Human breast milk miRNA, maternal probiotic supplementation and atopic

dermatitis in offsrping

Melanie Rae SimpsonPhD candidate

Department of Public Health and General PracticeNorwegian University of Science and Technology

ProPACT Study

-4 wks

Lactobacillus rhamnosus GG (LGG; 5 x 1010 CFU) Bifidobacterium animalis subsp. lactis Bb-12 (Bb-12; 5 x 1010 CFU) Lactobacillus acidophilus La-5 (La-5; 5 x 109 CFU).

Probiotic milk (n=211)

Placebo milk (n=204) Cultured milk, sterilised after culture process

n=415

Probiotics in the Prevention of Allergies among Children in Trondheim

ProPACT Study

Birth-4 wks 3 mths

Intervention period

Lactobacillus rhamnosus GG (LGG; 5 x 1010 CFU) Bifidobacterium animalis subsp. lactis Bb-12 (Bb-12; 5 x 1010 CFU) Lactobacillus acidophilus La-5 (La-5; 5 x 109 CFU).

Probiotic milk (n=211)

Placebo milk (n=204) Cultured milk, sterilised after culture process

n=415

ProPACT Study

2 yrs1 yrBirth-4 wks 6 wks 3 mths

Intervention period n=287n=415

QUESTIONNAIRES

• 6 weeks, 1 year, 2 years, (6 years)

CLINICAL EXAMINATION

• 2 and 6 years• Offered earlier with persistent itchy rash

ProPACT Study

2 yrs1 yrBirth-4 wks 6 wks 3 mths

Intervention period n=287n=415

• 40% fewer cases of atopic dermatitis at 2 years

• No effect on other allegy related diseases

ProPACT Study

2 yrs1 yrBirth-4 wks 6 wks 3 mths

Intervention period n=287n=415

BREAST MILK SAMPLES

• Collected at 10 days and 3 months post-partum

• Only 3 month samples used in this analysis

Breast milk and miRNAs• Breastfeeding may have long lasting immunological

consequences– Relationship between breastfeeding and allergy related diseases

(atopic dermatitis, asthma, allergic rhinoconjunctivitis, allergic sensitisation) is controversial

• miRNAs– Short non-coding RNAs – Approx. 22 nucleotides (nt)– Regulate gene expression at post-transcriptional level

• Human breast milk contains high concentration of miRNA– Stable under harsh conditions – Potentially active after ingestion

Objectives1. Describe miRNA profile of human breast milk

2. Investigate associations between maternal probiotic supplementation and miRNAs

3. Investigate associations between miRNAs, as mediators of the effect of probiotics, on the development of atopic dermatitis

Methods

SAMPLE SELECTION

• 54 breast milk samples

• Randomly selected

• Criteria ensured sufficient number with atopic dermatitis

Methods

SAMPLE PREPARATION

• Enriched for extracellular vesicles (Exoquick™)

• RNA isolated (Quigen miRNeasy kit)

Methods

UCAGUGCACUACAGAACUUUGU

AAGCUGCCAGUUGAAGAACUGU

SEQUENCING

• 11-28 nucleotides

• Library preparation (ScriptMinder™ Small RNA-Seq Library Prepartion Kit)

• Sequencing (Illumina HiSeq 2000)

• 50 base pair single end reads

Methods

UCAGUGCACUACAGAACUUUGU

AAGCUGCCAGUUGAAGAACUGU

hsa-miR-148a-3p

hsa-miR-22-3p

BIOINFORMATICS• Raw reads were processed, filtered• Mapped to the human genome (hg 19)• miRNAs mapped to miRBase version 20.0 • Differential expression of miRNAs using

limma (R package)• Probiotic vs placebo• Atopic dermatitis vs no atopic

dermatitis

Methods

UCAGUGCACUACAGAACUUUGU

AAGCUGCCAGUUGAAGAACUGU

hsa-miR-148a-3p

hsa-miR-22-3p

Target genes and

their functions

FUNCTIONAL PREDICTIONS

• TargetScan v7.0

• Database for Annotation, Visualisation and Integrated Discovery (DAVID) v6.7

• Enrichment Map app in Cytoscape

ResultsFifty-four (54) mother-infant pairs

• 32 from the probiotics group (11 with atopic dermatitis)

• 22 from the placebo group (18 with atopic dermatitis)

Representative of original population, except

• Higher proportion with maternal and family history of

allergy related disease

Small RNA profile

0

10

20

30

40

50

Perc

enta

ge o

f rea

ds

miRNA tRNA rRNA

Short RNAs up to approx. 1000nt

After sequencing (11-28nt):

Highly expressed miRNAs

0

50,000

100000

150000

200000

250000

CP

M m

atur

e m

iRN

As

miR

-148

a-3p

miR

-22-

3pm

iR-3

0d-5

ple

t-7b-

5pm

iR-2

00a-

3ple

t-7a-

5ple

t-7f-5

pm

iR-1

46b-

5pm

iR-2

4-3p

miR

-21-

5pm

iR-3

0a-5

pm

iR-2

9a-3

pm

iR-3

35-5

pm

iR-2

6a-5

pm

iR-4

29m

iR-3

0b-5

pm

iR-1

93a-

3pm

iR-3

75m

iR-2

7a-3

pm

iR-1

41-3

p

TOP 5

miR-148a-3p

miR-22-3p

miR-30d-5p

Let-7b-5p

miR-200a-3p

125 miRNAs 98.5% of reads6568 targets

Top 20 miRNAs 76.0% of reads3498 targets

Top 10 miRNAs 63.0% of reads

1759 targets

Top 5 miRNAs53.8%

1371 predicted targets

Functional predictions – broad range of potential functionsa) the positive and negative regulation of metabolic processes involving nitrogen

compounds, RNA, DNA and macromolecules and the positive regulation of transcription and gene expression,

b) embryonic development, c) angiogenesis, d) catabolic processes e) cell migration and localisation.

Target predictions

Enrichment Map of top 50 Gene Ontology (GO) Terms

Differential expression: Probiotic vs Placebo

miRNA Fold change p-value FDRProbiotic vs Placebo

miR-574-3p 0.640 0.016 0.818miR-340-5p 0.697 0.040 0.818let-7d-3p 1.401 0.044 0.818miR-218-5p 0.690 0.050 0.818

miRNA Fold change p-value FDRAtopic dermatitis vs no-atopic dermatitis

miR-452-5p 0.660 0.001 0.107let-7d-3p 1.615 0.005 0.308miR-146b-5p 0.674 0.011 0.433miR-21-5p 0.752 0.016 0.433miR-22-3p 1.258 0.019 0.433miR-375 1.247 0.023 0.433miR-16-5p 0.686 0.026 0.433miR-511-5p 1.323 0.028 0.433miR-26b-5p 0.808 0.041 0.461let-7f-5p 0.802 0.041 0.461miR-30e-5p 0.844 0.042 0.461miR-374a-5p 0.797 0.044 0.461miR-335-5p 1.343 0.049 0.468

Differential expression: AD vs no-AD

Conclusions• Stable group of core breast milk miRNAs

– At least partially conserved across a number of mammalian species

• Predicted functional consequences suggest a broad range of biological processes and molecular functions

• Individual miRNAs are unlikely to play a major role in the prevention of atopic dermatitis in infancy by probiotics ingested in the perinatal period.

Further research• Mediators of beneficial effect of probiotic

supplementation– Stool samples– Peripheral blood mononuclear cells (PBMC)– Bacterial swabs from mouth and vagina

• miRNA and other short RNA in breast milk– In vitro experiments and computational modelling– Paired fresh and frozen samples

Thanks• Supervisors

– Dr Torbjørn Øien– Dr Ola Storrø

• Co-authors– BioCore: Jostein Johansen and Prof Pål Sætrom– Laboratory: Dr Gaute Brede

• Participants and co-workers

• International Milk Genomics Consortium

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