[PPT] - The early-life gut microbiome and pediatric asthma Marie-Claire PowerPoint Presentation

SLIDE 1

Marie-Claire Arrieta Ph.D

Departments of Physiology & Pharmacology and Pediatrics

The early-life gut microbiome and pediatric asthma

SLIDE 2

Graham-Rowe et al. 2011. Nature. Nov.;S2-S4.

The global distribution is disproportionate.

SLIDE 3

Animal models

Intranasal OVA 0 7 14 21 22 23 24 25 26 OVA IP OVA IP Sacr.

Sensitization Challenge

Ovalbumin Model of Allergic Asthma

14 21 22 23 25 HDM IN HDM IN HDM IN Sacr.

House Dust Mite Model of Allergic Asthma

SLIDE 4

BREASTFEEDING FARMS C-SECTIONS ANTIBIOTICS

Epidemiology

HELMINTHS

SLIDE 5

Laforest-Lapointe and Arrieta, 2017. Front Immun.

Low resilience during critical immune development stage

SLIDE 6

Intranasal OVA

0 7 14 21 22 23 24 25 26 OVA IP OVA IP Sacr.

Sensitization Challenge

*

Birth (Neonatal)

Neonatal Abx

7 wks (Adult)

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(((,#$-.#/0123"(/$(((*%"2/0123"(

(((4566(0789:(((((((45660789:(

Adult Abx

Experimental evidence

Ovalbumin Model of Allergic Asthma

SLIDE 7

Neonatal antibiotic treatment affects experimental asthma severity

*

5 10 15 20 25 n.s.

Adult Abx

PBS OVA OVA + Vanco OVA + Strep

5

1

Total cells x 10 ml

5

1

Total cells x 10 ml

PBS OVA OVA + Vanco OVA + Strep

Neonatal Abx

A

5 10 15 20 25

Russell, et. al. EMBO Rep. 2012.

n.d.

4

1

cells x 10 ml PBS OVA OVA + Vanco OVA + Strep

5 10 15 20 25

**

n.s.

Eosinophils

OVA-specific IgE

25 50 75 100 n.d.

*

PBS OVA OVA + Vanco OVA + Strep ng ml

1

SLIDE 8

Hill, et. al. Nature Med. 2012.

SLIDE 9

Hill, et. al. Nature Med. 2012.

SLIDE 10

1 5 15 Risk of asthma at school age

[Asthma Predictive Index (Odds Ratio)]

87 subjects 136 subjects

22 subjects

Wheeze Atopy

Positive allergen skin prick test at 1-year ≥ 1 episode of wheezing by 1- year of age

74 subjects

Controls Atopy Only Wheeze Only

Atopy + Wheeze

CHILD Study:

16S rRNA analysis at 3 months and one year of age

10

SLIDE 11

D

Lachnospira spp.

atopy wheeze controls 0.00001 0.0001 0.001 0.01 0.1 1 10 100 1000 10000

Bifidobacterium spp.

atopy wheeze controls 0.0001 0.001 0.01 0.1 1 10 100 1000 10000 100000

1-year 3-months

Veillonella spp.

a t

p

y w h e e z e c

n

t r

l

s 0.00001 0.0001 0.001 0.01 0.1 1 10 100

***

Veillonella spp.

a t

p

y + w h e e z e c

n

t r

l

s 0.00001 0.0001 0.001 0.01 0.1 1 10 100 1000

Rothia spp.

Atopy + Wheeze Controls 0.001 0.01 0.1 1 10 100 1000

Faecalibacterium spp.

atopy + wheeze controls 0.000001 0.00001 0.0001 0.001 0.01 0.1 1 10 100 1000 10000

Lachnospira spp.

A t

p

y + W h e e z e C

n

t r

l

s 0.00001 0.0001 0.001 0.01 0.1 1 10 100 1000 10000

Bifidobacterium spp.

Atopy + Wheeze Controls 0.001 0.01 0.1 1 10 100 1000

Rothia spp.

a t

p

y w h e e z e c

n

t r

l

s 0.00001 0.0001 0.001 0.01 0.1 1 10 100 1000

***

Rothia spp. Rothia spp. Veillonella spp. Veillonella spp. Faecalibacterium spp. Lachnospira spp. Bifidobacterium spp. Bifidobacterium spp.

Relative Quantification (log10)

Faecalibacterium spp.

a t

p

y w h e e z e c

n

t r

l

s 0.001 0.01 0.1 1 10 100 1000 10000

*

Faecalibacterium spp. Lachnospira spp.

Veillonella spp.

atopy wheeze controls 0.00001 0.001 0.1 10 1000

***

Veillonella spp.

a t

p

y + w h e e z e c

n

t r

l

s 0.000001 0.0001 0.01 1 100 10000

Rothia spp.

atopy wheeze controls 0.00001 0.001 0.1 10 1000

***

Rothia spp.

Atopy + Wheeze Controls 0.0001 0.01 1 100 10000

Faecalibacterium spp.

atopy wheeze controls 0.0001 0.01 1 100 10000

*

Faecalibacterium spp.

a t

p

y + w h e e z e c

n

t r

l

s 0.000001 0.0001 0.01 1 100 10000

Lachnospira spp.

A t

p

y + W h e e z e C

n

t r

l

s 0.00001 0.001 0.1 10 1000 100000

Lachnospira spp.

a t

p

y w h e e z e c

n

t r

l

s 0.00001 0.001 0.1 10 1000 100000

Bifidobacterium spp.

Atopy + Wheeze Controls 0.0001 0.01 1 100 10000

Bifidobacterium spp.

atopy wheeze controls 0.0001 0.01 1 100 10000 1000000

Lachnospira spp. Atopy + Wheeze Controls Controls Atopy + Wheeze

Veillonella spp.

CTRL AW

1.0×10-08 1.0×10-06 1.0×10-04 1.0×10-02 1.0×1000

*** Veillonella spp.

Controls AW

1.0×10-10 1.0×10-08 1.0×10-06 1.0×10-04 1.0×10-02 1.0×1000

* Lachnospira spp.

Controls AW 1.0×10-10 1.0×10-08 1.0×10-06 1.0×10-04 1.0×10-02

* Rothia spp.

Controls AW 1.0×10-10 1.0×10-08 1.0×10-06 1.0×10-04

Faecalibacterium spp.

Controls AW 1.0×10-10 1.0×10-08 1.0×10-06 1.0×10-04 1.0×10-02 1.0×1000

Bifidobacterium spp.

Controls AW 1.0×10-10 1.0×10-08 1.0×10-06 1.0×10-04 1.0×10-02

Lachnospira spp.

C T R L A W

1.0×10-10 1.0×10-08 1.0×10-06 1.0×10-04 1.0×10-02 1.0×1000

*** Rothia spp.

CTRL AW

1.0×10-10 1.0×10-08 1.0×10-06 1.0×10-04 1.0×10-02 1.0×1000

*** Faecalibacterium spp. CTRL AW

1.0×10-10 1.0×10-08 1.0×10-06 1.0×10-04 1.0×10-02

*** Bifidobacterium spp.

CTRL AW

1.0×10-10 1.0×10-08 1.0×10-06 1.0×10-04 1.0×10-02 1.0×1000

Veillonella spp. Veillonella spp. Lachnospira spp. Lachnospira spp. Rothia spp. Rothia spp. Faecalibacterium spp. Faecalibacterium spp. Bifidobacterium spp. Bifidobacterium spp.

Relative Quantification 3-months 1-year Arrieta et al. 2015 Sci. Transl. Medicine.

SLIDE 12

Gut bacterial metabolism: short-chain fatty acids (SCFA)

SCFA:

Used as energy source by intestinal and mucosal immune

cells.

Transported to the liver where they are involved in numerous

biochemical pathways of lipid and energy metabolism.

Involved in immune response regulation.

SLIDE 13

Pathway

Secondary bile acid metabolism Hemoglobin metabolism Phenylalanine metabolism Histidine metabolism Food component

B

glycolithocholate sulfate 2 4 6

*

I-urobilinogen CTRL AW 20 60 80

*

glycocholenate sulfate 2 4 6 8

*

phenylacetylglutamine CTRL AW 2 4 6

*

glycohyocholate 2 4 6 8 10

*

imidazole propionate CTRL AW 5 10 15 20 25

*

tauroursodeoxycholate 1×105 2×105 3×105

*

ferulate C T R L A W 0.0 0.5 1.0 1.5

*

Urine metabolites

* ** * ** ** * *

P = 0.052 glycohyocholate

ursodeoxycholate

A *

Acetate umol/g feces CTRL A + W 10 20 30 40

*

Propionate umol/g feces CTRL A + W 2 4 6 8 Butyrate umol/g feces C T R L A + W 0.0 0.5 1.0 1.5 2.0

*

Arrieta, et al 2015 Sci. Transl. Medicine.

SLIDE 14

From correlation to causation

breeding mice

ffspring

Asthmatic Microbiota (AW) (i) Control birth 3 wks 7 wks OVA

Ovalbumin

(AW) AW + FLVR (ii) FLVR birth 3 wks 7 wks OVA

Hu Human man mic microb

biot

iota a tran ansfer er (inoculated with feces from AW subject)

14

SLIDE 15

100

25 50 75 100 CTRL VLRF

Fa Family

AW AW + FLV FLVR

Bacteroidaceae Clostridiaceae Enterobacteriaceae Enterococcaceae Lachnospiraceae Micrococcaceae Prevotellaceae Ruminococcaceae S24-7 Turicibacteraceae

Clostr Bacteroidaceae Enterobacter Enterococcaceae Lachnospir Micrococcaceae Peptostreptococcaceae Pre Ruminococcaceae S24 Tur

Abundance ce (%)

25 50 75

AW AW

Peptostreptococcaceae

A B

AW + FLVR

AW

60
40
20

20

60
40
20

20 60

PC 2 (24.8%)

40 60 40

PC 1 (41.5%)

1.0 1.5 40 60

C

Abundance ce (%)

0.5

Lach chnosp spira

20

Faeca calibact cterium Ro Rothia Ve Veillonella

Lachnospira 20 40 60

0.0 0.2 0.4 0.6 Faecalibacterium Rothia Veillonella

Microbial intestinal colonization in F1 mice

Arrieta, et al 2015 Sci. Transl. Medicine.

SLIDE 16

FLVR reduces lung inflammation

Na Naive AW AW AW AW + FLVR

Bronchoalveolar lavage

16

Naive AW AW + FLVR 50 100 150 200

Macrophages Lymphocytes Neutrophils Eosinophils

BAL Total Cell Differential

**** *

cells (x103)ml

BAL Counts

Naive AW AW + FLVR

20 40 60 80 cells(x104)/ml

* **

Arrieta, et al 2015 Sci. Transl. Medicine.

SLIDE 17

What about asthma elsewhere?

Prof. Phil Cooper

Las Esmeraldas, Ecuador 29 71 AW Controls 3 months

Forno et al. Thorax 2015

SLIDE 18

Ru Rural t to u urban tr transiti tion

SLIDE 19

Ecuador cohort: epidemiology

Categorical variable Odds ratio P Maternal antibiotics during pregnancy 2.39 0.02 Vaginal birth

2.42

0.03 Breastfeeding over 5 months

0.17

0.02 Child Ascaris infection 3.82 0.05 Child Trichuris infection

8.18

0.004 Respiratory infections 2.21 0.001 Potable water 2.49 0.02

SLIDE 20

Microbiota in babies at risk of asthma

Bacteria

8
6
4
2

2 4 6 8

Bifidobacterium_179 Bacteroides_7 Ruminococcus_gnavus_18 Streptococcus_10 Streptococcus_9 Streptococcus_5 Veillonela_26 Veillonella_4

log2 Fold Change CTRL:AW Firmicutes Bacteroidetes Actinobacteria Phylum

Acetate

µmol/g feces

AW CTRL 50 100 150

*

Arrieta, et al 2017 J Allergy Clin Immunol.

SLIDE 21

CTRL AW

10-9 10-8 10-7 10-6 10-5 10-4 10-3

DNA (ng/ul)

***

Pichia kudriavzevii

8
6
4
2

2 4 6 8

1269 __uncultured_fungus_Saccharomycetales 717 __uncultured_fungus_Saccharomycetales 716 __uncultured_fungus_Saccharomycetales 1295 __uncultured_fungus_Saccharomycetales 643 __uncultured_fungus_Saccharomycetales 642 __uncultured_fungus_Saccharomycetales 638 __uncultured_fungus_Saccharomycetales 639__uncultured_fungus_Saccharomycetales 1328__Saccharomyces 58 __Debaryomyces_hansenii 558 __Candida_tropicalis 1541 __Pichia_kudriavzevii 1540 __Pichia_kudriavzevii 1529 __Pichia_kudriavzevii 1539 __Pichia_kudriavzevii 1534 __Pichia_kudriavzevii 1531 __Pichia_kudriavzevii 1538 __Pichia_kudriavzevii 1533 __Pichia_kudriavzevii

log2FoldChange

Decreased in CTRL Increased in CTRL

A B

Arrieta, et al 2017 J Allergy Clin Immunol.

SLIDE 22

Relative abundance CTRL AW

0.0 0.2 0.4 0.6

*

Fungal relative abundance CTRL AW

10-10 10-9 10-8 10-7 10-6 10-5 10-4

DNA (ng/ul)

*

Fungal 18S A B

Arrieta, et al 2017 J Allergy Clin Immunol.

SLIDE 23

Atopic wheeze Healthy 3 months Microbial metabolites Acetate Bacteria Faecalibacterium sp. Lachnospira sp. Veillonella sp. Rothia sp. Streptococcus sp. Bacteroides sp. Ruminococcus gnavus Bifidobacterium sp. Bacteria Fungi Pichia kudriavzevii Saccharomycetales

SLIDE 24

+

Antibiotics13m ation OTU_1598_Saccharomycetales OTU_1603_Saccharomycetales OTU_1131_uncultured.stramenopile OTU_1130_uncultured.stramenopile OTU_48_uncultured.freshwater.cercozoan OTU_30_Gregarina OTU_1323_Meyerozyma.guilliermondii OTU_1319_Meyerozyma.guilliermondii OTU_635_Meyerozyma.guilliermondii OTU_1213_Saccharomycetales TU_704_Saccharomyces.cerevisiae..baker.s.yeast. OTU_703_Saccharomycetales OTU_1627_Eurotiales OTU_1697_Pleosporales OTU_1696_Pleosporales OTU_1682_Pleosporales OTU_1327_Capnodiales OTU_1653_Fungi OTU_1484_Fungi OTU_1482_Fungi OTU_1860_Insecta OTU_1782_Insecta OTU_1531_Aspergillus.niger OTU_1729_Prymnesiales OTU_1242_Saccharomycetales OTU_1164_Issatchenkia.terricola OTU_1566_Eurotiales OTU_1356_Cladosporium.sp..6027 OTU_1358_Cladosporium.sp..6027 OTU_263_Geotrichum.candidum OTU_571_Saccharomycetales OTU_1651_Leotiomycetes OTU_1612_Leotiomycetes OTU_1554_Eurotiales OTU_105_uncultured.Eimeriidae OTU_841_Bacillariophyceae OTU_315_Amb.18S.431 OTU_314_Amb.18S.431 OTU_1309_uncultured.cercozoan OTU_1305_uncultured.cercozoan OTU_1429_Pichia.kudriavzevii OTU_1419_Pichia.kudriavzevii OTU_1591_Candida.tropicalis OTU_1588_Candida.tropicalis OTU_1866_Eurotiales OTU_1830_Eurotiales OTU_1392_Jatropha.curcas OTU_1391_Capnodiales OTU_1071_Prymnesiophyceae OTU_85_Chlorochytrium.willei

++ Child_ABX Mom_ABX

Hypothesis

Antibiotic-driven alterations in the fungal intestinal microbiota affect immune development and asthma.

SLIDE 25

Can fecal fungal overgrowth influence atopy?

CTLR ABX ABX+Y

0.00 0.05 0.10 0.15

IL-10 + cells

% Parental

*

CTLR ABX ABX+Y

0.0 0.5 1.0 1.5 2.0 2.5

CD4 + IL-4 +

% Parental

P=0.102

CTLR ABX ABX+Y

0.0 0.5 1.0 1.5 2.0 2.5

CD4 + IL-5 +

% Parental

*

CTLR ABX ABX+Y

0.0 0.5 1.0 1.5 2.0 2.5

CD4 + IL-13 +

% Parental

*

CTRL ABX ABX+Y

2×104 4×104 6×104

CFU/ml Fecal fungal growth

*

CTLR ABX ABX+Y

0.0 0.2 0.4 0.6 0.8

CD4 + IL-6 +

% Parental

P=0.06

2 wk 1 wk

Abx
Abx + C. albicans

and P. kudriavzevii

untreated

birth 7 wk OVA + Alum IP 9 wk 11 wk Immune assessment

A B C

P=0.1

IL-10+ CD4+ IL-13+ CD4+ IL-5+

P=0.06

CD4+ IL-4+ CD4+ IL-6+

SLIDE 26

B

Does fungal gut colonization influence immune development?

B + F B

+ +

A

FITC-Dextran Colon B B + F

200 400 600 800 FITC-Dextran Fluorescence (AFU)

*

I L

1

I L

9

T N F

α

20 40 60 80 Colon Culture

pg/ml

B B+F

* * *

Jejunum culture

IFN-λ IL-17E/IL-25 IL-4 IL-9 0.0 0.2 0.4 0.6 0.8 1.0

pg/ml

p = 0.07

* *

p = 0.07

IL-10 IL-17F TNF-α 5 10 15 20

pg/ml * * *

D C

SLIDE 27

Does fungal gut colonization influence immune development?

B B + F

30 35 40 45 % Parental

T cells (CD3+)

B B+F

0.0 0.2 0.4 0.6 0.8

IL-13 + cells

% Parental

B B + F

20 25 30 35 % Parental

CD8+ T cells

B B + F

2 4 6 8

IL-10 + cells

% Parental

B B + F

45 50 55 60 % Parental

B cells (CD19+)

B B + F

0.0 0.5 1.0 1.5

CD25+ FoxP3+ T regs

% Parental

B B + F

0.00 0.05 0.10 0.15

IL-4 + cells

% Parental

B B+F

0.00 0.05 0.10 0.15

IL-13 + cells

% Parental

SPLENOCYTES

* * * * * * * * IL-6 + cells

SLIDE 28

Summary

Changes in the gut microbiome can influence asthma

development.

Early life is the most critical stage for the

development of the microbiome.

The first 100 days of human life represent an early

‘critical window’ in which microbial dysbiosis enhances the risk of asthma and allergic disease.

SLIDE 29

Summary

Microbial dysbiosis can change with geographical

location.

Asthma-related microbial dysbiosis is both bacterial and

fungal.

Fungal overgrowth may also explain immune

alterations that lead to asthma.

Prospective observational microbiome studies are

critical to study dysbiotic markers that precede and may be involved in asthma pathogenesis.

SLIDE 30

Arrieta Lab Erik Bernardes Haley Jamison Isabelle Laforest-Lapointe Jumana Samara Shen Yen Tsou Joseph Girgis Finlay Lab Brett Finlay Shannon Russell Lisa Thorson Sophie Yurist Miriam Nieves Heidi Britton Rozlyn Boutin Ecuador infant study Phil Cooper Andrea Arevalo Turvey Lab Mohn Lab Bill Mohn Pedro Dimitriu Stuart Turvey Leah Stiemsma Wegener-Parfrey Lab Laura Wegener-Parfrey Evan Morien