Prebiotics and food allergies

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1 Prebiotics and food allergies Cathryn Nagler, Ph.D. Committee on Immunology University of Chicago Workshop: Prebiotics Quantifying impact on host health Probiota Americas, Chicago, IL May 31, 2016

2 Oral tolerance - physiological induction of mucosal and systemic non-responsiveness to dietary antigens Why is the induction of tolerance to food failing in increasing numbers of patients with food allergies?

4 Antibiotic use Western high fat, low fiber diet Elimination of enteropathogens (H. pylori, helminths) Vaccination/reduced exposure to infectious disease Alteration of commensal microbiota dysbiosis genetically susceptible individual Inflammatory Bowel Disease Obesity Food Allergy Diabetes Autism Asthma Caesarean birth/formula feeding Feehley et al Seminars in Immunopathology 2012, 34; 671

5 The gastrointestinal microbiota changes throughout life Dominguez-Bello, MG et al, Gastroenterology 2011, 140;1713

6 A model of food allergy C3H/HeJ Sacrifice D0 D2 D7 D14 D21 D28 D35 D36 Intragastric sensitization with peanut+ cholera toxin (CT) Challenge Li et al JACI 2000, 106; 150

7 TLR4 signaling influences susceptibility to allergic responses to food Bashir et al J. Immunol. 2004, 172; 6978

8 Does neonatal administration of oral antibiotics alter the composition of the fecal microbiota? C57BL/6 mice D-7 Weaning Dilute antibiotics in drinking water Sacrifice D0 D2 D7 D14 D21 D28 D35 D36 Daily antibiotic gavage Intragastric sensitization with peanut+cholera toxin (CT) Challenge Antibiotic cocktail: kanamycin, gentamicin, colistin, metronidazole, vancomycin

9 Abundance Antibiotic treatment dramatically alters the composition of the microbiota and increases sensitization to food NT Abx NT Abx Other Phyla/Unclassified Bacteria All Members Actinobacteria All Members Proteobacteria Desulfovibrionales Others Firmicutes Others Erysipelotrichi All Members Bacilli Others Lactobacillaceae Clostridia Others Lachnospiraceae Ruminococcaceae Bacteroidetes Others Bacteroidaceae Rickinellaceae Prevotellaceae S24-7 Feces Ileal Contents

10 Abundance Log Copies 16S rrna gene / g content Antibiotic treatment dramatically alters the composition of the microbiota and increases sensitization to food NT Feces Abx NT Ileal Contents Abx Other Phyla/Unclassified Bacteria All Members Actinobacteria All Members Proteobacteria Desulfovibrionales Others Firmicutes Others Erysipelotrichi All Members Bacilli Others Lactobacillaceae Clostridia Others Lachnospiraceae Ruminococcaceae Bacteroidetes Others Bacteroidaceae Rickinellaceae Prevotellaceae S NT *** Abx Feces *** ** NT * Abx Ileal Contents

To identify allergy protective bacterial populations we selectively colonized germ free mice with representatives of the numerically predominant taxa in the murine colon 2

11 To identify allergy protective bacterial populations we selectively colonized germ free mice with representatives of the numerically predominant taxa in the murine colon 2 Bacteroides are enriched among the transient bacteria associated with digesta 1 Clostridia are resident bacteria associated with the apical mucosa Nava & Stappenbeck 2011, Gut Microbes 2; 99

12 Selective colonization of gnotobiotic mice differentially induces fecal IgA and colonic Foxp3 + Tregs Stefka, Feehley et al PNAS 2014, 111; 13145

13 The composition of the colonizing commensal microbiota directs protection against allergic responses to food Stefka, Feehley et al PNAS 2014, 111; 13145

14 Relative Expression Clostridia colonization uniquely induces expression of a subset of genes in the colonic epithelium B. uniformis-induced Common Clostridia-induced ** Germ free B. uniformis Clostridia GF B. uniformis Clostridia Cyp2c55 Tmod4 Arg2 Reg3b ProS1 Wfs1 Vegfc Fbn1 Col16a1 Gadd45a Pdk4-3 SD Row Mean +3 SD 0 Reg3b Reg3g Stefka, Feehley et al PNAS 2014, 111; 13145

15 Relative expression Il22 Expression of IL-22 is significantly increased upon Clostridia colonization 10 GF B. uniformis Clostridia ** *** 5 0 Small Intestine Colon Stefka, Feehley et al PNAS 2014, 111; 13145

16 IL-22 is a barrier protective cytokine Sabat, Ouyang & Wolk Nat Rev Drug Disc 2014, 13;21

17 Ara h 6 in serum (ng/ml) ** ** ** Clostridia-induced IL-22 is necessary to reduce intestinal permeability to food antigen C57BL/6 mice D-7 Weaning D0 D6 ELISA for Ara h 6 Daily Abx gavage Clost. or IL-22Fc Challenge Arachis hypogaea NT Abx AbxClostridia Abx+IL-22Fc Pre 1 hr 3 hr Stefka, Feehley et al PNAS 2014, 111; 13145

18 Ara h 6 in serum (ng/ml) ** ** ** Ara h 6 in serum (ng/ml) Clostridia-induced IL-22 is necessary to reduce intestinal permeability to food antigen C57BL/6 mice D-7 Weaning D0 D6 ELISA for Ara h 6 Daily Abx gavage Clostridia Isotype or αil-22 Challenge Arachis hypogaea NT Abx AbxClostridia Abx+IL-22Fc AbxClostridia+isotype PN AbxClostridia+aIL-22 PN * 0 Pre 1 hr 3 hr 0 Pre 1 hr 3 hr

19 Relative expression Il22 Ara h 6 in serum (ng/ml) Ara h 2 in serum (ng/ml) A consortium of Bacteroides+Akkermansia does not induce IL- 22 and enhances intestinal permeability to food antigen Germ free C57BL/6 mice RNA D0 D4 D6 ELISA for Ara h 6/2 Colonization with B. uniformis or consortia of Clostridia or Bacteroides Challenge Arachis hypogaea Germ free B. uniformis Bacteroides+Akkermansia Clostridia Small Intestine *** *** *** Colon Germ free B. uniformis Bacteroides+Akkermansia Clostridia * *** Pre 15 min 45 min 3000 *** Pre 15 min 45 min Bacteroides+Akkermansia consortium (Elaine Hsiao, Cal Tech) 70% Bacteroides (uniformis, thetaiotaomicron, acidifaciens), 25% A. muciniphila, 3% Parabacteroides

20 Peanut sensitization did not induce a Th2 skewed T cell or antibody response in mice treated with anti-il-22 C57BL/6 mice D-7 Weaning/ Clostridia Clostridia Sacrifice D0 D7 D14 D21 D28 D35 D36 Daily antibiotic gavage 150µg i.p. αil-22 (8E11, Genentech) or isotype control Intragastric sensitization with peanut+cholera toxin (CT) Challenge Stefka, Feehley et al PNAS 2014, 111; 13145

21 Depletion of IL-22 primed for an IL-17 response and an increase in peanut-specific IgG C57BL/6 mice D-7 Weaning/ Clostridia Clostridia Sacrifice D0 D7 D14 D21 D28 D35 D36 Daily antibiotic gavage 150µg i.p. αil-22 (8E11, Genentech) or isotype control Intragastric sensitization with peanut+cholera toxin (CT) Challenge Stefka, Feehley et al PNAS 2014, 111; 13145

22 Clostridales abundance Bacteroidales abundance Relative expression Reg3b Relative expression Reg3b Treatment with anti-il-22 decreases expression of anti-microbial peptides (RegIIIb) and increases Clostridia abundance AbxClostridia+isotype AbxClostridia+aIL-22 ** * AbxClostridia+isotype AbxClostridia+aIL-22 ** * Small Intestine Colon AbxClostridia+isotype AbxClostridia+aIL-22 Small Intestine Colon AbxClostridia+isotype AbxClostridia+aIL Days after weaning Days after weaning

23 PC2 (5.2% of var. expl.) The composition of the fecal microbiota is altered in anti-il-22 treated mice PC1 (12.7% of var. expl.) Day AbxClostridia+isotype AbxClostridia+aIL-22 Stefka, Feehley et al PNAS 2014, 111; 13145

24 What are the mechanism(s) by which Clostridia elicit a barrier protective response?

25 Commensal bacteria ferment insoluble dietary fiber to produce short chain fatty acids Berni Canani, Gilbert & Nagler, Curr. Opin. Allergy Clin Immunol. 2015, 15; 243

26 SCFA concentration (µmol/g feces) Clostridia produce high levels of the short chain fatty acids acetate and butyrate GF Conventionalized Clostridia * *** ****** * ** Acetate Propionate Butyrate

27 Relative expression Il22 Ara h 6 in serum (ng/ml) Butyrate, but not acetate, induces IL-22 expression and reduces intestinal permeability RNA Germ free C57BL/6 mice D1 D3 D4 Ara h 6 ELISA Butyrate or acetate gavage Challenge Arachis hypogaea 8 6 GF Butyrate Acetate *** *** 150 GF Butyrate Acetate Ileum Colon 0 Pre 15 min 45 min

Ara h 6 in serum (ng/ml) Abundance ** High fiber diets reduce intestinal permeability to food allergens 3 week old C3H/HeJ mice D1 D7 D14 ELISA for Ara h 2 Ad libitum feeding Wean on to diet

28 Ara h 6 in serum (ng/ml) Abundance ** High fiber diets reduce intestinal permeability to food allergens 3 week old C3H/HeJ mice D1 D7 D14 ELISA for Ara h 2 Ad libitum feeding Wean on to diet Challenge Arachis hypogaea Pre 15 min 1 hr 3 hr AIN-76A Potato Starch Wheat Bran Rice Bran Mixed Starch Fiber Blend AIN-76A Potato StarchWheat Bran Rice Bran Mixed Starch Fiber Blend Other Phyla/Unclassified Bacteria Others Actinobacteria Other Actinobacteria Proteobacteria Other proteobacteria Tenericutes Tenericutes Firmicutes Other/Unclassified Firmicutes Erysipelotrichi Erysipelotrichi Bacilli Other Bacilli Lactobacillaceae Enterococcacea Streptococeceae Clostridia Other Clostridia Lachnospiraceae Ruminococcaceae Bacteroidetes Other Bacteroidetes Bacteroidaceae Rickinellaceae Prevotellaceae S24-7 Bruce Hamaker, Whistler Center for Carbohydrate Research, Purdue

29 Tolerance to dietary allergen requires the induction of a bacteria-induced barrier protective response

30 Can microbiome-modulating therapeutics be used to treat food allergy?

Supplementation of extensively hydrolyzed casein formula (EHCF) with Lactobacillus GG accelerates acquisition of tolerance in children with

and APT Oral food challenge (OFC) Full clinical evaluation Full clinical evaluation SPT and APT Oral food challenge (OFC) 100 80 P<0.001 P<0.

31 Supplementation of extensively hydrolyzed casein formula (EHCF) with Lactobacillus GG accelerates acquisition of tolerance in children with cow s milk allergy % of children acquiring tolerance after 12 months Visit 1 Visit 2 6 months Visit 3 12 months Full clinical evaluation SPT and APT Oral food challenge (OFC) Full clinical evaluation Full clinical evaluation SPT and APT Oral food challenge (OFC) P<0.001 P<0.001 P<0.001 P< EHCF EHCF + LGG RHF SF AAF casein rice soy amino acids Berni Canani R, et al. The Journal of Pediatrics 2013, 163; 771

32 The cow s milk allergic (CMA) infant microbiome exhibits significantly increased diversity Berni Canani, Sangwan, Stefka et al ISMEJ 2016, 10; 742

33 The composition of the fecal microbiota is altered in cow s milk allergic (CMA) infants Berni Canani, Sangwan, Stefka et al ISMEJ 2016, 10; 742

34 Demographic variables do not explain differences in bacterial abundance Age of Solid Food Introduction Lachnospiraceae Bodyweight Exclusive Breastfeeding Period Age at Sampling * Allergy Mode of Birth Sex Berni Canani, Sangwan, Stefka et al ISMEJ 2016, 10; 742

35 Treatment with EHCF plus LGG is associated with increased levels of fecal butyric acid and enhanced tolerance to CMA Tolerance to CMA at 12 months EHCF 0/7 EHCF + LGG 5/12* Berni Canani, Sangwan, Stefka et al ISMEJ 2016, 10; 742

36 Genus level differential feature selection revealed a relationship between the abundance of bacterial strains, increased fetal butyrate concentation and acquisition of tolerance Berni Canani, Sangwan, Stefka et al ISMEJ 2016, 10; 742

37 food IL-22 M. Velasquez-Manoff, Nature 2015, 518: S4

38 Is sensitization to a food allergen increased in mice colonized with an allergic infant microbiota? Healthy infant microbiota Non-allergic Sensitize with b lactoglobulin (BLG)/CT Cow s milk allergic (CMA) infant microbiota Allergic

39 DT C BLG-specific IgE (ng/ml) A humanized pre-clinical food allergy model to screen microbiome-modulating therapeutics 0 Healthy Colonized Allergic Colonized n=6 n=7 40 * = * * * * * * Time (min.) Healthy CMA 50mg BLG gavage 50mg BLG gavage

40 A humanized pre-clinical food allergy model to screen microbiome-modulating therapeutics Candidate therapeutic bacterial species (or mixtures) Healthy infant microbiota Cow s milk allergic (CMA) infant microbiota Sensitize with b-lactoglobulin/ct Non-allergic Pre-biotic dietary fiber (to expand butyrate producing Clostridia) Encapsulated nanoformulations of butyrate Allergic

University of Chicago Yang-Xin Fu Aly Khan Digestive Diseases Research Center Eugene Chang Betty Theriault Dion Antonopoulos Institute for Molecular Engineering Jeff Hubbell

Andrew Stefka Rosita Aitoro Diana Bouhassira Prabhanshu Tripathi Goo-young Seo Severine Cao Stefano Guandalini Roberto Berni Canani & team University of Naples Liang Zhou

41 University of Chicago Yang-Xin Fu Aly Khan Digestive Diseases Research Center Eugene Chang Betty Theriault Dion Antonopoulos Institute for Molecular Engineering Jeff Hubbell Argonne National Laboratories Jack Gilbert Naseer Sangwan Acknowledgments Taylor Feehley Vince Guzzetta Caitlin Yee Emily Beltran Songhua Quan Pedro Belda-Ferre Cat Plunkett Andrew Stefka Rosita Aitoro Diana Bouhassira Prabhanshu Tripathi Goo-young Seo Severine Cao Stefano Guandalini Roberto Berni Canani & team University of Naples Liang Zhou (Northwestern) Wenjun Ouyang (Genentech) Bruce Hamaker Bin Zhang (Purdue/Whistler Center for Carbohydrate Research) Sarkis Mazmanian (Cal Tech) Elaine Hsiao (Cal Tech) Kathy McCoy (University of Bern)

Hygiene Hypothesis: 10 years later. Christina Ciaccio MD, MSc Assistant Professor The University of Chicago

Hygiene Hypothesis: 10 years later Christina Ciaccio MD, MSc Assistant Professor The University of Chicago none Disclosures Definitions Microbe: Microscopic organism Microbiota: Microbial community Definition