Methanobrevibacter smithii, Methanosphera stadmanae, Bacteroides ovatus, Bacteroides thetaiotaomicron, Bacteroides acidifaciens, Bacteroides vulgatus, Bacteroides fragilis, Bacteroides firagilis subsp. firagilis, and Peptostreptococcus species.

[0070] Additionally or alternatively, in some embodiments, the probiotic compositions of the present technology include at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least

24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least

32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least

40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least

48, at least 49, or 50 bacterial species selected from the group consisting of Lactobacillus sp. strain 100-14, Lactobacillus sp. strain 100-100, Lactobacillus acidophilus, Lactobacillus delbrueckii, Lactobacillus fermentum, Lactobacillus sp. strain 100-93, Lactobacillus plantarum, Lactobacillus salivarius, Lactobacillus johnsonii, Lactobacillus amylovorous, Lactobacillus amylophilus, Lactobacillus brevis, Lactobacillus buchneri, Lactobacillus casei, Lactobacillus crispatus, Lactobacillus farciminis, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus hilgardii, Lactobacillus kefir anofaciens, Lactobacillus lindneri, Lactobacillus paracasei, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus sanfranciscensis, L. salivaricus strain JCM 1046, Lactobacillus casei Shirota, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus mucosae, Lactobacillus rhamnosus GG, Bifidobacterium longum, Bifidobacterium adolescentis, Bifidobacterium animalis,

[0071] The prebiotic or probiotic compositions of the present technology may optionally be co-administered with bile acid sequestrants such as cholestyramine, colestipol, and colesevelam.

Therapeutic Methods

[0072] The following discussion is presented by way of example only, and is not intended to be limiting.

[0073] One aspect of the present technology includes methods of treating inflammatory bowel disease or nonalcoholic fatty liver disease. Additionally or alternatively, in some embodiments, the present technology includes methods of treating reduced gut barrier function mediated, at least in part, by elevated levels of conjugated bile acids.

[0074] In one aspect, the present disclosure provides a method for treating inflammatory bowel disease or nonalcoholic fatty liver disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of at least one prebiotic or probiotic composition disclosed herein. In some embodiments, the subject is diagnosed as having, suspected as having, or at risk of having a gastrointestinal or liver disease or condition characterized by elevated levels of conjugated bile acids. Additionally or alternatively, in some embodiments, the subject is diagnosed as having inflammatory bowel disease or nonalcoholic fatty liver disease.

[0075] In therapeutic applications, compositions or medicaments comprising a prebiotic or probiotic composition disclosed herein are administered to a subject suspected of, or already suffering from such a disease or condition (such as, a subject diagnosed with a

gastrointestinal or liver disease or condition characterized by elevated levels of conjugated bile acids and/or a subject diagnosed with inflammatory bowel disease or nonalcoholic fatty liver disease), in an amount sufficient to cure, or at least partially arrest, the symptoms of the disease, including its complications and intermediate pathological phenotypes in development of the disease.

[0076] The present methods are useful for treating inflammatory bowel disease or nonalcoholic fatty liver disease at any level or degree of disease activity or severity. In certain embodiments, the methods of the present technology are useful for treating

inflammatory bowel disease or nonalcoholic fatty liver disease in a subject having mild disease activity. In other embodiments, the methods of the present technology are useful for treating inflammatory bowel disease or nonalcoholic fatty liver disease in a subject having moderate disease activity. In yet other embodiments, the methods of the present technology for treating inflammatory bowel disease or nonalcoholic fatty liver disease in a subject having severe disease activity. Such methods provide benefit by reducing the level or degree of inflammatory bowel disease or nonalcoholic fatty liver disease activity in a subject, and include, for example, reducing the level or degree of disease activity in a subject having severe disease activity to a level or degree of moderate disease activity, mild disease activity, or no disease activity; reducing the level or degree of disease activity in a subject having moderate disease activity to a level or degree of mild disease activity or no disease activity; and, reducing the level or degree of disease activity in a subject having mild disease activity to a level or degree of no disease activity. In some embodiments, the methods of the present technology may be applied to a subject having inflammatory bowel disease or nonalcoholic fatty liver disease during a period of active disease. Such methods provide benefit by reducing the duration of the period of active disease, reducing or ameliorating one or more symptoms of inflammatory bowel disease or nonalcoholic fatty liver disease, or treating inflammatory bowel disease or nonalcoholic fatty liver disease.

[0077] Subjects suffering from a gastrointestinal or liver disease or condition characterized by elevated levels of conjugated bile acids and/or a subject diagnosed with inflammatory bowel disease or nonalcoholic fatty liver disease can be identified by any or a combination of diagnostic or prognostic assays known in the art. For example, typical symptoms of inflammatory bowel disease include, but are not limited to, abdominal pain, diarrhea, rectal bleeding, weight loss, fever, loss of appetite, dehydration, anemia and malnutrition. Typical symptoms of nonalcoholic fatty liver disease include, fatty liver, nonalcoholic steatosis, tiredness and fatigue, including muscle weakness and a lack of energy, discomfort and/or swelling in the upper abdomen, weight loss, low appetite, nausea, cirrhosis, liver failure, vomiting, and diarrhea, tarry stools, abdominal swelling and pain, jaundice, itchy skin, confusion, difficulty focusing, memory loss, and hallucinations.

[0078] In some embodiments, the subject may exhibit one or more mutations in NOD2, IRGM , ATG16L1, IL23R , IL10 , IL10RA , IL10RB and PTPN2, and/or may comprise IBD risk loci such as those described in Liu JZ et al ., Nat Genet 47(9):979-86 (2015) and McGovern D et al., Gastroenterology 149(5): 1163-76 (2015), and are detectable using techniques known in the art. In some embodiments, the subject may exhibit one or more mutations in CLOCK , STAT3 , ABCC2 , PXR, PPP1R3B , FDFT1, ERLIN1, LTBP3, PARVB , PNPLA3, TM6SF2 and GCKR and/or variants in the NCAN/TM6SF2/CILP2/PBX4 multilocus. See also Sookoian et al., Clin Mol Hepatol. 23(1): 1-12 (2017).

[0079] In some embodiments, subjects with a gastrointestinal or liver disease or condition characterized by elevated levels of conjugated bile acids, and/or subjects suffering from inflammatory bowel disease or nonalcoholic fatty liver disease that are treated with the at least one prebiotic or probiotic composition disclosed herein will show amelioration or elimination of one or more of the following symptoms: abdominal pain, diarrhea, rectal bleeding, weight loss, fever, loss of appetite, dehydration, anemia, malnutrition, fatty liver, nonalcoholic steatosis, tiredness and fatigue, including muscle weakness and a lack of energy, discomfort and/or swelling in the upper abdomen, weight loss, low appetite, nausea, cirrhosis, liver failure, vomiting, and diarrhea, tarry stools, abdominal swelling and pain, jaundice, itchy skin, confusion, difficulty focusing, memory loss, and hallucinations.

Additionally or alternatively, in some embodiments of the methods, administration of the at least one prebiotic or probiotic compositions disclosed herein results in decreased levels in one or more of Akkermansia muciniphila, and Lactococcus lactis in the colon of the subject.

[0080] In certain embodiments, subjects with a gastrointestinal or liver disease or condition characterized by elevated levels of conjugated bile acids, and/or subjects suffering from inflammatory bowel disease that are treated with the at least one prebiotic or probiotic composition will show reduced colorectal tumorigenesis compared to untreated inflammatory bowel disease subjects.

[0081] In certain embodiments, subjects with a gastrointestinal or liver disease or condition characterized by elevated levels of conjugated bile acids, and/or subjects suffering from inflammatory bowel disease or nonalcoholic fatty liver disease that are treated with the at least one prebiotic or probiotic composition will show reduced levels of one or more bile acids including glycocholic acid, glycochenodeoxycholic acid, glycodeoxycholic acid, taurocholic acid, taurochenodeoxycholic acid and taurodeoxycholic acid compared to untreated inflammatory bowel disease or nonalcoholic fatty liver disease subjects.

[0082] In one aspect, the present disclosure provides a method for monitoring the therapeutic efficacy of at least one prebiotic or probiotic composition of the present technology in a subject diagnosed with inflammatory bowel disease or nonalcoholic fatty liver disease comprising: (a) detecting conjugated bile acid levels in a test sample obtained from the subject after the subject has been administered the at least one prebiotic or probiotic composition; and (b) determining that the at least one prebiotic or probiotic composition is effective when the conjugated bile acid levels in the test sample are reduced compared to that observed in a control sample obtained from the subject prior to administration of the at least one prebiotic or probiotic composition. The test sample may be tissues, cells or biological fluids (blood, plasma, saliva, urine, stool, serum, colonic mucosa, etc) present within a subject.

[0083] Alternatively, fecal bacterial bile salt hydrolase levels of the subject may be used to determine efficacy of the at least one prebiotic or probiotic composition in the subject (see Examples described herein).

[0084] Accordingly, in certain embodiments, the method further comprises detecting expression levels of one or more fecal bacterial bile salt hydrolases in the subject, wherein an increase in expression levels in one or more fecal bacterial bile salt hydrolases relative to those observed in the subject prior to treatment is indicative of the therapeutic efficacy of the at least one prebiotic or probiotic composition of the present technology.

Prophylactic Methods

[0085] In one aspect, the present technology provides a method for preventing or delaying the onset of a gastrointestinal or liver disease or condition characterized by elevated levels of conjugated bile acids. Additionally or alternatively, in some aspects, the present technology provides a method for preventing or delaying the onset of inflammatory bowel disease or nonalcoholic fatty liver disease.

[0086] Subjects at risk or susceptible to a gastrointestinal or liver disease or condition characterized by elevated levels of conjugated bile acids, and/or subjects at risk or susceptible to inflammatory bowel disease include those that exhibit one or more mutations in NOD2, IRGM , ATG16LJ IL23R , IL10 , IL10RA , IL10RB and PTPN2, and/or may comprise IBD risk loci such as those described in Liu JZ et al ., Nat Genet 47(9):979-86 (2015) and McGovern D et al., Gastroenterology 149(5): 1163-76 (2015). Subjects at risk or susceptible to nonalcoholic fatty liver disease include those that exhibit one or more mutations in CLOCK , STAT3 , ABCC2 , AR, PPP1R3B , D 77, ERLIN1, LTBP3, PARVB , PNPLA3, TM6SF2 and GCKR and/or variants in the NCAN/TM6SF2/CILP2/PBX4 multilocus. Such subjects can be identified by, e.g ., any or a combination of diagnostic or prognostic assays known in the art.

[0087] In prophylactic applications, pharmaceutical compositions or medicaments comprising at least one prebiotic or probiotic composition disclosed herein are administered to a subject susceptible to, or otherwise at risk of a gastrointestinal or liver disease or condition characterized by elevated levels of conjugated bile acids, and/or a subject susceptible to, or otherwise at risk of inflammatory bowel disease or nonalcoholic fatty liver disease, in an amount sufficient to eliminate or reduce the risk, or delay the onset of the disease, including biochemical, histologic and/or behavioral symptoms of the disease, its complications and intermediate pathological phenotypes presenting during development of the disease. Administration of the at least one prophylactic prebiotic or probiotic

composition can occur prior to the manifestation of symptoms characteristic of the disease or disorder, such that the disease or disorder is prevented or, alternatively, delayed in its progression. In certain embodiments, the prophylactic methods of the present technology may be applied to a subject having inflammatory bowel disease or nonalcoholic fatty liver disease during a time period of remission (i.e., inactive disease). In such embodiments, the present methods provide a benefit by extending the time period of remission (e.g., extending the period of inactive disease) or by preventing, reducing, or delaying the onset of active disease.

[0088] In some embodiments, treatment with the at least one prebiotic or probiotic composition will prevent or delay the onset of one or more of the following symptoms:

abdominal pain, diarrhea, rectal bleeding, weight loss, fever, loss of appetite, dehydration, anemia, malnutrition, fatty liver, nonalcoholic steatosis, tiredness and fatigue, including muscle weakness and a lack of energy, discomfort and/or swelling in the upper abdomen, weight loss, low appetite, nausea, cirrhosis, liver failure, vomiting, and diarrhea, tarry stools, abdominal swelling and pain, jaundice, itchy skin, confusion, difficulty focusing, memory loss, and hallucinations. In certain embodiments, (a) subjects with a gastrointestinal or liver disease or condition characterized by elevated levels of conjugated bile acids, and/or (b) subjects with inflammatory bowel disease or nonalcoholic fatty liver disease that are treated with the at least one prebiotic or probiotic composition will show bile acid levels (e.g., one or more of glycocholic acid, glycochenodeoxycholic acid, glycodeoxycholic acid, taurocholic acid, taurochenodeoxycholic acid and taurodeoxycholic acid) that resemble those observed in healthy control subjects. Additionally or alternatively, in certain embodiments, (a) subjects with a gastrointestinal or liver disease or condition characterized by elevated levels of conjugated bile acids, and/or (b) subjects with inflammatory bowel disease or nonalcoholic fatty liver disease that are treated with the at least one prebiotic or probiotic composition will show fecal bacterial bile salt hydrolase expression levels that resemble those observed in healthy control subjects. Additionally or alternatively, in certain embodiments, (a) subjects with a gastrointestinal or liver disease or condition characterized by elevated levels of conjugated bile acids, and/or (b) subjects with inflammatory bowel disease or nonalcoholic fatty liver disease that are treated with the at least one prebiotic or probiotic composition exhibit fecal microbiota that resemble those observed in healthy control subjects.

[0089] For therapeutic and/or prophylactic applications, a composition comprising at least one prebiotic or probiotic composition disclosed herein, is administered to the subject. In some embodiments, the at least one prebiotic or probiotic composition is administered one, two, three, four, or five times per day. In some embodiments, the at least one prebiotic or probiotic composition is administered more than five times per day. Additionally or alternatively, in some embodiments, the at least one prebiotic or probiotic composition is administered every day, every other day, every third day, every fourth day, every fifth day, or every sixth day. In some embodiments, the at least one prebiotic or probiotic composition is administered weekly, bi-weekly, tri-weekly, or monthly. In some embodiments, the at least one prebiotic or probiotic composition is administered for a period of one, two, three, four, or five weeks. In some embodiments, the at least one prebiotic or probiotic composition is administered for six weeks or more. In some embodiments, the at least one prebiotic or probiotic composition is administered for twelve weeks or more. In some embodiments, the at least one prebiotic or probiotic composition is administered for a period of less than one year. In some embodiments, the at least one prebiotic or probiotic composition is

administered for a period of more than one year. In some embodiments, the at least one prebiotic or probiotic composition is administered throughout the subject’s life.

[0090] In some embodiments of the methods of the present technology, the at least one prebiotic or probiotic composition is administered daily for 1 week or more. In some embodiments of the methods of the present technology, the at least one prebiotic or probiotic composition is administered daily for 2 weeks or more. In some embodiments of the methods of the present technology, the at least one prebiotic or probiotic composition is administered daily for 3 weeks or more. In some embodiments of the methods of the present technology, the at least one prebiotic or probiotic composition is administered daily for 4 weeks or more. In some embodiments of the methods of the present technology, the at least one prebiotic or probiotic composition is administered daily for 6 weeks or more. In some embodiments of the methods of the present technology, the at least one prebiotic or probiotic composition is administered daily for 12 weeks or more. In some embodiments, the at least one prebiotic or probiotic composition is administered daily throughout the subject’s life.

Determination of the Biological Effect of the Prebiotic or Probiotic Compositions of the Present Technology

[0091] In various embodiments, suitable in vitro or in vivo assays are performed to determine the effect of a specific prebiotic or probiotic composition disclosed herein and whether its administration is indicated for treatment. In various embodiments, in vivo assays can be performed with representative animal models, to determine if a given prebiotic or probiotic composition exerts the desired effect on reducing or eliminating signs and/or symptoms of inflammatory bowel disease or nonalcoholic fatty liver disease. Compounds for use in therapy can be tested in suitable animal model systems including, but not limited to rats, mice, chicken, cows, monkeys, rabbits, and the like, prior to testing in human subjects. Similarly, for in vivo testing, any of the animal model systems known in the art can be used prior to administration to human subjects. In some embodiments, in vitro or in vivo testing is directed to the biological function of one or more prebiotic or probiotic compositions.

[0092] Measures for determining efficacy of treatment of IBD in clinical practice have also been described and include, for example, the following: symptom control; fistula closure; extent of corticosteroid therapy required; and, improvement in quality of life. Heath-related quality of life (HRQL) can be assessed using the Inflammatory Bowel Disease Questionnaire (IBDQ), which is extensively used in clinical practice to assess quality of life in a subject with IBD. See Guyatt et al., Gastroenterology 96:804-810 (1989). Briefly, the IBDQ consists of 32 questions grouped into four categories as follows: bowel symptoms (10 items, including, for example, frequent or loose bowel movements, abdominal cramps, pain, etc.),· systemic symptoms (5 items, including, for example, fatigue, lack of energy, poor sleep patterns, etc .); social functioning (5 items, including, for example, avoiding social events, canceling engagements, etc),· and emotional functioning (12 items, including, for example, anger, frustration, depression related to chronic disease, worry about surgery, etc). For example, IBDQ scores for healthy subjects are generally about 210; IBDQ scores for subjects with active Crohn's disease are generally between about 125 and 130; IBDQ scores for subjects with Crohn's disease during quiescent ( e.g ., inactive disease) intervals are generally about 170. (See, e.g., Irvine et al., Am J Gastroenterol 92 (supp): l8S-24S (1997)).

Improvements in any of the foregoing response criteria are specifically provided by the methods of the present technology.

[0093] Animal models of inflammatory bowel disease or nonalcoholic fatty liver disease may be generated using techniques known in the art (for instance, see Examples described herein). Such models may be used to demonstrate the biological effect of the prebiotic or probiotic compositions disclosed herein in the prevention and treatment of gastrointestinal conditions arising from increased levels of conjugated bile acids, and for determining what comprises a therapeutically effective amount of the one or more prebiotic or probiotic compositions disclosed herein in a given context.

Modes of Administration and Effective Dosages

[0094] Any method known to those in the art for contacting a cell, organ or tissue with one or more prebiotic or probiotic compositions disclosed herein may be employed. Suitable methods include in vitro, ex vivo, or in vivo methods. In vivo methods typically include the administration of one or more prebiotic or probiotic compositions to a mammal, suitably a human. When used in vivo for therapy, the one or more prebiotic or probiotic compositions described herein are administered to the subject in effective amounts (i.e., amounts that have the desired therapeutic effect). The dose and dosage regimen will depend upon the degree of the disease state of the subject, the characteristics of the particular prebiotic or probiotic composition used, e.g. , its therapeutic index, and the subject’s history.

[0095] The effective amount may be determined during pre-clinical trials and clinical trials by methods familiar to physicians and clinicians. An effective amount of one or more prebiotic or probiotic compositions useful in the methods may be administered to a mammal in need thereof by any of a number of well-known methods for administering pharmaceutical compounds. The prebiotic or probiotic compositions may be administered systemically or locally.

[0096] The one or more prebiotic or probiotic compositions described herein can be incorporated into pharmaceutical compositions for administration, singly or in combination, to a subject for the treatment or prevention of inflammatory bowel disease or nonalcoholic fatty liver disease. Such compositions typically include the active agent and a

pharmaceutically acceptable carrier. As used herein the term“pharmaceutically acceptable carrier” includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds can also be incorporated into the compositions.

[0097] Pharmaceutical compositions are typically formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral ( e.g ., intravenous, intradermal, intraperitoneal or subcutaneous), oral, rectal, inhalation, transdermal (topical), intraocular, iontophoretic, and transmucosal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. For convenience of the patient or treating physician, the dosing formulation can be provided in a kit containing all necessary equipment (e.g., vials of drug, vials of diluent, syringes and needles) for a treatment course (e.g, 7 days of treatment).

[0098] Pharmaceutical compositions suitable for injectable use can include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, CREMOPHOR EL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, a composition for parenteral administration must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.

[0099] The pharmaceutical compositions having one or more prebiotic or probiotic compositions disclosed herein can include a carrier, which can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.

Glutathione and other antioxidants can be included to prevent oxidation. In many cases, it will be advantageous to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate or gelatin.

[00100] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, typical methods of preparation include vacuum drying and freeze drying, which can yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[00101] Oral compositions generally include an inert diluent or an edible carrier. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules, e.g ., gelatin capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash.

Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

[00102] For administration by inhalation, the compounds can be delivered in the form of an aerosol spray from a pressurized container or dispenser, which contains a suitable propellant, e.g. , a gas such as carbon dioxide, or a nebulizer. Such methods include those described in U.S. Pat. No. 6,468,798.

[00103] Systemic administration of a therapeutic compound as described herein can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal

administration can be accomplished through the use of nasal sprays. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art. In one embodiment, transdermal administration may be performed by iontophoresis.

[00104] A therapeutic agent can be formulated in a carrier system. The carrier can be a colloidal system. The colloidal system can be a liposome, a phospholipid bilayer vehicle. In one embodiment, the therapeutic agent is encapsulated in a liposome while maintaining the agent’s structural integrity. One skilled in the art would appreciate that there are a variety of methods to prepare liposomes. (See Lichtenberg, et al, Methods Biochem. Anal., 33:337-462 (1988); Anselem, et al. , Liposome Technology , CRC Press (1993)). Liposomal formulations can delay clearance and increase cellular uptake (See Reddy, Ann. Pharmacother ., 34(7- 8):9l5-923 (2000)). An active agent can also be loaded into a particle prepared from pharmaceutically acceptable ingredients including, but not limited to, soluble, insoluble, permeable, impermeable, biodegradable or gastroretentive polymers or liposomes. Such particles include, but are not limited to, nanoparticles, biodegradable nanoparticles, microparticles, biodegradable microparticles, nanospheres, biodegradable nanospheres, microspheres, biodegradable microspheres, capsules, emulsions, liposomes, micelles and viral vector systems.

[00105] The carrier can also be a polymer, e.g. , a biodegradable, biocompatible polymer matrix. In one embodiment, the therapeutic agent can be embedded in the polymer matrix, while maintaining the agent’s structural integrity. The polymer may be natural, such as polypeptides, proteins or polysaccharides, or synthetic, such as poly a-hydroxy acids.

Examples include carriers made of, e.g. , collagen, fibronectin, elastin, cellulose acetate, cellulose nitrate, polysaccharide, fibrin, gelatin, and combinations thereof. In one embodiment, the polymer is poly-lactic acid (PLA) or copoly lactic/glycolic acid (PGLA). The polymeric matrices can be prepared and isolated in a variety of forms and sizes, including microspheres and nanospheres. Polymer formulations can lead to prolonged duration of therapeutic effect. (See Reddy, Ann. Pharmacother ., 34(7-8):915-923 (2000)). A polymer formulation for human growth hormone (hGH) has been used in clinical trials. (See Kozarich and Rich, Chemical Biology, 2:548-552 (1998)). [00106] Examples of polymer microsphere sustained release formulations are described in PCT publication WO 99/15154 (Tracy, et al), U.S. Pat. Nos. 5,674,534 and 5,716,644 (both to Zale, et al. ), PCT publication WO 96/40073 (Zale, et al. ), and PCT publication WO 00/38651 (Shah, et al). U.S. Pat. Nos. 5,674,534 and 5,716,644 and PCT publication WO 96/40073 describe a polymeric matrix containing particles of erythropoietin that are stabilized against aggregation with a salt.

[00107] In some embodiments, the therapeutic compounds are prepared with carriers that will protect the therapeutic compounds against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Such formulations can be prepared using known techniques. The materials can also be obtained commercially, e.g ., from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to specific cells with monoclonal antibodies to cell-specific antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.

[00108] The therapeutic compounds can also be formulated to enhance intracellular delivery. For example, liposomal delivery systems are known in the art, see, e.g., Chonn and Cullis, “Recent Advances in Liposome Drug Delivery Systems,” Current Opinion in Biotechnology 6:698-708 (1995); Weiner,“Liposomes for Protein Delivery: Selecting Manufacture and Development Processes,” Immunomethods , 4(3):20l-9 (1994); and Gregoriadis,“Engineering Liposomes for Drug Delivery: Progress and Problems,” Trends BiotechnoL, 13(12):527-37 (1995). Mizguchi, et al. , Cancer Lett., 100:63-69 (1996), describes the use of fusogenic liposomes to deliver a protein to cells both in vivo and in vitro.

[00109] Dosage, toxicity and therapeutic efficacy of any therapeutic agent can be

determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g, for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds that exhibit high therapeutic indices are advantageous. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.

[00110] The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds may be within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the methods, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (z.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to determine useful doses in humans accurately. Levels in plasma may be measured, for example, by high performance liquid chromatography.

[00111] Typically, an effective amount of the one or more prebiotic or probiotic

compositions disclosed herein sufficient for achieving a therapeutic or prophylactic effect, range from about 0.000001 mg per kilogram body weight per day to about 10,000 mg per kilogram body weight per day. Suitably, the dosage ranges are from about 0.0001 mg per kilogram body weight per day to about 100 mg per kilogram body weight per day. For example dosages can be 1 mg/kg body weight or 10 mg/kg body weight every day, every two days or every three days or within the range of 1-10 mg/kg every week, every two weeks or every three weeks. In one embodiment, a single dosage of the therapeutic compound ranges from 0.001-10,000 micrograms per kg body weight. In one embodiment, one or more prebiotic or probiotic composition concentrations in a carrier range from 0.2 to 2000 micrograms per delivered milliliter. An exemplary treatment regime entails administration once per day or once a week. In therapeutic applications, a relatively high dosage at relatively short intervals is sometimes required until progression of the disease is reduced or terminated, or until the subject shows partial or complete amelioration of symptoms of disease. Thereafter, the patient can be administered a prophylactic regime.

[00112] In some embodiments, a therapeutically effective amount of one or more prebiotic or probiotic compositions may be defined as a concentration of the composition at the target tissue of 10 ³² to 10 ⁶ molar, e.g, approximately 10 ⁷ molar. This concentration may be delivered by systemic doses of 0.001 to 100 mg/kg or equivalent dose by body surface area. The schedule of doses would be optimized to maintain the therapeutic concentration at the target tissue, such as by single daily or weekly administration, but also including continuous administration ( e.g ., parenteral infusion or transdermal application).

[00113] The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to, the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of the therapeutic compositions described herein can include a single treatment or a series of treatments.

[00114] The mammal treated in accordance with the present methods can be any mammal, including, for example, farm animals, such as sheep, pigs, cows, and horses; pet animals, such as dogs and cats; laboratory animals, such as rats, mice and rabbits. In some

embodiments, the mammal is a human.

Combination Therapy

[00115] In some embodiments, one or more of the prebiotic or probiotic compositions disclosed herein may be combined with one or more additional therapies for the prevention or treatment of inflammatory bowel disease or nonalcoholic fatty liver disease. Additional therapeutic agents include, but are not limited to, anti-inflammatory agents such as corticosteroids and aminosalicylates, immunosuppressive agents such as tumor necrosis factor (TNF)-alpha inhibitors, pain relievers, iron supplements, calcium, and vitamin D supplements.

[00116] In some embodiments, the one or more prebiotic or probiotic compositions disclosed herein may be separately, sequentially or simultaneously administered with at least one additional therapeutic agent selected from the group consisting of anti-inflammatory agents such as corticosteroids and aminosalicylates, immunosuppressive agents such as tumor necrosis factor (TNF)-alpha inhibitors, pain relievers, iron supplements, calcium, vitamin D supplements, mesalamine, sulfasalazine, infliximab, adalimumab, prednisone, budesonide, 6- mercaptopurine, 5-aminosalicylic acid (5-ASA), azathioprine, cyclosporine, golimumab, balsalazide, olsalazine, methotrexate, natalizumab, vedolizumab, ustekinumab,

acetaminophen, ibuprofen, naproxen sodium (Aleve), diclofenac sodium, orlistat,

sibutramine, pioglitazone, rosiglitazone, metformin, atorvastatin, pravastatin, rosuvastatin, gemfibrozil, ursodiol, vitamin E, vitamin C, pentoxifylline, betaine, and losartan.

[00117] In any case, the multiple therapeutic agents may be administered in any order or even simultaneously. If simultaneously, the multiple therapeutic agents may be provided in a single, unified form, or in multiple forms (by way of example only, either as a single pill or as two separate pills). One of the therapeutic agents may be given in multiple doses, or both may be given as multiple doses. If not simultaneous, the timing between the multiple doses may vary from more than zero weeks to less than four weeks. In addition, the combination methods, compositions and formulations are not to be limited to the use of only two agents.

Kits

[00118] The prebiotic or probiotic compositions described herein may, in some

embodiments, be assembled into kits to facilitate their use in treating inflammatory bowel disease or nonalcoholic fatty liver disease. A kit may include one or more containers housing the prebiotic or probiotic compositions of the present technology and instructions for use. Specifically, such kits may include one or more prebiotic or probiotic compositions described herein, along with instructions describing the intended therapeutic application and the proper administration of these prebiotic or probiotic compositions. In certain embodiments, the prebiotic or probiotic compositions in a kit may be in a pharmaceutical formulation and dosage suitable for a particular application and for a method of administration of the prebiotic or probiotic compositions.

[00119] Each of the compositions of the kit, where applicable, may be provided in liquid form ( e.g ., in solution), or in solid form, (e.g., a dry powder). In certain cases, some of the compositions may be constitutable or otherwise processable (e.g., to an active form), for example, by the addition of a suitable solvent or other species (for example, water or a cell culture medium), which may or may not be provided with the kit.

[00120] The kits may include written instructions on or associated with its packaging.

Instructions also can include any oral or electronic instructions provided in any manner such that a user will clearly recognize that the instructions are to be associated with the kit, for example, audiovisual (e.g., videotape, DVD, etc.), Internet, and/or web-based

communications, etc. The written instructions may be in a form prescribed by a

governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, and can also reflect approval by the agency of manufacture, use or sale for human administration.

[00121] The kit may contain any one or more of the prebiotic or probiotic compositions described herein in one or more containers. As an example, in one embodiment, the kit may include instructions for mixing one or more compositions of the kit and/or isolating and mixing a sample and applying to a subject. The kit may include a container housing the prebiotic or probiotic compositions described herein. The prebiotic or probiotic compositions may be in the form of a liquid, gel or solid (powder). The prebiotic or probiotic compositions may be prepared sterilely, packaged in syringe and shipped refrigerated. Alternatively it may be housed in a vial or other container for storage. A second container may have other prebiotic or probiotic compositions prepared sterilely. Alternatively the kit may include the active prebiotic or probiotic compositions premixed and shipped in a syringe, vial, tube, or other container. The kit may have one or more or all of the components required to administer the prebiotic or probiotic compositions to a patient, such as a syringe, topical application devices, or iv needle tubing and bag.

[00122] The kit may have a variety of forms, such as a blister pouch, a shrink wrapped pouch, a vacuum sealable pouch, a sealable thermoformed tray, or a similar pouch or tray form, with the accessories loosely packed within the pouch, one or more tubes, containers, a box or a bag. The kit may be sterilized after the accessories are added, thereby allowing the individual accessories in the container to be otherwise unwrapped. The kits can be sterilized using any appropriate sterilization techniques, such as radiation sterilization, heat

sterilization, or other sterilization methods known in the art. The kit may also include other components, depending on the specific application, for example, containers, cell media, salts, buffers, reagents, syringes, needles, a fabric, such as gauze, for applying or removing a disinfecting agent, disposable gloves etc.

EXAMPLES

[00123] The present technology is further illustrated by the following Examples, which should not be construed as limiting in any way.

Example 1: Materials and Methods

[00124] Induction o/DSS colitis. To induce colitis, mice were administered 1-2% DSS (MP Biochemical, Santa Ana, CA) in drinking water for 6-7 days, as indicated. For comparisons of colitis severity, measurements of body weight changes, and the severity of rectal bleeding and diarrhea were carried out as previously described. Montrose et al ., Journal of lipid research 54:843-51 (2013). Briefly, bleeding was assessed by detection of heme in stool using the Hemoccult Sensa test (Beckman Coulter, Brea, CA) or evidence of gross bleeding on a scale from 0 to 3. Diarrhea was assessed by measuring the softness or appearance of the stool on a scale from 0 to 3. At the end of the experiment, mice were euthanized and colons were excised, length measured and then flushed with ice-cold phosphate-buffered saline (PBS). Colons were either snap-frozen in liquid nitrogen or fixed in 10% formalin or 4% paraformaldehyde for 4-6 hours, Swiss-rolled, paraffin-embedded and then sectioned.

Sections were stained with hematoxylin and eosin (H&E) to evaluate the severity of histologic injury, which was adapted from. O'Connor et al ., Nat Immunol 10:603-9 (2009). All animal studies were approved by the Institutional Animal Care and Use Committees at Weill Cornell Medicine (WCM).

[00125] Measurement of Fecal Fructose levels. Fecal samples were snap-frozen after collection and stored at -80°C before use. Feces were dried for 4 hrs using an Eppendorf Vacufuge Concentrator 5301. Based on the dry weight, fecal samples were reconstituted in distilled water (40 mg/ml) and mechanically disrupted with a TissueLyser II (using stainless steel beads). After centrifugation, clarified supernatants were used to measure fructose levels using EnzyChrom Fructose Assay Kit.

[00126] Quantitative Real-Time PCR. RNA was extracted from tissue and reverse transcribed to make cDNA using the qScript cDNA Synthesis Kit. The resulting cDNA was amplified using primers specific to the genes of interest Glyceraldehyde 3 phosphate dehydrogenase (GAPDH) was used as an endogenous normalization control. Quantitative real-time PCR (qRT-PCR) was performed using Fast SYBR Green Master Mix on a

StepOnePlus real-time PCR system. Relative fold induction was determined using the ddCT (relative quantification) analysis protocol.

[00127] Immune Cell Profiling of the Colonic Lamina Propria. Colons were removed and flushed with ice-cold PBS, slit open longitudinally and cut into 1 cm pieces. Tissues were placed into dissociation solution (Ca/Mg free HBSS, 2% FBS, 5mM EDTA, lmM DTT) and put into a shaking incubator for 20 min at 37°C. The solution and tissue was vortexed and strained and the dissociation step was repeated. The remaining tissue was placed into digestion solution (RPMI 1640 medium, 10% FBS, lmg/mL Collagenase III, 0.4u/mL dispase, 0. lmg/mL DNAse) and incubated at 37°C in a shaking incubator for 45 min.

Solution and tissue was strained and the flow-through was centrifuged and pellet was resuspended in 40% percoll and centrifuged. Debris was removed and the pellet containing lamina propria cells was used for staining.

[00128] Cells were first stained with brilliant violet 570 viability dye to identify live and dead cells. Blocking of non-specific binding of isolated cells was then carried out by addition of anti-Fc monoclonal antibody followed by incubation with the following fluorescently conjugated monoclonal antibodies at a dilution of 1 :200 on ice for 30 minutes: CD45- Alexa700, CD1 lc-Pacific Blue, CD1 lb- peridinin chlorophyll protein complex Cyan5.5, F/480-phycoerythrin-Cyan7, Ly6G-allophycocyanin, MCHII-fluoroscienisothiocyanate NK1.1 -biotin. Phycoerythrin-Texas Red secondary antibody was used to detect NK1.1.

[00129] Flow cytometry was conducted on an LSRII flow cytometer with data analyzed using FlowJo software. Comparisons of inflammatory cell types based on surface markers using the antibodies described above were made between control diet fed and HFrD fed mice to determine the average percentage of inflammatory cells expressing these markers in individual mice.

[00130] 16S rRNA sequencing. Feces were collected from individual mice, snap-frozen in liquid nitrogen, and shipped to Molecular Research for 16S rRNA profiling. DNA extraction from feces was carried out using the Powersoil DNA Kit per manufacturer’s instructions. Following DNA extraction, the 16S rRNA gene V4 variable region primers 515/806 with barcode on the forward primer were used in a 30 cycle PCR using the HotStarTaq Plus Master Mix Kit (under the following conditions: 94°C for 3 minutes, followed by 30-35 cycles of 94°C for 30 seconds, 53°C for 40 seconds and 72°C for 1 minute, after which a final elongation step at 72°C for 5 minutes was performed. Pooled samples were purified using calibrated Ampure XP beads and the pooled and purified PCR product was used to prepare illumina DNA library. Sequencing was performed using an Illumina HiSeq.

Sequence data were processed using MR DNA analysis pipeline (Shallowater, TX,). In summary, sequences were joined, depleted of barcodes then sequences <l50bp or with ambiguous base calls were removed. Sequences were denoised, OTUs generated and chimeras removed. Operational taxonomic units (OTUs) were defined by clustering at 3% divergence (97% similarity). Final OTUs were taxonomically classified using BLASTn against a curated database derived from RDPII and NCBI. Principal coordinate analysis was carried out on unweighted UniFrac data using Emperor software.

[00131] RNA sequencing (RNA-seq). The mucosa of the distal colon was excised under visualization through a dissecting microscope. Total RNA was isolated from frozen colon tissue using the RNeasy mini kit (Qiagen). Following RNA isolation, total RNA integrity was checked using a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). RNA

concentrations were measured using the NanoDrop system (Thermo Fisher Scientific). RNA library construction and RNA-seq were performed by the Genomics Core Laboratory at WCM. Messenger RNA was prepared using TruSeq Stranded mRNA Sample Library Preparation kit (Illumina), according to the manufacturer’s instructions and was sequenced on an Illumina HiSeq4000 sequencer with pair-end 50 bp cycles. Sequencing quality was assessed using FastQC (Babraham Bioinformatics, Cambridge, UK). Raw sequenced reads were aligned to the Mouse reference genome (Version mmlO from UCSC) using STAR (Version 2.4.2) aligner. Aligned reads were quantified against the reference annotation (mmlO from UCSC) to obtain Fragments per Kilobase per million (FPKM) and raw counts using CuffLinks (v 2.2.1) and HTSeq , respectively. Differential expression analysis was carried out based on the Limma/Voom with Benjamini Hochberg correction for multiple testing. Principal component analysis (PCA) was performed using the prcomp function in R (Version 3.5.0).

[00132] Pathway analysis using GSEA (Gene Set Enrichment Analysis Software) Software from Broad institute with GO Ontology pathways as the reference database was used to identify functions of differentially expressed genes. Genes were ranked by the t-statistic value obtained from comparisons and the pre-ranked version of the tool was used to identify significantly enriched biological pathways and test enrichment of gene signatures from other studies. Pathways enriched with FDR < 0.25 were considered to be significant.

[00133] Fecal metabolomic analysis. Samples were shipped to Metabolon (Morrisville, NC) for detection of metabolites using a database of over 4,500 named molecules. Fecal samples were lyophilized and then resuspended in water (20 pL/mg of dried sample) for

homogenization. Following homogenization, 100 pL of the fecal suspensions was used for extraction. Extracts were prepared using the automated MicroLab STAR system (Hamilton Company, Reno, NV). A recovery standard was added prior to the first step in the extraction process for QC purposes. To remove proteins, dissociate small molecules bound to proteins or trapped in the precipitated protein matrix, and to recover chemically diverse metabolites, proteins were precipitated with methanol under vigorous shaking for 2 minutes followed by centrifugation. The resulting extract was divided into five fractions: two (i.e., early and late eluting compounds) for analysis by ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS; positive ionization), one for analysis by UPLC-MS/MS (negative ionization), one for the UPLC-MS/MS polar platform (negative ionization), and one sample was reserved for backup. Detection of metabolites was performed using Ultrahigh Performance liquid chromatography-tandem mass spectroscopy. Briefly, The UPLC-MS/MS platform utilized a Waters Acquity UPLC with Waters UPLC BEH Cl 8-2. l x 100 mm, 1.7 pm columns and a Q-Exactive high resolution/accurate mass spectrometer (Thermo Fisher Scientific,) interfaced with a heated electrospray ionization (HESI-II) source and Orbitrap mass analyzer operated at 35,000 mass resolution. The sample extract was dried then reconstituted in acidic or basic LC-compatible solvents, each of which contained 8 or more injection standards at fixed concentrations to ensure injection and chromatographic consistency. One aliquot was analyzed using acidic, positive ion-optimized conditions and the other using basic, negative ion-optimized conditions in two independent injections using separate dedicated columns (Waters UPLC BEH C18-2.1x100 mm, 1.7 pm). Extracts reconstituted in acidic conditions were gradient eluted using water and methanol containing 0.1% formic acid, while the basic extracts, which also used water/methanol, contained 6.5 mol/L ammonium bicarbonate. A third aliquot was analyzed via negative ionization following elution from a HILIC column (Waters EIPLC BEH Amide 2.1x150 mm, 1.7 pm) using a gradient consisting of water and acetonitrile with lOmM ammonium formate. The MS analysis alternated between MS and data-dependent MS2 scans using dynamic exclusion, and the scan range was from 80-1000 m/z. Metabolites were identified by automated comparison of the ion features in the experimental samples to a reference library of chemical standard entries that included retention time, molecular weight (m/z), preferred adducts, and in-source fragments as well as associated MS spectra and curated by visual inspection for quality control using software developed at Metabolon. Dehaven et al ., Journal of cheminformatics 2:9 (2010). Identification of known chemical entities is based on comparison to metabolomic library entries of purified standards. Commercially available purified standard compounds have been acquired and registered into LIMS for determination of their detectable characteristics. Additional mass spectral entries have been created for structurally unnamed biochemicals, which have been identified by virtue of their recurrent nature (both chromatographic and mass spectral). These compounds have the potential to be identified by future acquisition of a matching purified standard or by classical structural analysis. Peaks were quantified using area-under-the-curve. Raw area counts for each metabolite in each sample were normalized to correct for variation resulting from instrument inter-day tuning differences by the median value for each run-day, therefore, setting the medians to 1.0 for each run. This preserved variation between samples but allowed metabolites of widely different raw peak areas to be compared on a similar graphical scale. Missing values were imputed with the observed minimum after normalization.

[00134] Citrobacter rodentium infection. C. rodentium strain DBS100 (ATCC 51459;

American Type Culture Collection, Manassas, VA) was cultured overnight aerobically at 37°C in Lennox (LB) broth (Invitrogen, Carlsbad, CA). Bacterial concentration was evaluated by measuring absorbance at 600 nm. Prior to inoculation, mice were fasted for 8 hours then inoculated with 5x10⁹ C. rodentium in a total volume of 200 pL. Mice were given access to food after the inoculation. The same culture was used to inoculate all mice in a single experiment to rule out effects of growth variation on disease pathogenesis. Disease severity was assessed over 6 days by monitoring body weight changes and diarrhea and rectal bleeding (as described above). On day 6 after infection, mice were sacrificed and colon length was determined. The severity of colonic injury was determined using a histology score as previously described. O'Connor et al., Nat Immunol 10:603-9 2009. Each section was evaluated for pathological changes in the mucosa, submucosa, muscularis externa and serosa, including inflammation (location and extent), edema, mucosal changes of ulceration, hyperplasia and attenuation, with crypt loss or abscess noted by examination of H&E stained slides assessed by a gastrointestinal pathologist. Severity scores ranged from 0 to 5 as follows: 0, within normal limits or absent; 1, minimal; 2, mild; 3, moderate; 4, marked; and 5, severe. Cytokine expression in the colon was measured by qRT-PCR. To evaluate lethality, mice were treated as described for the 6 day study; however following infection, mice were observed for 14 days and death was recorded.

[00135] Statistical analysis. To evaluate difference in study endpoints, including colon length, histologic score, stool score, the expressions of cytokines, abundance of a particular bacteria strain measured using qPCR, and mucus thickness, between two experimental groups, the non-parametric Wilcoxon rank-sum test was used. Welch’s t-test was used to compare the number of immune cells. Difference in the continuous endpoints across multiple experimental groups was examined using ANOVA. Linear contrasts of interest was examined using simultaneous tests of general linear hypotheses and p-values were adjusted for multiple comparisons by using Bonferroni-Holm method. Log transformation was used where appropriate to ensure the underlying model assumptions were satisfied. For relative mouse body weight, and the occurrence of severe bleeding (score >=2) or severe diarrhea (score >=2) measured over time, generalized linear mixed effects model was used and contrasts of interest for parameters of interest including the rate of change in weight and average proportion of mice with severe bleeding or diarrhea was examined using simultaneous tests of general linear hypotheses and p-values were adjusted for multiple comparisons by controlling the experiment- wise error rate using the“single-step” implementation in the R multcomp package. All the above described analyses were carried out in R. [00136] Broad-spectrum antibiotic treatment. To reduce bacterial abundance in the gastrointestinal tract of mice, drinking water containing ampicillin (0.5 g/L) (Millipore- Sigma, Burlington, MA), gentamicin (0.5 g/L) (Millipore- Sigma), metronidazole (0.5 g/L) (Millipore-Sigma), neomycin (0.5 g/L) (Millipore- Sigma) and vancomycin (0.25 g/L) (VWR International, Radnor, PA) was given for 3 weeks. Splenda (4 g/L) (Heartland Food Products Group, Carmel, IN) was included to mask the bad taste of the antibiotics and therefore maintain adequate water consumption. Control water contained Splenda only. This antibiotic cocktail has been successfully used to reduce the microbial population in other studies. Abt et al., Immunity 37: 158-70 (2012). Mice were continued on their respective treatments during DSS exposure.

[00137] Germ-free mice. Germ-free (GF) mice were bred in the gnotobiotic facility at WCM. To carry out experiments, mice were transferred from the gonotobiotic facility to isolator cages and fed irradiated diet and autoclaved water over the duration of the experiment. Mice were assessed for parameters of disease severity in a sterile biosafety cabinet using sterile gloves in order to maintain them in a GF state.

[00138] Quantitative real-time PCR. In the DSS and C. rodentium models of colitis, total RNA was isolated using the RNeasy Mini Kit (Qiagen) and RNA concentrations were measured using the NanoDrop system (Thermo Fisher Scientific). For RNA extraction of tissues exposed to DSS, messenger RNA (mRNA) was isolated using Oligotex mRNA Mini Kit (Qiagen), according to the manufacturer’s instructions. RNA was reverse transcribed to make cDNA using the qScript cDNA Synthesis Kit (Quantabio, Beverly, MA). The resulting cDNA was amplified using custom primer sequences or QuantiTect Primer Assays (Qiagen). Glyceraldehyde 3 phosphate dehydrogenase (Gapdh) was used as an endogenous

normalization control for both designed and commercial primers. The amplified products of designed primers were verified by sequencing. Quantitative real-time PCR (qRT-PCR) was performed using Fast SYBR Green Master Mix (Applied Biosystems, Foster City, CA) on a StepOnePlus real-time PCR system (Applied Biosystems). Relative fold induction was determined using the ddCT (relative quantification) analysis protocol.

[00139] 16S rRNA analysis. Feces were collected from individual mice, snap-frozen in liquid nitrogen, and shipped to Molecular Research (Shallowater, TX) for 16S rRNA profiling. DNA extraction from feces was carried out using the Powersoil DNA Kit (Qiagen) per manufacturer’s instructions. Following DNA extraction, the 16S rRNA gene V4 variable region PCR primers 515/806 with barcode on the forward primer were used in a 30 cycle PCR using the HotStarTaq Plus Master Mix Kit (Qiagen,) under the following conditions: 94°C for 3 minutes, followed by 30-35 cycles of 94°C for 30 seconds, 53°C for 40 seconds and 72°C for 1 minute, after which a final elongation step at 72°C for 5 minutes was performed. Pooled samples were purified using calibrated Ampure XP beads and the pooled and purified PCR product was used to prepare the Illumina DNA library. Sequencing was performed using an Illumina HiSeq (Illumina, San Diego, CA). Sequence data were processed using MR DNA analysis pipeline. In summary, sequences were joined, depleted of barcodes then sequences <l50bp or with ambiguous base calls were removed. Sequences were denoised, OTUs generated and chimeras removed. Operational taxonomic units (OTUs) were defined by clustering at 3% divergence (97% similarity). Final OTUs were

taxonomically classified using BLASTn against a curated database derived from RDPII and NCBI.

[00140] Measurements of colonic mucus thickness. A segment of colon containing feces was harvested and immediately preserved in fresh Camoy’s fixative

(methanol:chloroform:glacial acetic acid (60:30: 10)) for 4-5 days. Tissues were washed twice in 100% methanol followed by 2 washes in 100% ethanol. The fixed tissues were then washed in a 1 : 1 ratio of 100% ethanol: xylenes for 15 minutes followed by two 15 minute washes in xylenes and subsequently embedded in paraffin with a vertical orientation.

Paraffin sections (5 pm thick) were cut and deposited on glass slides. Alcian blue/periodic acid Schiff staining was performed to stain mucus. Following staining, measurements of mucus thickness in 40-60 regions of each colon were carried out in a blinded manner using Fiji/ImageJ software (National Institutes of Health (NIH) as previously described. Jiao et al ., Digestive Diseases and Sciences 60:3590-602 (2015).

[00141] Quantification of goblet cells. Following PAS/AB staining of tissue sections for goblet cells, the percent of mucosa occupied by goblet cells was calculated using the

Fiji/ImageJ (NIH) through the WCM Microscopy and Image Analysis Core Facility. The entire region of interest was outlined and copied onto a separate file, and then subjected to a color deconvolution algorithm. The color-deconvolved image representing the mucosa was saved, and used to obtain two different measurements. To determine the total mucosal area, the color-deconvolved mucosa image was blurred with a large Gaussian blur (sigma = 20), and thresholded using the auto-threshold function“Huang.” This area was masked and measured, and labeled as“mucosal area.” To obtain the Goblet cell area, the same color- deconvolved image was smoothed with a small Gaussian blur (sigma = 2), and thresholded using the auto-threshold function“Default.” This area was then masked and measured, and labeled as“goblet cell area.” The ratio of the two measurements provided the proportion of mucosal surface occupied by goblet cells.

Example 2: High Fructose Diet Exacerbates Experimental Colitis

[00142] To determine whether elevated fructose consumption could be linked with increased incidence of IBD, the effect of a high fructose diet (HFrD) on the pathogenesis of

experimental colitis was examined. Mice were fed either a HFrD or a high glucose diet (HGD), and subsequently compared to mice that were fed a control diet, AIN-93G, containing a mixture of carbohydrates. As shown in Figure 1A, all three diets (HFrD, HGD, and AIN-93G) were isocaloric and contained 64 kcal% carbohydrate. Mice were fed one of these diets for 2 weeks, followed by the administration of 1% dextran sodium sulfate (DSS) in drinking water to induce colitis while continuing to be fed their respective diets (Figure 2). The severity of colitis in male mice fed the HGD was not different from those fed the control diet (AIN-93G); while mice fed the HFrD developed markedly worse colitis. As shown in Figures 3A-3E, the increased severity of the colitis included more severe weight loss, rectal bleeding, diarrhea, enhanced colonic shortening and more extensive colonic ulceration. The effects of a HFrD on experimental colitis in female mice mirrored the findings in male mice. Feeding mice a HFrD for only 1 week prior to the administration of DSS was as effective in causing more severe colitis as administering a HFrD for 2 weeks.

[00143] To complement the clinic-pathological measurements, and to gain insight into immunological alterations occurring in HFrD-fed mice, the in vivo expression of pro- inflammatory cytokines was measured. Cytokine gene expression was measured by qRT- PCR in the colons of mice that received the control diet (AIN-93G) or the HFrD, followed by DSS administration in drinking water for seven days. Control mice were fed either diet (HFrD or AIN-93G), followed by plain drinking water for seven days. As shown in Figures 4A-4E, the magnitude of the increase of several cytokines (INF, IL1B, IL6, IL17A , and IL22 ) was greater in the colons from mice fed a HFrD followed by seven days of DSS exposure compared to control diet fed mice that were exposed to DSS. Without wishing to be bound by theory, it is believed that, this cytokine expression pattern suggests a TH17 polarization of immune cells.

[00144] The number of immune cells in the lamina propria of colons from mice fed control diet (AIN-93G) or HFrD for 1 week followed by DSS administration in drinking water for 7 days was measured by FACS. Corresponding to the increased expression of pro- inflammatory cytokines observed upon HFrD feeding, flow cytometry of the colonic lamina propria revealed significantly greater numbers of myeloid cells including CD45+ cells, neutrophils, macrophages, dendritic cells and NK cells (Figure 5).

[00145] Although a 64 kcal% fructose diet was used in the above studies to mimic the carbohydrate content of AIN-93G diet, it is uncommon for humans to consume more than 15 kcal% of daily calories as fructose. Therefore, another experiment was carried out to determine whether administering a 15 kcal% fructose diet led to worse colitis than mice fed control diet. Male C57BL/6J mice were fed control diet (AIN-93 G) or diet containing 15 kcal% fructose for one week (Figure IB) followed by administration of 2% DSS in drinking water for one week. As shown in Figures 8A-8B, male mice fed a 15 kcal% fructose diet demonstrated more severe colitis compared to mice given the control diet.

[00146] Carbohydrate content of the diet was then modulated to serially increase fructose levels to determine whether fructose feeding induced a dose-dependent increase in colitis severity. Mice were fed one of the four diets shown in Figure 6, and were exposed to DSS while being continued on their respective diets. Figures 7A-7D demonstrate that the severity of colitis increased with the rise of fructose concentration in the diet.

[00147] Taken together, these results demonstrate that compared to other carbohydrates, a HFrD increases severity of DSS-induced colitis in mice, and that the severity of colitis worsens when higher concentrations of fructose were added to the diet.

[00148] Without wishing to be bound by theory, it is believed that acute colitis induced by high fructose feeding would increase colitis-associated tumorigenesis because colitis is a risk factor for colorectal cancer (CRC). To determine if there is a link between acute colitis and CRC, mice were administered one injection of the colon specific carcinogen azoxymethane (AOM, l2.5mg/kg) then given a control diet (AIN-93G) or a HFrD for one week followed by three rounds of DSS administration (each round consisted of five days of DSS followed by 14 days of plain drinking water) while being maintained on the respective diets. As shown in Figures 26A-26B, the quantification of tumor number showed a dramatic increase in mice fed an HFrD. These data demonstrate that feeding a diet high in fructose exacerbates experimental colitis and markedly worsens subsequent tumorigenesis. Thus, increased dietary fructose can affect the severity of colitis and colitis-associated tumorigenesis in mice. Example 3: Fructose-Mediated Exacerbation of Colitis is Preceded by Microbial Shift and Reduced Gut Barrier

[00149] Without wishing to be bound by theory, it is believed that elevated levels of dietary fructose worsens colitis and enhances colitis-associated tumorigenesis by altering gut microbiota and disrupting colonic immune homeostasis (Figure 9). Additionally, dietary alterations may attenuate the harmful effects of fructose, including protecting against colitis- associated colorectal tumorigenesis. To determine this, fructose in the feces from mice fed a HFrD or control diet (AIN-93G) for one week was measured to determine whether colonic microbes are exposed to higher levels of fructose. It was determined that HFrD feeding increased fecal fructose levels by approximately 20-fold ( P < 0.001) (see also Figure 10). Next, 16S rRNA microbial profiling was used to determine whether feeding a HFrD to mice altered the colonic bacteria. Male C57BL/6J mice were given a control diet (AIN-93G) and co-housed for two weeks. Mice were then individually housed and continued on the control diet or switched to a HFrD for one week. Feces were collected following co-housing (Day 0) then again following seven days of diet intervention (Day 7) (see Figure 11). 16S rRNA microbial sequencing was carried out on the feces and the results were plotted by principal coordinate analysis. It was determined that feeding a HFrD to mice for one week markedly shifted the fecal bacterial profile (Figure 12, Figure 13). Quantitative analysis is shown in Figure 34. The shift in the bacterial profile included an increase in the proportion of

Akkermansia muciniphila , a bacterial species that is known to degrade the protective mucus (Ottman et al., Appl Environ Microbiol 18: e0l0l4-l7 (2017); Seregin et al., Cell Rep 19:2174 (2017)) and has been shown to induce colitis in IL10 knockout mice. As shown in Figure 14, HFrD feeding also led to an increased abundance of Lactococcus lactis as well as a decreased abundance of Clostridium disporicum, Lactobacillus johnsonii, and Bacteroides acidifaciens . These data demonstrate that feeding a HFrD for one week altered luminal colonic microbes.

[00150] Without wishing to be bound by theory, it is believed that, the increased abundance of Akkermansia muciniphila suggests that the gut barrier may be defective, given its ability to degrade surface mucus. To determine whether the observed microbial changes are associated with changes in the gut barrier, the thickness of the mucus was measured, and a 22% decrease in mucus thickness was observed for mice fed a HFrD for one week ( P < 0.01) (Figures 22A- 22C). [00151] To determine whether inflammation in human patients with Crohn’s disease was associated with reduced GLUT5 expression, GLUT5 gene expression was measured in the ilea of Crohn’s disease patients vs. healthy controls. In the epithelial preparation of the ilea, an approximately 50% reduction in GLUT5 gene expression was observed in individuals with ileitis compared to healthy individuals (1.16+/-0.57 vs. 0.49+/-0.29; P = 0.008). Without wishing to be bound by theory, it is believed that reduced GLUT5 expression in the small intestine would lead to increased levels of fructose entering the colon and thus, Crohn’s disease patients may be at greater risk than healthy individuals for the harmful effects of dietary fructose.

[00152] To determine whether reducing the bacterial load ameliorates HFrD-mediated exacerbation of colitis, mice were administered an antibiotic cocktail. Five week-old

C57BL/6J WT mice were given the control diet (AIN-93G) and administered drinking water containing Splenda (4g/L) (Figure 18, Groups 1 & 2) or water containing ampicillin (0.5g/L), metronidazole (0.5g/L), vancomycin (0.25g/L), neomycin (0.5g/L), gentamicin (0.5g/L) and Splenda (4g/L) (Figure 18, Groups 3 & 4) for three weeks (h=10 mice per group). Feces from mice in each group was collected in week three to measure the bacterial abundance by PCR to ensure that treatment with the antibiotics was effective. As shown in Figure 19, this formulation of antibiotics led to approximately a 500-fold reduction in the gut bacterial load of the mice. As shown in Figure 18, following treatment with the antibiotics, mice in Groups 2 & 4 were given the HFrD, and mice in group 1 & 3 were given the control diet (AIN-93G) for one week. Subsequently, mice were administered 1% DSS in drinking water for seven days, while continuing on the respective diets. Clinical signs of colitis were examined for during DSS exposure including changes in body weight, severity of diarrhea and bleeding. Treatment with the broad spectrum antibiotics demonstrated attenuated weight loss (Figure 20A), colonic shortening (Figure 20B), and colonic ulceration (Figure 20C) in mice that were given the HFrD. These data demonstrate that HFrD-induced changes in the microbiota were responsible for the worsening of the experimental colitis.

[00153] The administration of broad spectrum antibiotics is an effective method to reduce bacterial abundance, however, it does not completely eliminate the presence of microbes and the drugs used may have direct effects on the intestinal epithelium. Therefore, germ-free (GF) mice were used to complement the broad spectrum antibiotic approach. GF mice and specific-pathogen-free (SPF) mice were given the control diet (AIN-93G) or the HFrD for one week, followed by the administration of 1% DSS in drinking water. SPF mice, which have a normal gut microbiota developed worse colitis when fed the HFrD (64 kcal%, Figures 21A-21E). By contrast, GF mice that were fed the HFrD did not exhibit worse colitis

(Figures 21A-21E). These data demonstrate that the exacerbation of colitis by the HFrD is microbiota-dependent.

[00154] Without wishing to be bound by theory, it is believed that elevated levels of dietary fructose leads to enhanced severity of colitis as well as increased risk of colorectal cancer in IBD patients. The following study was conducted to determine if dietary interventions could reverse the harmful effects of fructose. Male C57BL/6J mice were given AIN-93G control diet or HFrD for 1 week. Following this period, half of the mice given HFrD were switched to control diet for 1 week while the other 2 groups remained on their original diets. (See Figure 15). Mice were then challenged with 1% DSS for 1 week and changes in body weight, colon length and ulceration were analyzed. As expected, mice that were fed a HFrD for two weeks demonstrated greater weight loss (Figure 16A), had shorter colons (Figure 16B), and more colonic ulceration (Figure 16C) ( P < 0.001 for all endpoints) compared to control diet-fed mice. As shown in Figures 16A-16C, the colitis phenotype induced by HFrD was rapidly reversed by switching mice back to the control diet for 1 week. HFrD mice also exhibited a rapid change in the gut microbiota (Figure 16D). Switching mice from a HFrD to a control diet led to“normalization” of the fecal microbiota (switched group was similar to control group and both were distinguishable from the HFrD groups) (See Figure 17)

[00155] In order to assess global gene expression changes induced by HFrD, RNA-seq was performed on colonic mucosa of mice fed HFrD vs. control diet. RNA-seq was carried out on colonic mucosa from mice given control or HFrD for 1 week (n=4/group) prior to DSS- induced colitis. Differentially expressed genes were input into the Gene Ontology

Consortium database to determine significantly altered pathways. Pathway analysis of the RNA-seq profiling data revealed that a HFrD resulted in enrichment of processes involved in immune cell activation (See Figure 23).

Example 4: HFrD-induced Increase in the Ratio of Conjugated to Unconjugated Fecal Bile Acid Levels and Reduction in Bile Salt Hydrolase

[00156] Gut luminal metabolites are derived from both the microbes and the host, and there is increasing evidence that metabolites from the microbes can impact the host intestinal physiology. Targeted metabolite profiling studies were carried out on mouse fecal samples from the experiment described in Figure 11 to determine the mechanism by which a HFrD sensitizes mice to experimental colitis. The detection of metabolites was performed at Metabolon (Morrisville, NC) using high-performance liquid chromatography-tandem mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS). Detected metabolites were identified through comparison to a known database of over 3,000 named molecules. Analyses of mice fed the HFrD vs. control diet revealed a dramatic increase in relative levels of conjugated bile acids, including taurocholate, taurochenodeoxycholate, tauro-P-muricholate, taurodeoxycholate, taurolithocholate, tauroursodeoxycholate, taurohyodeoxycholic acid, and taurocholenate sulfate compared to mice fed control diet (Figure 24). These data demonstrate that HFrD can induce changes in the gut metabolome.

[00157] An increase in conjugated bile acids may be caused by a loss of microbes that express bile salt hydrolase, an enzyme important in converting conjugated bile acids to unconjugated bile acids. Lactobacillus johnsonii is one type of bacteria that expresses bile salt hydrolase and was found to be significantly decreased in HFrD fed mice. Consistent with the reduced population of Lactobacillus johnsonii in the feces of HFrD fed mice, a reduction in Lactobacillus johnsonii bile salt hydrolase gene expression was observed in HFrD fed mice (See Figure 25).

[00158] Bifidobacterium pseudolongum is another type of bacteria that expresses bile salt hydrolase and was found to be significantly reduced in HFrD fed mice (Figure 32A).

Consistent with the reduced population of Bifidobacterium pseudolongum in the feces of HFrD fed mice, a reduction of Bifidobacterium pseudolongum bile salt hydrolase gene expression was observed in feces of HFrD fed mice (Figure 32B). There was a strong correlation between the abundance of L. Johnsonii and B. pseudolongum and the expression of their respective BSH genes (rho=0.9l; P<0.00l and rho=0.98; P<0.00l for L. Johnsonii and B. pseudolongum , respectively).

Example 5: Fructose Exposure Alters Immune Homeostasis

[00159] Without wishing to be bound by theory, it is believed that elevated levels of dietary fructose can disrupt immune homeostasis by exposure to gut luminal content ( e.g. , bacteria or bacterial products) that can occur as a result of a reduced barrier function. Previous measurements of pro-inflammatory cytokines and flow-cytometry analysis demonstrate that elevated levels of dietary fructose increased the pro-inflammatory immune response during acute experimental colitis. Chronic colitis has been shown to contribute to tumorigenesis; therefore it was determined whether a HFrD also enhanced inflammation during chronic colitis. Mice were administered one injection of the colon specific carcinogen azoxymethane (AOM, l2.5mg/kg) then given a control diet (AIN-93G) or a HFrD for one week followed by three rounds of DSS administration to induce chronic inflammation (each round consisted of five days of DSS followed by 14 days of plain drinking water) while being maintained on the respective diets. Mice that were fed elevated dietary levels of fructose failed to gain weight normally (Figure 27A), had significantly shorter colons Figure 27B), and demonstrated more severe histological damage (Figure 27C). Consistent with the HFrD-induced severe chronic inflammation, levels of multiple pro-inflammatory cytokines were significantly increased in the colons of the mice (Figure 28). These data demonstrate that elevated levels of dietary fructose induces significant immune alterations during chronic colitis.

Example 6: Prebiotic Compositions of the Present Technology Protect Against Experimental Colitis

[00160] To determine if supplemental interventions could reverse the harmful effects of fructose, psyllium fiber supplements were examined to determine whether they would exert a protective effect against HFrD-mediated worsening of colitis. Mice were fed either a control diet (AIN-93G) or a HFrD for one week with either cellulose or psyllium as the fiber source. Psyllium fiber strongly protected against fructose-mediated exacerbation of weight loss ( P < 0.001) (Figure 29A), severe bleeding (Figure 29B; P < 0.001), as well as colonic shortening (Figure 29C) and colonic ulceration (Figure 29D). Psyllium also attenuated these disease manifestations in control diet-fed mice (weight loss: P < 0.001; severe bleeding: P = 0.015) and colon shortening (Figures 29A-29D).

[00161] Accordingly, the prebiotic compositions of the present technology are useful in methods for treating inflammatory bowel disease in a subject in need thereof.

Example 7: Elevated Dietary Fructose Promotes Growth of Citrobacter rodentium and Worsens Citrobacter rodentium-Induced Colitis

[00162] The fecal microbiota data indicated that the HFrD was associated with reduced amounts of beneficial probiotic bacterial species (/.. Johnsonii and B. pseudolongum). It is possible that a loss of probiotic bacteria could facilitate the expansion of pathogenic species leading to worse infectious colitis. To test this concept, Citrobacter rodentium inoculation, a commonly used model of infectious colitis in mice, was utilized. Zheng et al., Nat Med 14:282-9 (2008). After administration of the HFrD or control diet for 1 week, WT mice were inoculated with 5xl0⁹ CFU C. rodentium and continued on their respective diets. The abundance of C. rodentium was measured by qRT-PCR in feces from mice every 2 days over a 6 day period following infection. As shown in Figure 33A, a significant increase in abundance was detected in HFrD vs. control diet fed mice on days 2 and 4 post-inoculation. To determine whether this increased abundance resulted in more severe infectious colitis, the same mice were examined for signs of colitis during the 6 day period. As shown in Figure 33B, the mice that were fed a HFrD manifested attenuated weight gain and a higher incidence of diarrhea and bleeding. As shown in Figures 33C-33E, these changes were paralleled by shortening of the colon, worse histologic injury and elevated expression levels of III 7a and 1122. In a separate experiment, mortality was tracked over a 14 day period following C. rodentium infection which revealed that 40% of mice fed a HFrD died, while no deaths occurred in mice fed the control diet (P=0.029). Taken together, these findings demonstrate that high dietary fructose consumption enhances the growth of a pathogenic bacterium and worsens infectious colitis.

[00163] Dysbiosis occurs frequently in IBD and is believed to contribute to disease pathogenesis. Interestingly, Lactobacilli and Bifidobacteria, reduced in abundance in HFrD- fed mice, are also decreased in IBD patients. The observed reductions in fecal L.johnsonii and B. pseudolongum in mice fed dietary fructose were paralleled by decreased expression of BSH, a critical enzyme for the deconjugation of BAs. Consistent with this finding, a significant increase in levels of fecal conjugated BAs was observed. Glut5 in the plasma membrane of enterocytes is responsible for fructose absorption in the small intestine.

Without wishing to be bound by theory, it is believed that feeding a HFrD saturates the absorptive capacity of Glut5 resulting in elevated levels of fructose in the colon, dysbiosis, elevated conjugated BAs and worsening of experimental colitis.

[00164] These results can also be logically extended to nonalcoholic steatohepatitis (NASH) because HFrD-induced changes in both the microbiota and gut barrier contribute to experimental NASH. See Sellmann C et al ., J Nutr Biochem 2015; 26: 1183-92.

[00165] Accordingly, the prebiotic and probiotic compositions of the present technology are useful in methods for treating inflammatory bowel disease or nonalcoholic fatty liver disease in a subject in need thereof.

Example 8: Effects of Dietary and/or Pharmacolosical Interventions on HFrD-enhanced Colitis-Associated Tumor Burden

[00166] The data demonstrate that in addition to worsening both acute and chronic experimental colitis, feeding mice a diet high in fructose increases colitis-associated tumor burden (Figures 26A-26B). Additionally, both the removal of a HFrD (Figures 16A-16C), and supplementing psyllium fiber ameliorates HFrD-induced worsening of acute colitis (Figures 29A-29D). 5-aminosalicylic acid (5-ASA) is a standard therapy for maintaining remission in IBD patients and has been shown to suppress tumor development in mouse models of colitis-associated neoplasia. Without wishing to be bound by theory, it is believed that reducing fructose consumption or feeding psyllium during HFrD consumption will suppress inflammation-associated tumorigenesis, and further, may enhance the tumor suppressive efficacy of 5-ASA.

[00167] Impact of reduced fructose consumption on tumor suppressive efficacy of 5-ASA. One injection of AOM (l2.5mg/kg) will be administered to mice. Next, the mice will be fed either a control diet (AIN-93G) or HFrD for one week and exposed to 1% DSS for five days, followed by one week of plain drinking water. Mice will then be randomized into the following five groups:

1) Continued on control diet (AIN-93 G) (n = 20 mice);

2) Continued on HFrD (n = 20 mice);

3) Switched from HFrD to control diet (AIN-93G) (n = 20 mice);

4) Continued on HFrD and administered 5-ASA (75mg/kg in drinking water) (n = 20 mice);

5) Switched from HFrD to control diet (AIN-93G) and administered 5-ASA (75mg/kg in drinking water) (n = 20 mice)

[00168] Mice will continue on plain drinking water for one week, and will then be administered two cycles of DSS, during which the respective diets/treatments (Groups 1 through 5) will be given. The experimental design is shown in Figure 30. Five weeks after the last day of DSS exposure, or as determined by tumor burden by colonoscopy, mice will be sacrificed to assess the tumor burden and the level of chronic inflammation. Histological assessments of tumors and FISH-based examination of the microbial interaction with the tumors will also be made, along with 16S rRNA microbial analyses of mice feces collected during the study.

[00169] The effect on tumor suppressive activity of 5-ASA with psyllium supplementation. One injection of AOM (l2.5mg/kg) will be administered to mice. Subsequently, the mice will be fed either a control diet (AIN-93 G) or HFrD containing cellulose for one week, followed by the administration of DSS for five days and then one week of plain drinking water. Mice will then be randomized into five groups: 1) Control diet (AIN-93 G) containing cellulose (n = 20 mice);

2) HFrD containing cellulose (n = 20 mice);

3) HFrD containing psyllium (n = 20 mice);

4) HFrD containing cellulose and administered 5-ASA (75mg/kg in drinking water) (n = 20 mice);

5) HFrD containing psyllium and administered 5-ASA (75mg/kg in drinking water) (n = 20 mice)

[00170] Mice will continue on plain drinking water for one week, followed by the administration of 2 cycles of DSS, during which the respective diets/treatments (Groups 1 through 5) will be given. Five weeks after the last day of DSS exposure, or as determined by tumor burden by colonoscopy, mice will be sacrificed to assess the tumor burden and the level of chronic inflammation. Histological assessments of tumors and FISH-based examination of the microbial interaction with the tumors will also be made, along with 16S rRNA microbial analyses of mice feces collected during the study.

[00171] It is anticipated that reducing fructose consumption or feeding psyllium during HFrD consumption will suppress inflammation-associated tumorigenesis, and further, may enhance the tumor suppressive efficacy of 5-ASA.

[00172] Accordingly, the prebiotic compositions of the present technology are useful in methods for treating inflammatory bowel disease in a subject in need thereof.

Example 9: Changes in Gut Microbiota in Response to HFrD

[00173] To determine whether bacterial gene expression changes in response to HFrD, meta- transcriptomic analyses will be performed on fecal samples from mice fed a control diet (AIN-93G) or a HFrD diet. Briefly, RNA will be extracted from fecal samples, followed by the extraction of rRNA from the total RNA. TruSeq-barcoded RNA-seq libraries will be generated and the samples will be sequenced on a NextSeq500 instrument (Illumina, San Diego, CA) with a minimum of 20 million single-end 75 base pair reads. For meta- transcriptomic analysis, reads will be pre-processed to trim low quality reads, adapter sequences and small RNA reads using bowtie2v2.2. Reads will be mapped using tophatv.2.1. Normalized read counts (FPKM) values will be generated using cufflinks v2.2 and

differential gene expression between the test and the reference groups of each pairwise contrast will be identified using DESeq2. [00174] To further characterize if alterations in the luminal content contribute to worse colitis, bacterial transfer experiments will be performed. Conventionally-housed mice will be fed either the control diet (AIN-93G) or the HFrD for one week. Following this, cecal content and feces will be collected in an anaerobic chamber (to preserve anaerobic bacteria), homogenized in sterile phosphate buffered saline (PBS). The clarified supernatant from mice given the control diet (AIN-93G) or the HFrD will be given to GF mice by oral gavage. One week post inoculation, the recipient mice will be given DSS to induce colitis and be kept on the control diet (AIN-93G). Colitis severity will be compared in the GF mice that received the cecal content/feces from the control diet-fed mice vs. the HFrD-fed mice, as described above. The fecal microbial profile of the donor and recipient mice will be compared using 16S rRNA profiling to ensure that the bacterial transfers were performed successfully.

Example 10: Impact of Fructose on Immune Cell Functions

[00175] Intestinal tissue damage which results from the release of factors from resident and recruited immune cells is a major contributor to IBD pathogenesis. To determine whether a HFrD will provoke immune-related alterations that could promote colorectal tumorigenesis, high-throughput single cell RNA-seq will be used to generate a high-dimensional profile of the gut immunoenvironment and the associated transcriptional landscape, comparing mice fed a control diet (AIN-93G) or a HFrD before colitis onset and during chronic colitis. Mice will be fed (1) a control diet or a HFrD for one week; (2) a control diet or a HFrD for one week followed by one round of DSS administration (5 days of 1% DSS, followed by 14 days of plain drinking water); (3) a control diet or a HFrD for one week followed by two rounds of DSS administration; (4) a control diet or a HFrD for one week followed by three rounds of DSS administration. These time points will allow for the study of how HFrD feeding impacts the immunoenvironment of the gut prior to and during the evolution of colitis, and whether changes in immune cell populations or in immune-intrinsic transcriptional programs could be associated with the increased colorectal tumorigenesis provoked by this diet (Figures 26A- 26B). Previously performed pathway analysis of RNA-seq profiling carried out on the colonic mucosa from mice fed a control diet (AIN-93G) or a HFrD for one week prior to colitis revealed that feeding a HFrD resulted in the enrichment of processes involved in immune cell activation (Figure 23). Following this, 1,000-2,000 viable CD45⁺ leukocytes will be sorted from the colonic lamina propria of each mouse and analyzed with the 10^c Chromium platform to generate single cell RNA-seq data (n=4 samples/group). The RNA- seq studies will be complemented by Multiplex assays to determine the range of colonic cytokines whose expression and secretion are modulated by a HFrD before and during chronic colitis. The immune cell alterations described above may result from changes in the bacterial diversity and/or localization, it will be determined whether treatment with broad- spectrum antibiotics during the HFrD feeding can restore the immune homeostatic processes in the gut. Taken together, these data will demonstrate how elevated levels of dietary fructose may alter immune cell populations and/or immune-intrinsic processes in the gut as a mechanism to promote colonic inflammation and colorectal tumorigenesis.

[00176] Without wishing to be bound by theory, it is believed that the potential effects of a HFrD on immune cell function could occur via the microbiota, however, fructose exposure may directly influence immune cell processes by acting as a nutrient source which can modulate their metabolic processes. To test this concept, in vitro studies of bone marrow- derived myeloid cells isolated from mice fed a control diet will be conducted. Bone marrow progenitors will be cultured in GM-CSF or M-CSF to induce differentiation to dendritic cells (DCs) or macrophages, respectively, in the absence of exogenous fructose or in media containing fructose at increasing concentrations. FACS analyses will be used to determine the effects of fructose on DCs or macrophage differentiation, polarization and function.

[00177] DCs and macrophages already differentiated in normal medium, as well as primary bone marrow-resident neutrophils from naive mice, will be stimulated with diverse microbial products, such as TLR agonists, in the presence or absence of exogenous fructose to determine the impact of direct exposure on their function and metabolism. A combination of enzyme-linked immunosorbent assays (ELISA) and FACS analyses will be used to define whether fructose enhances the expression of pro-inflammatory mediators by TLR-activated myeloid cells. Further, extracellular Flux Analyzers will be used to determine whether fructose impacts the bioenergetics profile of these cells. This system will allow for the determination of real-time measurement of DC, macrophage, or neutrophil metabolism by simultaneously quantifying rates of extracellular acidification (ECAR) and oxygen consumption (OCR) as measures of glycolysis and mitochondrial respiration, respectively. This technology will be used in combination with pharmacological inhibitors to determine how fructose exposure may influence glucose uptake and glycolysis, as well as the use of mitochondrial energy sources such as pyruvate, glutamine, and fatty acids.

[00178] To complement these analyses, the intracellular fate of fructose will be determined within the myeloid cell populations by using metabolic tracing experiments and ¹³C-labeled fructose. Briefly, DCs, macrophages, and neutrophils will be exposed to ¹³C -labeled fructose in the presence or absence of TLR agonists, and targeted metabolomics will be used to monitor fructose influx and utilization in these cells over time. These analyses will reveal the main metabolic pathways influenced (inhibited or activated) by fructose in myeloid cells, and will demonstrate how this process may impact the expression of pro-inflammatory mediators that may contribute to the exacerbation of colitis and colitis-associated

tumorigenesis. In addition to examining myeloid cell populations, the direct effects of fructose on lymphoid cells will be evaluated by examining whether fructose can modulate CD4 T helper differentiation in vitro. The impact that fructose has on the expression of master regulators of CD4 T cell polarization such as T-bet, GAT A3 and RORyT, and their association to the expression of IFNy, IL-4 and IL-17, respectively will be assessed.

Example 11: Treatment with Bile Salt Hydrolase Expressing Probiotics Prevents or

Ameliorates Experimental Colitis

[00179] The findings indicate that a HFrD leads to a rapid change in the microbiota and more specifically in a reduction in bile salt hydrolase expressing bacteria including

Lactobacillus johnsonii and Bifidobacterium pseudolongum. The reduction in bile salt hydrolase expressing bacteria is associated with an increase in the ratio of conjugated to unconjugated bacteria in feces along with a thinning of colonic mucus which contributes to barrier function. Changes in the microbiota are causally linked to the exacerbation of experimental colitis. Given this background, mice will be treated with bile salt hydrolase expressing bacteria to evaluate whether this reduces the exacerbation of DSS induced colitis mediated by HFrD.

[00180] A probiotic mixture to reduce conjugated bile acid (BA) levels will be tested in multiple animal models of IBD. In one such instance, mice will be administered a mixture of 1c10⁸-1c10¹⁰ CFU of mixtures of different species of Lactobacillus, Bifidobacterium, Bacteroides, Enterococcus and Clostridium or vehicle, twice daily by oral gavage for 1 week while being fed a HFrD. Various combinations in type, amount and duration of treatment of these different species will be tested. Mice will then be administered dextran sodium sulfate (DSS) in drinking water to induce acute colitis for 1 week while continuously being inoculated with the probiotic mixture. Severity of experimental colitis will be compared between those mice being given the probiotic mixture vs. vehicle during the week of DSS exposure. Colitis severity will be assessed by evaluating changes in body weight, severity of diarrhea and rectal bleeding, changes in colon length, histologic injury of the colon and expression of inflammatory cytokines in colonic tissue. Additional endpoints to assess efficacy of therapy will be measuring levels of conjugated BAs in feces as well as evaluating the abundance of the inoculated species in feces.

[00181] In another instance, the effect of the probiotic mixtures will be evaluated in the context of chronic colitis. For this approach, HFrD will be given for 1 week to mice followed by 1 week of administration of DSS (while being continued on HFrD), followed by 1 week of plain drinking water (while being continued on HFrD). Following this period, mice will be administered the probiotic mixture (as described above) or vehicle along with an additional week of plain drinking water. Mice will be continued on the probiotic mixture or vehicle while mice are then given 2 more rounds of DSS (1 round = 1 week of DSS followed by 2 weeks of plain drinking water). Mice will be assessed for the same endpoints as described above during the 2 rounds of DSS.

[00182] In a third instance, germ-free 1110 knockout mice will be inoculated with fecal material from conventionally-housed mice (to initiate T-cell mediated colitis). One week later, 1110 knockout mice will be given HFrD and continuously inoculated with the probiotic mixture as described above for approximately 6 weeks while being tracked for disease severity.

[00183] It is anticipated that animals receiving the probiotic compositions of the present technology will show amelioration of colitis and reduced levels of conjugated BAs compared to the vehicle-treated IBD control animals.

[00184] Accordingly, the prebiotic and probiotic compositions of the present technology are useful in methods for treating inflammatory bowel disease in a subject in need thereof.

EQUIVALENTS

[00185] The present technology is not to be limited in terms of the particular embodiments described in this application, which are intended as single illustrations of individual aspects of the present technology. Many modifications and variations of this present technology can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. Functionally equivalent methods and apparatuses within the scope of the present technology, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing descriptions. Such modifications and variations are intended to fall within the scope of the present technology. It is to be understood that this present technology is not limited to particular methods, reagents, compounds compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. [00186] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[00187] As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as“up to,”

“at least,”“greater than,”“less than,” and the like, include the number recited and refer to ranges which can be subsequently broken down into subranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member.

Thus, for example, a group having 1-3 cells refers to groups having 1, 2, or 3 cells. Similarly, a group having 1-5 cells refers to groups having 1, 2, 3, 4, or 5 cells, and so forth.

[00188] All patents, patent applications, provisional applications, and publications referred to or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent they are not inconsistent with the explicit teachings of this specification.

Claims

1. A method for treating inflammatory bowel disease in a subject in need thereof

comprising administering to the subject an effective amount of a probiotic composition comprising bacterial cells belonging to one or more genera selected from the group consisting of Lactobacillus , Bifidobacterium, Bacteroides, Enterococcus and Clostridium.

2. A method for treating inflammatory bowel disease in a subject in need thereof

comprising administering to the subject an effective amount of a probiotic composition including one or more of Lactobacillus sp. strain 100-14, Lactobacillus sp. strain 100-100, Lactobacillus acidophilus, Lactobacillus delbrueckii,

Lactobacillus fermentum, Lactobacillus sp. strain 100-93, Lactobacillus plantarum, Lactobacillus salivarius, Lactobacillus johnsonii, Lactobacillus amylovorous, Lactobacillus amylophilus, Lactobacillus brevis, Lactobacillus buchneri,

Lactobacillus casei, Lactobacillus crispatus, Lactobacillus farciminis, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus hilgardii, Lactobacillus

kefir anofaciens, Lactobacillus lindneri, Lactobacillus paracasei, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus sanfranciscensis, L. salivaricus strain JCM1046, Lactobacillus casei Shirota, Lactobacillus

delbrueckii subsp. bulgaricus, Lactobacillus mucosae, Lactobacillus rhamnosus GG, Bifidobacterium longum, Bifidobacterium adolescentis, Bifidobacterium animalis, Bifidobacterium thermophiles, Bifidobacterium pseudolongum, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium bifidum, Bifidobacterium lactis, Enterococcus faecium, Clostridium disporicum, Clostridium perjringens,

Methanobrevibacter smithii, Methanosphera stadmanae, Bacteroides ovatus, Bacteroides thetaiotaomicron, Bacteroides acidifaciens, Bacteroides vulgatus, Bacteroides firagilis, Bacteroides firagilis subsp. fragilis , and Peptostreptococcus species.

3. The method of claim 1 or 2, wherein the subject exhibits at least one symptom

selected from the group consisting of abdominal pain, diarrhea, rectal bleeding, weight loss, fever, loss of appetite, dehydration, anemia and malnutrition.

4. The method of any one of claims 1-3, wherein the subject exhibits at least one mutation in one or more genes selected from the group consisting of NOD2, IRGM, ATG16L1, I 23P, IL10, IL10RA, IL10RB, and PTPN2.

5. A method for treating nonalcoholic fatty liver disease in a subject in need thereof comprising administering to the subject an effective amount of a probiotic composition comprising bacterial cells belonging to one or more genera selected from the group consisting of Lactobacillus , Bifidobacterium, Bacteroides, Enterococcus and Clostridium.

6. A method for treating nonalcoholic fatty liver disease in a subject in need thereof comprising administering to the subject an effective amount of a probiotic composition including one or more of Lactobacillus sp. strain 100-14, Lactobacillus sp. strain 100-100, Lactobacillus acidophilus, Lactobacillus delbrueckii,

Methanobrevibacter smithii, Methanosphera stadmanae, Bacteroides ovatus, Bacteroides thetaiotaomicron, Bacteroides acidifaciens, Bacteroides vulgatus, Bacteroides firagilis, Bacteroides firagilis subsp. firagilis, and Peptostreptococcus species.

7. The method of claim 5 or 6, wherein the subject exhibits at least one symptom

selected from the group consisting of fatty liver, nonalcoholic steatosis, tiredness, fatigue, including muscle weakness, discomfort or swelling in the upper abdomen, weight loss, low appetite, nausea, cirrhosis, liver failure, vomiting, and diarrhea, tarry stools, abdominal swelling and pain, jaundice, itchy skin, confusion, difficulty focusing, memory loss, and hallucinations.

8. The method of any one of claims 1-7, wherein the probiotic composition is

administered orally or rectally.

9. The method of any one of claims 1-8, wherein the probiotic composition is co- administered with a bile acid sequestrant.

10. The method of claim 9, wherein the bile acid sequestrant is cholestyramine,

colestipol, or colesevelam.

11. The method of any one of claims 1-10, wherein the probiotic composition is

sequentially, simultaneously, or separately administered with at least one additional therapeutic agent selected from the group consisting of an anti-inflammatory agent, a corticosteroid, an aminosalicylate, an immunosuppressive agent, a tumor necrosis factor (TNF)-alpha inhibitor, a pain reliever, an iron supplement, calcium, and a vitamin D supplement.

12. The method of any one of claims 1-11, wherein the probiotic composition is

sequentially, simultaneously, or separately administered with one or more agents selected from the group consisting of mesalamine, sulfasalazine, infliximab, adalimumab, prednisone, budesonide, 6-mercaptopurine, 5-aminosalicylic acid (5- ASA), azathioprine, cyclosporine, golimumab, balsalazide, olsalazine, methotrexate, natalizumab, vedolizumab, ustekinumab, acetaminophen, ibuprofen, naproxen sodium (Aleve), diclofenac sodium, orlistat, sibutramine, pioglitazone, rosiglitazone, metformin, atorvastatin, pravastatin, rosuvastatin, gemfibrozil, ursodiol, vitamin E, vitamin C, pentoxifylline, betaine, and losartan.

13. The method of any one of claims 1-12, wherein administration of the probiotic

composition results in reduced levels of one or more bile acids compared to an untreated subject suffering from inflammatory bowel disease or nonalcoholic fatty liver disease.

14. The method of claim 13, wherein the one or more bile acids are selected from the group consisting of glycocholic acid, glycochenodeoxycholic acid, glycodeoxycholic acid, taurocholic acid, taurochenodeoxycholic acid and taurodeoxycholic acid.

15. A method for monitoring the therapeutic efficacy of a probiotic composition in a subject diagnosed with inflammatory bowel disease or nonalcoholic fatty liver disease comprising:

(a) detecting conjugated bile acid levels in a test sample obtained from the subject after the subject has been administered the probiotic composition; and

(b) determining that the probiotic composition is effective when the conjugated bile acid levels in the test sample are reduced compared to that observed in a control sample obtained from the subject prior to administration of the probiotic composition,

wherein the probiotic composition includes one or more of Lactobacillus sp. strain 100-14, Lactobacillus sp. strain 100-100, Lactobacillus acidophilus, Lactobacillus delbrueckii, Lactobacillus fermentum, Lactobacillus sp. strain 100-93, Lactobacillus plantarum,

Lactobacillus salivarius, Lactobacillus johnsonii, Lactobacillus amylovorous, Lactobacillus amylophilus, Lactobacillus brevis, Lactobacillus buchneri, Lactobacillus casei, Lactobacillus crispatus, Lactobacillus farciminis, Lactobacillus gasseri, Lactobacillus helveticus,

Lactobacillus hilgardii, Lactobacillus kefir anofaciens, Lactobacillus lindneri, Lactobacillus paracasei, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus sanfranciscensis, L. salivaricus strain JCM1046, Lactobacillus casei Shirota, Lactobacillus

16. The method of claim 15, wherein the test sample is feces.

17. The method of claim 15 or 16, further comprising detecting bacterial bile salt

hydrolase levels in the feces of the subject.

8. A kit comprising a probiotic composition and instructions for using the probiotic composition to treat inflammatory bowel disease or nonalcoholic fatty liver disease, wherein the probiotic composition includes one or more of Lactobacillus sp. strain 100-14, Lactobacillus sp. strain 100-100, Lactobacillus acidophilus, Lactobacillus delbrueckii, Lactobacillus fermentum, Lactobacillus sp. strain 100-93, Lactobacillus plantarum, Lactobacillus salivarius, Lactobacillus johnsonii, Lactobacillus amylovorous, Lactobacillus amylophilus, Lactobacillus brevis, Lactobacillus buchneri, Lactobacillus casei, Lactobacillus crispatus, Lactobacillus farciminis, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus hilgardii, Lactobacillus kefir anofaciens, Lactobacillus lindneri, Lactobacillus paracasei, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus sanfranciscensis, L. salivaricus strain JCM1046, Lactobacillus casei Shirota, Lactobacillus

delbrueckii subsp. bulgaricus, Lactobacillus mucosae, Lactobacillus rhamnosus GG, Bifidobacterium longum, Bifidobacterium adolescentis, Bifidobacterium animalis, Bifidobacterium thermophiles, Bifidobacterium pseudolongum, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium bifidum, Bifidobacterium lactis, Enterococcus faecium, Clostridium disporicum , Clostridium perfir ingens,