Transcript Microbiome, Epigenetics and Allergy

Microbiome, Epigenetics and
Allergy
Zhengyi Deng, Yu Li
Jiwon Lee, Rui Yang
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Introduction
1
Background Introduction
2
Literature Review
3
Biological Evidence
4
Future Studies
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Introduction
Allergy Concepts
• Allergy—hypersensitivity of
immune system
• Hay fever, food allergies, atopic
dermatitis, allergic asthma,
and anaphylaxis
• Symptoms—red eyes, an itchy
rash, runny nose, shortness of
breath, or swelling
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Introduction
Allergy Mechanisms
• Three steps:
a. Sensitization and memory
induction
b. type I reaction
c. allergic inflammation
• Type I reaction:
a. Allergens bind to IgE on
mast cell
b. Cell will be triggered and
release inflammatory
substances like histamine
c. Those substances can cause
pathological changes
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Introduction
Allergy Mechanisms
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Introduction
Allergy Causes
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Multifactor causes
Genetic factors
Environmental exposure
Gene-environmental interaction
• Development factors
- Gene and environment
- Epigenetic alterations
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Introduction
Allergy Causes
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Introduction
Allergy and Microbiome
• Microbiome (microbiota): is the ecological community
of commensal, symbiotic and pathogenic microorganisms that
share our body space.
• Postnatal: microbiome facilitates the normal age-related
maturation of both Th1 and T regulatory (Treg) pathways
• Prenatal: maternal microbial transfer to the offspring begins
during pregnancy, providing a pioneer microbiome.
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Introduction
Prenatal and Postnatal Exposure
Borre. et al. (2014)
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Introduction
Environmental Factors and Epigenetics
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Prescott et al. (2011)
Introduction
Why focus on epigenetics?
• It is a possible mechanism
• Mitigate, cure or prevent allergic disease development.
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Introduction
1
Background Introduction
2
Literature Review
3
Biological Evidence
4
Future Studies
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Literature Review
• What do we know about the connection between microbiome,
epigenetic modification, and allergic diseases?
• Not so much for all three!
• Separately looking at the association between two of the three..
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Literature Review
Microbiota and Immunity
Clinical & Experimental Allergy
Volume 38, Issue 4, pages 629-633, 22 JUL 2007 DOI: 10.1111/j.1365-2222.2007.02780.x
http://onlinelibrary.wiley.com/doi/10.1111/j.1365-2222.2007.02780.x/full#f1
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Literature Review
Microbiota and Immunity
• Bisgaard 2011:
- Bacterial diversity in the early intestinal flora 1 and 12 months
after birth
- Followed for the first 6 years of life
- Allergic sensitization (serum specific IgE; skin prick test),
peripheral blood eosinophils, and allergic rhinitis.
- Inverse association
•
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Olszak 2012:
Mice study
Invariant natural killer T (iNKT) cells expression
Hypermethylation in CXCL16
We will talk more about it!
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Literature Review
Microbiota and Epigenetics
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Literature Review
Microbiota and Epigenetics
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Literature Review
Epigenetics and Immunity
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Literature Review
Epigenetics and Immunity
• Substantial epigenomic difference in asthma-susceptible
neonatal dendritic cells, by genome-wide DNA methylation
scanning (Fedulov 2009)
• Increased DNA methylation of repetitive elements Alu may be
associated with allergen sensitization (Sordillo 2013)
• DNA methylation in HLA-DR and –DQ gene region pose
significant genetic risk for peanut allergy (Hong 2015)
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Literature Review
Combination
• Review articles: recognize the impact of microbiome on
epigenetic modification, as well as the association between
epigenetics and allergic diseases
- Suggests the connection between microbiomes and immunemediated diseases
• Primary Research: rarely assess the pathway
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Introduction
1
Background Introduction
2
Literature Review
3
Biological Evidence
4
Future Studies
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Biological Evidence
Study I
Possible interactions between genome, epigenome,
transcriptome, and microbiome
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Biological Evidence
Study I Summary
• Intestinal microbiota and host physiology (inflammatory bowel
diseases)
• Toll-like receptor 2 (Tlr2): important role in mammalian gut
inflammation
• Tlr2 -/- mouse vs. WT Genome
Colonic mucosa scrapings isolated
Methylation-specific amplification microarray
Pyrosequencing
Epigenome
Expression microarray
Quantitative real-time PCR
Transcriptome
Massively parallel bacterial tag-encoded FLXTitanium amplicon pyrosequencing (bTEFAP)
Bacteria diversity data analysis
Microbiome
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Biological Evidence
Result 1—DNA Methylation Changes
Anpep: involved in final digestion of peptides. Defects in the gene
have been implicated in diseases such as leukemia, lymphoma, upper
respiratory tract infections, etc.
Ifit2: interferon-induced antiviral protein. Inhibits some viral
mRNAs.
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Biological Evidence
Result 2—Gene Expression Changes
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Biological Evidence
Result 3—Tlr2 deficiency associated with colonic
mucosal microbiomic changes.
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Biological Evidence
Study I—Significance
• First study to employ high throughput technologies testing
epigenome, transcriptome, and metagenome concomitantly in
relation to a single protein loss in mammals
• Showed changes on each level:
– Epigenomic changes: ~1.4% of colonocyte genome is
modified
– Transcriptomic changes: ~1.8% of all murine transcript
levels altered
– Metagenomic changes: significant portion of colonic mucosaassociated microbiome modified both in number and
composition
• Many molecular and cellular interactions from genotype to
phenotype
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Biological Evidence
Study II
 Relationship between microbiome exposure, epigenetics,
and health outcomes
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Biological Evidence
Study II—Summary
• Age-dependent regulation of invariant natural killer T (iNKT)
cells in relation to microbes in mouse models of inflammatory
bowel disease (IBD) and asthma
(1) Number of iNKT cells in GF and SPF mice (IBD and asthma
models)
(2) Susceptibility to disease (colitis and asthma)
(3) Whether susceptibility to disease changes when microbiota
established in adult GF vs. neonatal GF mice
(4) Epigenetic analysis of CXCL16
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CXCL16: ligand for a chemokine receptor on iNKT (increases in inflammation)
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Biological Evidence
Result 1—IBD Model, Oxazolone-Induced Colitis
Higher inflammation
(Similar results obtained for asthma model and ovalbumin-driven allergic asthma)
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Biological Evidence
Result 2—Results of microbial colonization
during early life
(Similar results for asthma model/ lymphocytes of the lungs)
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Biological Evidence
Result 3—Epigenetic modification of CXCL16
Bisulfite pyrosequencing of 5 CpG sites of CXCL16
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Biological Evidence
Study II—Significance
• CXCL16 is an age and organ-dependent microbially regulated
factor
– Modulates the quantities and function of iNKT cells in colon
and lungs
• Support for hygiene hypothesis
– Early life microbial exposure induces long-lasting effects on
iNKT cells
– In absence, exposure to stimulants may induce
autoinflammatory response.
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Introduction
1
Background Introduction
2
Literature Review
3
Biological Evidence
4
Future Studies
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Potential Study Design
Research Question & Strategy
• Research question:
Early life microbiome—DNA methylation—Later allergy
• Strategies
DNA methylation—Allergy
a) EWAS
b) Specify significant CpG
sites
CpG sites—Microbiome
a) Repeated sampling of
individuals
b) Bacteria diversity
analysis and clustering
c) Microbiome category
and methylation
differences
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Potential Study Design
Study Design
• Pilot Study
—Define the effect size and sample size
—Current study: 8 samples
• Main Study (sample collection sequence)
Infancy
Bacterial
Diversity
Fecal sample—
multiple sampling
within 1 years old
DNA
Methylation
Level
Disease
Outcome
Blood sample—
born, 1 year old
Disease at 6 years
old
Kumar. et al. 2014
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Potential Study Design
Research Question & Strategy
• Allergy—Methylation
- EWAS: specify significant CpG sites
- Manhattan plot (adjust for multiple comparison)
• Microbiome—methylation
- Bisulfate Pyrosequencing
- Model: model CpG sites=microbiome categories+covariates
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Potential Study Design
Population and Samples
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Study population:
prospective birth cohort
Allergy free but high risk (mother allergy)
No antibiotic use, no chronic lung diseases at birth
Sample Collection
Microbiome: repeated fecal samples—stable dominant bacteria
DNA methylation: blood samples
Disease Outcome
Blood IgE level and skin test
Diagnosis by pediatric allergist
Other covariates
mother’s use of antibiotics, breast feeding, pets raised at home,
etc.
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Potential Study Design
Techniques & Platforms
• Allergy Analysis
- Specific IgE levels and skin prick test
• DNA methylation analysis
- DNA methylation—allergy: Illumina Infinium
HumanMethylation450 BeadArray platform for EWAS
- DNA methylation—microbiome: bisulfite pyrosequencing
• Microbiome Composition Analysis
- 16s rRNA gene sequencing
- Dominant Bacteria or Principal Components
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Potential Study Design
Microbiota Diversity Analysis
• Study Subject
- Animals? Human Beings?
• Conduct Study
- Sample collection—(culturing)—
extract DNA—PCR and sequencing
• Microbiome analysis
- 16S rRNA gene analysis
• Categorize
- Bacteria dominant
- Clustering
Goodrich. et al. 2014
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Potential Study Design
Microbiome Statistical Analysis
• Classification and Clustering
Supervised
•Determine which taxa differ between predefined
groups of samples (subject knowledge)
•Build model and predict the classification of new
samples.
Unsupervised
•No prior knowledge
•Clustering based on abundances of specific taxa
•Cluster on human: enterotypes
• Dominant Bacteria Categorize people
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Potential Study Design
Microbiome Statistical Analysis
• Classification and Clustering
• Dominant Bacteria Categorize people
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Picture from Google
Potential Study Design
Research Question & Strategy
• Research question:
Early life microbiome—DNA methylation—Later allergy
• Strategies
DNA methylation—Allergy
a) EWAS
b) Specify significant CpG
sites
CpG sites—Microbiome
a) Repeated sampling of
individuals
b) Bacteria diversity
analysis and clustering
c) Microbiome category
and methylation
differences
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Potential Study Design
Advantages and Challenges
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Advantages
Longitudinal prospective study design
Summation scores or dominant of bacterial diversity
Study connections among all three factors
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Challenges
The biggest challenge: later life exposure influence
Hard to follow-up
Bacteria dominant: identifying all bacteria species
Principal component: hard to explain
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Potential Study Design
Summary
• Studied the association
Early life microbiome exposure—DNA methylation after
exposure—later life allergy onset
• Eliminate the inheritance factors by including only healthy
infants at the beginning
• Still challenges
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Conclusion
• Microbiome are responsible for acquired allergy symptoms
• Hygiene hypothesis
• Epigenetics may modify the influence
• Emerging area and not so much studies so far
• Calls for More Studies!!!
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Picture from Google
References
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Thavagnanam, S., J. Fleming, A. Bromley, M. D. Shields, and C. R. Cardwell. "A meta‐analysis of the association
between Caesarean section and childhood asthma." Clinical & Experimental Allergy 38, no. 4 (2008): 629-633.
Bisgaard, Hans, Nan Li, Klaus Bonnelykke, Bo Lund Krogsgaard Chawes, Thomas Skov, Georg Paludan-Müller, Jakob
Stokholm, Birgitte Smith, and Karen Angeliki Krogfelt. "Reduced diversity of the intestinal microbiota during
infancy is associated with increased risk of allergic disease at school age."Journal of Allergy and Clinical
Immunology 128, no. 3 (2011): 646-652.
Olszak, Torsten, Dingding An, Sebastian Zeissig, Miguel Pinilla Vera, Julia Richter, Andre Franke, Jonathan N.
Glickman et al. "Microbial exposure during early life has persistent effects on natural killer T cell
function." Science 336, no. 6080 (2012): 489-493.
Alenghat, Theresa, and David Artis. "Epigenomic regulation of host–microbiota interactions." Trends in
immunology 35, no. 11 (2014): 518-525.
Obata, Yuuki, Yukihiro Furusawa, and Koji Hase. "Epigenetic modifications of the immune system in health and
disease." Immunology and cell biology 93, no. 3 (2015): 226-232.
Program, C. A. M., Ober, C., Nicolae, D. L., & Mexico City Childhood Asthma Study (MCAAS. (2011). Meta-analysis
of genome-wide association studies of asthma in ethnically diverse North American populations. Nature
genetics,43(9), 887-892.
Fedulov, Alexey V., and Lester Kobzik. "Allergy risk is mediated by dendritic cells with congenital epigenetic
changes." American journal of respiratory cell and molecular biology 44, no. 3 (2011): 285-292.
Sordillo, Joanne E., Nancy E. Lange, Letizia Tarantini, Valentina Bollati, Antonella Zanobetti, David Sparrow, Pantel
Vokonas et al. "Allergen sensitization is associated with increased DNA methylation in older men."International
archives of allergy and immunology 161, no. 1 (2013): 37-43.
Hong, Xiumei, Ke Hao, Christine Ladd-Acosta, Kasper D. Hansen, Hui-Ju Tsai, Xin Liu, Xin Xu et al. "Genome-wide
association study identifies peanut allergy-specific loci and evidence of epigenetic mediation in US
children." Nature communications 6 (2015).
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