Targeting the Intestinal Microbiota in the Treatment of Functional Gastrointestinal Disorders

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1 Targeting the Intestinal Microbiota in the Treatment of Functional Gastrointestinal Disorders Yehuda Ringel, MD. Professor of Medicine Chief, Division of Gastroenterology Rabin Medical Center, Beilinson Hospital Affiliated with Tel-Aviv University Petach Tikva, Israel The Israeli Neurogastroenterology Group Workshop June 21, 2015 Felsenstein Medical Research Center (FMRC) Rabin Medical Center, Petach Tikva, Israel

2 The Intestinal Microbioa in Functional GI Disorders Presentation Outline: The intestinal microbiota What do we know about the intestinal microbiota in patients with FGIDs? - Microbiology data Can the intestinal microbiota affect symptoms and physiological functions relevant to FGIDs? - Clinical and Physiology data Can the intestinal microbiota be targeted in the treatment of FGIDs? - Examples from clinical trials Conclusions and personal perspective

3 The Human Microbiota: Multiple Body Sites The microbiota colonizes every surface of the human body that is exposed to the external environment. External Auditory Canal Hair on the Head Mouth Nostril Esophagus The gastrointestinal tract contains about 70% of all microbes in the human body. Skin Gastrointestinal Tract With the exception of defined pathogens, there is wide agreement that the microbiota is important to human health. Penis Vagina Firmicutes Actinobacteria Bacteroidetes Verrucomicrobi a Fusobacteria Proteobacteria Spor A et. al. Nature Reviews Microbiology 2011

4 The Human Intestinal Microbiota The human intestine contains approximately bacteria 10 times the number of human cells 100 times the number of genes in the human genome There are over 1,000 different species Less than a quarter of the intestinal bacteria have been identified The composition varies along the digestive tract Affected by host (GI motility) and environmental factors (diet, medications) aerobes aerobes anaerobes anaerobes duodenum jejunum ileum colon adapted from Lin et al., JAMA 2006

5 The Intestinal Microbiota Two Ecosystems I. Luminal microbiota Metabolic effects II. Mucosal microbiota Immunologic effects Modified from Parkes et al. Am J Gastroenterol 2008 The two ecosystems have distinct microenviroments Each has the potential to play different functions

6 The Intestinal Microbiota Two Ecosystems Fecal and Mucosal Microbiota - High Throughput 454 Sequencing Mucosa Principal component analysis Firmicutes Bacteroidetes Proteobacteria Tenericutes Actinobacteria Fusobacteria Elusimicrobia Feces Microbial diversity Firmicutes Bacteroidetes Proteobacteria Tenericutes Overall average abundance of bacterial taxa (genus level) Ringel Y et al., Gut Microbes 2015

7 Development of the Intestinal Microbiota Intestinal microbiota in healthy young children and adults High throughput microarray analysis of fecal samples Intestinal microbiota diversity Principal component analysis The development of the intestinal microbiota is a dynamic process and it is not fully established until the age of 4. Ringel-Kulka et al., PLoS ONE 2013

8 The Human Intestinal Microbiota The composition of the intestinal microbiota Sekirov I et al., Physiol Rev 2010

9 The Human Intestinal Microbiota Defining a healthy human intestinal microbiota Currently, there is no widely accepted description of a healthy gut microbiota A healthy microbiota can be described in terms of desirable: (1) Composition (2) Function (3) Ecologic stability - the ability to resist changes under stress (4) Resilience - the ability to return to baseline following stress conditions Fredrik Backhed; Claire M Fraser; Yehuda Ringel; Mary Ellen Sanders; R. Balfour Sartor; Philip M Sherman; James Versalovic; Vincent Young; Brett Finlay. The International Life Sciences Institute (ILSI) North America Committee on Gut Microbes and Health consensus conference on Defining a Healthy Gut Microbiome. 2012, Washington, DC. Cell Host & Microbe 2012

10 The Intestinal Microbiota and Human Diseases Intestinal dysbiosis is associated with various intestinal and systemic diseases Obesity Metabolic syndrome Antibiotic-associated Diarrhea (AAD) Functional GI Disorders (FGIDs) Inflammatory bowel diseases Liver diseases Atherosclerosis Type 1 diabetes Autism Allergy Asthma Celiac disease Emerging data demonstrate alterations in intestinal microbiota in various disease conditions (compared to healthy controls) although a causal relationship has not been established. Bäckhed et al., 2005; Sartor, 2010; Ringel, 2009; Honda and Littman, 2011

11 The Intestinal Microbiota and Human Diseases Intestinal dysbiosis is associated with various intestinal and systemic diseases Obesity Metabolic syndrome Antibiotic-associated Diarrhea (AAD) Functional GI Disorders (FGIDs) Inflammatory bowel diseases Liver diseases Atherosclerosis Type 1 diabetes Autism Allergy Asthma Celiac disease Emerging data demonstrate alterations in intestinal microbiota in various disease conditions (compared to healthy controls) although a causal relationship has not been established. Bäckhed et al., 2005; Sartor, 2010; Ringel, 2009; Honda and Littman, 2011

12 The Intestinal Microbioa in Functional GI Disorders Presentation Outline: The intestinal microbiota What do we know about the intestinal microbiota in patients with FGIDs? - Microbiology data Can the intestinal microbiota affect symptoms and physiological functions relevant to FGIDs? - Clinical and Physiology data Can the intestinal microbiota be targeted in the treatment of FGIDs? - Examples from clinical trials Conclusions and personal perspective

13 The Intestinal Microbiota in Patients with D-IBS Terminal restriction fragment length polymorphism (T-RFLP) analysis Fecal samples from patients with IBS show significantly lower microbial diversity compared to samples from healthy controls. Carroll, Ringel-Kulka et al., Am J Physiol Gastrointest Liver 2012

14 The Intestinal Microbiota in Patients with D-IBS Terminal restriction fragment length polymorphism (T-RFLP) analysis Multidimensional analysis (nmds) Hierarchical cluster analysis of T-RF data The majority of fingerprint profiles from fecal samples (blue circles) cluster separately from the majority of the colonic mucosal samples (red circles) Carroll, Ringel-Kulka et al., Am J Physiol Gastrointest Liver 2012

15 The Intestinal Microbiota in Patients with D-IBS High throughput 454 sequencing on fecal samples D-IBS patients Faecalibacterium genus Healthy controls Faecalibacterium prausnitzii Concentrations of specific bacterial species Carroll, Ringel-Kulka et al., Neurogastroenterol and Motil 2012

16 The Intestinal Microbioa in IBS High throughput 454 sequencing on fecal samples Clustering of Bacterial Communities Abundance of Taxa The intestinal mictobiota differ between patients with IBS and HC Ringel Y et al., DDW 2014 (MS under review)

17 The Intestinal Microbioa in IBS Clustering of Bacterial Communities By IBS Subtypes By Bloating Symptoms The intestinal mictobiota differ among patients with IBS and HC based on characteristics of their bowel habits and the presence of symptoms of abdominal bloating. Ringel Y et al., DDW 2014 (MS under review)

18 The Intestinal Microbioa in Functional GI Disorders Presentation Outline: The intestinal microbiota What do we know about the intestinal microbiota in patients with FGIDs? - Microbiology data => Microbiology Can the intestinal investigations microbiota suggest affect compositional symptoms and changes physiological in the functions relevant to FGIDs? intestinal microbiota of patients with functional GI disorders. - Clinical and Physiology data Can the intestinal microbiota be targeted in the treatment of FGIDs? - Examples from clinical trials Conclusions and personal perspective

19 The Intestinal Microbioa in Functional GI Disorders Presentation Outline: The intestinal microbiota What do we know about the intestinal microbiota in patients with FGIDs? - Microbiology data Can the intestinal microbiota affect symptoms and physiological functions relevant to FGIDs? - Clinical and Physiology data Can the intestinal microbiota be targeted in the treatment of FGIDs? - Examples from clinical trials Conclusions and personal perspective

20 Intestinal Microbiota and FGI Symptoms Significant associations (p<0.05) between specific bacterial taxa and clinical variables. Several bacterial groups correlated positively with pain and a negatively with average wellbeing and IBS-QOL. Bifido correlated negatively with IBS severity and positively with IBS-QOL. Akkermansia correlated negatively with pain and IBS severity, and positively with average wellbeing. Akkermansia has been suggested to have a role in increasing barrier function in mice (Everard et al PNAS) and human cell lines (Reunanen et al. AEM 2015). IBS Pain Bloating Pain Freq. Severity W-B QOL

21 Intestinal Microbiota - Effect on Gut Physiology Suggested Pathophysiological Model Ringel Y et al. Am J Physiol 2013

22 Intestinal Microbiota - Effect on Gut Physiology Intestinal Microbiota and Barrier Function The intestinal microbiota is a major source of intestinal proteases. Gibson et al., Appl. Environ. Microbiol Intestinal proteases can increase intestinal permeability through PAR or disruption of tight junctions. Steck N, et al. Postgrad Med J 2013 Fecal Serine Protease (FSP) in patients with IBS-D and UC: Gecse et al., Gut 2011 Annaházi et al., Pain 2009

23 Intestinal Microbiota - Effect on Gut Physiology Intestinal Microbiota and Barrier Function Fecal Cysteine Protease (FCP) in IBS: p< p=0.02 p=0.01 FCP activity is increased all subtypes of IBS FCP activity strongly correlated with IBS symptoms severity and pain scores Annahazi et al., Am J Gastroenterl 2013

24 Intestinal Microbiota Effects on Gut Physiology Intestinal Microbiota and Motor Function Summery of main changes in intestinal functions in germ-free animals Normal flora Germ Free Husebye et al., Am J Physiol Gastroint 2001 Barbara et al., Am J Gastroenterol 2005 The intestinal microbiota play an important role in maintaining normal intestinal motility Changes in intestinal microbiota can lead to significant alterations in gut function

25 Normalised barostat data (%) Intestinal Microbiota Effects on Gut Physiology Pain thresholds by rectal balloon distensions in humans p< HC (n=24) IBS (n=83) Patients with IBS had significantly lower pain thresholds than healthy subjects. Pain thresholds significantly correlated with: - Pain severity (rho= -0.70, p<0.01) - Pain frequency (rho= -0.65, p=0.016) - Bloating severity (rho= -0.67, p=0.013) Ringel Y et al., DDW 2015

26 Intestinal Microbiota Effects on Gut Physiology Bacterial groups associated with pain thresholds by rectal balloon distensions Random Forest analyses identify intestinal bacteria most contributing to the normalised barostat data. A set of 3 bacterial groups that explain 53.3% of variation in the barostat data: - Clostridium orbiscindens - Anaerotruncus colihominis - Anaerovorax odorimutans. Ringel Y et al., DDW 2015

27 Normalised barostat data Normalised barostat data Pain data Normalised barostat data Intestinal Microbiota and Sensory Function Bacterial groups associated with pain thresholds by rectal balloon distensions Clostridum orbiscindes r=0.64 p= Anaerovorax odorimutans r=0.46 p=0.054 Anaerotrucus colihominis r=0.59 p= Relative abundance (%) Relative abundance (%) Relative abundance (%) The hypersensitivity to colonic balloon distension in IBS patients is associated with specific groups of intestinal bacteria. Ringel Y et al., DDW 2015

28 The Intestinal Microbioa in Functional GI Disorders Presentation Outline: The intestinal microbiota What do we know about the intestinal microbiota in patients with FGIDs? - Microbiology data Can the intestinal microbiota affect symptoms and physiological functions relevant to FGIDs? - Clinical and Physiology data => Physiological Can the intestinal data demonstrate microbiota that be targeted the intestinal the microbiota treatment can of FGIDs? affect relevant - GI Examples symptoms from brain-gut clinical trials functions (barrier, motility, sensation, brain functions) and therefore can be an important factor in the pathogenesis of IBS. Conclusions and personal perspective

29 The Intestinal Microbioa in Functional GI Disorders Presentation Outline: The intestinal microbiota What do we know about the intestinal microbiota in patients with FGIDs? - Microbiology data Can the intestinal microbiota affect symptoms and physiological functions relevant to FGIDs? - Clinical and Physiology data Can the intestinal microbiota be targeted in the treatment of FGIDs? - Examples from clinical trials Conclusions and personal perspective

30 Targeting the Microbiota in the Treatment of FGIDs Factors that can influence the gut microbiome Bäckhed et al., Cell Host & Microbe 2012

31 Targeting the Microbiota in the Treatment of FGIDs Factors that can influence the gut microbiome Fecal Microbiome Transplant (FMT) - C. difficile - Ulcerative colitis? - IBS? - Other conditions? Bäckhed et al., Cell Host & Microbe 2012

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33 Targeting the Microbiota in the Treatment of FGIDs Interventions targeting the intestinal microbiota Diet, probiotics, antibiotics Effects of these interventions on: - The intestinal microbiome - Relevant intestinal physiology/functions - Clinical outcome

34 Targeting the Microbiota in the Treatment of FGIDs Interventions targeting the intestinal microbiota Diet, probiotics, antibiotics Effects of these interventions on: - The intestinal microbiome - Relevant intestinal physiology/functions - Clinical outcome

35 Intestinal Microbiota Effects of Diet A controlled-feeding study of 10 subjects with high-fat/low-fiber or low-fat/high-fiber diet. Intestinal microbiota was investigated by 454 sequencing of the 16S rdna gene. Correlation of diet with changes in the gut microbial taxa over 10 days Columns - bacterial taxa Rows - nutrients measured by dietary questionnaire Red - positive association / Blue - negative association The intensity of the colors - the degree of association between the taxa abundances and specific nutrients Wu GD et al., Science 2012

36 Intestinal Microbiota Effects of Diet Time and magnitude of effect Diet can significantly change the composition of the intestinal microbiome but the overall changes are not greater than the interindividual variability Wu GD et al., Science 2012

37 Intestinal Microbiota Effects of Probiotics Fecal samples from healthy adults pre- and post-intervention with commercially available probiotics containing either Bifidobacterium or Lactobacillus strains. Species belonging to the Firmicutes were affected by both Lactobacillus and Bifidobacterium Species belonging to the Bacteroidetes were affected only by Lactobacillus probiotics Probiotics affect the composition of the intestinal microbiota and this effect is strain specific. Kim S-W et al., DNA Research 2013

38 Targeting the Microbiota in the Treatment of FGIDs Interventions targeting the intestinal microbiota Diet, probiotics, antibiotics Effects of these interventions on: - The intestinal microbiome - Relevant intestinal physiology/functions - Clinical outcome

39 Targeting the Microbiota in the Treatment of FGIDs Synbiotic effects on intestinal motility (BB12 + inulin) Colonic transit time in the active arm Ascending Transverse Left side Total Pre p=0.056 post p=0.016 Probiotic intervention significantly shortened the CTT in the active group Pre-to-post changes in colonic transit time p=0.064 p=0.04 Placebo Active CTT significantly shortened in the probiotic (n=30) vs. the placebo (n=30) group The mean change was equivalent to 30% decrease in CTT Shortening in CTT was associated with: - softer stool (r=0.4,p=0.02) - a trend for improvement in ease of defecation (r=-0.4 p=0.08) Ringel-Kulka et al., ANMS 2014

40 Targeting the Microbiota in the Treatment of FGIDs Probiotics effects on intestinal sensation L. NCFM effect on intestinal pain pathways in humans MOR expression by PCR P=0.014 pstat3 expression by IHC Baseline Post- Intervention Increased expression of MOR receptors. Post receptor opioid signaling effect that has been linked to anti-inflammatory and mucosal healing responses in animals. Probiotic can affect opioid-mediated pain and anti-inflammatory pathways in human Ringel-Kulka et al., AP&T 2014 Goldsmith, et al. AJP 2011

41 Targeting the Microbiota in the Treatment of FGIDs Probiotic Effect on Brain Response to Emotional Stimuli Fermented milk with probiotic product (FMPP) decreases brain responses to emotional attention tasks. Gut brain communication (microbiota-to-brain signaling) exists and is modifiable, even in healthy women. Tillisch K et al., Gastroenterology 2013

42 Targeting the Microbiota in the Treatment of FGIDs Interventions targeting the intestinal microbiota Diet, probiotics, antibiotics Effects of these interventions on: - The intestinal microbiome - Relevant intestinal physiology/functions - Clinical outcome

43 Responders rate Targeting the Intestinal Microbiota Clinical Effects FODMAP Diet in IBS FODMAPs = Fermentable oligo- (fructans and galactans), di-(lactose), monosaccharide (fructose), and polyols; rapidly fermentable short-chain carbohydrates Low FODMAP vs. standard diet in IBS The low FODMAP diet can significantly improve functional GI symptoms Staudacher HM, et al. J Hum Nutr Diet. 2011

44 Targeting the Intestinal Microbiota Clinical Effects Low FODMAP vs. Australian Diet in IBS Low FODMAP diet is an effective treatment of functional GI symptoms in IBS. Halmos EP, et al. Gastroenterology 2014

45 Targeting the Intestinal Microbiota Clinical Effects FODMAP Diet in IBS Effect of low FODMAP Diet on total and specific bacterial abundance * P The low FODMAP diet have a marked effects on gut microbiota composition. Halmos EP,. et al. GUT 2015

46 Targeting the Intestinal Microbiota Clinical Effects FODMAP Diet in IBS Effect of fermentable carbohydrate restriction diet on intestinal microbiota Control 2 Intervention 3 P Control 2 Intervention 3 P Concentration, log 10 cells/g feces Proportion of total bacteria, % Total bacteria 9.7 ( ) 9.7 ( ) 0.52 Bacteroides- Prevotella 8.7 ( ) 8.8 ( ) ( ) 15.2 ( ) 0.72 E. rectale-c. coccoides 8.8 ( ) 8.7 ( ) ( ) 11.8 ( ) 0.27 F. prausnitzii 8.8 ( ) 8.8 ( ) ( ) 13.1 ( ) 0.16 Bifidobacteria 8.2 ( ) 7.4 ( ) < ( ) ( ) <0.001 Lactobacillus, enterococcus 7.4 ( ) 7.4 ( ) ( ) 0.6 ( ) 0.17 The low FODMAP/carbohydrate restriction diet may have long term nutritional and microbiota implications. Staudacher et al. J Nutr. 2012

47 Targeting the Intestinal Microbiota Clinical Effects Probiotics in IBS Relative risk of persistent IBS Symptoms probiotics vs. placebo Lactobacillus n=3; 140 pts Combination n=4; 302 pts Bifidobacterium n=2; 422 pts Streptococcus n=1; 54 pts Overall RR of not improving Global Symptoms Scores (GSS) 0.72 (95% CI ) Moayyedi P et al., Gut 2010

48 Targeting the Intestinal Microbiota Clinical Effects Probiotics in IBS: The currently available data, from well-designed randomized controlled clinical trials, is still limited and is not sufficient to support a general recommendation for the use of probiotics or a specific probiotic bacteria/product in patients with IBS. In view of the paucity of available treatments for IBS, the overall safety of probiotics lowers the bar for trying probiotics in patients with IBS and possibly other functional GI disorders. When choosing a product for such patients, it is recommended that providers and patients look for products that were specifically tested in IBS. Ringel Y, Ringel-Kulka T. J Clin Gastroenterol 2011 Ringel Y et al, Am J Gastroenterl 2012

49 Targeting the Intestinal Microbiota Clinical Effects Antibiotics in IBS Antibiotics can provide short-term clinical benefits in some patients with IBS The mechanisms by which antibiotics induce beneficial effect/s and the benefit vs. risk of recurrent/long term use are not yet clear. DuPont HL. AP&T 2014

50 Targeting the Intestinal Microbiota Clinical Effects Antibiotics in IBS Xifaxan can be taken orally three times a day for 14 days, for the treatment of abdominal pain and diarrhea in patients with IBS-D. Patients who experience a recurrence of symptoms can be retreated with a 14 day treatment course, up to two times.

51 The Intestinal Microbiota in Functional GI Disorders Presentation Outline: Does the intestinal microbiota play a role in the pathogenesis of FGIDs? - Suggestions from epidemiological data What do we know about the intestinal microbiota in patients with FGIDs? - Microbiology data Can the intestinal microbiota affect relevant physiological functions? - Physiology data Can the intestinal microbiota be targeted in the treatment of FGID? - Clinical data Conclusions and personal perspective

52 The Intestinal Microbiota in Functional GI Disorders Conclusions and Personal Perspective Interventions targeting the intestinal microbiota e.g., diet, probiotics and antibiotics, have a potential to: - Alter the composition (and function) of the intestinal microbiota - Affect intestinal functions that are relevant to the pathogenesis of certain GI disorders (motility, sensation, innate immunity, metabolic activity, brain and gut response to stress) - Induce beneficial clinical effect/s There is a rationale for targeting the intestinal microbiota in the treatment of FGIDs.

53 The Intestinal Microbiota in Functional GI Disorders Limitations and gaps in knowledge There is a discrepancy in the quality of the data coming from in-vitro and animal studies and the data from clinical human studies. Most of the clinical trials on interventions targeting the intestinal microbiota focused of clinical endpoints and did not include microbiota investigations. Most of the human microbiome research has focused on compositional (phylotyping) research and less on microbial functionality, mechanisms of effect and clinical relevance.

54 The Intestinal Microbiota in Functional GI Disorders Unresolved questions: Are the observed altered microbial composition a cause or a bystander? What are the mechanisms by which the altered intestinal microbiota lead to FGI symptoms and disorders? Who are the preferred targeted patients? What are the preferred, most effective and safe interventions and regimens? What is the expected magnitude of benefit? Need more high quality clinical, microbiome and mechanistic research!

55 Acknowledgements UNC-Chapel Hill Tamar Ringel-Kulka, MD, MPH Ian Carroll, PhD Michael Wu, PhD Balfour sartor, MD Olafur S Palsson, PsyD William E Whitehead, PhD Nitsan Maharshak, MD Katharine Thurlow, MS Jennica Siddle, BA Wageningen University, The Netherlands Willem de Vos, PhD University of Helsinki, Finland Jarkko Salojärvi, PhD Reetta Satokari PhD University of Nebraska Andrew K Benson, PhD INRA, Toulouse France Lionel Bueno, PhD Laurent Ferrier, PhD University of Aberdeen Karen Scott, PhD DK (YR), DK075621(YR), DK (WEW) CGIBD P30 DK34987 UL1RR025747

56 Thank you