INFANT GUT MICROBIAL MARKERS OF FOOD SENSITIZATION AT AGE 1

Transcription

1 INFANT GUT MICROBIAL MARKERS OF FOOD SENSITIZATION AT AGE 1 Anita Kozyrskyj, PhD, Professor Dept Pediatrics, Faculty of Medicine & Dentistry School of Public Health, University of Alberta kozyrsky@ualberta.ca Committee on Food Allergies Workshop, Aug 2015

2 The Microbial Deprivation Hypothesis Early-Life Infant Microbial Exposures Pregnancy & Birth (maternal microbes) Infant Diet: breast milk (microbes & promoters) Antibiotics (birth, postnatal) (selective microbe killing) Pre/Probiotics (microbe enrichment) Environment (microbes from pets, siblings, house dust, daycare ) Gut Microbiota Immune System Tolerance, Gut Permeability Allergic (food, ezcema) & Metabolic Disorders

3 CIHR Canadian Microbiome Initiative SyMBIOTA (Synergy in Microbiota) research program was one of the 7 Canadian Microbiome Initiative team grants funded ($2.5 million over 5 yrs) The Emerging Team Grant: Canadian Microbiome Initiative was launched in 2010 to enable Canadian researchers to join others on the international scene, including their US colleagues working with the Human Microbiome Project and the International Human Microbiome Consortium

4 Prepared by Meghan Azad, postdoctoral fellow Meconium, 3 mo, 1 yr Using metadata and infant fecal samples from the Canadian Healthy Infant Longitudinal cohort ( MiSeq

5 Canadian Healthy Infant Longitudinal Development (CHILD) study: n=3, GENERAL POPULATION MULTI-ETHNIC COHORT OF WELL CHARACTERIZED PREGNANCIES, BIRTHS & EARLY LIFE EXPOSURES FOR FULL-TERM INFANTS SEEN AT 3 MO, 1 YR, 3 YR, 5 YR PI, MALCOLM SEARS, McMASTER UNIVERSITY The Indoor Air Exposures, Genes, and Gene-Environment Interactions in the etiology of Asthma and Allergy in Early Childhood partnership is led by CIHR- IHDCYH, in partnership with AllerGen NCE, The Canada Mortgage and Housing Corporation, Environment Canada, Health Canada, the Healthy Indoors Partnership, Natural Resources Canada, the National Research Council and four other CIHR institutes..

6 Canadian Healthy Infant Longitudinal Development Study Exposures Pollution Home environment Work & School environment Stress Genetics Immune phenotypes Clinical phenotypes Infant Pulmonary Function Microbiome Viruses Diet SES Pets

7 CHILD a 6-year study of 3,300 children In-utero Delivery - recruitment: maternal, paternal studies; clinical, stress, nutrition and environment questionnaires - delivery: outcomes, cord blood, meconium 3 months - home visit: health questionnaires, home inspection, dust lung sampling, breast-milk, urine, nasal swab, stool, infant Moraes TJ et al. The Canadian Healthy Infant Longitudinal Development Birth Cohort Study: Biological function, Samples stress and (sub-cohorts) Biobanking. Paediatr Perinatal Epidemiol : months - questionnaire follow-up 1 year - clinic: skin tests, blood, lung function, infections, urine, nasal swab, stool; maternal studies 1 ½ years - questionnaire follow-up Subbarao PJ et al. Canadian Healthy Infant Longitudinal Development (CHILD) Study: examining developmental origins of allergy and asthma. Thorax 2015 Jun years - questionnaire follow-up 2 ½ years - questionnaire follow-up 3 years - clinic: questionnaires, clinical assessment, skin tests, lung function, urine 4 years - questionnaire follow-up 5 years - clinic: questionnaires, clinical assessment, skin tests, lung function, blood, physician assessment

8 Feeding and Delivery Mode on Microbiota in 24 infants Insert SyMBIOTA findings Azad et al. CMAJ 2013;185

9 INFANT GUT DYSBIOSIS, EARLY LIFE EXPOSURES & FOOD SENSITIZATION Azad et al. Clin Exp Allergy 2015;45:632-43

10 Context of research Food sensitization is not a good indicator for food allergy in infants. In the CHILD cohort, food allergy was diagnosed in: 11% of food sensitized infants at age 1 Many infants outgrow their food allergy BUT food-sensitized infants are: twice as likely to experience the atopic march

11 Methods: Microbiota profiling Fecal samples at 3 months & 1 year from166 full-term infants in Winnipeg with MiSeq at V4 of 16S rdna Sequences were clustered at 97% sequence similarity against the Greengenes database (closed-picking) Operational taxonomic units (OTUs) with relative abundance below were excluded 3.1x105 reads per sample representing 1127 OTUs Data were rarefied to 80,000 sequences per sample OTU relative abundance and diversity indices generated in QIIME

12 Operational Taxonomic Unit (OTU) x80million Streptococcus Escherichia OTU 1 Naser et al., 2007 Lachnospiraceae (Family) OTU 2 Unknown: OTU34 12

13 Food sensitization type (7%, n=12) Food allergen peanut 27% egg 60% milk 13%

14 Gut microbes (OTU) longitudinal changes by age, Principle Coordinates Analysis (M. Azad)

15 Species richness was significantly lower at 3 months but not 1 year in sensitized infants Chao1 species richness at 3 months p< Sensitized- 3 mo (n=12) Not sensitized- 3 mo (n=154) Sensitized- 1 yr (n=12) Not sensitized- 1 yr (n=154) Shannon diversity Chao1 richness

16 Taxonomy 101 (Biological Classification) Human E. coli C. difficile Kingdom Animalia Bacteria Bacteria Phylum Chordata Proteobacteria Firmicutes Class Mammalia Gammaproteobacteria Clostridia Order Primates Enterobacteriales Clostridiales Family Hominidae Enterobacteriaceae Clostridiaceae Genus Homo Escherichia Clostridium Species Homo sapiens Escherichia coli Clostridium difficile

17 Relative abundance of phyla at 3 months and 1 year: Lower Bacteroidetes & higher Proteobacteria

18 Relative abundance (family level) at age 3 mo & 1 yr: Lower Bacteroidacea & higher Enterobacteriaceae

19 FOOD SENSITIZATION & INFANT GUT DYSBIOSIS; WHAT CAME FIRST? Incident analyses

20 Infants with food allergy diagnosis (with testing) prior to 1 year of age excluded from the analysis

21 LOW MICROBIOTA DIVERSITY DOES NOT ALWAYS INDICATE AN ADVERSE OUTCOME Critical windows during infancy

22 Role of breastfeeding in modifying gut microbiota (M. Azad) 3 months 12 months Lower diversity is expected with breastfeeding Shannon Diversity: Mean 2.08 (Range ) Shannon Diversity: Mean 2.75 (Range )

23 FOOD SENSITIZATION & GUT DYSBIOSIS EXPLAINED BY OTHER FACTORS? Model adjustment

24 Lower species richness among sensitized infants at 3 months but not at 1 year; trend for lower diversity Breastfed, vaginally-delivered infants not exposed to antibiotics by 3 months

25 Lower Bacteroidacea/higher Enterobacteriaceae at 3 mo & 1 yr sensitized infants (vaginal birth, breastfed, no antibiotics)

26 FOOD SENSITIZATION & INFANT GUT MICROBIOTA INTERACTIONS Mutual adjustment

27 E/B ratio and low species richness associations with sensitization are independent of each other Likelihood of Sensitization at 1 Year

28 Comparison of findings to the human and animal literature Nguyen, T.L.A.; Vieira-Silva, S.; Liston, A.; Raes, J. How informative is the mouse for human gut microbiota research? Disease Models Mech. 2014, 8, Food sensitization and infant gut microbiota changes Most Escherichia associations are with atopic dermatitis Higher Clostridium coccoides at 6 months in confirmed allergy to cow s milk (Thompson-Chagoyan 2011;156) Higher Firmicutes & Escherichia/lower Bacteroidetes in 5-month old Chinese infants with confirmed food allergy (Ling 2014;80) Food allergy & infant antibiotic exposure (Molloy 2013;10) Highest risk with maternal intrapartum & infant cephalosporin use in prescription database study (Metsala 2013;24) In rodent models (Morin 2012;56; Schwab 2014;8) altered gut immune responses when microbiota colonization delayed; gut microbiota changes precede inflammation

29 Conclusions & Future Directions Lower species richness predicted food sensitization that could not be explained by: Taxon relative abundance (E/B ratio) findings Birth mode, breastfeeding status and antibiotic exposure HOWEVER, diversity measures do not take into account which microbes are present Higher E/B ratio at 3 months & 1 years was a marker for food sensitization: Presence before outcome indicates suitable predictor?

30 What can be done to prevent allergy Achievement of a microbiota profile later in infancy that reverses the E/B ratio may be helpful in preventing allergic disease, According to Dogra et al. (2015;6), 6-month old infants with low fecal abundance of Proteobacteria and Streptococcus but high % of bifidobacteria were less like to have high adiposity at 18 months O Donovan et al 2015;epub show that infant adiposity is a strong risk factor for atopic dermatitis

31 ROLE OF MATERNAL INTRAPARTUM & INFANT ANTIBIOTICS IN FOOD ALLERGY Early antibiotics cause NB dysbiosis

32 IV antibiotic use during delivery for CS or GBS prophylaxis (39%), % 2% Positive vaginal culture group B Streptococcus Cesarean Delivery (1 dose) 18% Cefazolin/cefoxitin Penicillin G Ampicillin Clindamycin 17% Persaud RR, Azad MB, Chari RS, Sears MR, Becker AB, Kozyrskyj AL et al. Perinatal antibiotic exposure of neonates in Canada and associated risk factors: a population-based study. J Matern Fetal Neonat Med 2014: 1-6.

33 Bacteroidaceae abundance is lower and Clostridiaceae is higher with IAP (penicillin G) in vaginal delivery versus no IAP Relative abundance at the phylum, order and family level at infant age 3 months Dominant Taxa IAP Method of Birth no IAP Vaginal Microbiota at 3 months (N=176) IAP Vaginal IAP Elective CS IAP Emergency CS (% BF at sample collection) (82% BF) (83% BF) (78% BF) (80% BF) N=96 N=40 N=17 N=23 Bacteroidetes * 0.4 *** 0.2 *** Bacteroidaceae * 0.3 ** 0.2 *** Firmicutes ** 52.1 *** Clostridiales * 49.3 *** Clostridiaceae * ** Veillonellaceae *** Lachnospiraceae Ruminococcaceae ~ 0.1 Proteobacteria ~ * Enterobacteriaceae ~ * Actinobacteria Bifidobacteriaceae Verrucomicrobia Azad et al. Impact of maternal intrapartum antibiotics, method of birth and breastfeeding on gut microbiota during the first year of life. BJOG 2015 in press

34 Greatest difference in OTU abundance with IAP is with emergency CS wrt significance level and families affected Relative abundance at the phylum, order and family level at infant age 3 months Dominant Taxa IAP Method of Birth no IAP Vaginal Microbiota at 3 months (N=176) IAP Vaginal IAP Elective CS IAP Emergency CS (% BF at sample collection) (82% BF) (83% BF) (78% BF) (80% BF) N=96 N=40 N=17 N=23 Bacteroidetes * 0.4 *** 0.2 *** Bacteroidaceae * 0.3 ** 0.2 *** Firmicutes ** 52.1 *** Clostridiales * 49.3 *** Clostridiaceae * ** Veillonellaceae *** Lachnospiraceae Ruminococcaceae ~ 0.1 Proteobacteria ~ * Enterobacteriaceae ~ * Actinobacteria Bifidobacteriaceae Verrucomicrobia Azad et al. Impact of maternal intrapartum antibiotics, method of birth and breastfeeding on gut microbiota during the first year of life. BJOG 2015 in press

35 Within the Firmicutes, abundance of Enterococcus, Clostridium and Veillonella is higher with IAP (cefazolin) in emergency CS compared to vaginal delivery without IAP IAP, Emergency CS (vs. no IAP) no IAP IAP, Emergency CS n=96 n=23 p FDR p Bacteroides (100) (100) <0.001 <0.001 *** Bacteroides Enterococcus (97) (100) <0.001 <0.001 *** Prevotella Veillonella (100) (100) <0.001 <0.001 *** Ruminococc Clostridium (100) (100) * Clostridiacea Atopobium (67) (91) * Lactobacillal Parabacteroides (100) (100) ~ Ruminococc Streptococcus (100) (100) Pasteurellac Clostridiales uncl (52) (74) Blautia Collinsella (90) (83) Streptococc Streptococcaceae uncl (79) (87) Lachnospira Relative abundance at 3 months (% fecal samples colonized)

36 Greater impact of timing of antibiotic exposure than number of courses

37 IMMUNOGLOBULIN A (IGA), ORAL TOLERANCE AND FOOD ALLERGY

38 Immunoglobulin A siga = first line of defense and maintains mutualism with gut microbiota Secretory IgA a class of antibodies critical in mucosal immunity Newborn infants unable to produce siga until ~ 1 month Marker for immune maturation in infants

39 Role of gut siga in food allergy.. immune exclusion is performed by SIgA to control epithelial colonization with microbes, inflammation and inhibit the penetration of food antigens number of intestinal IgA+ plasma cells is increased after exposure of germ-free mice to conventional gut microbiota. Bacteroides and Escherichia coli strains are particularly immuno-stimulatory but lactic-acidproducing bacteria also contribute.

40 Ig A c o n c e n tra tio n ( g /g to ta l p ro te in ) Ig A c o n c e n tra tio n ( g /g to ta l p ro te in ) Breastfeeding, Fecal IgA & Pets (Bridgman et al. J Dev Orig Health Dis 2015 in press) Extent of breastfeeding is correlated with fecal IgA levels Evidence of effect modification by pets (p=0.04 for interaction) 8 0 p = Microbial stimulation p = E xc lus ive (n= 2 1 ) P a rtia l (n= 1 3 ) N o ne (n= 1 2 ) 0 B F (n= 1 8 ) N o t B F (n= 1 0 ) B F (n= 1 7 ) N o t B F (n= 2 ) B re a s tfe e d in g s ta tu s a t 3 m o n th s N=46. Comparison by Spearman correlation. E x p o s u r e to p e ts N o e x p o s u re to p e ts N=47. BF, any breastfeeding (exclusive or partial) at 3 months. Comparison by Mann Whitney Test.

41 Infant fecal IgA & C. difficile, marker of colonization resistance in infants & mediates association between cesarean section and development of atopic dermatitis % colonized with C. difficile % colonized with C. difficile C. difficile colonization negatively associated with extent of breastfeeding and fecal IgA Reduced likelihood of colonization independent of breastfeeding, parity and birth mode p< Exclusive Partial None Breastfeeding status at 3 months Yes p=0.03 High IgA Unadjusted OR (95% CI) Mutually adjusted aor (95% CI) Mutual adjustment with addition of parity and birth mode aor (95% CI) High IgA 0.25 ( )* 0.36 ( ) 0.17 ( ) Breastfed at 3 months 0.15 ( )* 0.20 ( ) 0.10 ( ) 64.5 No Multivariate logistic regression; OR, odds ratio; CI, confidence interval. IgA in highest tertile.

42 Microbiota production of short-chain fatty acids such as acetate, butyrate Acetate levels negatively correlated with Clostridium abundance and positively correlated with abundance of Enterobacteriaceae; isobutyrate was correlated with Bacteroidetes

43 AND Mom matters Kozyrskyj AL et al. Fetal programming of overweight through the microbiome: boys are disproportionately affected. J Dev Orig Health Dis A. Potential mechanisms programming fetal/infant gut microbiota Maternal gut and vaginal microbiota dybiosis Leaky gut and/or placenta Vertical transmission of pathogenic bacteria Epigenetic effects of microbial metabolites Infant sex, ethnicity Male-female Caucasian-other Altered Gut Microbiota & Immunity Bacteroides Enterobacteria Staphylococcus Bifidobacterium Lactobacillus Fecal IgA Prenatal diet and health Birth mode Vaginal Cesarean Antibiotics Maternal Infant Infant diet Breastfeeding status Breast milk microbiota Childhood Overweight & Atopy B. Postnatal factors influencing infant gut microbiota Koleva PT et al. The infant gut microbiome: evidence for obesity risk and dietary intervention. Nutrients 2015;7:

44 SyMBIOTA (Synergy in Microbiota) PIs: Anita Kozyrskyj & James Scott MICROBIOTA THEME 1: James Scott Tedd Konya, David Guttman, Sylva Donaldson (U of Toronto); Dean Befus (U of Alberta) Antibiotic use Cesarean birth Infant diet EXPOSURE THEME 2: Anita Kozyrskyj Catherine Field, Radha Chari, Meghan Azad, Sarah Bridgman (University of Alberta) OUTCOMES THEME 3: Piush Mandhane Allan Becker, Kent HayGlass, Stuart Turvey (Universities of Alberta & Manitoba, UBC) ETHICS THEME 4: Sarah Bridgman Maternal survey on probiotic product knowledge

45 Acknowledgements Lead: Anita Kozyrskyj: Data & Sample Collection CHILD Study Staff & Families Project Management Sarah Bridgman, Sylva Donaldson DNA Extraction, Sequencing & Bioinformatics (Toronto) Co-PI James Scott, Tedd Konya, David Guttman Trainees & Research Assistants Petya Koleva, Meghan Azad, Rose Kalu, Farzana Yasmin Funding (Postdoctoral Fellowships) CIHR, Banting, AllerGen NCE, Alberta Innovates, Parker B. Francis

46 Acknowledgements Lead: Anita Kozyrskyj: Petya Koleva Rose Kalu Meghan Azad Farzana Yasmin Angela Chow Sarah Bridgman ECHA Rm 5-063