human milk oligosaccharides: benefits for the breastfed

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COMMUNITIES AND HOSPITALS ADVANCING MATERNITY PRACTICES

Wednesday Webinar: February 2019

Human Milk Oligosaccharides: Benefits for the breastfed infant and beyond

Presented byDr. Lars Bode, Associate Professor of Pediatrics

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COMMUNITIES AND HOSPITALS ADVANCING MATERNITY PRACTICES

Upcoming CHAMPS 4-Hour Clinical SkillsTrainings

• (4- hour) Anderson Regional Medical Center• Wednesday, February 20th, 2019 (morning session)

• Trainings are open to all CHAMPS hospitals and CHAMPS community partners. You can register for the trainings at CHEERequity.org/trainings

2/14/19

Human Milk OligosaccharidesBenef i ts for the breast fed infant and beyond

Lars Bode, PhD

L a r s s o n - R o se n q u i s t F o u n d a t i o nM o t h er - M i l k - I n f a n t C e n t e r o f R e s e a r c h E x c e l l e n c e

U n i v e r s i t y o f C a l i f o r n ia , S a n D i e g o

DisclosuresResearch in the Bode lab and the UC San Diego Mother-Milk-Infant Center of Research Excellence (LRF MOMI CORE) is currently supported by

• National Institutes of Health (NIH)• National Science Foundation (NSF)• Bill & Melinda Gates Foundation (BMGF)• Family Larsson-Rosenquist Foundation (FLRF)• Gerber Foundation (PI: Dr. Goran)

Dr. Bode receives travel reimbursements from Universities, foundations, and societies to speak at national and international grand rounds and conferences.

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DisclosuresNone of the work would be possible without amazing lab members as well as collaborators from across the US and around the world!

The Power of Human Milk

Saving Lives

“The deaths of 823,000 children and 20,000 motherseach year could be averted through universal breastfeeding, along with economic savings of US$300 billion.”

Editorial Commentary, The Lancet - Breastfeeding Series 2016

WHY?HOW?

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Fat

Protein

Lactose

Oligosaccharides

12

35

65

5-15 !

35

35

450.05

[g/L]

Macromolecules in Human Milk

What areHuman Milk Oligosaccharides

(HMOs)?

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≠

Glucose (Glc)

Galactose (Gal) N-acetylneuraminic acid (Neu5Ac) sialic acid

N-acetyl-glucosamine Fucose (Fuc) (GlcNAc)

Human Milk Oligosaccharides (HMOs) are Complex Sugars

β1-6

n=0-15

β1-3

β1-4

β1-3 β1-4

α1-2α1-3α1-4

α2-3α2-6

lactoselacto-N-biose

N-acetyllactosamineβ1-4 β1-4

α1-2 α1-3

2’-fucosyllactose (2’FL)

β1-4

3-fucosyllactose (3FL)

α2-3α2-6 β1-4

3’-sialyllactose (3’SL)


β1-4

β1-3

β1-3

Lacto-N-tetraose (LNT) Lacto-N-neotetraose(LNnT) (type 1 chain) (type 2 chain)

β1-4

β1-3β1-4 β1-4

β1-3

β1-3

β1-4

β1-6

Lacto-N-hexaose(LNH)

β1-4

β1-3

β1-3

β1-4

β1-6

β1-3

iso-Lacto-N-octaose

β1-4

β1-3β1-4

β1-3

para-Lacto-N-octaose

β1-3

β1-3

β1-4

β1-3

β1-4

β1-3

β1-3

β1-4

β1-6β1-4 β1-3

iso-Lacto-N-neooctaose

α1-2

Lacto-N-fucopentaoseI (LNFP I)

β1-4

β1-3

β1-3

β1-4

β1-3

β1-3

Lacto-N-fucopentaoseII (LNFP II)

α1-4

β1-4

β1-3

α1-3

Lacto-N-fucopentaoseIII (LNFP III)

β1-4

β1-4

β1-3

β1-3

Lacto-N-fucopentaoseV (LNFP V)

α1-3

Glucose (Glc) Galactose (Gal) N-acetylglucosamine (GlcNAc) Fucose (Fuc) N-acetylneuraminic acid (NeuAc)

α2-3

β1-4

β1-3

β1-3

LS-Tetrasaccharide a (LST a)

α2-6

β1-4

β1-3

β1-3

LS-Tetrasaccharide b (LST b)

α2-6β1-3

β1-4

β1-4

LS-Tetrasaccharide c (LST c)

α2-6

β1-4

β1-3

β1-3

α2-3

Disialyllacto-N-tetraose (DSLNT)

150 – 200 different HMO

HMO Blueprint and Structural Diversity

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A) Absolute HMO concentration (nmol/mL)

Personalized complex HMO composition(HMO ”fingerprint”)

preliminary data from CHILD study cohort (in collaboration with Dr. Meghan Azad)

n=1,206

B) Relative HMO composition (% of total HMOs)

Personalized complex HMO composition(HMO ”fingerprint”)

preliminary data from CHILD study cohort (in collaboration with Dr. Meghan Azad)

n=1,206

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Human milk oligosaccharides (HMO)

! 1 - 3 ! 1-4

! 1 -6

n=0-15

! 1-3

! 1-4″ 1-2/3/4

″ 2-3/6n=1-15

Glucose (Glc)

Galactose (Gal) N-acetylneuraminic acid (Neu5Ac) sialic acid

N-acetyl-glucosamine Fucose (Fuc) (GlcNAc)

!!! #

!

#$% !$%

Oligosaccharides currently added to infant formula

…are structurally distinct from HMOs

HMO MetabolismHMO

resistant to• low pH• pancreatic and

brush border enzymes

small intestine:>1% absorbed,appear in the urine

colon:bacterial degradation,10-80% excreted w/ feces

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SummaryWhat are HMOs?

• third most abundant component in human milk

• composition follows basic blueprint

• more than 150 different HMOs identified

• inter- and intrapersonal differences in HMO composition

• infant formula oligosaccharides (GOS/FOS) structurally (and functionally) different from HMOs

• reach distal GI tract intact, but are also absorbed and reach systemic circulation and organs other than the gut

Which maternal factors contribute to variation in

Human Milk Oligosaccharides (HMOs) composition?

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Maternal Drivers

Genetics

>5,000 milk samples

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Study nCHILD 1,143

STEPS 803

UCSD Biorep 609

Botswana 498

MACS 477

INSPIRE 411

India 298

Uganda 126

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>5,000 milk samples

~95% explained by one SNP

GWAS reveals genes involved inHMO biosynthesis

CHILD cohort data - In collaboration with Dr. Meghan Azad (Manitoba), Dr. Qingling Duan, Le Chang (Ontario)

2’FL

-Log

10(p

)

Chromosomes

Chromosome 19FUT2

10

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Study nCHILD 1,143

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α1-4 β1-R

Lewis b (Leb)

Structural Diversity and GeneticsGroup 1: Lewis-positive Secretor (Se+Le+)Lewis a-b+

β1-R FUT2 β1-R FUT3

Group 2: Lewis-positive Nonsecretor (Se-Le+)Lewis a+b-

α1-4 β1-Rβ1-R F✗UT2

FUT3

Lewis a (Lea)

Group 3: Lewis-negative Secretor (Se+Le-)Lewis a-b-

β1-R FUT2 β1-RF✗UT3

Group 4: Lewis-negative Nonsecretor (Se-Le-)Lewis a-b-

β1-R F✗UT2 F✗UT3

inte

nsity

[mV

]

Secretor Non-Secretor

α1-2

(2) 2’-fucosyllactose

α1-2

(7) Lacto-N-fucopentaose I

Structural Diversity and Genetics

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2’FL LNFP1FUT2_rs601338 (“Caucasian SNP”)

wt het homo

α1-2β1-3

α1-2

P<0.0001 P<0.000115,000 4,000

3,00010,000

2,0005,000

1,000

0 00 1 2 0 1 2

LNFP2 LNFP3

2’FL

[nm

ol/m

L]

β1-4β1-4

β1-3

LNFP

1[n

mol

/mL]

β1-4 β1-4

α1-4 β1-4β1-3

β1-3

0 1 2 0 1 20

2,000

4,000

6,000 300

LNFP

2[n

mol

/mL]

P<0.0001

β1-3

α1-3

0

100

200

LNFP

3[n

mol

/mL]

P<0.0001


FUT2_rs601338 (“Caucasian SNP”)

wt het homo

0 1 2

1,000

0

3,0002,000

2,000

4,000

LNT

[nm

ol/m

L]

β1-4

β1-3

β1-3

P<0.0001

LNT LNnT

0 1 20

1,000

3,000

LNnT

[nm

ol/m

L]

β1-4

β1-3β1-4

P<0.04

400 400

300 300

200 200

100 100

0 00 1 2 0 1 2

LSTb

[nm

ol/m

L]

α2-6

β1-4

β1-3

β1-3

P<0.0001 P<0.0001

LSTb

LSTc

[nm

ol/m

L]

α2-6β1-3

β1-4

β1-4LSTc

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FUT2_rs601338 (“Caucasian SNP”)

wt het homo

HMO sum

P<0.000130,000

20,000

10,000

00 1 2

HMO-bound FucP<0.0001

SUM

[nm

ol/m

L]

0 00 1 2 0 1 2

25,000 6,000

20,0004,000

15,000

10,0002,000

5,000

HM

O-b

ound

Fuc

[nm

ol/m

L]

HM

O-b

ound

Sia

[nm

ol/m

L]

HMO-bound SiaP<0.0001

What’s normal?Oligosaccharide concentrat ions and prof i les in milk

produced by healthy women vary geographical ly

Secretor [%]

McGuire M et al. Am J Clin Nutr 2017;105:1086–100

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Canadian CHILD cohort: (n=427)

Asian: 60% SecretorsCaucasian: 74% Secretors First Nation: 76% Secretors

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Maternal Drivers

Genetics• Secretor/Lewis• other?genomics, transcriptomics, HMO-mics• in silico pathway modeling• validation through in vitro enzyme

characterization and manipulation

Environment• diet

Diet and exercise alter mouse milk oligosaccharide concentration (3’-sialyllactose, 3’SL)

2,500

2,000

1,500

500

0

1,000

3'SL

[ug/

mL]

n=8

** ***

less sialylated HMO on high fat diet

In collaboration with Dr. Kristin Stanford, Ohio State University

Maternal Drivers of HMO Composition

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0

5000

10000

15000

20000

0

1000

2000

3000

4000

Conc

entr

atio

n(n

mol

/mL)

Conc

entr

atio

n(n

mol

/mL)

Sialylated HMOs Fucosylated HMOs


Maternal diet alters HMO composition

less sialylated HMO on high fat dietIn collaboration with Dr. Morey Haymond, Dr. Mahmoud Mohammad,

and Dr. Kjersti Aagaard, Baylor College of Medicine

Maternal Drivers of HMO CompositionMaternal use of probiotics during late pregnancy is associated with variation in HMO composition

In collaboration with Dr. Antti Seppo and Dr. Kirsi Järvinen-Seppo, University of Rochester

Seppo A et al. JAMA Pediatrics (In Press)

n.s. p = 0.0*

3

carb high fat glucose galactose

p = 0.02*

n.s.

carb high fat glucose galactose

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Maternal Drivers of HMO CompositionMaternal use of probiotics during late pregnancy is associated with variation in HMO composition

In collaboration with Dr. Antti Seppo and Dr. Kirsi Järvinen-Seppo, University of Rochester

Seppo A et al. JAMA Pediatrics (In Press)

Maternal Drivers



Environment• diet • supplements••

lifestyle (e.g. physical activity) exposures (e.g. smoking, drugs, etc.)

Time• lactation stage

Health / Disease

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Azad MB et al. J Nutr 2018;148:1–10

Maternal Drivers



Environment• diet • supplements••

lifestyle (e.g. physical activity) exposures (e.g. smoking, drugs, etc.)

Time• lactation stage• parityHealth / Disease

• season

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Azad MB et al. J Nutr 2018;148:1–10

Maternal Drivers



Environment• diet • supplements• lifestyle (e.g. physical activity)• exposures (e.g. smoking, drugs, etc.)

Time

Health / Disease• lactation stage• parity• age

• season

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Maternal Drivers and Infant Outcomes

gut microbiome necrotizing enterocolitis

diarrhea

HIV transmission

respiratory infections

gut-brain axisviral and bacterial infections asthma

brain development

cognitionIQ

food allergies

body composition

obesitycancer

lung developmentchronic inflammatory diseases

metabolic diseases

sepsis

What areHuman Milk Oligosaccharides

(HMOs) doing?

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HMO at the Interface of Host-Microbe Interactions

prebiotics

prebiotics

Shaping microbial community development

• structure-specific effects of HMO ex vivo (infant feces)

Dr. Karsten ZenglerUCSD

β1-4

β1-3 α1-2

β1-3

Lacto-N-tetraose (LNT) 2’-fucosyllactose(type 1 chain) (2’FL)

β1-4 β1-4

α2-3


!! !

#

!

#$% !$%

HMO as Prebiotics

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HMO as Prebiotics

prebiotics

• prebiotic effects of HMOs are structure-specific !

• different HMOs have different effects on microbial communities

• mixture of HMOs vs individual HMOs?

• short- and long-term consequences?(DOHaD: Developmental Origins of Health and Disease)

prebiotics bacteriostatic, bactericidal

antimicrobials


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prebiotics

Dr. Victor Nizet UCSD

bacteriostatic, bactericidal

antimicrobials

HMO Exhibit a Unique Antimicrobial Property Against Group B Streptococcus

• identified structure-specific HMO(LNT)

GBS+HMO

GBS

Lin A et al. (2017) J Biol Chem 292(27):11243-11249

• identified potential GBS target (glycosyltransferase)

• suggest HMO as cell wall substrate that inhibits growth

• other bacteria? / pathogens?

HMO as Antimicrobials

prebiotics

Dr. Victor Nizet UCSD


antimicrobials

HMO Exhibit a Unique Antimicrobial Property Against Group B Streptococcus

Vancomycin IC50 = 0.25 µg/mLVancomycin IC50 (+HMO) = 0.00602 µg/mL

Ciprofloxacin IC50 = 1.37 µg/mLCiprofloxacin IC50 (+HMO) = 0.0021 µg/mL

I =0.30I =0.29

Log Ciprofloxacin (µg/ml)

Gro

wth

at 4

h (C

FU)

Log Vancomycin (µg/ml)

Lin A et al. (2017) J Biol Chem 292(27):11243-11249

HMO as Antimicrobials

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antimicrobials

antiadhesives


HMO as Antiadhesives

bacterial lectin

“glycocalyx”

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prebioticsDr. Lars Eckmann UCSD


antimicrobials

antiadhesivesHMO Protect Against EnteropathogenicE. coli (EPEC) Attachment in vitro andColonization in Suckling Mice

attachment in vitro


prebioticsDr. Lars Eckmann UCSD


antimicrobials

antiadhesivesHMO Protect Against EnteropathogenicE. coli (EPEC) Attachment in vitro andColonization in Suckling Mice

attachment in vitro

Control pHMO treated0.0

0.5

1.0

Control pHMO treated

A/E

lesi

onco

unt/c

ell

A/E lesions (phalloidin staining) *


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antimicrobials

antiadhesivesepithelial cell modulators



antimicrobials

antiadhesives

epithelial cell modulators

HMO Protect Bladder Epithelial Cells Against Uropathogenic Escherichia coliInvasion and Cytotoxicity

Lin A et al. (2014) J Infect Dis 209:389-98• other epithelial cell responses?

HMO as Epithelial Cell Modulators

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antimicrobials

antiadhesivesepithelial cell modulators

immune cell modulators



antimicrobials

• identified specific HMOsthat block macrophage inflammation

epithelial cell modulators

Dr. Philip GordtsUCSD

antiadhesives

immune cell modulators

HMO as Immune Cell Modulators

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SummaryWhat are the potential benefits of HMOs?

• indirect effects on the infant (through effects on the microbiome)

• prebiotic• anti-microbial• anti-adhesive

• direct effects on the infant• epithelial cell response modulators• immune cell response modulators

• multiple potential other mechanisms

• health implications throughout the life cycle

HMO health implicat ions throughout the life cycle

effects at every stage of the life cycle (“synthetic” HMOs)

Jantscher-Krenn E et al.AJP EndoMetabolism effects on pregnant and Nov 14, 2018 breastfeeding women

immediate immediate short-term short-term

effects effects

Wise A et al. Front. Pediatr.Oct 2, 2018

lasting, long-term effects (DOHaD)

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Necrotizing Enterocolitis (NEC) in the Preterm Infant

immediate short-term

effects

inci

denc

e [%

]

Necrotizing Enterocoli t isN Engl J Med 2011

• one of the most common and fatal GI disorders in preterm infants• ~5% of all VLBW infants (<1,500g)• high mortality (>25%)

• ~6- to 10-fold higher risk in formula-fed infants (FF) compared to breast-fed infants (BF)

HMOs?which one?

Lucas A and Cole TJ (1990) Lancet 336: 1519-1523

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HMO isolation from humanmilk

In vivo efficacy testing in preclinical models

Necrotizing Enterocoli t is

Jantscher-Krenn E et al. (2012) GUT 61:1417-25

A 100

1.0

0.0

4.0

3.0

2.0

DF, Normal (score: 0) FF, Complete Destruction (score: 4)

*** ***

Villus Edema Epithelial Sloughing Misaligned Nuclei

C D

DF

Pat

holo

gyS

core

FF FF+HMO +GOS

FF

90

80FF+GOS76.5% (13/17)

FF70 73.1% (19/26)

0 24 48 72 96time post-partum [h]

Sur

viva

l[%

]

DF100% (18/18)

FF+HMO95% (19/20)



Yes No

Identification of most effective oligosaccharides



0.0

1.0

2.0

3.0

4.0

A

FF+HMO-2

Pat

holo

gyS

core

***

***

DF

FF 0 -

3-4

-1

0.0

1.0

2.0

3.0

4.0

Pat

holo

gyS

core

E *****

*

FFFF

+HMO 2 HMO 3+4

DF

FF+

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Yes No


β1-4α2-6

β1-3

β1-3

α2-3

DSLNT


Jantscher-Krenn E et al. (2012) GUT 61:1417-25Yu H et al. (2014) Angew Chem Int Ed Engl. 53(26):6687-91 Autran CA et al. (2016) Br J Nutr 116(2):294-9Yu H et al. (2017) J Org Chem. 82(24):13152-13160



Yes No


Clinical intervention study

Mother-infantpair cohorts

HMO composition Identification of associationsanalysis between HMO and clinical metadata

Match!

Mechanism?

inform

α2-6

β1-4

β1-3

β1-3

α2-3

DSLNT

Van Nierkerk E et al. (2014) J Nutr 144(8):1227-33 Autran CA et al. (2018) GUT 67:1064–1070

Jantscher-Krenn E et al. (2012) GUT 61:1417-25Yu H et al. (2014) Angew Chem Int Ed Engl. 53(26):6687-91 Autran CA et al. (2016) Br J Nutr 116(2):294-9Yu H et al. (2017) J Org Chem. 82(24):13152-13160 Etzold S et al. (in preparation)


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Necrotizing Enterocoli t isHMO effect is highly structure-specific !

1.0

0.0

4.0

3.0

2.0

Path

olog

y Sc

ore

***

FF FF+ FF+ FF+ DSLNT DSLNT DSLNT

-α2-3/6 -α2-3

β3 β3 β3β3 β3 β3

α3


β4α

6

β4

β4α

6

Autran CA et al. (2018) GUT 67:1064–1070

Food Sensitization and Allergy

lasting long-term effects (DOHaD)

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Miliku K et al. (2018) Allergy

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Miliku K et al. (2018)

Individual HMOs or HMO profiles?What we’ve learned so far…

• sometimes specific individual HMOs are effective

• effects are highly structure-specific

• dose-response effect

• mechanisms likely receptor-mediated (host and/or microbe)

• sometimes specific mixtures (composition profiles) are effective

• relative abundance of HMOs, ratio of HMOs

• mechanisms likely indirect through shaping microbial communities, microbial metabolites, and/or direct on host (immune system and other)

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What is the ’optimal’Human Milk Oligosaccharide

(HMO) composition?

Is there an ’optimal’Human Milk Oligosaccharide

(HMO) composition?

Infant(genetics)

Time

EnvironmentGeographic Location

Personalized, Context-dependent Composition

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Conclusion

• HMOs are the third most abundant component of human milk

• maternal (genetic and environmental) factors driving variation in complex HMO composition not fully understood

• combination of preclinical, cohort and clinical studies required to fully assess effects, functions and potential claims

• sometimes individual HMOs are effective; sometimes complex mixtures of HMOs in specific relative abundancies are required (personalized?)

• HMOs benefit the breastfed infant and potentially the fetus

• HMOs may have potential long-term effects (DOHaD)

• HMOs may benefit maternal health

• HMOs serve as template for novel therapeutics way beyond the first 1,000 days

www.bodelab.com www.milk.ucsd.edu

[email protected]

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http://www.bodelab.com/

http://www.milk.ucsd.edu/

mailto:[email protected]

human milk oligosaccharides: benefits for the breastfed

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