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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
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Neuro-gastroenterology an emerging field of research!
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1!
10!
100!
1000!
10000!
2000! 2001! 2002! 2003! 2004! 2005! 2006! 2007! 2008! 2009! 2010! 2011! 2012! 2013! 2014! 2015!
# of
publ
icat
ions!
Year!
microbiome or microbiota!
in multiple sclerosis!
Autism!
Epilepsy!
Stroke!
Alzheimer!
Parkinson!
dementia!
Literature review (up to 2015)
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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
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THE ENTERIC NERVOUS SYSTEM
“Second brain”
400 million neurons (=spinal cord)
Extends from esophagus to anus
Controls digestive system
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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
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If we are our genes... then we are more our microbiome than our genome
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MICROBIOME VARIES ACCORDING TO ANATOMICAL LOCATION OF THE HUMAN
BODY
Lee et al. Science 2010 The human Microbiome Project Consortium. Nature 2012
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WHILE DIFFERENCES IN MICROBIAL TAXA EXIST, METABOLIC PATHWAYS REMAIN CONSTANT
The human Microbiome Project Consortium. Nature 2012
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MICROBIAL COMMUNITIES AND THEIR PATHWAYS ARE ASSOCIATED WITH ETHNICITY
The human Microbiome Project Consortium. Nature 2012
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MICROBIOME CORRELATES WITH DIET AND TAXONOMY
HerbivoresCarnivoresOmnivores Ley et al. Science 2008
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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
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THE HUMAN MICROBIOME HELPS BALANCE IMMUNE RESPONSES
Lee and Mazmanian. Science 2010
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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
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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
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CROSSTALK BETWEEN MICROBIOTA AND THE IMMUNE SYSTEM
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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
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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
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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
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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
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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
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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
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• 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
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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
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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
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Autologous stimulation of PMBC with MS microbiota results in impaired Treg
differentiation
*
Egle Cekanaviciute
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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
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Population-based microbiome analysis
• ~150 MS patients (50% untreated) and household controls• Collected stool via overnight mail• Purified DNA• Sequenced 16S rRNA gene
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No global differences in microbiota between MS and CTRLs
PC1 (9.55%)
PC3 (4.13%)
PC2 (5.65%)
Control MS
A B C
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Significant differences in relative abundance of several OTUs
P = 0.05
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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
*
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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+
**
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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
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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
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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
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Mice respond differently to MS gut bacteria
Sarkis Mazmanian (Caltech)
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The iMSMS
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An International Collaboration
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The goal
To identify gut microbes associated with MS
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How?
• Humans
• Sequence bacterial DNA to characterize over/under represented communities in MS
• Germ-free mice
• Test causality of MS associated microbes
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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)