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The gut-brain axis The gut microbiome in MS!

Sergio E Baranzini, PhDProfessor

Department of NeurologyInstitute for Human Genetics

Program in BioinformaticsUCSF

Neuro-gastroenterology an emerging field of research!

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# of

publ

icat

ions!

Year!

microbiome or microbiota!

in multiple sclerosis!

Autism!

Epilepsy!

Stroke!

Alzheimer!

Parkinson!

dementia!

Literature review (up to 2015)

A FEW DEFINITIONS

  Microbiota: A collection of microbial communities living in a specific niche

  Microbiome: The collective set of genes from a given microbiota

  Probiotic bacteria: Live microorganisms which when administered in adequate amounts confer a health benefit on the host

  Prebiotics: Selectively fermented food ingredients that allow specific changes, both in the composition and/or activity in the gastrointestinal microbiota that confer benefits upon host health

THE ENTERIC NERVOUS SYSTEM

  “Second brain”

  400 million neurons (=spinal cord)

  Extends from esophagus to anus

  Controls digestive system

COMPOSITION OF THE HUMAN GUT MICROBIOTA

  1013-1014 microorganisms

  collective microbiome = 100X human genome

  humans are super-organisms whose metabolisms represents an amalgamation of microbial and human attributes

Gill et al. Science 2006

  If we are our genes... then we are more our microbiome than our genome

MICROBIOME VARIES ACCORDING TO ANATOMICAL LOCATION OF THE HUMAN

BODY

Lee et al. Science 2010 The human Microbiome Project Consortium. Nature 2012

WHILE DIFFERENCES IN MICROBIAL TAXA EXIST, METABOLIC PATHWAYS REMAIN CONSTANT

The human Microbiome Project Consortium. Nature 2012

MICROBIAL COMMUNITIES AND THEIR PATHWAYS ARE ASSOCIATED WITH ETHNICITY

The human Microbiome Project Consortium. Nature 2012

MICROBIOME CORRELATES WITH DIET AND TAXONOMY

HerbivoresCarnivoresOmnivores Ley et al. Science 2008

THE HUMAN-MICROBE RELATIONSHIP

  microbes are human symbionts more than pathogens

  gut microbes shape immune responses through the interaction of their metabolism with that of humans

  B. fragilis activates TLR2 through PSA and stimulates Treg to enable its colonization

  short-chain fatty acids regulate size and function of colonic Treg population and protect against colitis

Lee and Mazmanian. Science 2010Round et al. Science 2011Smith et al. Science 2013

THE HUMAN MICROBIOME HELPS BALANCE IMMUNE RESPONSES

Lee and Mazmanian. Science 2010

MICROBIOTA AND OBESITY

  Microbiota from twins discordant for obesity can transmit phenotype when transferred into germ-free mice

  When co-housed, mice recipient of the lean microbiota were dominant.

  Obesity is associated with reduced bacterial diversity and altered representation of bacterial genes and metabolic pathways

Ridaura et al. Science 2013Turnbaugh et al. Nature 2009

MICROBIOTA AND CANCER THERAPY

  Elimination of the microbiota impairs the response of subcutaneous tumors to CpG-oligonucleotide immunotherapy and platinum chemotherapy

  elimination of microbiota results in resistance to cyclophosphamide in tumor-bearing mice

Iida et al. Science 2013Viaud et al. Science 2013

CROSSTALK BETWEEN MICROBIOTA AND THE IMMUNE SYSTEM

NORMAL GUT MICROBIOTA MODULATES BRAIN DEVELOPMENT AND BEHAVIOR

  GF mice display increased motor activity and reduced anxiety, compared with SPF

  behavior is related to expression of genes involved in second messenger and LTP

  GF co-housed with SPF mice adopted the behavior of SPF mice

Diaz-Heijtz et al. PNAS 2011

INGESTION OF LACTOBACILLUS MODULATES EMOTIONAL BEHAVIOR VIA THE VAGUS NERVE

  L. rhamnosus induces changes in expression of GABAB1b gene in cortex, hippocampus, and amygdala

  L. rhamnosus reduced corticosterone levels and anxiety- and depression-related behavior

  These effects were abrogated by vagotomy

Bravo et al. PNAS 2011

MICROBIOTA AND AUTISM

  children with ASD have GI disturbances

  severity of GI dysbiosis is associated with severity of autism

  ASD microbiome =! heathy microbiome (hbacteroidetes, ifirmicutes)

  Toxins produced by Clostridia affect behavior in ASD

  Urinary metabolites (from gut bacteria) characterize ASD subjects from healthy siblings

  Oral treatment of MIA offspring with B. fragilis corrects gut permeability, alters microbial composition, and ameliorates defects in communicative, stereotypic, anxiety-like and sensorimotor behaviors

Hsiao et al. Cell 2013

LACTATION PROMOTES DEVELOPMENT OF HEALTHY MICROBIOTA

  Lactating mice develop a different microbiota than non-lactating mice

  milk contains SIgA, which prevents translocation of aerobic bacteria from the gut to draining lymph nodes

  Maternal IgA ameliorated colonic damage after dextran sulfate administration

Rogier et al. PNAS 2014

THE EFFECT OF GUT MICROFLORA IN AUTOIMMUNE DEMYELINATION

•  Mice treated with wide-spectrum antibiotics are resistant to EAE

•  Effect associated to ↓inflammatory cytokines, ↑ IL-10 and IL13 and ↑Treg population.

•  Also, ↑CD5+ B cells (regulatory role)

•  Oral administration of PSA (from B.fragilis) protects mice from EAE in an IL-10 dependent fashion.

•  Exposure of DCs to PSA, promotes Treg

•  Monocolonization with SFB restored Th17 responses and EAE susceptibility in GF mice. Ochoa-Reparaz et al. J Immunol. 2009

Ochoa-Reparaz et al. Gut Microbes. 2010Ochoa-Reparaz et al. Mucosal Immunol. 2010Lee et al. PNAS. 2011

GUT MICROBIOTA IS REQUIRED FOR SPONTANEOUS EAE MODEL

•  GF TCR/BCR TG mice do not develop spontaneous EAE

•  This effect is mediated by impaired Th17 differentiation

•  Also B cell recruitment is impaired

Berer et al. Nature. 2011

•  T cells from NMO patients recognize an AQP4 epitope with high homology to C. perfringens ABC transporter permease.

•  C. perfringens type B was isolated from a CIS patient.

•  ε toxin from C. perfringens (ETX) cross BBB and binds OL

•  C. perfringens type A is less prevalent in MS, while reactivity against ETX is 10x higher in MS

THE CLOSTRIDIUM CONNECTION

Varrin-Doyer et al. Ann Neurol. 2012Rumah, et al. PLoS One. 2013

THE CLOSTRIDIUM CONNECTION

•  Stool sample from healthy human was treated with Chloroform and transferred to GF mice

•  Mouse stool showed enrichment in Clostridia

•  Treg cell induction by human intestinal bacteria is transmitted horizontally and vertically

•  Treatment with 17-mix suppresses experimental colitis

Atarashi, et al. Nature. 2013

THE CLOSTRIDIUM CONNECTION!continued

  T cells from NMO patients recognize an AQP4 epitope with high homology to C. perfringens ABC transporter permease.

  PhyloChip analysis of NMO (n=16), MS (n=16) and CTRLS (n=15) revealed enrichment of C. perfringens in demyelinating disorders

Varrin-Doyer et al. Ann Neurol. 2012Cree BA, et al. Ann. Neurol. 2016

p = 1.37 x 10-4

p = 0.044

Autologous stimulation of PMBC with MS microbiota results in impaired Treg

differentiation

*

Egle Cekanaviciute

Microbiota transfer into germ-free mice

  Microbiota from 3 pairs of subjects (MS:CTRL) was transferred to germ-free mice (n=8 per group)

  Mice were immunized with MOG35-55 or CFA

  Disease scores were recorded over 30 days

Population-based microbiome analysis

•  ~150 MS patients (50% untreated) and household controls•  Collected stool via overnight mail•  Purified DNA•  Sequenced 16S rRNA gene

No global differences in microbiota between MS and CTRLs

PC1 (9.55%)

PC3 (4.13%)

PC2 (5.65%)

Control MS

A B C

Significant differences in relative abundance of several OTUs

P = 0.05

Significant differences in relative abundance of several OTUs

P = 0.05

Control MS0.0000

0.0002

0.0004

0.02

0.04

0.06

0.08

0.10

Acinetobacter

*

Control MS0.001

0.01

0.1

1

Akkermansia

*

Control MS0.001

0.01

0.1

1 Parabacteroides

*

A. calcoaceticus inhibits Treg differentiation in vitro

A BNo bacteria A.calc

CD25

FoxP

3

C D

A. calc

No bact.No bacteria A.calc

1.5

2.0

2.5

3.0

3.5

4.0

TG

F-b

eta1

, ng

/ml *

*

No bacteria A.calc0

10

20

30

40

50

CD25+ FOXP3+ ,%CD3+ CD4+

**

A. calcoaceticus stimulates Th1 and Th2 differentiation in vitro

AB

No bacteria A.calc

IFNg

IL4

No bacteria A.calc0

10

20

30

40

IL4+, %CD3+ CD4+

***

No bacteria A.calc0

1

2

3

4

IFNg+, %CD3+ CD4+

**

A. calcNo bacteriaC

E Acin.No Acin.

D

IL4

GATA3

FSC-A

FSC-A

No bacteria A. calc0

10

20

30

40

GATA3+, % CD3+ CD4+

**

No Acin. Acin.0

10

20

30

IL4+, % CD3+ CD4+

*F

A. muciniphila stimulates Th1 differentiation in vitro

A. muciNo A.muci

A

C

No bacteria A.muci

FSC-A

IFN

g

D

IFN

g

FSC-A

No bacteria A.muci0

5

10

15

IFNg+, %CD3+ CD4+

*

No A.muci A.muci0

5

10

15

IFNg, %CD3CD4

*

B

No bacteria P.dist0

20

40

60

IL10+, %CD3+ CD4+ CD25+

**

P. distasonis promotes IL-10+ Treg differentiation in vitro

No bacteria P.dist Own bacteria0

10

20

30

40 CD4+ CD25+, %CD3+

***

CD4

CD

25

No bacteria P. dist Own bacteria

A B C

D E

IL10

FSC-A

No bacteria P.dist

F

Mice respond differently to MS gut bacteria

Sarkis Mazmanian (Caltech)

The iMSMS

An International Collaboration

The goal

To identify gut microbes associated with MS

How?

•  Humans

•  Sequence bacterial DNA to characterize over/under represented communities in MS

•  Germ-free mice

•  Test causality of MS associated microbes

Acknowledgements

UCSFEgle Cekanaviciute

Ann ThomannTessel Runia

Daniel HimmelsteinBruce Cree

Scott ZamvilLiz Crabtree

Lothith MadireddyRachel Kanner

Sneha SinghCuquita GomezJorge Oksenberg

Stephen L Hauser Funding :

MSMC & iMSMSRob Knight (UCSD)

Sarkis Mazmanian (Caltech)Patrizia Casaccia (Mt Sinai)

Howard Weiner (B&W)Dennis Kasper (Harvard)Jorge Correale (FLENI)

Hartmut Wekerle (Max Planck)Siddharthan Chandran (Edinburgh)