Maternal stress and the microbiome:

programming of offspring
neurodevelopment
-

Tracy L. Bale, Ph.D.

Professor of Neuroscience
University of Pennsylvania

Sex-specific mechanisms
F1

F0

F2
Primordial

Bale Nat Rev Neuro 2015

Nature Reviews | Neuroscience

Early pregnancy stress and offspring with

stress sensitivity through 2 generations
Changes in behavioral stress responses

Changes in HPA axis response to stress

Cognitive deficits

Reduced weight gain
Mueller and Bale, 2008, J Neurosci
Morgan and Bale, 2011, J Neurosci
Howerton et al, 2013, PNAS
Howerton and Bale, 2014, PNAS

Corticosterone (ng/mL)

Changes in stress regulatory genes

Ctrl

400
350

E-PS

300
250
200
150
100
50
0

15

30
Time (min)

60

Getting a mouthful....

how the vaginal microbiome

impacts neurodevelopment

Proteobacteria

Firmicutes
Tenericutes

Bacteroidetes

Prenatal

Postnatal

Childhood

Puberty

Adulthood

Aging

Gastrointestinal tract

Bacterial
colonization

Dietary
transition

Mucosal
thinning

IEC
proliferation

Stages of brain development

Neuronal proliferation

Neuronal loss

Dendritic arborization

Synaptic loss

Neuronal migration

Apoptosis
Synaptic pruning
Excitatory synapses
Inhibitory synapses

Jasarevic, Morrison, Bale (2016) Phil Trans B

Does our EPS

model change the
maternal vaginal
microbiome?

Jasarevic 2014 Neuro Stress

Early pregnancy stress changes

vaginal microbiome
100%

Low Abundance Genus

Enterobacteriaceae;g__
Staphylococcus
S24-7;g__
Lactobacillus
Clostridiales;f__;g__
Flexispira
Bradyrhizobiaceae;g__
Helicobacter
Mannheimia
Prevotella
Gemella
Streptococcus
Aggregatibacter

90%
80%
70%
60%
50%
40%
30%
20%
10%
0%
Nonpregnant

Ctrl
Control

Stress
EPS

E7.5

Control
Ctrl

Stress
EPS

PN2

Impact of EPS on vaginal host proteomics

0
-2

Ctrl

Ctrl

EPS

EPS

Orthogonal component score

20

CTL
EPS

15
10
5
0
-5
-10
-15

-20
-20 -15 -10

-5

10

15

20

Stress component score

Maintenance of location
Leukocyte migration
Actin filament-based process
IMMUNE RESPONSE
Actin cytoskeleton organization
IMMUNE EFFECTOR PROCESS
Cytoskeleton organization
LEUKOCYTE MIGRATION
Defense response
LEUKOCYTE ACTIVATION
Response to wounding
LEUKOCYTE HOMEOSTASIS
Immune effector process
MYELOID CELL HOMEOSTASIS
Monosaccharide catabolic process
COMPLEMENT ACTIVATION
Hexose catabolic process
HEMOPOISESIS
Glucose catabolic process
0
4
8
12
Glycolysis
-80 -60 -40 -20

E7.5

Time
E7

PN2

PN2

20 40 60 80

DAVID Fold Enrichment

Enrichment (-log[P-value])

Are our EPS changes

in the maternal
vaginal microbiome
passed on to the
offspring gut?

Jasarevic 2014 Neuro Stress

Similar effects of EPS on maternal

vaginal and offspring gut microbiota

Jasarevic 2015 Endo

Does colonization by a stress-altered

microbiota impact neonate gut composition?
Control
EPS

Lactobacillus

Relative Abundance

0.4
0.3
0.2
0.1
0.0

*
Male

*
Female

Clostridia

Relative Abundance

0.020
0.015

B: Deferribacteraceae

C: Deferribacterales

D: Lactobacillus

E: Lactobacillaceae

F: Clostridiales

G: Clostridium

H: Haemophilus

CTL
EPS

0.010
0.005
0.000

A: Mucispirillum

Male

Female

Jasarevic et al. (in2015

press) Endocrinology
Jasarevic
Endo

What are the long-term consequences of

altered neonate gut microbiota composition?
Enriched taxa in PN28 colon
Control Male
Control Female
EPS Male
EPS Female

i
cill
Ba

Ba
cte
roi
de
B
te
5

A: Clostridiales
B:Flavobacteria
C: Bacteroidia
Deferribacteres
E: Leptospirae
F: Gammaproteobacteria

1: Ruminococcaceae
2: Mogibacteriaceae
3: Lachnospiraceae
4: Dehalobacteriaceae
5: Paenibacillaea

17

Clo

str
idi
a
A

G: Deltaproteobacteria
H: Rhiboziales
I: Erysipelotrichales

Erysipelotrichi

H
E

11

10

16

Pro
te

12

15

ob

14

ac
ter
i

13

6: Flavobacteriales
7: Bacteroidia
8: Coriobacterales
11: Leptospirlalles
12: Pasteuellales
13: Desulfovibrionaceae
14: Burkholderiales
15: Burkholderiales
16: Phyllobacteriaceae
17: Eryspelotrichales

Are our EPS changes

in the offspring gut
associated with effects
on metabolism or
neurodevelopment?

Jasarevic 2014 Neuro Stress

10

-1.0

-0.5

0.0

0.5

Propionylcarniti

Isovalerylcarniti

MSEA Fold Enrichment

-5

Indolelactic A

Hippuric A

Xanthosi

-20
-10

Creati

Glycerophosphocholi

-15

4-pyridoxic a

GLUCOSE-ALANINE CYCLE

Ribofla

1.0

Variable

Ortho

-10

GLUCOSE-ALANINE CYCLE

1.0

EPS alters plasma metabolites at PN2

MSEA Fold Enrichment

-10
-15
-20
-8

-6

-4

-2

0 2 4 6
EPS component score

PROSTACYCLIN SIGNALLING

1.0

0.0
0.5

-1.0

AMMONIA RECYCLING

INSULIN SIGNALLING

-0.5

0.0

p(corr)

0.5

1.0

MALATE-ASPARTATE SHUTTLE

Carnitine

ELECTRON TRANSPORT CHAIN

Imidazoleacetic Acid

-5

0.5

N-acetylaspartate

GLUTATHIONE METABOLISM

GLUCONEOGENESIS

Taurine

CITRIC ACID CYCLE

1.5

GLYCOLYSIS

GTP

RNA TRANSCRIPTION

STARCH AND SUCROSE METABOLISM

Pantothenic acid

INSULIN SIGNALLING

UREA CYCLE

UDP-glucuronate

MALATE-ASPARTATE SHUTTLE

2.0
1.0

UDP-D-glucose

AMMONIA RECYCLING

UDP-N-acetylglucosamine

PROSTACYCLIN SIGNALLING

Energy metabolism

GLUTAMATE METABOLISM

Glutamate

10

MSE

NUCLEOTIDE SUGARS METABOLISM

-ketoglutarate

ELECTRON TRANSPORT CHAIN

Succinic acid

Orthgonal component score

1.0

15

Sugar
metabolism

1.52.5

Malic acid

CTL
EPSGLUCONEOGENESIS

STARCH AND SUCROSE METABOLISM

ADP

GLYCOLYSIS

NAD+

UREA CYCLE

20

Mitochondrial metabolism

Glutathione Disulfide

NUCLEOTIDE SUGARS METABOLISM

2.0

Variable importance projection score

Plasma

GLUTAMATE METABOLISM

Fold Change (Relative to CTL)

EPS component score

p(corr)

RNA TRANSCRIPTION

CITRIC ACID CYCLE

GLUTATHIONE METABOLISM

Jasarevic 2015 Endo

Microbiota shaping nutrient

metabolism and transport: PN2 brain
Males

C
Paraventricular Thalamus

Paraventricular Nucleus

Hippocampus

Alanine
Arginine
Asparagine
Aspartate
Citrate
GABA
Glutamine
Glutamate
Glycine
Histidine
Isoleucine
Leucine
Lysine
Ornithine
Phenylalanine
Serine
Threonine
Tyrosine
Valine
-1

-2

-1

-2

-1

-2

-2

Females

Alanine
Arginine
Asparagine
Aspartate
Citrate
GABA
Glutamine
Glutamate
Glycine
Histidine
Isoleucine
Leucine
Lysine
Ornithine
Phenylalanine
Serine
Threonine
Tyrosine
Valine
-1

Log 2 [FC]

Log 2 [FC]

Jasarevic 2015 Endo

0

Log 2 [FC]

Demonstrating causality
Control

EPS

Does seeding with vaginal microbiota recover

microbial diversity in C-section offspring?
Microbial diversity

Bacterial Count
Shannon Diversity Index

3
2
1

C
C
-S
-S
ec
ec
tio
tio
n
n
-I
no
cu
la
te
d

Va
gi
na
lly

D
el
iv
er
ed

*
*

Vaginal

o
N
C IC
->
C C
->
E- E
>C
E>E

C
on
tro
EP l
S

Bacterial Count (Log 16S copies)

Cesaerean
18
Jasarevic et al. (unpublished)

Vaginal

0.1

0.01

0.001

Cesaerean

Lactobacillus

Vaginal

C o
->
C
C
->
E
E>C
E>E

C
on
tro
EP l
S

Relative Abundance (log 10)

C o
->
C
C
->
E
E>C
E>E

C
on
tro
EP l
S

Relative Abundance (log 10)

EPS programs fetal gut

Clostridia

0.1

0.01

0.001

0.0001

Cesaerean
19

Does the gut microbiome

contribute to HPA sensitivity?

300

30

20
10
0

Control EPS

200

100

400

CTL
EPS

300

15

30

120
Time (minutes)

40

CTL->CTL
EPS->CTL
CTL->EPS
EPS->EPS

30
20
10
0

CC CE EC EE

200

100

0
0

Caesarean delivered
AUC units (x 1000)

40

Corticosterone (ng/ml)

Vaginally delivered
AUC units (x 1000)

Corticosterone (ng/ml)

400

15

30

120
Time (minutes)

20

Proteobacteria

Firmicutes
Tenericutes

Bacteroidetes

Prenatal

Postnatal

Childhood

Puberty

Adulthood

Aging

Gastrointestinal tract

Bacterial
colonization

Dietary
transition

Mucosal
thinning

IEC
proliferation

Stages of brain development

Neuronal proliferation

Neuronal loss

Dendritic arborization

Synaptic loss

Neuronal migration

Apoptosis
Synaptic pruning
Excitatory synapses
Inhibitory synapses

Jasarevic, Morrison, Bale (2016) Phil Trans B

What about the EPS programming of

the gut itself?
Stress

E18.5&gut&

Embryonic Day 18.5

FC
class
class
class
Amica1
Amica1
Amica1
Camk2b
Camk2b
Camk2b
Mon1b
Mon1b
Mon1b
Piwil2
Piwil2
Piwil2
Limd1
Limd1
Limd1
Aim2
Aim2
Aim2
Pxylp1
Pxylp1
Pxylp1
Snx10
Snx10
Snx10
Ttc39a
Ttc39a
Ttc39a
Cdh4
Cdh4
Cdh4
NA.491
NA.491
NA.491
Fam173b
Fam173b
Fam173b
Ubn2
Ubn2
Ubn2
St6galnac1
St6galnac1
St6galnac1
Cyp2j13
Cyp2j13
Cyp2j13
Rbpms2
Rbpms2
Rbpms2
NA.29
NA.29
NA.29
Snhg18
Snhg18
Snhg18
Tmem204
Tmem204
Tmem204
Prss41
Prss41
Prss41
Fabp6
Fabp6
Fabp6
NA.466
NA.466
NA.466
Eif3j1
Eif3j1
Eif3j1
Dmtn
Dmtn
Dmtn
NA.473
NA.473
NA.473
Retsat
Retsat
Retsat
Iars2
Iars2
Iars2
Gm2115
Gm2115
Gm2115
1700084C01Rik
1700084C01Rik
1700084C01Rik
NA.532
NA.532
NA.532
Ly6d
Ly6d
Ly6d
NA.511
NA.511
NA.511
Gstt1
Gstt1
Gstt1
Yif1b
Yif1b
Yif1b
Krt4
Krt4
Krt4
Dnase1
Dnase1
Dnase1
Rdh5
Rdh5
Rdh5
NA.233
NA.233
NA.233
Dgat2
Dgat2
Dgat2
Cd68
Cd68
Cd68
Igfals
Igfals
Igfals
A930005H10Rik
A930005H10Rik
A930005H10Rik
Paqr4
Paqr4
Paqr4
4933404O12Rik
4933404O12Rik
4933404O12Rik
Rnaset2b
Rnaset2b
Rnaset2b
Atp6v0c
Atp6v0c
Atp6v0c
Rnps1
Rnps1
Rnps1
Rps2
Rps2
Rps2
NA.570
NA.570
NA.570
Hagh
Hagh
Hagh

FC

2
1
0

-1

-2

22
15.MCTL
15.MCTL
15.MCTL

11.MCTL
11.MCTL
11.MCTL

Male Ctrl
23.MCTL
23.MCTL
23.MCTL

19.MCTL
19.MCTL
19.MCTL

5.MEPS
5.MEPS
5.MEPS

Male EPS
13.MEPS
13.MEPS
13.MEPS

Female

10.MEPS
10.MEPS
10.MEPS

Male

222 class
class
class
MCTL
MCTL
MCTL
MEPS
MEPS
MEPS
111

Group
Male Control

000

-1
-1
-1

-2
-2
-2

Male EPS

Enrichment for immune genes in male

EPS E18.5 gut

23

Characterization of immune cell

infiltration prior to inoculation

25

SSC-A

33

Ly6G

Ly6C

51

% Ly6G+ Neutrophils
of CD45+ Cells

Male

36

25
20
15
10
5
0
80

% Ly6C+ Monocyes
of CD11b+ Cells

Female

Female

Male

60
40
20
0

Female

Male

CD11b%
Gating on total live singlet's CD45+ and then Ly6G- CD11b+ for bottom

24

Jasarevic,*Weinkopf,*et*al.,*(unpublished)*

EPS alters FoxP3+ trafficking in the

Early prenatal stress alters trafficking of FoxP3+ cell
male
gutgut
into
the fetal
male fetal

EPS

FoxP3

11.3

FoxP3 FMO
5.26

15

% FoxP3+ of CD4+ cells

Control

10

CD4
25

CON

EPS

2nd
hit
of
stress
reveals
EPS
male
Latent EPS programming to intestinal barrier dysfunction
vulnerability
tovariable
gut permeability
unmasked
by chronic
stress in adulthood

FITC intestinity Fold Change

(Relative to Control-No CVS)

Control-CTL
EPS-CTL
Control-CVS
EPS-CVS

26

Adrenal cortex
Vagus nerve

Gonads

Corticosterone
Catecholamines
Insulin
Glucose

Cytokines
Estrogen
Testosterone
Progesterone

IEC

Microbial
metabolites

Hormones
Neurotransmitters

Immune
cells

EEC

27

Animal welfare considerations

Importance of environment on study design
and interpretation, reproducibility

Shipping pregnant animals vs in-house breeding

28

Challenges in the field

Testing causality vs descriptive outcomes

Convincing neuroscientists that the
microbiome is a) real, and b) important to the
brain

29

Summary & Conclusions

EPS produces significant and lasting changes to the
maternal vaginal microbiome that are vertically passed
to the neonate gut

Changes in microbiota are associated with altered
plasma and gut nutrient metabolites & brain AA
transport in region-specific and sex-specific manner

C-section and oral inoculation suggest the gut is sexspecifically programmed, in part, prior to birth,
including the infiltration and activation of immune cells

Acknowledgements
Stefanie Bronson, Ph.D.

China Byrns

Jennifer Chan

Jessica Fluharty

Chris Howard

Chris Howerton, Ph.D.

Eldin Jasarevic, Ph.D.

Christopher Morgan

Katie Morrison, Ph.D.

Bridget Nugent, Ph.D.

Carly ODonnell

Ali Rodgers

SOVM Germ cell Ctr

Adrian Leu

Womens Behavioral Wellness Ctr

C. Neill Epperson, M.D.

!
Funding sources:

NIH MH073030, MH091258, MH087597,

SCOR P50-MH099910, MH104184, MH108286

Epigenetics Center

Benjamin Garcia, Ph.D.

Xiaoshi Wang, Ph.D.

CHMI

Dan Beiting, Ph.D.

Probiotics potential for vaginal

and babys health?

32

EPS alters PN2 metabolomics

3

-15
-20
-10

MSEA Fold Enrichment

-5

EPS component score

10

-1.0

-0.5

0.0

p(corr)

0.5

Propionylcarnitine

1.0-10

Isovalerylcarnitine

GLUCOSE-ALANINE CYCLE

RNA TRANSCRIPTIO

Indolelactic Acid

-5

GLYCOLYS

Hippuric Acid

GLUCONEOGENESIS

BCAA
catabolite

Xanthosine

Microbial
metabolite

UREA CYCL

ADP

RNA TRANSCRIPTION

Energy
metaboltes

Creatine

10

GLYCOLYSIS

Glycerophosphocholine

15

Vitamin
metabolism

Riboflavin

CTL
EPS

Variable importance projection score

Orthogonal component score

20

Fold Change
(Relative to CTL)

UREA CYCLE

4-pyridoxic acid

Colon
Colon

GLUCONEOGENES

GLUCOSE-ALANINE CYCL

1.0

Jasarevic 2015 Endo

EPS impact on PN2 hypothalamus

mitochondrial function

Howerton & Bale, PNAS 2014